Beneito

DEPARTAMENT DE QUIMICA ANALITICA
DESARROLLO DE MTODOS ANALTICOS PARA EL CONTROL DE CALIDAD DE SURFACTANTES Y ADITIVOS EN PRODUCTOS DE LIMPIEZA Y DE HIGIENE PERSONAL.
MIRIAM BENEITO CAMBRA
UNIVERSITAT DE VALNCIA Servei de Publicacions 2011
Aquesta Tesi Doctoral va ser presentada a Valncia el dia 28 doctubre de 2011 davant un tribunal format per: Dr. Michael Lmmerhofer Dra. Coral Barbas Arribas Dr. Alain Berthod Dr. Jos Javier Laserna Vzquez Dra. Mara Celia Garca lvarez-Coque
Va ser dirigida per: Dr. Guillermo Ramis Ramos Dr. Jos Manuel Herrero Martnez
Copyright: Servei de Publicacions Miriam Beneito Cambra
I.S.B.N.: 978-84-370-8800-6 Edita: Universitat de Valncia Servei de Publicacions C/ Arts Grfiques, 13 baix 46010 Valncia Spain Telfon:(0034)963864115
FACULTAT DE QUMICA DEPARTAMENT DE QUMICA ANALTICA
DESARROLLO DE MTODOS ANALTICOS PARA EL CONTROL DE CALIDAD DE SURFACTANTES Y ADITIVOS EN PRODUCTOS DE LIMPIEZA Y DE HIGIENE PERSONAL
Memoria para alcanzar el grado de Doctor (Doctorado Europeo) presentada por: Miriam Beneito Cambra
Directores: Dr. Guillermo Ramis Ramos Dr. Jos Manuel Herrero Martnez Valencia, 2011
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D. Guillermo Ramis Ramos, catedrtico del Departamento de Qumica Analtica de la Universidad de Valencia y D. Jos Manuel Herrero Martnez, profesor titular del mismo departamento,
Certifican
Que la presente memoria, que lleva por ttulo Desarrollo de mtodos analticos para el control de calidad de surfactantes y aditivos en productos de limpieza y de higiene personal constituye la Tesis Doctoral de Da. Miriam Beneito Cambra.
Asimismo, certifican haber dirigido y supervisado tanto los distintos aspectos del trabajo, como su redaccin.
Burjassot, Junio de 2011
Guillermo Ramis Ramos
Jos Manuel Herrero Martnez
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IV
Esta tesis se ha realizado gracias a una beca V-Segles-Empresa entre la Universitat de Valncia y Qumicas Oro
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A mi familia
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AGRADECIMIENTOS
La realizacin de esta Tesis Doctoral no habr sido posible sin la ayuda y el apoyo de un gran nmero de personas: En primer lugar, quisiera agradecer a mis Directores, el Dr. Guillermo Ramis Ramos y al Dr. Jos Manuel Herrero Martnez, por su ayuda y dedicacin. Su confianza, apoyo y paciencia han sido fundamentales para poder llevar a cabo este proyecto. A si mismo, quisiera dar las gracias al Dr. Ernesto F. Sim Alfonso, por su contribucin al desarrollo de mi trabajo y por haber estado dispuesto a apoyarme cuando lo he necesitado. Tambin quisiera agradecer a Da. Marisa Caellas y D. Miguel ngel Chamorro de Qumicas Oro por esta oportunidad que se me ha brindado y la paciencia durante estos aos. Cmo no tambin agradecer a todos aquellos que, de un modo u otro hicieron posible mi estancia en la Universidad de Viena, y principalmente al Dr. Wolfgang Lindner y Dr. Michael Lmmerhofer, DANKE SCHN. Por otro lado, agradecer a mis compaeros del Laboratorio 10, con los que he compartido tantas cosas. cosas buenas, otras no tan buenas, agobios, preocupaciones, trabajo y momentos de diversin (os acordis de Keops?): Aarn, Alex, Amparo, Anna, Claudia, Cristina, Elisa, Enrique, Hugo, Isabel, Ivn, Laura, Mara N., Mara V., Pascuali, Patty, Pietro, Roberto, Sandra, Tamara, Victoria, Virginia y Yanelis. A la Dra. M Jess (MariJesu!!!!! Que ja casi ho tinc, no masfixies!!! Moltes grcies per tot, pels consells, xarradetes i espentes quan fan falta). Todos y cada uno, estis muy presentes, muchas gracias por todo. A todos los dems compaeros y profesores del Departamento, con los
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que ha sido una verdadera satisfaccin relacionarme y trabajar. Tambin dar las gracias a toda la gente con la que compart muchos momentos en Viena, los compaeros de laboratorio y otra gente que estuvo ah, en especial al Dr. Xavier Subirats, moltes grcies per tot, el teu suport, nim i consells son molt valuosos per a mi. Tambin agradecer a mis amigos, los de siempre: Amara, Amparo, Enrique, John, Jose (Teuler), M Mar, Montse, Oscar, Rafa, Ramn, Rebeca, Vicente y sin olvidar las ltimas incorporaciones (Aharn, Alexis y Nayara), muchas gracias por estar siempre ah, apoyarme y motivarme para seguir hacia delante. A todos los amigos de la facultad, que tantas horas hemos compartido en los pasillos, clases, cafetera y por ah, gracias por entenderme, apoyarme y aguantarme. Dar las gracias tambin a Pilar y Rosa, que tantos aos compartiendo el mismo piso, al final une mucho, muchas gracias por todos aquellos aos y por continuar manteniendo esa amistad. Y como no, agradecer tambin a mi familia: mis abuelos, tos y primos, por demostrar siempre tanto inters en mi trabajo y por estar ah, y en especial a mis padres, quienes siempre han estado a mi lado y me han apoyado en los momentos difciles, muchas gracias por vuestra ayuda incondicional en todos los aspectos. Y como no, a mi to Batiste, tu no podies faltar, grcies per tot, ja veus, tenim algo positiu. A todos vosotros, y a los que aunque no haya nombrado han sido partcipes de que todo haya llegado a buen fin, GRACIAS.
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NDICE
Captulo I Introduccin.... I.1. Detergentes.... I.1.1. Introduccin...... I.1.2. Componentes en las formulaciones de los productos de limpieza....... I.2. Surfactantes.... I.2.1. Introduccin.. I.2.2. Clasificacin de los surfactantes... I.2.3. APGs. I.2.4. Alcoholes no-etoxilados y etoxilados... I.2.5. AESs.. I.3. Polmeros... I.3.1. Introduccin.. I.3.2. Polmeros en detergentes.. I.3.3. PVP-NO.... I.3.4. PVP I.3.5. PVA... I.3.6. Mtodos de anlisis de polmeros..... I.4. Enzimas.. I.4.1. Introduccin.. I.4.2. Proteasas.... I.4.3. Amilasas.... I.4.4. Lipasas...
xx1 xx3 xx3
xx3 x12 x12 x14 x20 x25 x29 x31 x31 x34 x36 x37 x37 x38 x41 x41 x43 x45 x46
XII
I.4.5. Celulasas........ I.5. Tcnicas analticas. I.5.1. LC.. I.5.1.1. Medida de parmetros cromatogrficos.... I.5.2. CE.. I.5.2.1. Mecanismo de separacin en CE... I.5.2.2. EOF I.5.2.3. Movilidad y tiempo de migracin.. I.5.2.4. Tcnicas de electroseparacin capilar.. I.5.3. CEC... I.5.3.1. Instrumentacin en CEC... I.5.3.2. Columnas empleadas en CEC... I.5.3.3. Polimerizacin de columnas monolticas basadas en steres de metacrilato y acrilato.. I.5.3.4. Caracterizacin de materiales monolticos... I.5.4. MS. I.5.4.1. Fuentes de iones. I.5.4.2. Analizadores de masas.. I.5.4.3. Acoplamiento de una tcnica de separacin a un MS... I.5.4.4. Resolucin. I.5.5. NMR.. I.5.5.1. Espectrmetro de NMR.. I.5.5.2. NMR de 1H. I.5.5.3. NMR de 13C....
x47 x49 x49 x52 x56 x56 x57 x58 x59 x60 x60 x62
x64 x65 x66 x68 x70
x72 x73 x73 x75 x76 x78
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I.5.6. Algunas tcnicas de tratamiento estadstico de datos I.5.6.1. Anlisis clasificatorio supervisado....... I.5.6.2. LDA...
x79 x79 x80
Captulo II Objetivos y plan de trabajo..... II.1. Surfactantes.. II.1.1. APGs... II.1.2. Alcoholes. II.1.3. AES.. II.2. Polmeros.. II.2.1. PVP-NO... II.2.2. PVP.. II.2.3. PVA. II.3. Enzimas.... II.3.1. CZE.. II.3.2. Aminocidos por infusin directa en MS.... II.3.3. Aminocidos derivatizados con OPA-NAC.... II.3.4. Digestos con tripsina II.4. Columnas monolticas.. II.4.1. Columnas monolticas de LM fotopolimerizadas usando LPO como iniciador..... II.4.2. Comparacin de iniciadores para la preparacin de columnas monolticas de metacrilato para CEC.....
x83 x85 x85 x86 x87 x87 x87 x88 x88 x89 x89 x89 x90 x90 x91
x91
x91
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Captulo III Materiales y mtodos.................................................. III.1. Surfactantes. III.1.1. APGs... III.1.1.1. Estndares, materias primas y otras muestras... III.1.1.2. Reactivos de uso general. III.1.1.3. Instrumentacin y condiciones de trabajo.. III.1.1.4. Preparacin de estndares y muestras... III.1.2. Alcoholes.... III.1.2.1. Estndares, materias primas y otras muestras... III.1.2.2. Reactivos de uso general. III.1.2.3. Instrumentacin y condiciones de trabajo.. III.1.2.4. Preparacin de estndares y muestras... III.1.3. AESs... III.1.3.1. Estndares, materias primas y otras muestras... III.1.3.2. Reactivos de uso general..... III.1.3.3. Instrumentacin y condiciones de trabajo.. III.1.3.4. Preparacin de estndares y muestras... III.2. Polmeros sintticos III.2.1. PVP-NO.. III.2.1.1. Estndares, materias primas y otras muestras... III.2.1.2. Reactivos de uso general. III.2.1.3. Instrumentacin y condiciones de trabajo.. III.2.1.4. Preparacin de estndares y muestras... III.2.2. PVP. III.2.2.1. Estndares, materias primas y otras muestras...
x93 x95 x95 x95 x95 x95 x96 x97 x97 x98 x98 x99 101 101 101 102 103 106 106 106 107 107 108 108 108
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III.2.2.2. Reactivos de uso general. III.2.2.3. Instrumentacin y condiciones de trabajo.. III.2.2.4. Preparacin de estndares y muestras... III.2.3. PVA.... III.2.3.1. Estndares, materias primas y otras muestras... III.2.3.2. Reactivos de uso general. III.2.3.3. Instrumentacin y condiciones de trabajo.. III.2.3.4. Preparacin de estndares y muestras... III.3. Enzimas... III.3.1. CZE. III.3.1.1. Estndares, materias primas y otras muestras... III.3.1.2. Reactivos de uso general. III.3.1.3. Instrumentacin y condiciones de trabajo.. III.3.1.4. Preparacin de estndares y muestras... III.3.2. Aminocidos por infusin directa en MS.. III.3.2.1. Estndares, materias primas y otras muestras... III.3.2.2. Reactivos de uso general.... III.3.2.3. Instrumentacin y condiciones de trabajo.. III.3.2.4. Preparacin de estndares y muestras... III.3.2.5. Construccin de las matrices de entrenamiento y evaluacin...... III.3.2.6. Seleccin de variables predictoras para la construccin de un modelo de LDA..... III.3.3. Aminocidos derivatizados con OPA-NAC... III.3.3.1. Estndares, materias primas y otras muestras... III.3.3.2. Reactivos de uso general....
108 109 110 111 111 111 111 114 114 114 114 116 116 117 118 118 118 119 119
121
122 123 123 124
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III.3.3.3. Instrumentacin y condiciones de trabajo.. III.3.3.4. Preparacin de estndares y muestras... III.3.4. Digestos con tripsina.. III.3.4.1. Estndares, materias primas y otras muestras... III.3.4.2. Reactivos de uso general.... III.3.4.3. Instrumentacin y condiciones de trabajo.. III.3.4.4. Preparacin de estndares y muestras... III.4. Columnas monolticas..... III.4.1. Monmeros, agentes entrelazantes, iniciadores, estndares y reactivos de uso general.... III.4.2. Tratamiento de columnas... III.4.2.1. Acondicionado de columnas... III.4.2.2. Preparacin de columnas monolticas... III.4.3. Instrumentacin y condiciones de trabajo..
124 125 126 126 126 126 127 128
128 129 129 130 132
Results.............. Chapter IV No ionic surfactants in cleaning products.. IV.1. APGs... IV.1.1. APGs by HPLC-MS... IV.1.1.1. Optimisation of working conditions using continuous infusion MS.... IV.1.1.2. Separation of APGs by HPLCMS in isocratic conditions...... IV.1.1.3. Separation of APGs by HPLCMS with gradient elution..... IV.1.1.4. Quantitation studies....
135 137 139 139
141
141
144 146
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IV.1.2. Fragmentation of D-Glucose and AMGs... IV.1.2.1. Optimization of the IT-MS detection conditions IV.1.2.2. Fragmentation of D-Glucose and their isotopic forms using MSn... IV.1.2.3. Fragmentation of AMGp using MSn...... IV.1.2.4. Fragmentation of AMGf using MSn....... IV.2. Non-ethoxylated and ethoxylated alcohols.... IV.2.1. Chromium(VI) oxide oxidation of non-ethoxylated and ethoxylated alcohols for determination by ESI-MS. IV.2.1.1. Optimization of the infusion medium.. IV.2.1.2. Relative sensitivity of carboxylic and ethoxy-carboxylic acids as a function of n and m.... IV.2.1.3. Oxidation yields of primary non-ethoxylated alcohols and extraction recoveries of the corresponding carboxylic acids.... IV.2.1.4. Influence of water concentration on the oxidation yield... IV.2.1.5. Oxidation yields of ethoxylated alcohols.... IV.2.1.6. Application to industrial and environmental samples..
150 152
153 157 159 162
162 164
166
168
169 171
173
Chapter V Anionic surfactants.... V.1. AESs..... V.1.1. Determination of FAEs and AESs by SAX separation, derivatization with a cyclic anhydride and HPLC... V.1.1.1. Derivatization and HPLC separation of the derivatives... V.1.1.2. Optimization of the SAX separation of the surfactant classes....
179 181
181
184
186
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V.1.1.3. Calibration studies... V.1.1.4. Application to industrial raw materials and commercial products. V.1.1.5. Seawater analysis.....
190
193 194
Chapter VI Synthetic polymers... IV.1. PVP-NO...... VI.1.1. Characterization of PVP-NO by FSCE and MEKC... VI.1.1.1. FSCE studies in the absence and presence of PEA. VI.1.1.2. Discussion on the results obtained by FSCE.. VI.1.1.3. MEKC studies in the absence and presence of PEA... VI.1.1.4. Discussion on the results obtained by MEKC. VI.1.1.5. Quantitation studies and application to real samples using FSCE.. VI.2. PVP..... VI.2.1. Characterization and determination of PVP by complexation with an anionic azo-dye and NECEEM.. VI.2.1.1. Electropherograms of PVP in the presence of anionic azo-dyes...... VI.2.1.2. Formation rate of the PVPCR complexes. VI.2.1.3. Application of NECEEM principles to the interpretation of the electropherograms of PVPdye mixtures.......................................... VI.2.1.4. Influence of MW on the location and shape of the peak of the PVPdye complexes... VI.2.1.5. Determination of the maximal stoichiometry of the PVPdye complexes......
197 199 199 202 206
208 210
215 217
217
219 222
223
226
228
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VI.2.1.6. Influence of MW on the mobility of the PVPCR complexes at low q values..... VI.2.1.7. Determination of PVPdye stability constants....... VI.2.1.8. Analytical applications.... VI.3. PVA..... VI.3.1. Evaluation of MW and tacticity of PVA by NECEEM of a polymer and a dye... VI.3.1.1. Effect of PVA on the UVVis absorption spectrum of CR..... VI.3.1.2. Selection of working conditions...... VI.3.1.3. Maximal stoichiometry of the PVACR complex and its relationship with log MW and tacticity.... VI.3.1.4. Structure of the complex..... VI.3.1.5. Influence of Mw and tacticity on the electrophoretic mobility of the complex. VI.3.1.6. Stability and dissociation rate constants of the complex.....
231 233 235 239
239
241 242
244 247
250
254
Chapter VII Enzymes... VII.1. Intact enzymes by CE... VII.1.1. Identification of enzymes for the detergent industry by CZE of the intact proteins... VII.1.1.1. CZE with a basic BGE... VII.1.1.2. CZE with an acid BGE.. VII.1.1.3. Relative sensitivities and application to enzyme identification. VII.1.1.4. Influence of surfactants commonly used in the formulation of cleaning products...
259 261
261 263 264
266
267
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VII.2. Classification of enzymes by MS.......................... VII.2.1. Rapid classification of enzymes in cleaning products by hydrolysis, MS and LDA. VII.2.1.1. Mass spectra and normalization of the variables.. VII.2.1.2. Construction of LDA models.. VII.2.1.3. Evaluation of the prediction capability of the optimal LDA model.. VII.3. Classification of enzymes by HPLC-UV-Vis.... VII.3.1. Enzyme class identification in cleaning products by hydrolysis followed by derivatization with o-phthaldialdehyde,HPLC and LDA..... VII.3.1.1. Optimization of OPANAC derivatization and chromatographic conditions.. VII.3.1.2. Data matrices and construction and evaluation of the LDA models. VII.4. Tryptic digests of enzymes, columns comparation... VII.4.1. Comparison of microparticulate and monolithic columns for RP-LC of tryptic digests of industrial enzymes in cleaning products.... VII.4.1.1. Study of intact enzymes and tryptic digests using a ProSwift polymeric monolithic column.... VII.4.1.2. Study of tryptic digests of enzymes using microparticulate and silica monolithic columns.... VII.4.1.3. Influence of matrixes commonly used in Cleaning product formulations...
269
269 271 272
276 276
276
278
280 284
284
287
290
295
Chapter VIII Monolithic columns... VIII.1. Monolithic columns of LM..... VIII.1.1. Photo-polymerized LMA monolithic columns for CEC using LPO as initiator...
297 299
299
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VIII.1.1.1. Preparation and characterization of columns photo-initiated with LPO.... VIII.1.1.2. Comparison of LPO and AIBN as initiators for photo-polymerization.. VIII.2. Comparison of photo-initiators for methacrylate monolithic columns. VIII.2.1. Comparison on photo-initiators for the preparation of methacrylate monolithic columns for CEC..... VIII.2.1.1. Preparation and characterization of LM columns photo-initiated with AIBN or DMPA.. VIII.2.1.2. Preparation and characterization of LMA columns photo-initiated with BPO or LPO...
301
311
316
316
317
324
Captulo IX Conclusiones generales... IX.1. Surfactantes. IX.1.1. APGs.. IX.1.1.1. Separacin y determinacin de APGs por LC con deteccin ESI-MS.... IX.1.1.2. Estudio de fragmentacin de la D-Glucosa y los AMG en presencia de iones de sodio en IT-MS.... IX.1.2. Alcoholes.... IX.1.2.1. Oxidacin de alcoholes no etoxilados y etoxilado con cromo(VI) y posterior determinacin por ESI-MS..... IX.1.3. AES.... IX.1.3.1. Determinacin de FAEs y AESs por separacin mediante SAX, derivatizacin con un anhdrido cclico y LC..
329 331 331
331
333 335
335 341
341
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IX.2. Polmeros sintticos.... IX.2.1. PVP-NO. IX.2.1.1. Caracterizacin de PVP-NO por CE y MECK... IX.2.2. PVP. IX.2.2.1. Caracterizacin y determinacin de PVP por complejacin con un azo-colorante aninico seguida de NEECEM.... IX.2.3. PVA.... IX.2.3.1. Evaluacin de la MW y tacticidad del PVA mediante NEECEM de mezclas polmero-colorante... IX.3. Enzimas.. IX.3.1. CZE.... IX.3.1.1. Identificacin de enzimas de la industria de la detergencia por CZE de enzimas intactas.... IX.3.2. Aminocidos por infusin directa en MS.. IX.3.2.1. Clasificacin rpida de enzimas en productos de limpieza por hidlisis aminocidos, MS y LDA. IX.3.3. Aminocidos derivatizados con OPA-NAC.. IX.3.3.1. Identificacin de la clase de las enzimas presentes en los productos de limpieza mediante hidrlisis seguida de derivatizacin con OPA-NAC, HPLC y LDA. IX.3.4. Digestos con tripsina.. IX.3.4.1. Comparacin de columnas microparticuladas y monolticas para RP-LC de digestos trpticos de enzimas industriales en productos de limpieza...
344 344 344 347
347 350
350 354 354
354 356
356 357
357 359
359
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IX.4. Columnas monolticas. IX.4.1. Columnas monolticas de LMA para CEC.... IX.4.1.1. Columnas monolticas de LMA para CEC utilizando LPO como iniciador....... IX.4.1.2. Comparacin de fotoiniciadores para la preparacin de columnas monolticas de metacrilato para CE.......................................
362 362
362
364
Captulo X Referencias.
367
Abreviaturas.
399
Anexo I Separation and determination of alkylglycosides by liquid cromatography with electrospray mass spectrometric detection.
xA3
Anexo II Study of the fragmentation of D-Glucose and Alkylmonoglycosides in the presence of sodium ions in an Ion-Trap Mass Spectrometer.......................................
A13
Anexo III Chromium(VI) oxide oxidation of non-ethoxilated and ethoxilated alcohols for determination by electrospray ionization mass spectrometry..
A31
Anexo IV Characterization of poly(4-vinylpyridine-1-oxide) by free-solution capillary electrophoresis and micellar electrokinetic chromatography..................................
A41
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Anexo V Characterization and determination of poly(vinylpyrrolidone) by complexation with an anionic azo-dye and nonequilibrium capillary electrophoresis....
A51
Anexo VI Evaluation of molecular mass and tacticity of polyvinyl alcohol by non-equilibrium capillary electrophoresis of polymer-dye equilibrium mixtures..
A61
Anexo VII Rapid classification of enzymes in cleaning products by hydrolysis, mass spectrometry and linear discriminant analysis.... A71
Anexo VIII Enzyme class identification in cleaning products by hydrolysis followed by derivatization with o-phthaldialdehyde, HPLC and linear discriminant analysis...
A79
Anexo IX Photo-polymerized lauryl methacrylate monolithic columns for CEC using lauroyl peroxide as initiator....
A87
Anexo X Comparison on photo-initiators for the preparation of methacrylate monolithic columns for capilary electrochromatography.....................................
A99
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CAPTULO I
INTRODUCCIN
Captulo I. Introduccin
I.1. Detergentes
I.1.1. Introduccin El jabn es conocido desde las culturas antiguas (egipcios y sumerios), siendo usado tanto para lavar la ropa como para el aseo personal. Los jabones se obtenan mediante saponificacin de grasas vegetales o animales con cenizas vegetales o minerales como la sosa custica. Hasta el siglo XV, uno de los principales ncleos de vida social en las ciudades europeas eran los baos pblicos. En los siglos posteriores, los baos fueron considerados inmorales y el jabn pas a ser algo a evitar. Se vesta la misma ropa durante semanas y los malos olores se tapaban con perfumes. En Europa, el jabn no volvi a apreciarse hasta bien entrado el siglo XVIII, cuando los mdicos se dieron cuenta de la importancia de la higiene para la salud. Por otro lado, la industrializacin y las importaciones de grasas baratas facilitaron la fabricacin de jabones a gran escala
[Ramrez-Corrales, 2006].
Los detergentes (en latn, detergere significa limpiar) son mezclas de diferentes sustancias cuya finalidad es disolver la suciedad o las impurezas de un objeto sin corroerlo. En 1907, una compaa alemana fabric el primer detergente al aadir sales sdicas de perborato, silicato y carbonato al jabn tradicional
[www.ambientum.com].
A partir de 1930 se empezaron a sintetizar
detergentes derivados de la industria del petrleo. Posteriormente se descubrieron otros ingredientes que daban al conjunto una mayor capacidad limpiadora.
I.1.2. Componentes en las formulaciones de productos de limpieza Para lograr su papel limpiador, un detergente debe producir numerosos fenmenos, los cuales dependen del tipo de sustrato y de las condiciones de lavado. As, se han diseado frmulas especficas capaces de actuar con
Miriam Beneito Cambra
eficiencia en casos particulares, y frmulas generales con resultados ms o menos satisfactorios en la mayora de los casos. En la composicin qumica de productos de limpieza, adems de los surfactantes, que constituyen la principal materia activa de la formulacin, se encuentran aditivos de muy distintos tipos. La composicin difiere en funcin del uso industrial o domstico del producto. En las formulaciones existe un gran nmero de componentes cuyos papeles se complementan uno a otro, a menudo con un efecto de sinergia. A continuacin se comentan los diferentes tipos de componentes que se encuentran en formulaciones de detergentes, as como el papel que stos desempean.
A) Surfactantes Los surfactantes actan como agentes humectantes del sustrato, modifican la tensin superficial del agua y emulsionan las partculas de suciedad. En algunas aplicaciones se usan las propiedades bactericidas de ciertos surfactantes en formulaciones desinfectantes [Showell, 2006].
B) Agentes secuestrantes (builders) Estos agentes tienen como propsito mejorar la accin limpiadora del surfactante mediante varios efectos. Su principal accin es secuestrar los cationes divalentes del agua dura (calcio, magnesio) para evitar su interaccin con los surfactantes. La eliminacin se hace en forma soluble (quelato), por precipitacin, o por intercambio inico. Otra de las acciones de los builders es mantener el pH de la disolucin del detergente a un valor alcalino, neutralizar los cidos grasos libres y formar jabones in-situ en la interfase. Tambin aumentan el potencial (negativo) de superficie de los textiles y de las manchas, y por tanto, inhiben la redeposicin de la suciedad. Dentro de los agentes secuestrantes se encuentran los compuestos inorgnicos solubles, como el tri(poli)fosfato
(Na5P3O10) y el pirofosfato (Na4P2O7) sdicos, as como los silicatos (xNa2O ySiO2) y el carbonato sdico (Na2CO3). Por su parte, los compuestos inorgnicos insolubles, como las zeolitas (alumino-silicatos naturales o sintticos), como el aluminosilicato sdico (Na2O Al3O3 2SiO2 xH2O) eliminan los cationes divalentes por intercambio inico [Showell, 2006].
C) Jabones como agentes dispersantes de calcio Muchas formulaciones detergentes lquidas y en polvo para lavadoras contienen sales alcalinas de cidos grasos, es decir jabones, cuyo papel es reducir la formacin de espuma. Los jabones tienen excelentes propiedades limpiadoras, son seguros y fcilmente biodegradables; sin embargo, son muy sensibles a los cationes divalentes, especialmente al calcio, con el cual producen sales insolubles en agua. Los agentes dispersantes de jabones de calcio son surfactantes aninicos o anfteros que se co-micelizan con los jabones para formar disoluciones que no precipitan en aguas duras [Salager, 1988].
D) Agentes antirredeposicin Estas sustancias impiden que las suciedades separadas de los tejidos durante el lavado vuelvan a depositarse sobre los mismos. Los agentes antirredeposicin ms utilizados son la carboximetilcelulosa, y otros derivados no inicos de la celulosa. Comercialmente tambin se utilizan polmeros sintticos como PVP, PVA y algunos copolmeros de stos [Salager, 1988].
E) Agentes espumantes y no espumantes Contrariamente a la creencia general, la produccin de espuma no tiene nada que ver con el poder detergente. En ciertos usos, como champs o detergentes para las manos, la generacin de grandes volmenes de espuma es un
factor deseable. Son muchos los surfactantes capaces de generar y mantener la espuma en ausencia de suciedad, sin embargo, sta fcilmente desaparece en presencia de suciedad, especialmente en presencia de grasa. Para mantener la espuma a lo largo del uso del detergente se aaden agentes espumantes como el lauril sulfato sdico, y surfactantes no inicos nitrogenados
Showell, 2006]. [Salager, 1988;
Sin embargo, en otras aplicaciones, la generacin de espuma es un
inconveniente. Por ejemplo, en los lavavajillas, un elevado volumen de espuma puede interferir en la rotacin del brazo, provocando una degradacin del rendimiento. En estos casos se utilizan agentes no espumantes y antiespumantes, que evitan la formacin o aceleran la desaparicin de la espuma. Entre los agentes utilizados para el control de la espuma suelen emplearse surfactantes etoxilados no inicos y jabones, y antiespumantes compuestos por partculas de slice coloidal en suspensin, junto con aceites de silicona (polidimetil siloxano)
[Salager, 1988; Showell, 2006].
F) Agentes blanqueadores La obtencin de blancura en textiles es quizs una de las propiedades ms importantes para el consumidor. En el mercado existen dos tipos de agentes blanqueadores para textiles, ambos con propiedades oxidantes: los hipohaluros, como el hipoclorito de sodio, y las sales inorgnicas peroxigenadas, como el perborato de sodio y el perxido de sodio estabilizado con carbonato sdico (percarbonato). Los agentes blanqueadores oxidantes deben ser intrnsecamente inestables para cumplir con su funcin (oxidar), paradjicamente, al mismo tiempo deben ser estables cuando estn almacenados, solos o en la mezcla detergente. El hipoclorito es un agente blanqueador ms activo y agresivo que el perborato, siendo particularmente eficaz en la oxidacin de sustancias que contienen nitrgeno. Adems de poseer una accin blanqueadora (an a baja
temperatura), es un efectivo bactericida. Por su accin sobre las sustancias nitrogenadas no puede conservarse en forma de polvo o de lquido en formulaciones detergentes que contengan sales de amonio, aminas, amidas, etc. Por ello se suele emplear como un ingrediente aparte. Respecto a las sales inorgnicas preoxigenadas, se usa el perborato de sodio tetrahidrato (NaBO34H2O), el percarbonato o carbonato de sodio peroxihidrato
(2Na2CO33H2O2), y el peroximonosulfato de potasio (KHSO5). El perborato y otras sales semejantes exhiben una accin blanqueadora menos agresiva que el hipoclorito, y son slidos compatibles con la gran mayora de los componentes de los detergentes en polvo [Salager, 1988; Showell, 2006].
G) Blanqueantes pticos El grado de blancura obtenido mediante agentes blanqueadores se puede exaltar mediane el blanqueado ptico. Los blanqueadores pticos son colorantes orgnicos poliaromticos que absorben luz ultravioleta y emiten una luz azulada en el visible mediante fluorescencia. A la luz del sol, aaden un tono azulado que compensa el tono amarillento de los tejidos, por lo que mejora la blancura o profundidad de los colores. Segn la funcin a la que se destinen, poseen grupos polares ms o menos importantes para adsorberse sobre fibras hidroflicas como el algodn, o hidrfobas como el polister. Se han desarrollado agentes fluorescentes solubles en agua y resistentes al hipoclorito u otros blanqueadores, gracias a que los tomos de nitrgeno de dichas sustancias estn situados en estructuras altamente aromticas de tipo benzotriazol o benzofurano
1988]. [Salager,
H) Suavizantes Despus de un lavado con detergentes sintticos formulados con agentes secuestrantes, el textil seco presenta una superficie que no es agradable al contacto con la piel. El residuo de los surfactantes sintticos adsorbidos aumenta la carga esttica de las fibras, y la ausencia de sustancias con accin lubricante vuelve al textil relativamente rgido. Los agentes suavizantes contrarrestan ambos fenmenos, ya que por una parte reducen la carga esttica, y por otra depositan una capa lubricante. Muchos surfactantes catinicos producen estos efectos, pero son incompatibles con los surfactantes aninicos utilizados en la mayora de las formulaciones, por lo que deben usarse por separado. Los mejores suavizantes de este tipo son las sales de alquil amonio cuaternario y de imidazolio. Existe una tendencia hacia la produccin de formulaciones que contengan agentes suavizantes compatibles con los agentes limpiadores. Estos suavizantes son surfactantes con cierto carcter catinico que se absorben sobre las fibras textiles, siendo a la vez compatibles con surfactantes aninicos comnmente empleados. Para este fin se usan surfactantes no inicos con un grupo nitrogenado, o tambin algunos surfactantes anfteros, normalmente conteniendo un grupo amido-amina (que tambin actan como dispersantes de jabones de calcio) [Salager, 1988; Showell, 2006].
I) Enzimas Se utilizan uso en las formulaciones de detergentes para eliminar las manchas, gracias a la rotura cataltica de componentes especficos de las mismas. Las proteasas, encargadas de la degradacin de las protenas, son las ms comunes en la mayora de los detergentes. Sin embargo, tambin se emplean otras enzimas como las amilasas (degradan el almidn), lipasas (degradan lpidos) y celulasas (degradan la celulosa). Estas enzimas son capaces de
degradar rpidamente manchas en un medio de pH alcalino y a temperaturas de hasta 60C. Estn diseadas para ser activas a temperatura ambiente, siendo particularmente utilizadas en detergentes lquidos [Salager, 1988; Showell, 2006].
J) Polmeros El uso de polmeros ha ido aumentando a medida que las formulaciones de detergentes han evolucionado. Los polmeros han venido a sustituir a los fosfatos que fueron cayendo en desuso a causa de su elevado impacto ambiental. Se han usado principalmente polmeros de tipo poliacrilato, con el fin de remplazar a agentes secuestrantes como el tripolifosfato de sodio. La carboximetilcelulosa fue tambin uno de los primeros polmeros utilizados para este fin. Los xitos obtenidos con los polmeros para otros propsitos ha impulsado la aparicin de un gran nmero de stos
[Zini, 1999].
Entre otros muchos usos, se han
comercializado nuevos dispersantes y polmeros que inhiben la transferencia de color entre los tejidos. Por cada polmero comercializado, existen otros muchos patentados, lo que pone de manifiesto el inters dentro de este rea [Showell, 2006;
Watson, 2006].
K) Espesantes A menudo es conveniente modificar la reologa, es decir, la consistencia de la formulacin del detergente en condiciones dinmicas, lo que tiene importantes consecuencias prcticas. As por ejemplo, los lavavajillas contienen espesantes que ayudan a mantener en suspensin el fosfato y otras sustancias que de otra forma quedaran separadas de la fase lquida. El espesado puede conseguirse a partir de electrolitos inorgnicos, como por ejemplo, NaCl, arcillas como la laponita o hectorita, o polmeros de un elevado peso molecular como la carboximetilcelulosa y las gomas de guar o xantana. Existen polmeros
particularmente efectivos como espesantes en las formulaciones de detergentes para uso domstico. Por ejemplo, los modificadores reolgicos de la serie Carbopol (Lubrizol) han demostrado ser excepcionales viscosantes, agentes de suspensin y estabilizadores en una gran variedad de productos de cuidado personal. La mayora de estos polmeros son homo- y copolmeros de cido acrlico de alta masa molecular, entrecruzados con un polialquenil politer
[Showell, 2006].
L) Perfumes En un sentido tcnico los perfumes no aaden un mayor poder detergente a los formulados, sin embargo, desde el punto de vista del consumidor representan un factor importante, ya que segn el aroma parece que el detergente funcione mejor. El olor es una caracterstica que debe ser cuidadosamente tratada en la formulacin de los detergentes para la aceptacin del producto por parte de los consumidores. Los perfumes son complejas mezclas de compuestos orgnicos, por ejemplo, el perfume de un detergente puede estar compuesto por 30, 50 o incluso ms de 100 compuestos. Esta compleja naturaleza puede dar lugar a complejas interacciones con el resto de componentes del detergente, que pueden afectar tanto al perfume como a las sustancias activas. En detergentes de uso domstico, particularmente para lavavajillas y desinfectantes, se incorporan perfumes, la mayora de los cuales son terpenos, cuyo esqueleto est compuesto de 2, 3 ms unidades del isopreno (2-metil-butadieno)
2006]. [Salager, 1988; Watson,
10
M) Disolventes La seleccin de los disolventes empleados en las formulaciones de detergentes depende de la naturaleza de las sustancias activas presentes en los formulados, de la aplicacin final del detergente y del factor econmico. El agua es el disolvente ms ampliamente empleado en muchas de las formulaciones de detergentes para uso domstico e industrial. Sin embargo, muchas de las sustancias activas comnmente empleadas en formulacin de detergentes presentan una limitada solubilidad en agua, por lo que se requiere la adicin de un co-solvente (como el etanol) o de un hidrtopo (como el cumeno sulfonato)
[Showell, 2006].
N) Hidrtopos En los detergentes lquidos, en ocasiones es necesario incluir hidrtopos para garantizar la estabilidad del detergente en un amplio intervalo de temperaturas. Los hidrtropos son sustancias hidroflicas con un grupo apolar, cuya funcin es aumentar la solubilidad de los surfactantes en formulaciones lquidas. Los hidrtropos no tienen propiedades surfactantes por ellos mismos, pero actan como co-solubilizadores a alta concentracin. Los ms utilizados son los sulfonatos de tolueno, etilbenceno y xileno [Salager, 1988; Showell, 2006].
O) Bactericidas Ciertas frmulas desinfectantes contienen bactericidas, los cuales pueden ser surfactantes anfteros que actan tambin como agentes dispersantes de jabones de calcio. Tambin se usan compuestos catinicos que presentan un efecto suavizante por sus propiedades antiestticas. Los desinfectantes pueden tambin contener productos clorados bactericidas y sustancias con propiedades desodorantes
[Salager, 1988].
En los detergentes lquidos, especialmente en los
11
ms diluidos, donde el agua constituye una gran parte del volumen total del producto, es normal que la formulacin incluya un agente antibacteriano (normalmente un bacteriosttico) que alargue el tiempo de vida del producto
[Watson, 2006].
P) Agentes anticorrosin En las formulaciones de detergentes tambin se pueden encontrar agentes anticorrosin, con el objetivo de proteger las partes metlicas de las mquinas o sistemas de lavado. Generalmente, se usa el silicato de sodio que adems posee un papel secundario como builder [Salager, 1988].
I.2. Surfactantes
I.2.1. Introduccin La palabra surfactante deriva de la contraccin de los trminos surfaceactive-agent (superficie-activo-agente) y engloba a una serie de especies qumicas capaces de modificar las propiedades de las interfases de los lquidos (acuoso o no acuoso) en los que estn presentes. Las propiedades caractersticas de estas molculas residen en su carcter anfiflico, esto es, de la presencia de una parte hidroflica y otra hidrofbica en la molcula. Los surfactantes se concentran en la superficie libre del lquido y en las interfases entre fases inmiscibles, disminuyendo la tensin superficial. El cambio en la tensin superficial afecta a la capacidad para formar emulsiones, a la mojabilidad, al poder dispersante, a la detergencia y a las propiedades solubilizantes. Los surfactantes poseen una estructura molecular tpica, esencialmente lineal y asimtrica, con dos zonas, una hidrofbica y la otra hidroflica. La parte hidrfobica es una cadena aliftica, lineal o ramificada, conteniendo en general
12
entre 10 y 18 tomos de carbono. En los productos naturales y en los de transformacin qumica predominan las cadenas no ramificadas, mientras que en los derivados del petrleo y los obtenidos por sntesis (usualmente a partir del carbn) existen multitud de cadenas ramificadas. Por su parte, el resto hidrfilo, determinante de la solubilidad en agua, puede ser un grupo polar de carcter cido tal como un grupo sulfato, sulfonato o carboxilato, o de carcter bsico como una amina, una sal de amonio cuaternario o el ion piridinio, aunque tambin puede ser un grupo polar no inico. Los surfactantes, ya sean naturales o sintticos, organizan el medio formando micelas y otras microestructuras, cambian la solubilidad de compuestos hidrofbicos y modifican el entorno de los constituyentes del medio. Sobre las propiedades surfactantes de un compuesto influyen, adems de la propia naturaleza del grupo hidrfilo, la situacin que ste ocupa en la molcula. En principio se puede distinguir: Posicin terminal: la estructura molecular es polar y totalmente asimtrica. El grupo hidrfilo puede estar unido directamente al hidrfobo, o entre ambos puede existir un resto de carcter aliftico o aromtico que posea cierto carcter hidrfilo. Si la cadena hidrfoba tiene una longitud adecuada, estas estructuras muestran bsicamente carcter detergente. Posicin central: el grupo hidrfilo se intercala en cualquier punto de la cadena hidrfoba, aunque si en ella existen puntos reactivos (enlaces dobles, grupos hidroxilo, etc.) tiende a ocupar esos lugares. Conforme el grupo hidrfilo est ms centrado en la cadena, ms mermada se encuentra la capacidad detergente del compuesto. Varios grupos hidrfilos: en una cadena pueden estar presentes varios grupos hidrfilos, lo que exalta notablemente la solubilidad del surfactante en agua. Sin embargo, esta estructura proporciona propiedades dispersantes al surfactante.
13
I.2.2. Clasificacin de los surfactantes Atendiendo a su carga, los surfactantes pueden dividirse en cuatro clases: aninicos, catinicos, no inicos y anfotricos
[Oldenhove de Guertechin, 1999].
Aunque todos los surfactantes en s mismos presentan sus particularidades, hay algunas caractersticas comunes a cada clase.
A) Aninicos Se caracterizan por tener un grupo hidrfilo cargado negativamente, es decir, el grupo hidrfilo tiene carcter cido y forma fcilmente un anin. Los ms antiguos y conocidos son los jabones. Los surfactantes aninicos son los ms utilizados y han sido considerados como el caballo de batalla en el mundo de los detergentes. En consecuencia, su produccin es elevada, siendo adems muy econmicos. Suelen distinguirse las siguientes familias: ABS, AS, AES, APES, AOS, alquil sulfonatos, -sulfonatos de cidos grasos (inicos y steres de alquilo), mono- y di-alquil sulfosuccinatos y sulfonatos derivados del petrleo (Fig. I.1). Los surfactantes aninicos son especialmente beneficiosos en cuanto a su accin limpiadora, que se apoya en el hecho de que muchas superficies se encuentran cargadas negativamente, por lo que no pueden quedarse adsorbidos sobre las mismas, impidiendo as la redeposicin de sustancias indeseables. En funcin de la naturaleza del grupo funcional que presenta la carga, muestran una resistencia variable hacia la hidrlisis. As por ejemplo, los sulfatos se hidrolizan con facilidad, mientras que los sulfonatos son muy estables. Algunos surfactantes aninicos, presentan la propiedad de generar fases acuosas viscosas, pudiendo ser empleados como espesantes. Una limitacin de los surfactantes aninicos es su tendencia a precipitar en presencia de iones calcio y magnesio, que abundan en las aguas duras, si bien, los AESs son mucho menos sensibles a los cationes
14
alcalinos que los ASs. Por otro lado, la baja solubilidad y las particulares propiedades de las interfaces de los precipitados de sulfato de magnesio son explotadas de forma positiva a la hora de optimizar el rendimiento de los detergentes.
CH3(CH2)m CH CH3(CH2)n n + m = 7 11
-
CH3(CH2)nCOO- Na+ n + 1 = 10 18 Jabones CH3(CH2)nOSO3 Na+ n + 1 = 12 18 AS
SO3 Na+
ABS CH3(CH2)n(OCH2CH2)mOSO3 Na+ AES

-
(OCH2CH2)mOSO3 Na+
CH3(CH2)nCH
CH(CH2)mSO3 Na+ AOS
m + n = 9 15 m = 1 8 R: cadena alqulica APES ROOCCH CH3(CH2)nSO3 Na+ Alquil sulfonatos

-
ROOCCH SO3 Na+ Alquil sulfosuccinatos
Fig. I.1. Estructura y nomenclatura de las principales familias de surfactantes aninicos.
B) Catinicos Los surfactantes catinicos tienen el grupo hidrfilo de carcter bsico. Suelen agruparse en derivados grasos de amida, amidoaminas, imidazolinas, derivados del petrleo, nitrilos cclicos alifticos, aromticos, compuestos no nitrogenados, polimricos catinicos y xidos de amina. En la Fig. I.2 se muestra la estructura y nomenclatura de las principales familias de surfactantes catinicos.
15

CH3 CH2NH+ CH3 n + 1 = 7 18 Sales de alquilbencildimetilamonio (CH2)nCH3 CH3 NH+ CH3 n + 1 = 7 18 Sales de alquilfenildimetilamonio (CH2)nCH3
CH3 N+ (CH2)nCH3 R1 N+ CH3 CH3 R1
CH3 N+ R2 CH3
n + 1 = 12 14 Sales de alquil piridinio
R1, R2: cadenas alqulicas Sales de alquil trimetil y dialquildimetil amonio
Fig. I.2. Estructura y nomenclatura de las principales familias de surfactantes catinicos.
Los surfactantes catinicos de importancia industrial son compuestos grasos nitrogenados y, especialmente, compuestos con nitrgeno cuaternario
[Wittcoff, 1987].
Son de poca utilidad en procesos de limpieza, ya que al presentar
la mayora de las superficies carga negativa, los cationes se retienen sobre ellas en lugar de solubilizar la suciedad adherida. Sin embargo, y debido a estas mismas propiedades, poseen numerosas aplicaciones especializadas. Por ejemplo, las aminas y los compuestos cuaternarios inhiben el crecimiento de microorganismos como bacterias y algas. Adems las aminas grasas primarias y las aminopropilaminas grasas se utilizan como inhibidores de la corrosin, y en la limpieza de metales, cuando se utiliza HCl para disolver el xido. La amina se orienta en la interfase entre el metal y la disolucin cida, con las colas hidrfobas comprimidas entre s, formando una capa protectora de una o dos molculas de espesor. Esta capa es tan cerrada que evita el ataque del metal limpio por parte del exceso de cido. Otra aplicacin ms de los compuestos grasos nitrogenados, y que depende de la actividad de la superficie y orientacin
16
de los iones del surfactante, es el suavizado de textiles. El surfactante catinico se adsorbe y orienta en la interfase formada entre el textil y el agua. Tambin, tienen afinidad por la superficie del cabello, utilizndose como acondicionadores y suavizantes en productos que se aplican despus del lavado, para contrarrestar as el efecto apelmazante de los surfactantes aninicos.
C) No inicos En esta clase de surfactantes, el grupo hidrfilo no es capaz de ionizarse y formar sales. Los surfactantes no inicos son especialmente tiles por su baja susceptibilidad a los iones Ca2+ y Mg2+ del agua dura. Aprovechando su compatibilidad con especies inicas, se suelen mezclar con surfactantes aninicos, dando lugar a asociaciones beneficiosas. Por ejemplo, los surfactantes no inicos ayudan a solubilizar las sales de calcio o magnesio de los surfactantes aninicos. El balance entre la parte hidroflica e hidrofbica en estos surfactantes se obtiene teniendo en consideracin la cantidad y naturaleza de las unidades polares y la parte hidrofbica de la molcula (longitud de la cadena hidrocarbonada). La parte hidrfila de la molcula es casi siempre una cadena de unidades de EO. Los grupos ter le proporcionan la polaridad necesaria para garantizar su solubilidad en agua por aceptacin de puentes de hidrgeno. Los FAEs son los surfactantes no inicos ms empleados en productos de limpieza, cosmticos, herbicidas, etc. Los APEs, principalmente OPEs y NPEs, tambin poseen una cadena de unidades de EO, pero a diferencia de los FAEs, absorben en el UV. La aplicacin de los APEs en detergentes est sometida a restricciones legales, debido a la difcil eliminacin biolgica (escasa biodegradabilidad) de los metabolitos ms hidrfobos, concretamente, alquifenoles no etoxilados y monoetoxilados. Los FAEs lineales se biodegradan ms rpidamente que los APEs. Adems, tienen mejores propiedades de detergencia que los ABS sobre
17
muchos tipos de suciedad y sobre la mayora de las telas, y son especialmente eficaces para eliminar la grasa de las fibras sintticas. Tambin actan bien en fro, por lo que actualmente aparecen en las formulaciones de los detergentes domsticos como uno de sus principales y ms ferecuentes componentes. Los derivados de aminas, amidas (FAA) y steres de cidos grasos, son tambin bastante empleados en productos de aseo corporal. As por ejemplo, la dietanolamida de coco posee buenas propiedades espumantes, estabilizando la espuma de los surfactantes aninicos. Finalmente, los surfactantes no inicos a base de azcares (APGs) tienen una biodegradabilidad sumamente rpida, baja toxicidad y alta tolerancia desde el punto de vista dermatolgico. Adems, pueden elaborarse a partir de materias primas naturales. En la Fig. I.3 se muestra la estructura y nomenclatura de las principales familias de los surfactantes no inicos.
CH3(CH2)nO(CH2CH2O)mH FAEs
(CH2CH2O)mH
m = 2 100 APEs O RCHN (CH2CH2O)mH R: cadena alqulica FAAs (CH2CH2O)nH
HOH2C HO HO O OH HOH2C O HO O (CH2)nCH3 OH n = 8 18 m = 0 3 APGs

Fig. I.3. Estructura y nomenclatura de las principales familias de surfactantes no inicos.
m
18
D) Anfotricos Finalmente las molculas que tienen simultneamente grupos con carcter cido y bsico son surfactantes anfteros o iones dobles. Son compuestos con una estructura con una carga positiva y otra negativa simultneamente sobre la misma molcula. Algunos de estos compuestos tienen grupos cidos o bsicos dbiles, por lo que pueden comportarse como aninicos o catinicos en funcin del pH. Usualmente se emplean junto con otros surfactantes (aninicos o catinicos) para resaltar las propiedades deseadas, como puede ser la detergencia o la formacin de espuma. Son especialmente utilizados en la formulacin de productos de aseo personal (gel de bao, champs, etc.) por su suavidad y compatibilidad con la piel, siendo menos irritantes que los surfactantes catinicos y aninicos. La formulacin de estos productos es complicada por la posible precipitacin del surfactante anftero cuando el pH est prximo a su punto isoelctrico. Pueden utilizarse, junto con NaOH, en limpiadores alcalinos para superficies grasas, y como limpiadores cidos junto con HCl para superficies oxidadas, debido a que son estables y funcionales en un amplio intervalo de pH. Un nmero importante de surfactantes anfteros son compuestos naturales ampliamente conocidos, como por ejemplo la lecitina. Una familia adicional de surfactantes anfteros que presentan un grupo amonio cuaternario son las alquilbetanas (Fig. I.4).
C17H35COOCH2 C17H35COOCH2 O CH3 CH3 R CH2OPCH2CH2N+CH3 OLecitina CH3 N+CH2COOCH3 R: cadena alqulica Alquil betanas
Fig. I.4. Estructura y nomenclatura de las principales familias de surfactantes anfotricos.
19
I.2.3. APGs Los APGs son surfactantes no inicos obtenidos a partir de materiales renovables (glucosa y cidos grasos)
1998; Balzer, 2000; Biermann, 1993]. [Koch, 1993; Balzer, 1996; Hill, 1997; Rybinski,
Se obtienen a partir de la alquilacin de
cadenas cortas de glucsidos procedentes de la alcoholisis cida de polisacridos como el almidn. Para su produccin se han estudiado dos vas, la sntesis directa o un proceso de transacetalizacin en dos pasos [Varvil, 2009]. Los oligmeros se distinguen por la longitud de la cadena de alquilo, el nmero de unidades de glucosa y la isomera
En la Fig. I.5 se muestran
los efmeros de anillo de los alquilmonoglucsidos.

CH2OH O OH
3
6 5 4
CH2OH O OH
3
HOHC
HOHC H H
2 1 5 4
OCnH2n+1 H
2 1
H H
H H
OCnH2n+1
OH
OH
Alquil--D-glucofuransido CH2OH
5
Alquil--D-glucofuransido CH2OH
5
H
4
O H H
2 1 4
H HO
O H
HO
H OH
3
H OH
3
OCnH2n+1
1
OCnH2n+1 H OH
H
2
OH
Alquil--D-glucopiransido
Alquil--D-glucopiransido
Fig. I.5. Estructura de los epmeros e ismeros del anillo de los alquilmonoglucsidos.
Tres tipos de isomera estn presentes en los APGs: estereoisomera (epmeros -y -), isomera del anillo (unidades de glucsido, que pueden ser glucopiransidos o glucofuransidos), y con excepcin de los AMGs, ismeros de posicin (con predominio de las uniones interglucosdicas 1,4 - y 1,6- [Beneito20
Cambra, 2007]).
Los diferentes oligmeros se pueden abreviar como CnGm,
donde n es el nmero de tomos de carbono en la cadena alqulica, y m el nmero de unidades de glucosa. Cuando es necesario distinguir entre piransidos y furansidos, en la presente memoria se emplea una "p" o una "f", al igual que se aadir - - para distinguir entre los diferentes epmeros, as por ejemplo, C8G1p, hace referencia al oligmero -octilmonoglucopiransido.
A) Propiedades y aplicaciones Los APGs se sintetizan controlando las condiciones para obtener principalmente estructuras donde la cadena glucosdica presente un DP comprendido entre 1,2 y 1,7 unidades, y cadenas alqulicas que se hallen en el intervalo de 8 a 14 tomos de carbono
[Willing, 2006].
Se obtienen as mezclas
industriales muy complejas que contienen principalmente AMGs, con cantidades importantes de alquildiglucsidos y menos importantes de otros APGs [Oldenhove
de Guertechin, 1999].
La presencia de numerosos grupos hidroxilo en las
molculas de glucosa asegura la completa solubilidad de la molcula en agua

[Partearroyo, 1991].
Los APGs con 16 o ms tomos de carbono en la cadena
alqulica son menos importantes, ya que son insolubles en agua cuando su DP es inferior a 2, siendo solubles cuando est por encima de 5 [Willing, 2006]. Adems de la buena solubilidad en agua, presentan puntos de nube (temperatura a la que los surfactantes se separan en dos fases) a temperaturas generalmente superiores a 100C; son poco sensibles a la presencia de electrolitos y raramente se ven influenciados por los cationes presentes en aguas duras
Este tipo de surfactantes son buenos
emulsificantes, especialmente para molculas como los aceites naturales y las grasas. Los APGs proporcionan buenas propiedades de mojado y formacin de espuma, siendo sus propiedades espumantes superiores a la de los alcoholes
21
etoxilados
Por estas propiedades favorables, as
como por la sinergia con otros surfactantes, y compatibilidad tanto medioambiental como dermatolgica
[Willing, 2006],
son ampliamente utilizados
en productos de limpieza e higiene personal

Biermann, 1993; Garca, 1997; Uppgard, 2000],
[Balzer, 1996; Rybinski, 1998;
aunque tambin se han utilizado en
la industria del petrleo y del gas [McGregor, 2004].
B) Mtodos de anlisis Dado que los APGs se estn convirtiendo en unos surfactantes cada vez ms interesantes por los diversos aspectos comentados anteriormente, tiene inters el desarrollo de mtodos para su caracterizacin y cuantificacin
2005]. [Thiele,
La cantidad total de APGs se puede determinar por hidrlisis seguida de
derivatizacin con antrona (9,10-dihidro-9-cetoantraceno) y colorimetra

[Buschmann, 1995; Buschmann, 1996-B], 1996-A],
valoracin potenciomtrica
[Kim, 2001]
[Buchmann,
espectrometra de infrarrojo cercano
e hidrlisis enzimtica
[Kroh, 1999].
Los APGs se han determinado en muestras ambientales mediante

[Meissner,
hidrlisis, seguida de destilacin de los alcoholes y derivatizacin

1999].
Para la concentracin de los APGs de muestras de agua se emplean

[Steber,
cartuchos de SPE C18, utilizando metanol para la elucin de los mismos

1995].
Se han descrito separaciones utilizando TLC con fases C8 y C18
[Buschmann, 1996-B; Buchmann, 1996-A; Buschmann, 1996-C; Spilker, 1996; Klaffke, 1998].
Los ismeros de los APGs pueden separarse sin derivatizar mediante GC a
alta temperatura, o bien, con una sililacin previa [Rybinski, 1998; Buchmann, 1996A; Spilker, 1996; Billian, 1998].
Las formas piransidas y furansidas pueden

[Billian,
distinguirse por los diferentes fragmentos obtenidos mediante GC-MS

1998].
Se ha empleado tambin MEKC con deteccin electroqumica para la

[Hbner, 2006].
determinacin de APGs en champ
Se han descrito distintos
procedimientos de HPLC con derivatizacin post-columna y deteccin

22
fotomtrica
[Kramer, 1992],
refractomtrica
[Klaffke, 1998],
ELSD
[Buschmann,
1996-B; Buchmann, 1996-A; Lafosse, 1992; Czichocki, 2002; Armari, 2003] [Czichocki, 2002; Eichhorn, 1999; Klaffke, 1999; Khn, 2004].
o por MS
Se ha descrito el
comportamiento cromatogrfico de los alquilglucopiransidos en columnas C8, C18, fenil y PVA

[Lafosse, 1992],
y se han realizado estudios comparativos
empleando columnas de slice, C18 y PVA [Czichocki, 2002]. Eichhorn y Knepper

[Eichhorn, 1999]
utilizaron HPLC-MS con ESI para determinar APGs en aguas
fluviales y residuales fortificadas. Utilizando una columna C18 y elucin en gradiente con ACN/agua, es posible resolver los epmeros - y - y los ismeros de anillo. Los alquilglucopiransidos pueden distinguirse de los
alquilglucofuransidos por su distinta afinidad por el NH + . Kuhn y Neubert 4

[Khn, 2004]
usaron HPLC-ESI-QTOF-MS con una columna C18, fase mvil
MeOH/agua y elucin en gradiente para caracterizar mezclas industriales de APGs; sin embargo, no llegaron a resolver los ismeros.
C) Estudios de fragmentacin de glucosa y oligosacridos Las tcnicas de fragmentacin como el CID son poderosas herramientas para la elucidacin estructural de oligosacridos. Con este fin se han empleado iones alcalinos y iones amonio
[Harvey, 2000-A; Harvey, 2000B; Chiarelli, 1987;
Cancilla, 1999; Harvey, 1997; Harvey, 1994; Kovik, 1995; Penn, 1996; Asam, 1997],
cationes divalentes [Harvey, 2001; Madhusudanan, 2005] y sales de hierro [Carlesso,

2000; Carlesso, 2001],
sin embargo, las fragmentaciones con mayor sensibilidad y
las que ms informacin permiten obtener son aqullas que emplean iones sodio y calcio
[Harvey, 2000-A; Harvey, 2000-B; Harvey, 2001].
La fragmentacin de los
[Zaia, 2004],
oligosacridos tambin se ha estudiado utilizando ITMS
y tambin
se han realizado anlisis de carbohidratos mediante MALDI-MS [Harvey, 2006].
23
Al emplear bajas energas de fragmentacin, y de acuerdo con la nomenclatura de Domon-Costello

[Zaia, 2004; Domon, 1988],
la rotura de los
[Asam,
enlaces glucosdicos produce principalmente fragmentos B e Y (Fig. I.6)

1997; Creaser, 2002].
Todas las uniones secuenciales de polisacridos lineales o

[Asam, 1997; Harvey,
ramificados pueden analizarse a partir de los fragmentos

2001; Zaia, 2004].
Por su parte, la utilizacin de altas energas de fragmentacin
produce roturas de anillo, lo cual proporciona informacin adicional de las uniones

[Harvey, 2000-A; Harvey, 2000-B; Harvey, 2001; Cancilla, 1999; Asam, 1997;
Zaia, 2004; Creaser, 2002].
CH2OH
5
O
1
0,2X
CH2OH
5
Y0
HO
4
O
1 2
HO
3
0,2A
OH
2
Z0
OH
3
OH
B1
O
C1
CnH2n+1
Terminacin reductora
CnH2n+1
OH
Fig. I.6. Estructura de los -epmeros de AMGp (izquierda) y AMGf (derecha). Se indican ejemplos de la nomenclatura de las fragmentaciones. Los enlaces del anillo estn numerados en el sentido de las agujas del reloj comenzando por cero tras el oxgeno. En los -epmeros, a diferencia de los -epmeros, los grupos H y OR en el tomo de carbono 1 estn invertidos.
En cuanto a los espectros CID de monosacridos y sus derivados, se han demostrado diferencias estereoqumicas de monosacridos en presencia de Zn2+
[Gaucher, 1998]
y ion cloruro
[Carlesso, 2000].
Tambin se han distinguido
ismeros posicionales y diastereoismeros de monosacridos sulfatados

[Minamisawa, 2005]
y se han comparado los espectros de la D-glucosa y 2[Macek, 2003].
fluorodesoxiglucosa
Utilizando el espectro de CID, varias
pentosas y hexosas, incluyendo sus esteroismeros, pueden diferenciarse en
24
funcin de la competicin en los procesos de fragmentacin
[March, 2005].
Berman y colaboradores utilizaron el anlisis multivariante de los datos de TOFSIMS para distinguir entre varias furanosas, as como entre varias piranosas
[Berman, 2006].
Sin embargo, la fragmentacin CID ha sido poco utilizada para
distinguir ismeros de anillo. El tamao del anillo de la ltima unidad en el extremo no reductor de los oligosacridos se ha relacionado con la abundancia de los iones [M + Na-90]+ y [M + Na-104]+ en el espectro CID
[Kovik, 2001].
Usando TOF-SIMS, los monosacridos con anillos de cinco miembros fueron ms estables que los correspondientes de seis miembros, dando diferentes perfiles de pico en la fragmentacin [Berman, 2006].
I.2.4. Alcoholes no etoxilados y etoxilados Los alcoholes primarios son componentes comunes en muchas muestras, tanto biolgicas como industriales, adems de estar presentes a nivel de trazas en el medio acutico. Los alcoholes grasos se obtienen mediante la hidrogenacin cataltica de los cidos grasos correspondientes o de sus steres. La industria actualmente se concentra en la hidrogenacin de steres de alcoholes grasos y cidos grasos. Debido a que los aceites y grasas usadas como materia prima son una mezcla de cidos grasos de distinta longitud de cadena, se obtienen como productos alcoholes de nmero de carbonos variable
[Sad, 2007].
Los alcoholes
grasos a su vez se emplean tambin como materia prima en la produccin de importantes clases de surfactantes, incluyendo AESs, FAEs y sulfonatos de alcoholes grasos etoxilados [Farm, 2006.]. Tal como se ha indicado anteriormente, los FAEs se obtienen en forma de complejas mezclas de oligmeros con la estructura: CH3(CH2)n-1(OCH2CH2)mOH que puede abreviarse como CnEm, donde n adopta valores entre 8 y 18 y m, entre 0 y 30, el valor medio de EO se representa como m .
25
A) Propiedades y aplicaciones En los ltimos aos, los surfactantes basados en alcoholes grasos han ganado importancia en el mercado de detergentes debido a sus excelentes propiedades de lavado y su superior biodegrabilidad. Los alcoholes con una cadena hidrocarbonada de entre 10 y 18 carbonos (alcoholes grasos) son potentes feromonas, mientras que los de mayor masa molecular son componentes esenciales en las ceras de las plantas
[Bianchi, 1995].
La importancia industrial de
los alcoholes deriva de su uso generalizado como disolventes y cargas (espesantes) en productos industriales y del hogar. Actualmente, los alcoholes grasos derivados de materias primas renovables son usados en la produccin de surfactantes no inicos, como los ASs, AESs, FAEs y APGs
Fielder, 1989; Sparham, 2005]. [Marcomini, 1996;
Otro de los campos de aplicacin de los alcoholes
grasos es la industria de los cosmticos, donde se emplean en la formulacin de jabones lquidos, champs, acondicionadores, cremas y lociones [Sad, 2007]. En cuanto a los FAEs, son los surfactantes no inicos ms empleados en productos de limpieza, siendo tambin utilizados en la formacin de cosmticos, estabilizantes o dispersantes de herbicidas, etc. Los grupos ter proporcionan la polaridad necesaria para garantizar su solubilidad en agua. Si bien la cadena de EO no es tan polar como un grupo ionizado, un conjunto de 5 a 10 unidades de EO puede alcanzar una notable capacidad hidrfila. Los FAEs son excelentes agentes humectantes, compatibles tanto con surfactantes aninicos como catinicos, y su detergencia no se reduce en presencia de iones metlicos como Ca2+ o Mg2+. Tienden a ser lquidos o ceras con bajo punto de fusin y, por consiguiente, son difcilmente utilizables en la formulacin de detergentes en polvo. Otro de sus inconvenientes es su tendencia a precipitar a temperaturas elevadas o fuerzas inicas altas, debido a la menor solvatacin de la cadena de EO en comparacin con los grupos inicos. Adems, a temperaturas ms
26
elevadas, se reduce el peso estadstico de las conformaciones polares de la cadena de EO, de manera que la zona polar pierde buena parte de su carcter hidroflico. En estas condiciones, el surfactante se separa del medio acuoso formando otra fase (punto de nube). Los FAEs lineales se caracterizan, a grandes rasgos, por el intervalo de valores de n (tambin llamado corte hidrofbico), y por el nmero medio de unidades de EO; sin embargo, tanto para el control de calidad industrial como medioambiental, se requiere informacin sobre la distribucin de los oligmeros. La caracterizacin de los FAEs es importante porque sus propiedades fisico-qumicas, as como el riesgo ambiental que comportan, vienen fuertemente condicionadas por las distribuciones de sus cadenas hidrofbica e hidroflica [Marcomini, 1996; Rudewicz, 1986; Eadsforth, 2006;
Belanger, 2006; Van Compernolle, 2006; Ribosa, 2007; Jurado, 2007].
Una clase especial de FAEs incluye compuestos de masa molecular baja (tanto n como m < 4). Estos compuestos conocidos como cellosolves, se utilizan por ejemplo, como aditivos anticongelantes en combustibles para aviacin y como disolventes en algunas formulaciones para limpiadores
2003]. [Cheremisinoff,
B) Mtodos de anlisis Los cellosolves y los alcoholes grasos con una cadena igual o menor a 26 tomos de carbono se han determinado con xito mediante GC [Nichols, 2006], sin embargo, los FAEs con m > 4, no pueden determinarse por GC debido a su limitada volatilidad
[Rudewicz, 1986; Crescenzi, 1995; Battersby, 2001].
El principal
inconveniente de los mtodos de HPLC para la determinacin de alcoholes grasos y FAEs ha sido la falta de un detector adecuado
2001]. [Rudewicz, 1986; Petrovic,
Este problema se ha resuelto en parte con la introduccin de detectores

[Kudoh, 1984; Mengerink, 1991; Cho, 2003;
como el de ndice de refraccin
27

Trathnigg, 2002]
y el ELSD
[Mengerink, 1991; Heinig, 1998; Bear, 1988; Miszkiewicz,
2000; Miszkiewicz, 1996-B; Kamiusuki, 2000],
sin embargo, los LODs en el detector
de ndice de refraccin son altos, mientras que la sensibilidad del ELSD se ve mermada para compuestos voltiles como alcoholes no etoxilados y oligmeros de FAEs con un bajo grado de etoxilacin (m < 3)
Zafn, 2006]. [Miszkiewicz, 1996-A; Bernab-
Los LOD ms bajos se consiguen mediante procesos de cromognicos y fluorognicos
derivatizacin pre-columna con agentes
[Marcomini, 1996; Schmitt, 1990; Kiewiet, 1995; Lemr, 1994; Zanette, 1996; Lemr, 1996; Sun, 1997; Hoffman, 2004-A; Lemr, 2003; Okada, 1991; Okada, 1992; Hoffman, 2004-B; Bachus, 2003; Desbne, 2005; Heinig, 1996-A; Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Zu, 2010].
En la determinacin de FAEs mediante MS, la formacin de aductos con iones positivos, ya sea utilizando la fuente ESI o APCI, resulta problemtica debido a la disminucin de la sensibilidad conforme disminuye m lo que es especialmente notable para m < 4. Finalmente, los alcoholes no etoxilados (m = 0) no pueden detectarse por MS [Rudewicz, 1986; Crescenzi, 1995; Petrovic, 2001; Zu,
2010; Chiron, 2000; Sherrard, 1994; Dunphy, 2001; Cassani, 2004; Sparham, 2005].
Para superar este inconveniente se han desarrollado procedimientos de derivatizacin, en los que se adiciona un cromforo y/o una carga permanente a los oligmeros de los FAEs
[Lemr, 1994; Lemr, 1996; Lemr, 2003; Desbne, 2005;
Heinig, 1996-A; Zu, 2010; Dunphy, 2001; Cassani, 2004; Sparham, 2005].
Sin
embargo, la derivatizacin de FAEs no es una tarea sencilla, ya que se requiere un medio anhidro [Okada, 1992; Zu, 2010; Dunphy, 2001; Barry, 2003], existiendo un mayor riesgo de interferencia tanto por el exceso de reactivo como por los subproductos de la reaccin
[Sun, 1997; Hoffman, 2004-A; Mic-Tormos, 2008-A;
Mic-Tormos, 2008-B; Dunphy, 2001].
Por otra parte y debido al alto riesgo de
perder oligmeros con bajos valores de m por volatilizacin, el contenido de agua de las muestras debe reducirse con precaucin, lo que aumenta el tiempo de
28
anlisis [Dunphy, 2001]. Sin embargo, en la derivatizacin con anhdridos cclicos se tolera la presencia de pequeas cantidades de agua en las muestras
Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Zu, 2010]. [Mic-
I.2.5. AESs Los AESs son una clase de surfactantes aninicos ampliamente utilizados en productos de limpieza e higiene corporal
[Fiedler, 1989].
Se obtienen por
esterificacin de FAEs, utilizando trixido de azufre o cido clorosulfnico. La estructura molecular de los AESs est formada por una cadena de hidrocarburo unida a una cadena de EO con un grupo sulfato en el extremo [Strain, 1959; Suter,
1944; Arthur D. Little, 1991].
Estos compuestos raramente son sustancias puras,
tratndose generalmente de mezclas, lo que es debido a la materia prima (alcoholes) y a que su grado de etoxilacin es un valor medio. Los AESs se obtienen como complejas mezclas de oligmeros con la estructura:
CH 3 (CH 2 ) n -1 (OCH 2 CH 2 ) m OSO3
que se puede abreviar como CnEmS, donde n adopta valores entre 12 y 16, mientras que m suele oscilar entre 0 y 3, el valor medio de EO se representa como m .
A) Propiedades y aplicaciones Las disoluciones acuosas de sulfatos muestran un comportamiento especial, debido a que su viscosidad, primero aumenta por adicin de electrolitos como el cloruro de sodio en bajas concentraciones, disminuyendo con adiciones posteriores. La mxima viscosidad, en cuanto a la concentracin de la sal a aadir, depende de la estructura del ter sulfato. En comparacin con los ASs, los correspondientes AESs son ms solubles en agua y muestran mejor la tolerancia a la presencia de iones Ca2+ y Mg2+. La introduccin de una cadena de EO en el
29
AS tiende a aumentar el poder espumante del surfactante, especialmente en agua dura, ya que permite un aumento de la adsorcin interfacial. Tambin se mejoran las propiedades humectantes y emulsionantes. Por ello se usan en las formulaciones de jabn en barras, en diversos productos cosmticos y farmacuticos y en el tratamiento de textiles [Salager, 2004; Spilker, 2005].
B) Mtodos de anlisis Las principales caractersticas de estos surfactantes (viscosidad,
detergencia, formacin de espuma y compatibilidad con la piel), as como su impacto ambiental, dependen de la distribucin de cadenas de alquilo y EO
[Arthur D. Little, 1991; Tadros, 2005; Marcomini, 1996; Rudewicz, 1986; Eadsforth, 2006; Belanger, 2006; Van Compernolle, 2006; Ribosa, 2007; Jurado, 2007].
Por lo
tanto, tiene inters el desarrollo de mtodos para su caracterizacin y determinacin. Sin embargo, debido a la complejidad de las muestras, la falta de cromforos, y los amplios intervalos de polaridad de los oligmeros, el anlisis de AESs no es fcil. Adems, la ausencia de estndares comerciales de AESs dificulta su determinacin. La determinacin de AESs puede realizarse con el mtodo del azul de metileno, en el que se determina el contenido total de surfactantes aninicos
[Llenado, 1983].
Para determinar ASs y AESs se ha utilizado la cromatografa de

[Boiani, 1987; Shamsi, 1995], [Pan, 1995; Nair, 1998]
pares inicos con deteccin UV indirecta
la
cromatografa inica con deteccin conductimtrica
y la
HPLC con derivatizacin post-columna y deteccin fluorimtrica

A].
[Smedes, 1982-
Para la deteccin UV indirecta de AESs con separacin previa por HPLC se
han utilizando tampones acuosos con agentes reveladores UV como el veronal

[Nielen, 1991],
el sulfonato de naftaleno
[Romano, 1991; Shamsi, 1994],
el sulfonato
de tolueno [Shamsi, 1995] o los cidos benzoico y p-hidroxibenzoico [Heinig, 1996B].
Tambin se ha llevado a cabo la determinacin de AESs mediante GC tras

30
hidrlisis de los analitos con cidos diluidos para obtener sus correspondientes alcoholes y alcoholes etoxilados, seguida de derivatizacin a trimetil sililteres
[Sones, 1979; Kirby, 1975].
En otro mtodo se utiliza GC-FID, con derivatizacin
previa de los AESs a sus correspondientes bromuros de alquilo [Neubecker, 1985]. La determinacin de AESs mediante HPLC-ESI-MS se ha aplicado a diversas matrices ambientales como aguas, lodos de depuradora y sedimentos marinos
[Popenoe, 1994; Bruno, 2002; Lara-Martn, 2005].
I.3. Polmeros
I.3.1. Introduccin Un polmero es una macromolcula constituida por la unin repetida de pequeas unidades (monmeros) a travs de enlaces covalentes. Ejemplos de polmeros de origen natural son las protenas (seda, enzimas, colgeno), los polisacridos (almidn, celulosa) y los cidos nucleicos, los cuales cumplen funciones especficas en los seres vivos. Dentro de los polmeros sintticos, el ms simple es el polietileno, siendo el etileno el monmero a partir del cual se forma. La unidad estructural que se repite a lo largo de la cadena polimrica se denomina unidad repetitiva, y la reaccin en la cual los monmeros se unen entre s para formar el polmero se denomina polimerizacin. Los polmeros consisten en mezclas de molculas de distintas longitudes de cadena, y por ello se habla de la MW promedia.
31
A) Clasificacin de los polmeros Existen diferentes formas de clasificar a los polmeros: i) Segn su composicin: - Homopolmeros: Formados por una nica unidad repetitiva. Por ejemplo, el polimetacrilato de metilo, donde el nico monmero se repite. - Copolmeros: formados por ms de una unidad repetitiva. Por ejemplo, aqullos que estn formados por 2 monmeros, pudiendo las unidades repetitivas distribuirse de distintas maneras a lo largo de la cadena del polmero. Por ejemplo, siendo A una unidad repetitiva y B otra, pueden disponerse: al azar (ABBBBABABBAAABBA), de forma alternada
(ABABABABABABABAB) o por bloques (AAAABBBAAAABBB). Los copolmeros presentan propiedades intermedias entre las de los homopolmeros que se formaran a partir de cada tipo de monmero por separado.
ii) Segn su estructura: - Lineales: formados por monmeros difuncionales. Ejemplo: polietileno, poliestireno. - Ramificados: se requiere el agregado de monmeros trifuncionales, por ejemplo, glicerol. - Entrecruzados: se forma un material compuesto por una molcula tridimensional continua, unida por enlaces covalentes. Por ejemplo, resinas urea- formaldehdo y fenol-formaldehdo.
iii) Segn la reaccin de polimerizacin: - Polimerizacin por reaccin en cadena (o adicin): se genera una partcula reactiva (radical, anin o catin) a partir de una molcula de monmero, y sta se adiciona a otro monmero de manera repetitiva.
32
- Polimerizacin por crecimiento en pasos (o condensacin): los monmeros que reaccionan tienen un grupo funcional reactivo en cada extremo de la molcula y la unin entre los monmeros requiere la prdida de una molcula pequea, normalmente agua.
B) Estereoisomera en polmeros vinlicos La tacticidad (del griego taktikos, orden) o estereoregularidad de los polmeros es la organizacin estructural espacial de un polmero, esto es la disposicin que adquieren los sustituyentes en el espacio. As cuando un par de sustituyentes R adyacentes apuntan en la misma direccin en el espacio, la pareja o dada se denomina m, mientras que si los sustituyentes apuntan en direcciones contrarias del espacio se tiene una pareja o dada r (Fig. I.7).
R H R H R H R H R H R H
Dada m R H H R H R R H H R H R
n B
Dada r R H H R R H R H H R R H
n C
n
Fig. I.7. Estructura de los polmeros isotcticos (A), sindiotcticos (B) y atcticos (C).
Cuando se considera un mayor nmero de grupos R adyacentes, existen tres ordenamientos posibles con respecto al plano del esqueleto carbonado del polmero:
33
i) Isotctico: con todos los grupos R hacia el mismo lado de la cadena polimrica extendida (Fig. I.7-A). ii) Sindiotctico: con los grupos R alternando a uno y otro lado (Fig. I.7-B). iii) Atctico: con los grupos R distribudos al azar (Fig. I.7-C). El tipo de estereoregularidad se establece en la reaccin de polimerizacin y no es posible convertir un estereoismero en otro por simple rotacin de los enlaces sigma a lo largo de la cadena polimrica.
I.3.2. Polmeros en detergentes Se pueden enumerar varias caractersticas importantes que los polmeros pueden ofrecer, y que en distinto grado, pueden ayudar a obtener como resultado final un buen lavado. Existen polmeros que se emplean en el formulado de detergentes que actan como agentes para la prevencin de la re-deposicin de suciedad, secuestradores de los iones calcio y magnesio, agentes dispersantes y otros que actan como DTI (inhibidores de la redeposicin de colorantes)
1999]. [Zini,
A) Agentes antirredeposicin El efecto de los polmeros en las operaciones de lavado se conoce desde los aos 60 del siglo XX. La eliminacin de las manchas de las superficies es una compleja combinacin de procesos fsico-qumicos como disolucin,
desplazamiento y desorcin. Estos procesos son reversibles y las partculas de suciedad pueden readsorberse sobre los tejidos tras permanecer en la disolucin de lavado. Para evitar esto, se utilizan en las formulaciones componentes que garanticen un rendimiento ptimo de la dispersin de la suciedad en la disolucin. Sin embargo, las propiedades de la superficie pueden variar considerablemente, existiendo diferentes afinidades entre las fibras y los
34
diferentes tipos de suciedad, dependiendo principalmente de la polaridad. As, las partculas que se eliminan fcilmente de fibras de polaridad intermedia pueden ser fuertemente adsorbidas por fibras tanto hidroflicas como hidrofbicas, provocando un envejecimiento de los distintos tejidos tras numerosos lavados
[Waldhoff, 2005].
Para evitar estos iconvenientes, las formulaciones contienen
polmeros que interactan fuertemente tanto con las partculas de suciedad en la disolucin de lavado, como con las fibras textiles, sobre las que se adsorben de manera irreversible. En ambos casos, se impide la redeposicin de la suciedad. Los polmeros derivados de la celulosa y del almidn son los ms utilizados como agentes antirredeposicin, aunque tambin se pueden emplear otros como el PVP o PVA.
B) Agentes secuestrantes Los agentes secuestrantes enmascaran los cationes divalentes del agua dura, evitando su interaccin con los surfactantes. Los polmeros tambin utilizados con este fin suelen ser homopoliacrilatos o copolmeros de acrlico y maleico [Zini, 1999].
C) Agentes inhibidores de la transferencia de color Desde los aos 90 del siglo XX, la industria de los detergentes ha lanzado al mercado varios polmeros solubles en agua con capacidad para inhibir parcialmente la transferencia de color de unos tejidos a otros durante el proceso de lavado
[Bertleff, 1998; Oakes, 2003-A].
Estos polmeros, denominados DTIs,
actan atrapando los colorantes desprendidos, mantenindolos en disolucin, y por lo tanto, previniendo su redeposicin en los tejidos. Estos polmeros muestran una fuerte afinidad con los grupos funcionales aromticos. Los DTI, quedan adsorbidos sobre los colorantes en las aguas de lavado, mediante una
35
combinacin de interacciones dipolo-dipolo inducido y -, previniendo eficazmente la redeposicin del color

[Jger, 1991].
Los DTI estn constituidos
por monmeros con residuos ricos en electrones y fuertemente bipolares o fcilmente polarizables, por lo general, heterociclos aromticos o alifticos con sustituyentes vinlicos. Los polmeros que se utilizan con este fin suelen ser PVP y PVP-NO y tambin el copolmero PVP-PVI.
I.3.3. PVP-NO El PVP-NO es un polmero empleado como DTI en las formulaciones de detergentes. Durante aos, se us principalmente PVP y sus copolmeros, sin embargo, la eficacia de estos polmeros para atrapar colorantes se reduce en presencia de surfactantes aninicos
[Oakes, 2003-A].
Por esta razn, se han
desarrollado nuevas generaciones de DTIs, con propiedades superiores de complejacin, y con mayor tolerancia frente a los surfactantes aninicos. Un DTI de nueva generacin relativamente comn es el PVP-NO, obtenido a partir de una polimerizacin radicalaria de la 4-vinil-piridina utilizando AIBN como iniciador, seguida de oxidacin con perxido de hidrgeno (Fig. I.8)
[Lee, 1996].
Los diferentes tipos de PVP-NO comercial para detergencia estn constituidos por especies con masas moleculares entre 9 y 36 kDa, que corresponden a cadenas de polmero que contienen desde 75 hasta 300 monmeros, respectivamente [Oakes, 2003-A; www.ispcorp.com].
CH2 - CH
CH2 CH
N+ N 4-vinilpiridina OPVP-NO n
Fig. I.8. Estructura de la 4-vinilpiridina y del PVP-NO.
36
I.3.4. Poli(vinilpirrolidona) De la primera polimerizacin de la N-vinilpirrolidona se obtuvo un polmero soluble, el PVP (Fig. I.9), patentado en 1939. La polimerizacin se realiza mediante radicales libres en agua o en 2-propanol, utilizando como iniciador perxido de hidrgeno o un perxido orgnico, respectivamente [Bhler,
2005].
C H C H2 N O
C H C H2 N O n
N-vinilpirrolidona
PVP
Fig. I.9. Estructura de la N-vinilpirrolidona y del PVP.
El PVP es un polmero no inico que puede aplicarse en gran variedad de campos gracias a caractersticas como su solubilidad en agua, as como en diversos disolventes orgnicos polares, buena afinidad con diferentes polmeros y resinas, alta higroscopicidad, capacidad de formacin de pelculas, buena adherencia a diversos sustratos y posibilidad de formar complejos/quelatos
[www.shokubai.co.jp].
El PVP es un polmero verstil utilizado en cosmticos,

[Frauenfelder, 1974; Sheth,
textiles, tintes, productos farmacuticos y adhesivos

1985].
En la formulacin de productos de limpieza, el PVP se emplea como DTI,
mantenimiento los colorantes desprendidos durante el lavado en disolucin, por lo que inhibe su transferencia a otros tejidos presentes en el lavado [Bertleff, 1998;
Oakes, 2003-A; Oakes, 2003-B; Oakes, 2005].
I.3.5. Alcohol polivinilico El PVA (Fig. I.10) es un polmero sinttico obtenido por primera vez en 1924 por Hermann Staudinger, mediante la hidrlisis del PVAcO con hidrxido
37
de potasio en etanol
[Gallardo, 1999].
El PVA presenta un amplio abanico de
aplicaciones en medicina, cosmtica, alimentacin, farmacia, detergencia, etc., como consecuencia de propiedades tales como su inocuidad, alta
biocompatibilidad y solubilidad en agua [Park, 2010]. Segn la ruta de sntesis, se obtienen PVAs con diferentes tacticidades, como corresponde a una estructura que presenta centros quirales adyacentes repetidos a lo largo de la cadena polimrica.
CH2 CH O C CH3 PVAcO O n PVA CH2 CH OH n
Fig. I.10. Estructura del PVAcO y del PVA.
El PVA es un polmero capaz de complejar cationes metlicos como Cu2+, y de formar steres con algunos aniones como borato, titanato, antimoniato y vanadato. En disolucin acuosa, los complejos polmero-catin tienen propiedades similares a las observadas en las disoluciones de polielectrolitos (especies con cargas positivas o negativas). La carga de los grupos PVA-catin puede provocar la ionizacin de la molcula de agua [Tsujimoto, 2002].
I.3.6. Mtodos de anlisis de polmeros Durante los ltimos aos, la CE se ha aplicado a la caracterizacin y determinacin de polmeros sintticos
[Gallardo, 1999; Cottet, 2005].
Principalmente, la FSCE y la CGE han sido empleadas para separar polielectrolitos y polianfolitos (especies con cargas tanto positivas como negativas), y la MEKC se ha usado para separar polmeros no cargados [Gallardo,
1999].
Tanto la FSCE como la MEKC proporcionan informacin sobre el
38
comportamiento
de
polmeros
en
disolucin,
permiten
estimar
cuantitativamente algunas de las disoluciones de polmeros
[Bohrisch, 2000].
Gallardo y col. [Gallardo, 1999] separaron por MEKC una fraccin mayoritaria en metacrilato y otra rica en vinilpirrolidona a partir de copolmeros no inicos. Tambin, el empleo de MEKC ha proporcionado informacin sobre el progreso de la sntesis, naturaleza y composicin de copolmeros inicos
[Aguilar, 2002].
Por otro lado, en CGE es posible conseguir la discriminacin por masa molecular de polmeros inicos mediante un proceso de tamizado molecular, a travs de un BGE que contiene un polmero no inico, como el PEG derivados de celulosa, como dextrano u otros polisacridos
1998; Starkweather, 2000; Welch, 2001]. [Grosche, 2000]
[Bohrisch, 2000; Clos,
En FSCE, la movilidad electrofortica para polielectrolitos de cadena corta es funcin de la longitud de la cadena, de modo que la movilidad se incrementa cuantos ms monmeros cargados existan en la misma. Sin embargo, la movilidad alcanza un mximo y despus comienza a decrecer cuando el polielectrolito cambia su conformacin extendida a la enrollada u ovillada. En el caso de polielectrolitos ms largos, la relacin masa/carga permanece constante, y el polmero migra con una movilidad que se denomina de drenaje libre
[Cottet, 2000], 2000].
proporcionando un pico nico en FSCE
[Bohrisch, 2000; Grosche,
No obstante, algunos polmeros pueden dar dos o ms seales a diferentes
tiempos de migracin en lugar de un pico nico. As por ejemplo, se ha demostrado la formacin de micelas en disolucin acuosa de copolmeros compuestos por cadenas largas polianinicas de polisulfonato de estireno y cadenas hidrofbicas cortas de polietileno-propileno
[Cottet, 2001].
En estos
copolmeros, las cadenas libres (unmeros) presentan una movilidad elevada (en trminos absolutos), mayor que la movilidad de los polmeros micelizados.
39
Adems, el pico del polmero micelizado es ms ancho que el pico del polmero libre, lo que se ha atribuido a cierta dispersin en el nmero de agregacin. Gyorffy y col.
[Gyrffy, 1998]
analizaron un copolmero aninico de PVP-
NO y cido maleico mediante FSCE, encontrando un pico estrecho seguido de uno ms ancho. Para explicar este comportamiento se propuso la existencia de dos componentes en el polmero. Utilizando SEC y FSCE, tepnek y col.
[tepnek, 2001]
demostraron que algunos copolmeros forman micelas en
disolucin, y que stas se encuentran en equilibrio dinmico con los unmeros. Adems, se demostr que el equilibrio micelas-unmeros est cinticamente ralentizado en medio acuoso, donde las micelas se comportan como nanopartculas independientes. Para la caracterizacin de polmeros mediante CE puede emplearse la tcnica descrita por S. N. Krylov y col.
[Berezovski, 2002; Krylov, 2003; Drabovich,
2006; Lin, 2008; Krylov, 2006; Krylov, 2007],
desarrollada inicialmente para estudiar
las interacciones entre una protena o un fragmento de DNA con un marcador fluorescente. En dicha tcnica, denominada NECEEM, los enlaces no covalentes de marcadores fluorescentes se equilibran con una protena diana o con DNA, y la mezcla se inyecta en el capilar. Se obtiene una banda debida al complejo seguida del pico producido por el exceso de marcador libre. Adems, entre ambas seales se obtiene una regin intermedia, con el aspecto de una cada exponencial, debida al marcador liberado por el complejo durante la separacin electrofortica siguiendo una cintica de pseudo-primer oden. La formacin de complejos entre polmeros solubles, como PVP, y azocolorantes, se conoce desde hace dcadas [Frauenfelder, 1974]. Otra tcnica empleada para la caracterizacin de los polmeros es la NMR, con la que puede determinarse la tacticidad o estereoregularidad de los polmeros
[Moritani, 1972.; Wu, 1977].
La espectrofotometra UV-Vis se ha utilizado para la
40
determinacin de copolmeros, siempre y cuando al menos uno de los monmeros presente un grupo cromforo. Por ejemplo, el copolmero de metacrilato de metilo y acenaftileno se pueden caracterizar por esta tcnica
[Snchez, 2000; Skoog, 1994].
Tambin se han caracterizado ciertos polmeros
naturales mediante la formacin de complejos polmero-ion coloreado [Berezovski,

2002; Krylov, 2003; Drabovich, 2006; Lin, 2008; Krylov, 2006; Krylov, 2007].
Una tcnica habitual para caracterizar especies de elevada masa molecular es la SEC capilar
2008], [Snchez, 2000; Skoog, 1994].
Otras tcnicas, como la viscosimetra

[Karimi,
[Snchez, 2000.; Skoog, 1994]
o la osmometra de presin de vapor
tambin pueden emplearse para la caracterizacin de polmeros, ya que los
resultados obtenidos pueden correlacionarse con la masa molecular y otras propiedades de los polmeros. Algunos polmeros empleados como DTI, en particular la PVP, tambin pueden determinarse mediante pirlisis seguida de GC-MS [Uchiyama, 1998].
I.4. Enzimas
I.4.1. Introduccin Las enzimas aparecieron en el mercado de los productos de limpieza en la dcada de 1960, para ayudar a eliminar las manchas proteicas. Hoy en da, la presencia de diferentes enzimas en las formulacioens mejora la detergencia, ya que facilitan la degradacin de grandes y complejas molculas tales como las protenas, almidones y grasas. Los productos de reaccin obtenidos tras la actuacin de las enzimas son ms solubles en las aguas de lavado, por lo que son arrastrados ms fcilmente por los surfactantes. Por otro lado, las enzimas tambin ayudan a mantener la blancura y brillo en los tejidos, as como a
41
clarificar los colores, ya que pueden eliminar las fibras desprendidas de los tejidos (pelusa) [Crutzen, 1999]. La incorporacin de las enzimas a los detergentes en polvo es muy habitual, siendo menos frecuente en detergentes lquidos, por la mayor dificultad para preservar su estabilidad. Tampoco suelen usarse en patillas de jabn ni en lavamanos, debido a su inestabilidad durante los procesos de fabricacin, y por posibles procesos de irritacin cutnea. Los detergentes enzimticos representan un 30% del consumo mundial total de detergentes, el 80% del mercado en USA
[Krawczyk, 1996]
y un 85% en Europa [Whalley, 1994].
El amplio uso de enzimas en detergentes se justifica por sus favorables caractersticas: - Su alto rendimiento a bajas concentraciones favorece el desarrollo de detergentes cada vez ms concentrados. - Su ventajosa relacin precio/efectividad. - Su excelente perfil medio-ambiental. Las enzimas son protenas que se producen en todas las clulas vivas. Son las responsables de la catlisis de muchas reacciones qumicas biolgicas, que generalmente tienen lugar a bajas temperaturas y a pH neutro o cercano al neutro, con una alta eficiencia y especificidad [Krawczyk, 1995]. Las enzimas presentes en los detergentes no son tan especficas. Por ejemplo, las enzimas proteolticas (proteasas) son menos discriminantes y aumentan la velocidad de rotura de muchas protenas. Del mismo modo, las enzimas amiolticas (amilasas) ayudan a fragmentar los almidones, las enzimas lipolticas (lipasas) catalizan la hidrlisis de los triglicridos y las enzimas celulticas (celulasas) ayudan a romper la celulosa. La mayor velocidad de rotura de molculas grandes, procedentes de comida, secreciones corporales, etc, a molculas ms pequeas y solubles hace que las enzimas faciliten la eliminacin de varias manchas que de otra manera
42
serian difciles de quitar slo con los detergentes. Tambin cabe mencionar que al actuar como catalizadores, las enzimas suelen ser muy efectivas a bajas concentraciones (menos de 0,1% en peso de enzima en el detergente)
1999]. [Crutzen,
Las enzimas utilizadas en los detergentes son producidas por cepas de baterias resitentes a medios alcalinos, que producen, por lo tanto, enzimas que tambin son resistentes a pHs altos. Adems se trata de enzimas exocelulares, es decir, que son secretadas por las bacterias en el medio circundante. Por lo tanto, estas enzimas pueden ser aisladas sin necesidad de romper las clulas bacterianas, lo que hace que su extraccin y purificacin sea fcil y poco costosa. Las enzimas son sensibles a algunos de los ingredientes presentes en los detergentes, tanto durante el tiempo de almacenaje del producto como en el agua de lavado. Dentro de ellos, los surfactantes catinicos son los ms perjudiciales, aunque stos no son muy utilizados
[Baas, 1997].
Los surfactantes aninicos,
especialmente los ABS, tambin degradan las enzimas, mientras que los surfactantes no inicos no las desestabilizan
[Bahn, 1987].
Las enzimas se
desactivan por la presencia de agentes oxidantes, sin embargo, son ms estables en productos que contienen slo perborato sin ningn tipo de activador. An en este caso, la oxidacin de las enzimas durante el tiempo de almacenaje puede resultar un problema.
I.4.2. Proteasas Las proteasas son las enzimas encontradas mayoritariamente en los detergentes. Como se ha descrito anteriormente, las proteasas catalizan la rotura de largas protenas en molculas ms pequeas, como pptidos o aminocidos, que pueden ser ms fcilmente eliminadas por los surfactantes. Segn la posicin activa, las proteasas pueden clasificarse en dos grupos:
43
i) Endopeptidasas: rompen los enlaces peptdicos dentro de la cadena polipeptdica, dando lugar a pptidos solubles en agua. ii) Exopeptidasas: rompen los enlaces peptdicos terminales, obtenindos aminocidos libres. Tambin pueden ser clasificadas en funcin de las diferentes cepas de Bacillus, las cuales exhiben diferentes sensibilidades en funcin del pH, y distintas resistencias frente a los dems ingredientes de los detergentes. Las enzimas procedentes del Bacillus licheniformis, como por ejemplo la Alcalase (Novo Nordisk) o la Maxatase (Genencor), ofrecen una excelente relacin coste/prestaciones, especialmente a bajas temperaturas y a un pH moderado (pH 7-10). Otras proteasas, procedentes del Bacillus lentus/alcalophilus, como la Esperase, la Savinase (Novo Nordisk) y el Maxacal (Genencor), presentan una mayor actividad a pH ms alto, y una mejor resistencia al pH, con un intervalo de pH ptimo entre 8 y 12. Tambin difieren de otras proteasas en su masa molecular y en el punto isoelctrico. Estas proteasas tambin presentan una mayor estabilidad y actividad a altas temperaturas, as como una mejor eficacia en presencia de perborato, pero tambin tienen una mayor sensibilidad a la presencia de surfactantes aninicos [Crutzen, 1999; Maurer, 2005]. La actividad proteoltica se puede estimar mediante una gran variedad de mtodos
[Sarath, 1989].
Numerosos mtodos utilizan la degradacin de la
hemoglobina o la casena como protenas estndar para medir dicha actividad

[Maurer, 1997].
Hasta ahora, todas las proteasas empleadas en detergentes

[Egmond, 1997; Bott, 1997].
pertenecen a la familia de la subtilisina
Las protenas
de esta familia tienen una estructura tridimensional y un tamao casi idnticos. Las diferentes variantes de la enzima se distinguen slo por pequeas variaciones en la secuencia de aminocidos producidos de forma natural, o bien, mediante ingeniera gentica
[Ballinger, 1998].
Estas modificaciones pueden dar lugar a
44
pequeas diferencias en el punto isoelctrico, y a ligeros cambios en los electroferogramas obtenidos mediante enfoque isoelctrico, electroforesis en gel o capilar
[Vinther, 1992-A].
Cuando estas pequeas diferencias no son suficientes
para la identificacin de la enzima, es necesario recurrir a un mtodo de asociacin o la secuenciacin completa de la protena para resolver el problema
[Christianson, 1994].
En algunas variantes de proteasas el residuo de metionina en
la posicin 222 se ha sustituido por un residuo de aminocido ms estable frente a la oxidacin, lo que conduce a un aumento de la estabilidad en el periodo de almacenaje. Estas proteasas se pueden identificar y determinar mediante oxidacin con perxido de hidrgeno en un tampn de cido brico [Estell, 1985].
I.4.3. Amilasas Las amilasas aumentan la eficacia de los detergentes ayudando a disolver las manchas constituidas mayoritariamente por almidn, si bien, la mejora en la eficacia limpiadora es menor que la obtenida con proteasas. Estas enzimas son amilasas de origen bacteriano (Bacillus licheniformis) que catalizan la hidrlisis de los enlaces -1,4 glicosdicos de la amilosa y la amilopectina en dextrinas, oligosacridos y azcares, dando lugar a molculas solubles. Las amilasas presentan una mayor estabilidad frente a pHs alcalinos y a temperaturas altas que las proteasas. Para mantener la actividad de las amilasas, el medio debe contener suficiente concentracin de ion Ca2+, necesaria para estabilizarlas frente a la desnaturalizacin y al ataque de las proteasas [Crutzen, 1999]. Las amilasas pueden detectarse mediante el uso de tabletas de Phadebas o productos similares constituidos por almidn entrecruzado, insoluble y coloreado; en presencia de amilasa se produce su degradacin, con formacin de dextrinas y liberacin de colorantes solubles. Hasta ahora, las principales amilasas usadas en detergentes derivan del Bacillus licheniformis, cuya enzima
45
natural es termoestable. Mediante ingeniera gentica, se han desarrollado variantes de dicha enzima que mejoran su estabilidad frente a la oxidacin (Duramyl, Purastar OxAmTM), mostrando adems un mejor rendimiento que la variante natural original, especialmente en los detergentes para lavavajillas. Estas variantes de la amilasa pueden identificarse por su capacidad de resistir una incubacin en tampones que contienen perxido de hidrgeno, perborato o percarbonato [Estell, 1985; Maurer, 2005].
I.4.4. Lipasas Los triglicridos son los principales constituyentes de las manchas procedentes de grasas animales y aceites vegetales. Las lipasas empleadas en detergencia catalizan la hidrlisis de los enlaces ster de los principales componentes de las grasas, los TAGs. Por lo tanto, las lipasas ayudan a eliminar las manchas grasas, las cuales se adhieren fuertemente a los tejidos, y adems evitan su redeposicin durante el lavado. Las lipasas son una buena muestra de la cooperacin enzima-surfactante. Estas enzimas actan a temperaturas a las cuales la solubilizacin de las grasas por parte de los detergentes es baja. La reaccin enzimtica conduce a una mezcla de diglicridos, monoglicridos y glicerol, los cuales son menos hidrofbicos que los materiales originales, siendo por tanto ms fcilmente arrastrados por los surfactantes [Crutzen, 1999]. La primera lipasa industrial, Lipolase, de la casa comercial Novo, y aislada del hongo Humicola lanuginosa, apareci en el mercado en 1988. Gracias a la ingeniera gentica, la Lipolasa puede ser producida con buenos rendimientos a partir del inofensivo microorganismo Aspergillus oryzae. En relacin con la accin limpiadora, la lipasa difiere de proteasas y amilasas, ya que se necesitan varios lavados para observar sus beneficios
Gormsen, 1991]. [Aaslyng, 1991;
La diferencia es debida al efecto del agua en la actividad lipdica:
46
la enzima es poco activa cuando el contenido de agua en los tejidos est por debajo del 5%, o por encima del 80%, siendo mxima con un contenido de agua del 80%
[Gormsen, 1991].
Este nivel de agua se alcanza durante el proceso de
secado, por lo que la cantidad de material graso que permanece en el tejido durante el primer lavado es alta, sin embargo, durante el segundo lavado los triglicridos que han sido hidrolizados son eliminados mucho ms fcilmente
[Crutzen, 1999].
En cuanto al anlisis cualitativo, las lipasas pueden detectarse mediante incubacin con laurato o palmitato de p-nitrofenilo, con la subsiguiente aparicin del color amarillo del p-nitrofenol [Brockmann, 1981; Novo Nordisk, 1995; Martinelle,
1996; Pencreach, 2001; Eggert, 2000].
El ensayo enzimtico es extremadamente
sensible y debe ser controlado por la elevada probabilidad de falsos positivos debidos a la actividad de fondo de la Humicola, o por la autolisis del sustrato. En electroforesis en gel, la enzima puede teirse con steres de naftilo, como por ejemplo el acetato de -naftilo
[Stander, 2000; Sims, 1965; Maurer, 2005].
Ya que
las lipasas hidrolizan especficamente a los TAGs en cidos grasos libres y glicerol, se pueden determinar por valoracin de los cidos grasos que se han liberado durante la incubacin de un estndar
1995; Martinelle, 1996; Maurer, 2005]. [Brockmann, 1981; Novo Nordisk,
Por otro lado, tambin pueden determinarse
por deteccin fluorimtrica los componentes alcohlicos (glicerol) previo marcaje con resorufina [Junge, 1983]. En todos los casos, es necesario calibrar los mtodos utilizando la correspondiente lipasa como estndar.
I.4.5. Celulasas Todas las enzimas consideradas hasta el momento solubilizan las manchas por la degradacin de los principales constituyentes. Las celulasas ejercen un rol bastante diferente y proporcionan otros beneficios: suavidad de las fibras,
47
luminosidad del color, propiedades antipelusa y antirredeposicin [Maurer, 1998]. Estos beneficios son resultado de la eliminacin de microfibras formadas en la superficie de los tejidos de algodn
[Christensen, 1987].
Estos efectos son
acumulativos, incrementndose de forma considerable con el nmero de ciclos de lavado realizados

[Whalley, 1994].
Las celulasas catalizan la hidrlisis de las
uniones 1,4--glucosdicas de la celulosa. Las celulasas empleadas en detergencia son mezclas de endocelulasas, las cuales degradan aleatoriamente las cadenas de celulosa, y exocelulasas, que atacan los extremos de las cadenas de celulosa, liberando glucosa y celobiosa (un disacrido). Puesto que este ltimo compuesto inhibe la actividad de la exocelulasa, las formulaciones tambin contienen -glucosidasas, que transforman la celobiosa en glucosa
Crutzen, 1999]. [Boyce, 1986;
Las celulasas son las enzimas con mayor grado de variabilidad
entre las distintas clases de enzimas utilizadas en detergentes. As, las celulasas pueden obtenerse a partir de Humicola insolens, diversas especies de los gneros Trichoderma, Thielavia o Melanocarpus, o varias especies de Bacillus, as como a partir de hongos. Las celulasas se pueden detectar mediante el uso de mtodos de tincin basados en el CR o en el azul de tripn. Estos mtodos, bien conocidos en el mbito de la Bioqumica Analtica
[Cantwell, 1983; Bartley, 1984],
se basan en la
formacin de complejos entre el colorante y las cadenas de glucosa. Tambin se han descrito mtodos de anlisis de celulasas basados en la derivatizacin cromognica de la celulosa
[Fernley, 1963; McHale, 1981].
La identificacin de
celulasas separadas previamente mediante electroforesis est basada en la utilizacin de 5-bromoindoxil--D-cellobiosido, que se hidroliza a 5bromoindoxilo, el cual reacciona con el colorante Rojo Rpido (Fast Red), previamente acoplado a la celulosa mediante un enlace azo
[Chernoglazov, 1989].
La carboximetilcelulosa, derivado soluble de la celulosa, es tambin ampliamente
48
utilizada para el ensayo de la actividad de la celulasa. Este sustrato es especfico para la endo-1,4--glucanasa. Slo la actividad de las endoglucanasas es relevante para la funcin que desempean las celulasas dentro de los detergentes, mientras que las exocelulasas (celobiohidrolasas) desempean una funcin limitada o nula [Ghose, 1987]. Adems, tampoco se ha observado sinergismo entre exocelulasas y endoglucanasas
[Eriksson, 1985].
Por tanto, la actividad
predominante de las endoglucanasas justifica la utilizacin de la carboxicelulasa como el estndar ms adecuado para la determinacin de la actividad de las celulasas. La carboxicelulasa se determina mediante la medicin del aumento de azcares reducidos o la disminucin de la viscosidad de la disolucin. El mtodo basado en la disminucin de la viscosidad tiene la ventaja de ser robusto, pero presenta el problema de ser laborioso y difcil de automatizar [Maurer, 2005].
I.5. Tcnicas analticas
I.5.1. LC La cromatografa lquida es un mtodo fsico de separacin en el cual los componentes a separar se distribuyen entre dos fases: la fase estacionaria, que permanece fija, y la fase mvil, que se mueve en una direccin definida. Un sistema cromatogrfico (Fig. I.11) se compone al menos de un sistema de bombeo, un dispositivo para la introduccin de la muestra o inyector, una columna y un detector, y un sistema adecuado para la adquisicin de datos y control. Las bombas empleadas en LC pueden ser de dos tipos: bombas isocrticas o bombas de gradiente. Los mdulos de bombas para gradiente generan, mediante su control programado, mezclas de composicin variable. Segn el tipo
49
de bomba, las mezclas se preparan a baja o a alta presin, siendo distintas las caractersticas, ventajas y limitaciones de cada tipo.
Columna particulada o monoltica
Fase mvil Inyector Sistema de reparto Inyeccin de la muestra Registro de datos Detector
Residuo Conc.
A B C
tR Cromatograma
Fig. I.11. Diagrama de bloques de los componentes esenciales de un cromatgrafo de lquidos.
Tanto en LC como en otras tcnicas analticas, la muestra se inserta en el flujo de corriente de la fase mvil a travs de un bucle de inyeccin mediante una vlvula de 6 vas y 2 posiciones, que puede ser manual o automtica. El contenido del bucle se intercala entre el mdulo de bombas y la entrada de la columna. El tipo de columna a emplear depender del mecanismo de retencin con el que se desee trabajar. Los tipos de LC ms habituales son: i) Cromatografa de reparto: se utiliza una columna con una fase lquida enlazada. Dependiendo de las polaridades relativas de las fases mvil y estacionaria, se distinguen dos modalidades: RP, cuando la fase estacionaria es apolar y la fase mvil es polar, o bien NP, cuando la fase estacionaria es polar y la fase mvil es apolar. Un modo especial de cromatografa en fase normal es el HILIC, en el que la fase mvil es menos polar que la fase estacionaria, si bien mantiene su miscibilidad con agua.
50
ii) Cromatografa inica: la fase estacionaria es un intercambiador catinico o aninico de tipo cido fuerte, base fuerte, cido dbil o base dbil. iii) Cromatografa de exclusin molecular: como fase estacionaria se utilizan los poros de un slido microporoso o de un gel. Por otro lado, es importante desgasificar los disolventes para prevenir la formacin de burbujas en el equipo. Para ello, los cromatgrafos lquidos suelen incluir un desgasificador en lnea, insertado antes del paso de la fase mvil por el mdulo de bombas. Las tcnicas de deteccin ms empleadas en LC son la espectrofotometra UV-Vis y la MS. Como detectores alternativos se utilizan los de ndice de refraccin, as como los detectores evaporativos. Para aplicaciones especficas se usan los detectores amperomtricos y fluorimtricos. Finalmente, la deteccin conductimtrica es habitual en cromatografa inica. En espectrofotometra UVVis, la seal es proporcional a la concentracin molar del soluto, cuya absortividad molar depende de la naturaleza del grupo o grupos absorbentes. Cuando los LODs no son lo suficientemente bajos, se utilizan tcnicas de preconcentracin, o se exalta la absortividad de los solutos aumentando su conjugacin por derivatizacin. Los detectores espectrofotomtricos obedecen a dos tipos de diseo: longitud de onda variable y DAD. En los primeros, se selecciona una longitud de onda de medida fija, mientras que los segundos son capaces de barrer todo el intervalo del espectro UV-Vis varias veces por segundo. En este ltimo caso, el monocromador, est situado despus del paso de la radiacin por la muestra, de modo que sobre sta incide radiacin de todas las frecuencias. Despus del paso del haz por la muestra, se dispersa la radiacin transmitida, de modo que cada fotodiodo mide la intensidad correspondiente a un pequeo intervalo de longitudes de onda.
51
En MS, el detector proporciona informacin de alto nivel sobre las estructuras moleculares de los analitos, permitiendo distinguir grupos funcionales, elementos qumicos e istopos, de acuerdo con los valores m/z de los iones y sus fragmentos inicos. Un detector de MS acoplado a un cromatgrafo puede diferenciar compuestos con caractersticas de retencin muy similares, siendo posible identificarlos y/o cuantificarlos aunque slo estn parcialmente resueltos, o incluso no resueltos en absoluto. Ms recientemente, se han introducido modalidades cromatogrficas que implican miniaturizacin, bien en el tamao de la columna o en el dimetro de partcula. Entre las ventajas de estas tcnicas, cabe citar la reduccin de los tiempos de anlisis, del consumo de reactivos, del volumen de residuos generados y del tamao de muestra. A su vez, se pueden obtiener mejoras en la sensibilidad (los LODs son menores), y mayores eficacias. Entre sus desventajas, hay que indicar las mayores exigencias instrumentales, como el uso microbombas y micro/nano-nebulizadores.
I.5.1.1. Medida de parmetros cromatogrficos Para llevar a cabo una separacin cromatogrfica, el analista debe establecer si se puede separar adecuadamente al analito del resto de componentes de la muestra, y si la cantidad en la que se halla es suficiente como para poderlo detectar y/o determinar. El tiempo que transcurre desde la inyeccin hasta la deteccin de un analito es su tiempo de retencin o tR. Por su parte, el factor de capacidad o retencin relativa (k) expresa la retencin neta en unidades de tiempo muerto, t0, o tiempo que tarda en eluir un compuesto que no presenta retencin:
ki = t R ,i t0 t0
(E.I.1)
donde tR,i es el tiempo de retencin del analito i. El intervalo ptimo de valores de k se sita entre 1 y 5, si bien valores entre 0,2 y 10 son aceptables. Valores de
52
k inferiores a 0,2 indican poca retencin, preferencia excesiva del soluto por la fase mvil. Por el contrario, valores de k superiores a 20 indican una retencin demasiado alta, producida por una preferencia excesiva del soluto por la fase estacionaria, lo que implica tiempos de anlisis muy largos y en general picos anchos y de baja altura, difciles de detectar y de medir con precisin, y por tanto, con LODs mayores de lo deseable. La capacidad de un sistema cromatogrfico para distinguir entre dos solutos se expresa mediante el factor de selectividad i,j, que se calcula como el cociente entre las retenciones relativas de ambos solutos:
i, j =
kj ki
(E.I.2)
siendo i y j dos picos adyacentes, e i el soluto menos retenido. El grado de separacin entre dos solutos se mide mediante la resolucin, R:
R= t R ,i t R , j 0,5( wi + w j ) (E.I.3)
siendo wi y wj las anchuras de las bases de los picos de los compuestos i y j. Por su parte, la eficacia describe el grado de ensanchamiento de bandas en relacin al volumen de retencin. Se obtiene una elevada eficacia cuando los picos se mantienen estrechos a pesar de haber requerido un volumen elevado de fase mvil para su elucin. La eficacia global de un sistema se describe mediante el nmero de platos tericos (N), y la eficacia por unidad de longitud por la altura equivalente a un plato terico (H), o por su inversa (1/H). Para un soluto determinado, N puede calcularse a partir de las expresiones:
t N = 16 R w
2
t N = 5,54 R w 1/ 2
(E.I.4)
donde tR es el tiempo de retencin del soluto y w y w1/2 son la anchura de la base
53
del pico y su anchura a media altura, respectivamente. Por su parte, H se relaciona con N a travs de la expresin:
N= L H
(E.I.5)
donde L es la longitud de la columna. La ecuacin de Van Deemter describe las contribuciones a H, esto es, indica como los diversos factores de construccin y funcionamiento de la columna influyen sobre la eficacia. Para el caso de columnas microparticuladas tanto en HPLC como en CEC, se tiene la expresin simplificada siguiente:
H = A+ B + C u u
(E.I.6)
donde u es la velocidad lineal promedia de la fase mvil. El trmino A de la ecuacin de van Deemter se conoce como difusin de remolino o torbellino, y se debe a la distinta longitud de los caminos recorridos y a las distintas velocidades de los solutos en su avance por el lecho cromatogrfico (Fig. I.12, parte A). La contribucin al ensanchamiento de bandas se debe a que las molculas se mueven a distinta velocidad segn la anchura del camino seguido. Adems, la fase mvil que avanza por el centro de los canales se mueve ms rpidamente que la que avanza pegada a las paredes. Esta contribucin a H es funcin slo de la geometra del relleno, esto es, no depende de u .
A A Trmino A B B

Fig. I.12. A) Difusin de remolino y B) difusin molecular longitudinal.
54
El trmino B representa la difusin molecular longitudinal (en la direccin axial), que es debida a la difusin de los solutos a nivel molecular (Fig. I.12, parte B). Esta difusin es proporcional a la difusibilidad de los solutos y al tiempo de residencia de la muestra en la columna. Conforme aumenta el tiempo de permanencia, mayor es la difusin, y por tanto el trmino B slo cobra importancia a velocidades de flujo bajas. Esta dependencia con el tiempo de permanencia se refleja en la proporcionalidad inversa de esta contribucin repecto a u . El trmino C corresponde a la contribucin combinada de las velocidades de transferencia de masa en la fase mvil y en la fase estacionaria (CM y CS). Este trmino es proporcional a u ya que el avance de la fase mvil compite en trminos de velocidad con las transferencias de masa del soluto entre ambas fases, de modo que la importancia del trmino C aumenta con la velocidad de la fase mvil al ser menor el tiempo de equilibrado entre ambas fases. La lentitud o retraso con la que se efectan las transferencias de masa despus de cada etapa de avance de la fase mvil origina un ensanchamiento de la zona ocupada por el soluto. Los trminos A y C de la ecuacin de van Deemter indican que la eficacia de la columna puede mejorarse utilizando partculas de fase estacionaria ms pequeas, o tambin rellenos ms uniformes. La forma de la curva de van Deemter proporciona informacin acerca de la calidad del empaquetado o relleno de la columna cromatogrfica. Cuanto menores sean las contribuciones de los trminos A y C mayor ser el nmero de platos tericos a una determinada velocidad de la fase mvil. En la Fig. I.13 se muestra una representacin tpica de esta ecuacin.
55
Altura equivalente a un plato terico
H = A+
B + C u u u
C u
B u
A Uptima Velocidad lineal promedia
Fig. I.13. Representacin de H frente a (curva de van Deemter).
I.5.2. CE La CE es una tcnica de separacin que se basa en la migracin diferencial de especies cargadas en el seno de un tubo capilar y en presencia de un campo elctrico. La fuerza que impulsa el flujo en CE se genera en la misma pared interna del capilar mediante el fenmeno conocido como electromosis
1994; Li, 1992; Camilleri, 1993; Heiger, 1992; Brown, 1995; Rogan, 1993]. [Landers,
I.5.2.1. Mecanismo de separacin en CE La velocidad que adquiere un soluto cargado en el seno de un campo elctrico es proporcional al campo: v = e E (E.I.7)
donde v es la velocidad del ion, e la movilidad electrofortica del mismo y E el campo elctrico aplicado. La movilidad electrofortica es una constante caracterstica del ion en un determinado medio. Viene determinada por el balance entre la fuerza electrosttica, Fe, y las fuerzas de rozamiento, Ff: Fe = q E Ff = 6rv (E.I.8) (E.I.9)
56
donde r es el radio efectivo del ion hidratado o solvatado y es la viscosidad de la disolucin. Cuando se alcanza el estado estacionario, ambas fuerzas son de la misma intensidad pero signo opuesto, por lo que: e = q/ (6r) (E.I.10)
De esta ecuacin se deduce que las partculas con mayor densidad de carga elctrica tendrn movilidades mayores. La movilidad se considera positiva cuando el ion se dirige al ctodo. La movilidad medida en funcin del tiempo de migracin se conoce como movilidad aparente o efectiva (ap), y difiere de la intrnseca o electrofortica cuando se observa en presencia de EOF no nulo. La movilidad aparente corresponde a la suma de las movilidades electrofortica y electroosmtica, siendo la velocidad total de las partculas cargadas la suma de sus velocidades electrofortica y electroosmtica: v = ve + vEOF = apE = (e +EOF)E (E.I.11)
I.5.2.2. EOF El EOF es uno de los principales fenmenos que afectan a las separaciones electroforticas. En contacto con un medio acuoso, la superficie del capilar de slice tiene generalmente un exceso de carga negativa que es debido a la ionizacin de los grupos silanol. Para capilares de slice fundida, el EOF se puede controlar reduciendo o aumentando el nmero de grupos silanol en forma ionizada. As, el EOF es prcticamente nulo por debajo de pH 3, y aumenta con el pH. Para la slice se tiene: log Kmedio 5,5. Los iones que se encuentran en la disolucin tienden a neutralizar la carga de la superficie del capilar, formando una doble capa elctrica, y creando una diferencia de potencial residual conocida como potencial zeta. La primera capa, o capa de adsorcin primaria, est fuertemente retenida, pero sobre ella se establece una capa difusa en la que predominan los iones del signo contrario al
57
potencial zeta de la superficie. Esta segunda capa est menos retenida, por lo que puede moverse por aplicacin de una diferencia de potencial, y en su movimiento arrastra a todo el lquido. En la Fig. I.14 se representa esquemticamente este fenmeno.
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
Capa de adsorcin primaria
+
+
+ + -
EOF +
Capa difusa
Fig. I.14. Esquema de la generacin de EOF por aplicacin de campo elctrico.
La velocidad del EOF (vEOF) es proporcional al campo: vEOF = EOFE (E.I.12)
donde EOF es la movilidad electroosmtica que es proporcional al potencial zeta sobre la superficie: EOF = / (E.I.13)
donde es el potencial zeta o densidad de carga residual sobre la superficie de la pared interna del capilar, y es la permisividad elctrica o constante dielctrica del medio. La EOF depende de la naturaleza de la pared del capilar y de la composicin del medio, especialmente del pH y de la fuerza inica. El potencial zeta es funcin de la carga por unidad de superficie, y depende tambin del pH, y de la concentracin y la naturaleza de todos los iones presentes.
I.5.2.3. Movilidad y tiempo de migracin El tiempo que un soluto necesita para migrar desde el punto de inyeccin hasta el punto de deteccin se denomina tiempo de migracin. El tiempo de migracin se relaciona con los siguientes parmetros experimentales:
58
+ +
+ +
+ +
+ + +
ap =
l lL = tE tV
(E.I.14)
donde la ap es la movilidad aparente, V es el voltaje aplicado, l es la longitud efectiva del capilar o distancia desde la entrada hasta el detector, L es la longitud total del capilar, t es el tiempo de migracin y E es el campo elctrico aplicado.
I.5.2.4. Tcnicas de electroseparacin capilar La electroseparacin capilar abarca diversas tcnicas caracterizadas por su gran versatilidad. Las tcnicas ms utilizadas son:
A) FSCE o CZE En la FSCE o CZE, el capilar se llena slo con el BGE, y la separacin tiene lugar gracias a las diferentes movilidades electroforticas de los solutos. La seleccin del BGE es extremadamente importante, ya que la selectividad de la separacin depende en gran medida de su naturaleza. Los reactivos utilizados deben cumplir las siguientes condiciones: i) Buena capacidad amortiguadora en el intervalo de pH requerido. ii) Baja absorbancia a la longitud de onda de deteccin. iii) Baja movilidad electrofortica de sus componentes para minimizar la corriente.
B) MEKC La MEKC fue introducida en 1984 por Terabe y col. [Terabe, 1984; Terabe,
1989; Terabe, 1992; Otsuka, 1989],
y se utiliza para separar especies sin carga
elctrica neta. La separacin se basa en las diferencias de las constantes de asociacin entre solutos y micelas. El BGE contiene un surfactante inico a concentracin superior a su CMC. Tanto las micelas como los iones libres del surfactante experimentan interacciones hidrofbicas y electrostticas con los
59
solutos. Las especies no cargadas que interaccionan con las micelas adquieren movilidad, distinguindose del EOF, y las que no interaccionan se mueven con el mismo. Se puede aplicar tambin a especies inicas, si bien, en este caso el mecanismo de separacin es mixto, cromatogrfico y electrofortico a la vez.
I.5.3. CEC La CEC es una tcnica analtica de separacin en fase lquida que combina la elevada eficacia de la CZE con la alta selectividad y reproducibilidad proporcionadas por la HPLC. En CEC, la separacin se lleva a cabo en columnas capilares rellenas total o parcialmente con una fase estacionaria. Como en CZE, el EOF es generado por el campo elctrico. Para que se genere EOF debe asegurarse la presencia de cargas sobre la superficie del relleno de la columna. Por otro lado, el EOF es el responsable del bombeo en CEC y en otras tcnicas de electroseparacin. El bombeo mediante el EOF da lugar a un perfil plano de velocidades en el seno de la fase mvil, a diferencia del perfil parablico que se obtiene cuando el bombeo es por presin externa, como en HPLC. El perfil plano del flujo es uno de los factores que permite obtener elevadas eficacias. En CEC el mecanismo de separacin es doble [Rathore, 1996]. Por un lado, hay un mecanismo cromatogrfico, ya que se produce un reparto de los solutos entre una fase mvil y una estacionaria. Por otro lado, los solutos inicos tambin se separan mediante un mecanismo electrofortico, esto es, en base a las diferencias de movilidad electrofortica, por lo que la naturaleza del relleno de la columna determina el EOF e influye sobre la selectividad de la separacin.
I.5.3.1. Instrumentacin en CEC Un instrumento para CEC (ver el esquema de la Fig. I.15) est constituido bsicamente por una fuente de alto voltaje, un sistema de suministro de
60
disolvente y/o muestras en los viales de entrada y de salida de la columna, una columna capilar con una fase estacionaria en la cual se genera el EOF y tambin tiene lugar la separacin electrocromatogrfica, un compartimento isotrmico para la columna capilar, y un sistema de deteccin capaz de registrar los perfiles de concentracin de los analitos en el eluyente.
Fuente de campo elctrico Detector
Capilar relleno Fuente de presin
BGE
Fig. I.15. Esquema de un instrumento de CEC.
La misma instrumentacin utilizada en CE sirve para CEC, si bien, en este caso se debe de disponer de un sistema de presurizacin de los viales de entrada y salida. Esta presurizacin es necesaria para evitar la formacin de burbujas, que podran interrumpir la corriente. Las burbujas pueden originarse por diversas causas, ya sea por diferencias locales en la velocidad del EOF [Rathore, 1998], en el campo elctrico, por prdida del gas atrapado en los poros de la fase estacionaria, o por gas formado electroqumicamente calentamiento
[Knox, 1988; Tsuda, 1987], [Carney, 1999],
por
o en el caso de columnas empaquetadas,
por la presencia de las fritas que mantienen la integridad estructural del relleno
[Rebscher, 1994].
La presurizacin se puede aplicar sobre el vial de entrada o en el
de salida, aunque generalmente se aplica sobre ambos viales, ya que as se asegura un flujo reproducible. Para presurizar los viales se usa un gas inerte, normalmente N2, a presiones prximas a 10 bares. Para obtener resultados reproducibles en CEC, es necesario el control de parmetros como la temperatura
61
de la columna, el voltaje aplicado y la presin. En los equipos comerciales de CE, estos parmetros se controlan automticamente, lo que da lugar a mejoras significativas de la reproducibilidad y seguridad de las separaciones. La espectrofotomtrica UV-Vis es la tcnica de deteccin ms utilizada en CEC
[Choudhary, 2000; Rozing, 2001; Devowsky, 2002; Cahours, 2002],
siendo
posible trabajar tambin en el modo de deteccin indirecto. La deteccin se realiza en la misma columna, utilizando como celda de deteccin una pequea seccin de la misma, adyacente al relleno, y de la cual se ha retirado la capa protectora de polmero. Otras tcnicas de deteccin tambin ampliamente utilizadas son la fluorescencia inducida por lser [Wall, 2002; Horstktter, 2002; Liu,
2001],
y la MS [Shamsi, 2004; Klampfl, 2004; Barcel-Barrachina, 2004].
I.5.3.2. Columnas empleadas en CEC Las columnas para CEC se preparan normalmente a partir de capilares de slice fundida con dimetros internos comprendidos entre 100 y 200 m. En base a las diferencias en los soportes cromatogrficos, se pueden distinguir tres tipos de columnas: abiertas, empaquetadas y monolticas. Las columnas monolticas, constituidas por un lecho continuo, poseen una estructura porosa que permite trabajar en HPLC con caudales elevados, y por tanto, obtener separaciones rpidas sin que ello conlleve un aumento excesivo de la presin necesaria para mantener el caudal, a diferencia de lo que sucede con las columnas particuladas. En CEC las columnas monolticas constituyen igualmente una alternativa a las empaquetadas, con algunas interesantes ventajas. As, dada su estructura continua, no es necesario el empleo de fritas en los extremos del lecho monoltico, ya que ste est anclado directamente sobre la pared del capilar mediante enlaces covalentes. Adems, pueden prepararse in situ, por lo que la fabricacin de lechos monolticos es relativamente sencilla en
62
comparacin con las tcnicas de empaquetado de partculas. Las columnas monolticas pueden clasificarse en dos categoras principales, de slice y polimricas. Las columnas de slice se preparan usando la tecnologa sol-gel. La estructura de un monolito de slice muestra esqueletos interconexionados que crean una distribucin determinada de poros. Por otro lado, la preparacin de las columnas monolticas polimricas se lleva a cabo fcilmente mediante el relleno de las columnas capilares con una mezcla de polimerizacin constituida por monmeros, un agente entrelazante (cross-linker), una mezcla porognica de disolventes y un iniciador radicalario. La hidrofobicidad del monolito resultante se puede controlar seleccionando la naturaleza del monmero [Liao, 1996; Palm, 1997]. El EOF se asegura mediante la presencia de monmeros derivados de los cidos acrlico o sulfnico, o mediante sales de amonio cuaternario en la mezcla de polimerizacin
[Ericson, 1999].
La
polimerizacin se inicia por medios trmicos, qumicos o mediante radiacin UV. Para evitar cualquier desplazamiento del monolito a lo largo de la columna, es necesario anclar el polmero a la pared interna del capilar. Para ello, antes de introducir en el capilar la mezcla de polimerizacin, se silaniza la pared interna del mismo. Con este fin, en la mayor parte de los casos, se utiliza 3(trimetoxisilil)propil metacrilato (silano binding). Para construir monolitos se han utilizado diferentes tipos de polmeros, pudiendo distinguirse principalmente entre los derivados de acrilamida, poliestireno y steres de metacrilato o acrilato. En las primeras columnas monolticas que fueron descritas se utiliz acrilamida y metacrilamida
1989; Fujimoto, 1995; Hoegger, 2001]. [Hjertn,
Estos polmeros se preparan por
polimerizacin de acrilamida, metacrilamida o sus derivados en presencia de metilenbisacrilamida o piperazina diacrilamida como agentes entrelazantes. Las
63
columnas monolticas basadas en poliestireno
[Gusev, 1999; Petro, 1996]
se
obtienen por polimerizacin de estireno o sus derivados con divinilbenceno como agente entrelazante. Los monolitos basados en steres de metacrilato
2003; Zhang, 2003; Peters, 1998-A; Peters, 1998-B] [Merthar,
se preparan mediante
polimerizacin de butilmetacrilato u otros steres derivados del metacrilato, empleando etilenglicol dimetacrilato como agente entrelazante.
I.5.3.3. Polimerizacin de columnas monolticas basadas en steres de metacrilato y acrilato Las columnas monolticas basadas en polimetacrilato son las ms extendidas y mejor caracterizadas, habiendo sido ampliamente desarrolladas por Svec y col. [Peters, 1998-A; Peters, 1998-B], quienes han descrito aplicaciones tanto en HPLC como en CEC. Los polmeros de metacrilato y acrilato poseen caractersticas mecnicas y qumicas que los hacen altamente apropiados como fases estacionarias. Son estables en un amplio intervalo de valores de pH (212), a diferencia de las fases estacionarias basadas en slice, que se degradan con gran facilidad por encima de pH 9. Su sntesis es rpida y sencilla, siendo posible partir de monmeros de polaridades muy diversas. La polimerizacin de los monolitos basados en steres de metacrilato y/o acrilato se realiza mediante una reaccin radicalaria, iniciada generalmente por una temperatura elevada, por irradiacin UV, o por agentes qumicos a temperatura ambiente. Para la iniciacin trmica de la polimerizacin se suele adicionar a la mezcla de monmeros AIBN
2001], [Peters, 1998-A; Peters, 1998-B; Chirica,
perxido de benzoilo [Xie, 1997], u otros perxidos [Cant-Mirapeix, 2008-D]. Svec y col.
[Peters, 1998-A; Peters, 1998-B]
han demostrado que las
propiedades cromatogrficas (eficacia, selectividad, permeabilidad, etc) de estos materiales pueden alterarse variando la composicin de la mezcla de
64
polimerizacin, lo que constituye una va interesante, no slo para el desarrollo y optimizacin de las separaciones cromatogrficas, sino para sus aplicaciones de inters ambiental, bioqumico e industrial.
I.5.3.4. Caracterizacin de materiales monolticos Los materiales monolticos se pueden caracterizar estudiando tanto sus propiedades morfolgicas como electrocromatogrficas. Existen numerosas tcnicas que proporcionan informacin acerca de la influencia de diversos factores sobre las propiedades morfolgicas de los materiales monolticos Para el estudio de las propiedades morfolgicas de los materiales monolticos, existen numerosos mtodos y herramientas analticas. Entre ellas cabe citar la SEM
[Doneanu, 2002], [Baeuml, 2002],
la porosimetra de intrusin de mercurio
la adsorcin/desorcin de nitrgeno, evaluada mediante la

[Brunauer, 1938]
ecuacin de Brunauer-Emmet-Teller
y la permeabilidad
cromatogrfica. Las columnas monolticas utilizadas a lo largo de esta Tesis Doctoral se han caracterizado principalmente mediante SEM, por lo que esta tcnica se comenta ms extensamente a continuacin. Mediante SEM se obtienen imgenes de la estructura de un material. Sobre la superficie del material se enfoca un haz de electrones. Este haz barre la superficie del material, produciendo principalmente la emisin de electrones secundarios de baja energa y electrones retrodispersados de mayor energa, recogindose ambos mediante adecuados sistemas de deteccin [Aballe, 1996]. La informacin obtenida vara segn las caractersticas del detector empleado. Los electrones secundarios se forman en una delgada capa superficial, del orden de 5 a 10 nm de espesor. La seal est constituida en parte por electrones que emergen de la muestra con una energa inferior a 50 eV. El nmero de electrones de este tipo es suficientemente elevado como para establecer un buen contraste entre las
65
estructuras que se quieren describir. Por otra parte, al tratarse de electrones de baja energa, pueden desviarse fcilmente de su trayectoria emergente inicial, y se puede obtener informacin de zonas que no estn a la vista del detector. Esta particularidad es fundamental para otorgar a la seal la posibilidad de aportar una informacin tridimensional de la topografa de la muestra, siendo quizs la caracterstica ms conocida de esta tcnica. Por otro lado, la principal utilidad de la seal de electrones retrodispersados, compuesta por aquellos electrones que emergen de la muestra con una energa superior a 50 eV, reside en que su emisin depende fuertemente del nmero atmico de los elementos de la muestra. Por esta razn, dos zonas con distinta composicin qumica se revelarn con distinta intensidad, aunque no exista ninguna diferencia de topografa entre ellas. Para aplicar SEM la muestra a analizar debe estar seca. En caso contrario, la baja presin existente en el microscopio causara la evaporacin de los componentes voltiles que saldran despedidos violentamente, alterando la estructura de la muestra. Adems, la superficie debe ser conductora, lo que se consigue recubrindola con una pelcula de un material conductor. Para ello, se utilizan tcnicas de pulverizacin catdica a alto vaco. Por otro lado, la reducida estabilidad trmica de los polmeros limita el voltaje que puede aplicarse para obtener las imgenes.
I.5.4. MS La MS es una tcnica de aplicacin general, capaz de suministrar informacin sobre la composicin cualitativa y cuantitativa, tanto de analitos orgnicos como inorgnicos, en muestras complejas. Los espectros de masas se obtienen por conversin de los componentes de una muestra en sus respectivos
66
iones en fase gas, que se separan en funcin de su relacin m/z, siendo la seal analtica la abundancia o intensidad para cada valor m/z. En la Fig. I.16 se muestra un esquema de los componentes principales de un espectrmetro de masas.
Muestra
10-5 - 10-8 torr Sistema de Sistema de entrada entrada Analizador Analizador de masas de masas Detector Detector
Fuente de iones Fuente de iones
Sistema Sistema de vaco de vaco
Procesador Procesador de la seal de la seal
Dispositivo Dispositivo de lectura de lectura
Fig. I.16. Componentes de un espectrmetro de masas.
Como se ilustra en la figura, un espectrmetro de masas est formado en primer lugar por un sistema de entrada, que permite la introduccin de una pequea cantidad de muestra en el espectrmetro. El sistema de entrada elegido ser diferente segn se quieran introducir muestras slidas, lquidas o gaseosas. La muestra se puede introducir de manera discreta mediante una jeringa o una sonda directa, o de forma continua mediante el acoplamiento con un sistema de inyeccin en flujo o mediante un sistema cromatogrfico o electrofortico. Junto al sistema de entrada, la fuente de iones es la encargada de convertir los componentes de la muestra en iones por bombardeo con electrones, molculas, fotones o por otros medios. En muchas ocasiones el sistema de entrada y la fuente de iones estn combinados en un nico componente. Una vez producidos los iones, stos son acelerados hacia el analizador por aplicacin de campos elctricos. En el analizador de masas los iones se separan en base a su relacin m/z, de forma que llegan al detector en diferentes momentos. Esta
67
pequea corriente de iones se amplifica mediante el transductor, normalmente un multiplicador de electrones que puede estar combinado con un multiplicador de fotones
[Rubinson, 2001; Skoog, 1996].
Estos cuatro componentes se encuentran,
generalmente, dentro de un sistema de vaco, a unas presiones de 10-7 10-10 atm, necesarias para evitar colisiones con el gas de fondo u otras molculas. Slo en algunos casos, el vaco se aplica tan slo al analizador de masas y al detector. Por ltimo, una vez registrada la seal analtica, se procede a su procesado y anlisis, obtenindose el espectro de masas. En el espectro de masas se muestran las cantidades de masa recogidas a valores crecientes de la relacin m/z. Cada pico del espectro proporciona informacin sobre la masa de un determinado fragmento de la molcula que lo origin. Para las fuentes habituales en HPLC y CE, y para los espectros obtenidos en modo positivo, el fragmento de mayor tamao suele ser la molcula sin fraccionar con prdida de un electrn, M+, o con la adicin de un protn, [M+H]+, u otro catin.
I.5.4.1. Fuentes de iones Las fuentes de iones ms habituales son las siguientes [Rubinson, 2001]: EI, CI, ESI, APCI, APPI, ICP y FAB. Dentro de estas, las fuentes ms utilizadas en el acoplamiento LC-MS, son la ESI, la APCI y la APPI. En esta seccin slo se explica la fuente ESI, que es la que se ha empleado en alguno de los trabajos de esta Tesis. Dos caractersticas importantes de la ESI (Fig. I.17) son la generacin de iones en fase gas en la misma forma en que se encuentran en disolucin, y la relativa facilidad para impedir la entrada de grandes cantidades de disolvente en el analizador de masas. Por esta ltima razn, la ESI es una fuente muy utilizada en los acoplamientos HPLC-MS. En ESI la disolucin de la muestra fluye a travs de una aguja hueca. La aguja se encuentra sometida a un campo elctrico
68
elevado respecto a las paredes de la cmara de nebulizacin, por lo que se forman pequeas gotas con carga elctrica. La aguja tiene dos flujos concntricos, el interior que lleva la muestra, y el exterior que lleva un gas que ayuda a la formacin del aerosol. Las gotas que poseen carga neta son atradas hacia un electrodo a travs del espacio abierto de la cmara de nebulizacin. En la cmara, las gotas se mueven en contra del gas de secado, que evapora parte del disolvente.
Iones Gas nebulizador
Gas de secado Aerosol
Entrada del capilar
Fig. I.17. Esquema de una fuente ESI.
Como consecuencia de este proceso, el volumen de las gotas disminuye y los iones de la superficie se ven forzados a aproximarse unos a otros. En un momento dado, la repulsin de los iones se hace mayor que la tensin superficial que mantiene unidas a las gotas y stas se rompen (explosin de Coulomb). De estas gotas se generan los iones en fase gas, que son atrados hacia el orificio de entrada del analizador de masas por la accin de un segundo campo elctrico. Dependiendo de la polaridad de este campo, en el analizador de masas entran slo los aniones (operacin en modo negativo) o slo los cationes (modo positivo). Los procesos de generacin de iones y evaporacin del disolvente tienen lugar en la superficie de las gotas, y cualquier cambio en la misma puede ser muy importante. La fuente de ionizacin por electronebulizacin puede
69
producir iones con carga mltiple, lo que permite el anlisis de compuestos de elevada masa.
I.5.4.2. Analizadores de masas La funcin de los analizadores de masas es la de separar iones con diferentes relaciones m/z
[Rubinson, 2001].
Existen diferentes tipos de
analizadores, y el poder de resolucin del espectrmetro de masas depender principalmente de las prestaciones del analizador. Los analizadores de masas ms ampliamente utilizados en HPLC y CE son el cuadrupolo (simple o triple), la IT y el de tiempo de vuelo. En esta seccin slo se explica la trampa de iones, por ser la empleada en algunos de los trabajos de esta Tesis. Una IT permite analizar cationes y aniones en un amplio intervalo de valores de m/z. Mediante la accin de campos elctricos y magnticos, los iones se pueden confinar en un espacio reducido durante largos periodos de tiempo. Un diseo sencillo de trampa de iones consiste en un electrodo en forma de anillo y un par de electrodos colectores (electrodos de entrada y de salida). Al electrodo anular se le aplica un potencial de radiofrecuencia variable, mientras que los electrodos colectores estn a potenciales alternos adecuados para estabilizar las rbitas de los iones en el plano transversal o plano del anillo. Los iones que tienen el valor apropiado de m/z se mueven en rbitas estables, cuyo radio depende de un valor de m/z, dentro de la cavidad formada por el anillo. Los iones procedentes de la fuente de ionizacin se introducen a travs de una abertura practicada en el electrodo colector superior, quedando atrapados en rbitas estacionarias (Fig. I.18). A continuacin, se desestabilizan mediante una rampa de potencial continuo superpuesta al potencial alterno del electrodo de salida. Los iones dejan la cavidad del anillo a travs del electrodo de salida,
70
pasando al detector. En una IT, el proceso de carga y transmisin de iones al detector es discontinuo.
Captura de iones
Eyeccin secuencial
Incremento de V
Fig. I.18. Esquema de funcionamiento de una IT.
Entrada Gas nebulizador
Nebulizador
Desecho
Gas de secado
Vaporizacin y ionizacin (ESI)
Enfoque
Analizador Deteccin (IT)
Fig. I.19. Esquema de un espectrmetro de masas con fuente ESI y analizador IT.
La capacidad de retener iones en la IT permite obtener conjuntos de iones hijos, que se pueden aislar o fragmentar para barrer espectros de segunda generacin o MS2. A su vez, los iones resultantes pueden ser de nuevo aislados y fragmentados para barrer espectros de tercera generacin o MS3, y as
71
sucesivamente. Las fragmentaciones en el interior de la IT se consiguen mediante colisiones con molculas de He. En la Fig. I.19 se muestra un esquema de las diferentes partes de un espectrmetro de masas compuesto por una fuente ESI y un analizador IT.
I.5.4.3. Acoplamiento de una tcnica de separacin a un MS Los MS se pueden acoplar con GC, LC, SFC o con sistemas de CE o CEC. Para mostrar todos los componentes eluidos en la separacin se representa un TIC. La intensidad en el TIC es la suma de las respuestas del detector a todos los iones presentes en cada momento. Tambin se pueden representar cromatogramas a determinados valores de m/z, uno o varios. Estos se pueden obtener de dos formas: como SIMs y, a partir de los datos almacenados en un TIC, como EICs. Cuando el cromatograma se registra a un valor m/z nico (o a unos pocos valores de m/z), en lugar de hacer un barrido en un intervalo de m/z, la tcnica se denomina SIM. Esta monitorizacin proporciona un lmite de deteccin considerablemente menor que un EIC, especialmente si el EIC se ha obtenido a partir de un barrido muy amplio de valores de m/z. Adems, un SIM permite simplificar la seal en cromatogramas complejos que presenten numerosas interferencias. Para introducir en el espectrmetro el efluente procedente de un cromatgrafo lquido o de una separacin electrofortica, es necesario utilizar una interfaz adecuada. La misin de la interfaz es eliminar en lo posible la fase mvil sin distorsionar el perfil de concentraciones de los analitos. Actualmente, las fuentes ESI, APCI y APPI son las interfaces ms utilizadas en los acoplamientos HPLC-MS, CE-MS y CEC-MS. Una limitacin de los acoplamientos LC-MS o CE-MS es que los modificadores de la fuerza eluyente,
72
los tampones o electrolitos de fondo deben ser voltiles, para reducir en lo posible los lmites de deteccin y minimizar las posibles interferencias en el espectrmetro.
I.5.4.4. Resolucin La capacidad de un espectrmetro de masas para distinguir entre masas similares se expresa normalmente en trminos de resolucin, R, que se define como: R = m/m (E.I.15)
Siendo m la masa nominal del primer pico, e m el poder resolutivo del espectrofotmetro que se define como la anchura del primer pico a una fraccin dada de la altura, con frecuencia a un 10% o a un 50%. La resolucin que se necesita en un espectrmetro de masas depende en gran parte de su aplicacin. Por ejemplo, para diferenciar entre iones de la misma masa nominal pero con diferentes masas exactas, se necesitan equipos de elevada resolucin.
I.5.5. NMR La espectroscopa de NMR fue desarrollada a finales de los aos cuarenta para estudiar los ncleos atmicos. En 1951, se descubri que la espectroscopa de resonancia magntica nuclear poda ser utilizada para determinar las estructuras de los compuestos orgnicos. Esta tcnica espectroscpica puede utilizarse slo para estudiar ncleos atmicos con un nmero impar de protones o neutrones (o de ambos). Esta situacin se da en ncleos de una docena de elementos, entre ellos los de 1H,
13
C,
19
F y
31
P. Este tipo de ncleos son
magnticamente activos, es decir poseen espn no nulo. En el seno de un campo
73
magntico estos ncleos se comportan como pequeos imanes, pudiendo adquirir un movimiento de precesin, cuyos niveles estn cuantizados. En ausencia de campo magntico, los espines nucleares se orientan al azar. Sin embargo, cuando una muestra se coloca en un campo magntico, los ncleos con espn positivo se orientan en la misma direccin del campo, en un estado de mnima energa denominado estado de espn , mientras que los ncleos con espn negativo se orientan en direccin opuesta a la del campo magntico, en un estado de mayor energa denominado estado de espn . Existen ms ncleos en el estado de espn que en el siendo esta diferencia suficiente para establecer las bases de la espectroscopa de NMR. La diferencia de energa entre los dos estados de espn y , depende de la fuerza del campo magntico aplicado H0. Cuanto mayor sea el campo magntico, mayor diferencia energtica habr entre los dos estados de espn. Cuando una muestra es irradiada brevemente por un pulso intenso de radiacin, los ncleos en el estado de espn son promovidos al estado de espn . Esta radiacin se encuentra en la regin de las radiofrecuencias (rf) del espectro electromagntico, por ello se le denomina radiacin rf. Cuando los ncleos vuelven a su estado inicial, emiten seales cuya frecuencia depende de la diferencia de energa (E) entre los estados de espn y . El espectrmetro de NMR detecta estas seales y las registra como una grfica de frecuencias frente a intensidad, que es el llamado espectro de NMR. El trmino NMR procede del hecho de que los ncleos estn en resonancia con la radiofrecuencia de la radiacin rf. Es decir, los ncleos pasan de un estado de espn a otro como respuesta a la radiacin rf a la que son sometidos. La siguiente ecuacin muestra la dependencia entre la frecuencia de la seal () y la fuerza del campo magntico H0 (medida en Teslas, T).
E = h = h
H0 2
74
(E.I.16)
donde h es la constante de Planck y el radio giromagntico. El valor de este ltimo depende del tipo de ncleo que se est irradiando.
I.5.5.1. Espectrmetro de NMR En la Fig. I.20 se muestran de forma esquemtica los principales componentes de un equipo para medidas de NMR.
Tubo con muestra Espectro de NMR
Detector y Amplificador Imn superconductor
Generador de radiofrecuencia y ordenador
Fig. I.20. Esquema de un espectrmetro de NMR.
Como se aprecia, el espectrmetro de NMR consta de cuatro partes: un imn con un controlador que produce un campo magntico preciso y estable, un transmisor de radiofrecuencias, un detector para medir la absorcin de energa de rf de la muestra, ordenador y un registrador para obtener el espectro de NMR. Para obtener un espectro de NMR, se coloca una pequea cantidad del analito en un disolvente en un tubo de vidrio largo que se sita dentro del campo magntico. El campo magntico se mantiene constante mientras un breve pulso de radiacin rf excita a todos los ncleos simultneamente. Dado que el corto pulso de radiofrecuencia cubre un amplio intervalo de frecuencias, los protones individualmente absorben la radiacin de frecuencia necesaria para entrar en resonancia (cambiar de estado de espn). A medida que dichos ncleos vuelven a
75
su posicin inicial emiten una radiacin de frecuencia igual a la diferencia de energa entre estados de espn. La intensidad de esta frecuencia disminuye con el tiempo a medida que todos los ncleos vuelven a su estado inicial. Un ordenador recoge la intensidad respecto al tiempo y convierte dichos datos en intensidad respecto a la frecuencia. La operacin matemtica conocida como transformada de Fourier (FT) permite realizar dicha conversin entre los dominios del tiempo y de la frecuencia.
I.5.5.2. NMR de 1H Hasta ahora se ha descrito el concepto de resonancia de un ncleo aislado dentro de un campo magntico, pero los ncleos se encuentran rodeados de electrones que los protegen parcialmente del campo magntico externo al que se ven sometidos. Los electrones se mueven generando un pequeo campo magntico inducido que se opone al campo magntico externo. En cualquier molcula, la nube electrnica que existe alrededor de cada ncleo acta como una corriente elctrica en movimiento que, como respuesta al campo magntico externo, genera una pequea corriente inducida que se opone a dicho campo. El resultado es que el campo magntico que realmente llega al ncleo es ms dbil que el campo externo, por tanto, se dice que el ncleo est apantallado. Este apantallamiento es importante desde el punto de vista experimental, ya que el campo magntico efectivo (Hef) que siente un protn dentro de una molcula es siempre menor que el campo externo, y por lo tanto, para que el ncleo entre en resonancia dicho campo externo debe ser mayor. Si todos los protones (1H) de una molcula estuvieran apantallados de igual forma, todos entraran en resonancia con la misma combinacin de frecuencia y campo magntico. Sin embargo, los protones se hallan dentro de entornos electrnicos diferentes y, por tanto, diferentemente protegidos o
76
apantallados. Por lo general, los efectos de apantallamiento de las nubes electrnicas que rodean a cada protn son diferentes, lo que provoca diferentes frecuencias de emisin. El resultado es un espectro de diversas frecuencias donde cada conjunto de ncleos especficos da origen a una seal nica de NMR. Las variaciones en las frecuencias de absorcin de NMR, que tienen lugar debido al distinto apantallamiento de los ncleos, reciben el nombre de desplazamientos qumicos (unidades ppm). En la prctica, es difcil medir con suficiente exactitud y en trminos absolutos el campo magntico al que un protn absorbe, lo que impide distinguir tipos de protones individuales, ya que las absorciones entre uno y otro tipo slo varan en unas pocas milsimas. Un mtodo ms exacto para expresar desplazamientos qumicos es determinar el valor respecto a un compuesto de referencia que se aade a la muestra. La diferencia en la intensidad del campo magntico necesario para la resonancia de los protones de la muestra y de los protones de referencia se puede medir con mucha exactitud. El compuesto de referencia ms comn en NMR es el TMS ((CH3)4Si). Como el silicio es menos electronegativo que el carbono, los grupos metilo del TMS son relativamente ricos en electrones, estando sus protones fuertemente apantallados. Como consecuencia, estos protones absorben a una intensidad de campo mayor que el resto de protones enlazados al carbono o a otros elementos, de manera que casi todas las seales de NMR aparecen a campos ms bajos. Adems, todos los protones del TMS absorben con el mismo desplazamiento qumico dando una nica absorcin intensa. Las escala ms comn de desplazamiento qumico es la escala , en la que la absorcin del TMS se define como 0,00 . La mayor parte de los protones absorben a campos menores que el TMS, de modo que la escala aumenta hacia los campos menores. La mayora de las seales de protones (1H) varan entre 0 y 12 , mientras que las seales del 13C varan de 0 a 250 .
77
I.5.5.3. NMR de 13C La resonancia magntica nuclear del

13
C es complementaria a la del 1H.
Esta ltima se utiliza para deducir la estructura del esqueleto carbonado, observando para ello los entornos magnticos de los tomos de hidrgeno, mientras que la espectroscopa de RMN del 13C determina el entorno magntico de los tomos de carbono. Aproximadamente el 99% de los tomos de carbono en una muestra natural son del istopo
12
C. Este istopo posee un nmero par de protones y un
nmero par de neutrones, por tanto, no tiene espn magntico y no puede dar lugar a seales de NMR. El istopo de
13
C menos abundante tiene un nmero
impar de neutrones, lo que le confiere un espn magntico de 172, igual al del protn. La espectroscopia de NMR de 13C es menos sensible que la de 1H, debido a que slo el 1% de los tomos de carbono presentes posee espn no nulo y a que, adems, la frecuencia de resonancia del 13C, para un campo magntico dado, es la cuarta parte de la que se da en la RMN del 1H. Los desplazamientos qumicos del carbono son de 15 a 20 veces mayores que los del hidrgeno, debido a que el carbono est directamente unido a los tomos que resultan ser bien apantallantes o desapantallantes. Adems, las seales en el espectro del
13
C son lneas verticales aisladas, es decir, no hay
desdoblamientos de espn-espn. Esto se debe a que slo el 1% de los tomos de

13
C entran en resonancia, ya que la probabilidad de que un ncleo de
13
C sea
adyacente a otro ncleo de 13C es muy pequea.
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I.5.6. Algunas tcnicas de tratamiento estadstico de datos
I.5.6.1. Anlisis clasificatorio supervisado En el anlisis clasificatorio supervisado, se construyen modelos capaces de pronosticar la pertenencia de un objeto a una categora a partir de variables cuantitativas o de escala. La matriz de datos contiene al menos una variable categrica, que indica la categora a la que pertenece cada objeto y que constituye la respuesta o variable que se quiere predecir, y una o ms variables de escala que describen otras tantas caractersticas de los objetos y que se utilizan como predictoras. Para construir el modelo, es necesario disponer de una muestra de objetos cuya categora sea conocida y para los que tambin se conozcan los valores de las variables predictoras. La pertenencia de los objetos a las categoras puede ser supuesta, esto es, puede tratarse de una hiptesis a comprobar. La asignacin de los objetos a las categoras debe ser exhaustiva (todos los objetos pertenecen a alguna categora) y mutuamente exclusiva (ningn objeto pertenece a ms de una categora). Estos objetos forman el conjunto de entrenamiento (training set), con el cual se construye el modelo de clasificacin. Una vez construido, el modelo se utiliza para predecir la categora de nuevos objetos a partir de la medida de las predictoras. La prediccin sobre un conjunto de evaluacin (evaluation set) permite validar el modelo, que luego se aplicar a predecir la categora de las muestras problema. Se utilizan diversos tipos de tcnicas clasificatorias, tales como el anlisis discriminante, que puede ser lineal (LDA) o cuadrtico (QDA) y la tcnica de las ANN.
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I.5.6.2. LDA En LDA se utiliza un algoritmo que busca funciones o vectores discriminantes, esto es, combinaciones lineales de las variables manifiestas que maximizan la varianza entre categoras, a la vez que minimizan las varianzas intra-categoras. Para construir el modelo, es necesario asignar los objetos del conjunto de entrenamiento a una categora dada. Para ello, se aade una variable categrica a la matriz de datos conteniendo tantas categoras como sean necesarias. El LDA estima los coeficientes a1, a2, , am de la funcin discriminante lineal, f, que es capaz de predecir la pertenencia de los objetos a una u otra categora:
f = a1 x1 + a2 x2 + ... + am xm
(E.I.17)
Las funciones discriminantes se contruyen de una en una, buscando las direcciones del espacio que hacen mxima la expresin:
'=
SC D SCI
(E.I.18)
donde SCD es la suma de cuadrados de las distancias eucldeas entre los objetos que pertenecen a distintas categoras en la direccin que indica la funcin discriminante buscada, y SCI es la suma de los cuadrados de las distancias eucldeas entre los objetos que pertenecen a la misma categora, tambin en la direccin de la funcin discriminante. A partir de q categoras se obtienen q-1 funciones discriminantes (aunque si el nmero de variables predictoras, N, es menor que q, se obtendrn N-1 funciones discriminantes). Las funciones discriminantes se obtienen en orden decreciente de su valor de , y manteniendo la ortogonalidad entre ellas. La funcin no est acotada, por lo que vara ampliamente con el nmero de objetos y con la separacin entre ellos. Por ello, en lugar de maximizar , se suele minimizar la lambda de Wilks, que se define como:
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W =
1 SCI = 1 + ' SCI + SCD
(E.I.19)
Esta funcin toma valores entre 0 y 1. Categoras bien resueltas proporcionan valores de w prximos a 0, mientras que categoras solapadas dan valores de w cercanos a la unidad.
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CAPTULO II
OBJETIVOS Y PLAN DE TRABAJO
Captulo II. Objetivos y plan de trabajo
La presente Memoria se enmarca dentro de una de las lneas de investigacin del grupo, financiada principalmente por el MICINN y fondos FEDER (proyecto CTQ2007-61445, cuyo investigador principal es el Dr. Guillermo Ramis), y cuyo objeto general es el desarrollo de mtodos analticos para el control industrial y ambiental de componentes de los productos de limpieza. En consecuencia, el objetivo principal de los trabajos presentados en esta Memoria es la puesta a punto de mtodos de anlisis rpidos y fiables para diferentes analitos de la industria de la detergencia. La presente Memoria puede dividirse en cuatro grandes bloques. El primer bloque est dedicado a los surfactantes, tanto no inicos como aninicos (captulos IV y V). El segundo y tercer bloque estn dedicados a los polmeros sintticos (captulo VI) y a las enzimas (captulo VII), utilizados en productos de limpieza. Por ltimo, y entroncando con otra de las lneas de investigacin del grupo, uno de los bloques de esta Memoria est dedicado a la preparacin de columnas monolticas para CEC. En particular, en este bloque se muestra el trabajo realizado en el campo de la optimizacin de columnas, con el objeto de aplicar dichas columnas a la separacin de digestos de enzimas obtenidos con tripsina, trabajo actualmente en desarrollo, y que por razones de tiempo no se incluye en esta Memoria.
II.1. Surfactantes
II.1.1. APGs Se propone el desarrollo de mtodos para la separacin y determinacin de APGs. El estudio de la separacin de mezclas de APGs se llev a cabo mediante HPLC-MS usando columnas de alquilamida y cianopropilo, con mezclas ACN/agua como fases mviles. Se optimizaron las condiciones de elucin para conseguir la resolucin completa de epmeros (- y -) e ismeros de anillo
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(piransidos y furansidos) de los APGs, tanto en materias primas industriales como en productos manufacturados. Asimismo, se estudi la influencia de distintos parmetros en los factores de respuesta de los APGs. Los procedimientos establecidos se aplicaron a la caracterizacin y determinacin de APGs en productos de aseo personal. Por otro lado, se propone el estudio de la fragmentacin secuencial de los AMGs, que son los principales componentes de los APGs industriales. La produccin industrial de APGs conduce a mezclas que, adems de contener importantes cantidades de AMGp, (ismeros de anillo con 6 miembros) presentan pequeas aunque significativas concentraciones de AMGf (ismeros de anillo con 5 miembros). As pues, se obtendrn y describirn los espectros CID tanto para los AMGp como para AMGf, utilizando diferentes formas isotpicas de la glucosa para ayudar a su interpretacin. Para la obtencin de los espectros, se emple infusin directa en MS-IT, o bien, HPLC-MS-IT.
II.1.2. Alcoholes Se pretende el desarrollo de un procedimiento de derivatizacin que permita aumentar la sensibilidad de los alcoholes no etoxilados y etoxilados por ESI-MS-IT. Para ello, los alcoholes primarios sern previamente oxidados a sus correspondientes cidos carboxlicos con el reactivo de Jones. Los extractos se infundirn directamente en ESI-MS-IT. El estudio se extender a FAEs y a los disolventes conocidos como Cellosolves, que al oxidarse proporcionan los correspondientes cidos etoxi-carboxlicos. Los procedimientos establecidos se aplicarn a la caracterizacin y determinacin de alcoholes grasos en muestras complejas, tales como cosmticos y productos de aseo personal, as como a muestras ambientales como agua de mar.
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II.1.3. AES En trabajos anteriores del grupo de investigacin en el seno del cual cual se ha realizado la presente Tesis, y en el marco de una de sus lneas de investigacin, se desarrollaron diferentes procedimientos para la determinacin de FAEs mediante RP-HPLC-UV, previa derivatizacin de los alcoholes con un anhdrido cclico
[Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009;
Mic-Tormos, 2010].
Sin embargo, se ha observado que los anhdridos cclicos
causan la derivatizacin tanto de FAEs como AESs, dando lugar a exactamente los mismos derivados, en concreto, los hemisteres del cido correspondiente al anhdrido utilizado. Se pretende desarrollar un mtodo para la separacin, caracterizacin y determinacin mezclas de FAEs y AESs en muestras tanto industriales como ambientales. Para ello la separacin de las dos familias de surfactantes se llevar a cabo mediante SPE utilizando un cartucho SAX. Tras la optimizacin de los parmetros de extraccin se emplearn las tcnicas de derivatizacin anteriormente publicadas con diferentes anhdridos cclicos. Finalmente se separarn los oligmeros obtenidos mediante HPLC-UV. El mtodo resultante se aplicar a la caracterizacin y determinacin de FAEs y AESs en muestras de diferente naturaleza.
II.2. Polmeros
II.2.1. PVP-NO Se estudiar la migracin caracterstica del PVP-NO, tanto en FSCE como en MEKC. Estas dos tcnicas pueden proporcionar informacin sobre el comportamiento cualitativo de polmeros en disolucin, as como informacin cuantitativa sobre muestras que contienen estos aditivos. Se discutir la naturaleza de las seales obtenidas en base a los estudios de la bibliografa, as
87
como a los nuevos resultados. Para prevenir la adsorcin del polmero sobre las paredes del capilar se utilizar PEA, que en disoluciones cidas existe como ion doble de baja masa molecular (sin carga elctrica neta). Este tipo de aditivo puede usarse en elevadas concentraciones sin causar un aumento significativo de la conductividad del BGE. Finalmente, se demostrar la utilidad de la FSCE para determinar este polmero en aditivos comerciales para el lavado de textiles.
II.2.2. PVP Se pretende desarrollar de un mtodo para la caracterizacin y determinacin de PVP en productos de limpieza mediante CZE. Dado que la movilidad electrofortica del PVP es casi cero, se emplearn colorantes azoicos que formen complejos con el polmero. Estos sistemas pueden interpretarse a la luz de los estudios de Krylov y col., con aplicacin de los principios en que se basa la tcnica conocida como NECEEM. A partir de los datos obtenidos y en base a los estudios de la bibliografa, se buscarn diferentes parmetros analticos. Posteriormente los procedimientos establecidos se aplicarn a la caracterizacin y determinacin de PVP en productos de limpieza y formulados farmacuticos.
II.2.3. PVA Siguiendo con la determinacin de polmeros no inicos mediante tcnicas de CE, se pretende el desarrollo de un mtodo para la caracterizacin y determinacin de este polmero. Dado que la movilidad electrofortica del PVA es casi cero, al igual que se ha indicado ms arriba para el PVP (apartado II.2.2), se emplearn colorantes que formen complejos coloreados y cargados con el PVA, seleccionando los colorantes que mejores resultados proporcionen;
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finalmente, se interpretarn los resultados aplicando los principios de la NECEEM.
II.3. Enzimas
II.3.1. CZE Se pretende examinar enzimas intactas (protenas nativas) mediante CZE con deteccin UV, como medio de clasificacin e identificacin de enzimas. Se estudiarn las cuatro clases principales de enzimas utilizadas en productos de limpieza: proteasas, amilasas, lipasas y celulasas. Para ello, las enzimas se precipitarn con acetona, se redisolvern e inyectarn en el equipo de CE. Dado que la carga y estructura de las protenas dependen en gran medida del pH, y con el objetivo de reunir ms informacin para la identificacin de enzimas, se emplearn dos BGEs diferentes, uno a pH cido y otro a bsico. Se utilizar el ensayo de Bradford para medir las concentraciones de protena en las diferentes enzimas industriales (seleccionando el BSA como protena de referencia).
II.3.2. Aminocidos por infusin directa en MS Se pretende desarrollar un mtodo rpido y simple para la clasificacin de enzimas presentes en productos de limpieza (proteasas, lipasas, amilasas y celulasas). Para ello, se llevar a cabo su precipitacin con acetona, seguida de hidrlisis cida, para obtener as los aminocidos constituyentes. Los correspondientes digestos de aminocidos sern infundidos en ESI-MS-IT. Las abundancias de los iones procedentes de los diferentes aminocidos se utilizarn para construir modelos de LDA, capaces de distinguir entre las diferentes clases de enzimas.
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II.3.3. Aminocidos derivatizados con OPA-NAC Se pretende desarrollar un mtodo para la identificacin de las clases de enzimas en la industria de los detergentes. Para ello, las enzimas concentradas industriales y las presentes en productos de limpieza se aislarn por precipitacin con acetona y se hidrolizarn con HCl. Los aminocidos resultantes se derivatizarn con OPA en presencia de NAC, y sus perfiles de concentracin sern establecidos por RP-HPLC con deteccin UV-Vis. A partir de las reas de los picos observados en los cromatogramas se construirn modelos LDA capaces de predecir la clase de enzima: proteasa, lipasa, amilasa o celulasa. Los conjuntos de entrenamiento se construirn a partir de los concentrados industriales de enzimas, como tambin mediante bases de detergente aditivadas con enzimas.
II.3.4. Digestos con tripsina Siguiendo en la lnea de intentar identificar las diferentes enzimas utilizadas en las formulaciones de productos de limpieza, se pretende buscar un mtodo alternativo basado en la digestin con tripsina, seguida de separacin de los pptidos resultantes por RP-HPLC con deteccin UV-Vis. Se compararn los perfiles peptdicos obtenidos en los digestos utilizando diferentes soportes cromatogrficos, incluyendo columnas monolticas comerciales (tanto
polimricas, como de slice) y columnas partculadas (tanto con relleno convencional como con relleno de partculas con tecnologa corteza-ncleo (shell-core). Bajo las condiciones ptimas, se llevar a cabo el anlisis de los digestos con tripsina de enzimas de diferentes clases. Se utilizarn parmetros como el nmero de picos, capacidad de pico y resolucin global para evaluar cada lecho cromatogrfico. El mtodo propuesto se aplicar tanto a la caracterizacin de las enzimas presentes en bases de detergentes aditivadas, como en detergentes comerciales.
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II.4. Columnas monolticas
II.4.1. Columnas monolticas de lauril metacrilato fotopolimerizadas usando LPO como iniciador Se pretende describir la preparacin de columnas monolticas para CEC usando LMA como base para sintetizar el polmero. La polimerizacin se llevar a cabo mediante radiacin UV utilizando LPO como iniciador. Para obtener resultados satisfactorios se optimizar la composicin de la mezcla de polimerizacin (es decir, relaciones de monmeros/porgenos y
monmeros/agente entrelazante, as como la composicin de los disolventes porognicos). La caracterizacin morfolgica de las columnas se llevar a cabo mediante fotografas SEM. Las prestaciones de estas columnas desde el punto de vista de CEC se realizar mediante medida de los factores de retencin y eficacias de una serie de analitos no cargados. Adems, se compararn las fases estacionarias obtenidas mediante fotopolimerizacin con las obtenidas con iniciacin trmica.
II.4.2. Comparacin de iniciadores para la preparacin de columnas monolticas de metacrilato para CEC Se describir la preparacin de columnas monolticas de LMA para CEC utilizando diferentes fotoiniciadores radicalarios. Los iniciadores a estudiar sern AIBN, DMPA, BPO y LPO. Se estudiar la influencia de cada iniciador y su contenido en una mezcla 1,4-butanodiol/1-propanol como disolventes
porognicos. La caracterizacin morfolgica de las columnas se realizar mediante fotografas SEM. Adems, se evaluarn sus prestaciones
cromatogrficas mediante la medida de la retencin y la eficacia de mezclas de analitos no cargados.
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CAPTULO III
MATERIALES Y MTODOS
Captulo III. Materiales y mtodos
III.1. Surfactantes
III.1.1. APGs
III.1.1.1. Estndares, materias primas y otras muestras Se utilizaron como estndares metil-- (>99%), metil-- (>99%), hexil-(>98%), octil-- (>98%), octil-- (>99%), decil-- (>99%) y dodecil--Dglucopiransidos (>99%, Fluka, Buchs, Suiza). En los estudios de determinacin de APGs se emple el nonil--D-glucopiransido (>99%, Glycon Bioch. GmbH. Biotechnol., Luckenwalde, Alemania) como patrn interno. En los estudios de fragmentacin de la glucosa y APGs tambin se emple D-glucosa anhidra (Fluka, Buchs, Suiza), D-glucosa-13C6 (99% tomos de
13
C), D-glucosa-6,6-d2
13
(98% de tomos deuterados), D-glucosa-1-13C (99% tomos
C) (Sigma-
Aldrich, Steinheim, Alemania). Tambin se usaron las mezclas comerciales de APGs Glucopone 215 CS UP y Plantacare 818 UP (Fluka). Los productos de cuidado personal como crema de manos y champ para nios se adquirieron en establecimientos locales.
III.1.1.2. Reactivos de uso general Los diferentes disolventes empleados fueron de grado HPLC. Se utiliz ACN, MeOH, HAcO, HCl, NaHCO3 (Scharlab, Barcelona, Espaa), NaAcO (SigmaAldrich, Steinheim, Alemania) y agua desionizada (desionizador Barnstead, Sybron, Boston, MA, USA).
III.1.1.3. Instrumentacin y condiciones de trabajo Se emple un espectrmetro de masas 1100 Series VL IT-MS, provisto de una fuente ESI (Agilent Technologies, Waldbronn, Alemania). Para infusin
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directa en MS se emple una bomba de jeringa (kdScientific, Holliston, MA, USA) con la cual se mantuvo un flujo constante de entrada de 0,3 mL h1 (5 L min1). En HPLC, se emple una columna de alquilamida (100 mm 2,1 mm, 3 m ID, Supelco, Bellefonte, USA) y una de cianopropilo (75 mm 3 mm, 3 m ID, Supelco). Como fases mviles se utilizaron mezclas de ACN/agua, que contenan 40 mM de tampn HAcO/NaAcO (pH 4,7), a un flujo de 0,2 mL min1. Para acelerar la elucin isocrtica con fases mviles que contenan un 20% de ACN tambin se emple un flujo de 0,4 mL min1. El intervalo de barrido de masas fue de m/z 50800, siendo el voltaje del capilar de 4 kV. Se utiliz el valor de m/z del ion ms abundante como masa objetivo cuando se inyectaban mezclas de estndares, y el valor m/z correspondiente a [M+Na]+ cuando se inyectaban los estndares. En todos los casos, el mximo de carga de la trampa de iones fue de 3104 cuentas, con un tiempo mximo de acumulacin de 300 ms. En los experimentos de infusin directa, los espectros se obtuvieron promediando la seal durante 2 min. Como gas de nebulizacin y de secado se emple nitrgeno (generador Gaslab NGLC MS 20, Equcien, Madrid, Espaa). La nebulizacin se efectu a 25 psi y 8 L min1, y la temperatura del gas de secado fue de 300 C. Como gas de colisin se utiliz He (C-50, Carburos Metlicos, Aranjuez, Espaa). Se emple el software Agilent LC/MSD ver. 4.2 para el tratamiento de datos. Para obtener los espectros de MS de alta resolucin se emple un VG Autospec (VG Analytical, Micromass Instruments, Manchester, UK) provisto de una fuente FAB.
III.1.1.4. Preparacin de estndares y muestras Las disoluciones de estndares y de los APGs comerciales se prepararon a una concentracin de 1000 g mL1 en MeOH/agua 50:50 (v/v), excepto para el estndar C12G1, cuya concentracin fue de 500 g mL1 por razones de
96
solubilidad. En infusin directa, las disoluciones fueron diluidas en proporcin 50:50 (v/v) con una mezcla de MeOH/agua 50:50 (v/v) conteniendo NaAcO 20 mM. En HPLC, se emplearon concentraciones de 2000 g mL1 de Glucopone y Plantacare en mezclas 50:50 (v/v) de MeOH/agua. Una crema de manos y un champ para nios se diluyeron en proporcin 1:100 con una mezcla 50:50 (v/v) de MeOH/agua. Las disoluciones inyectadas se pasaron a travs de filtros de nylon de 0,45 m de tamao de poro (Albet, Barcelona). Los volmenes de inyeccin en HPLC fueron de 20 L. Los espectros de alta resolucin se obtuvieron con disoluciones diluidas de D-glucosa y hexil, octil- y decil--Dglucopiransido en 50:50 (v/v) MeOH/agua.
III.1.2. Alcoholes
III.1.2.1. Estndares, materias primas y otras muestras Los alcoholes primarios no etoxilados y etoxilados se designarn como CnEm como se indica en la seccin I.2.4. Los cidos carboxlicos y etoxicarboxlicos provenientes de la oxidacin con xido de cromo(VI) de los alcoholes primarios no etoxilados y etoxilados, respectivamente, se designarn como CnEmA. Como estndares se emplearon los alcoholes no etoxilados: C3E0 (Scharlab, Barcelona, Espaa), C8E0, C10E0, C12E0, C14E0, C16E0 y C18E0 (Fluka, Buchs, Suiza); los alcoholes etoxilados: C2E1, C4E2, C8E1, C12E1, C12E2, C18E1 (Fluka), C12E3, C12E4, C12E5, C12E6 (suministrados por C. Solans, CSIC, Barcelona); los cidos carboxlicos: C2E0A, C3E0A, C8E0A, C10E0A, C12E0A (Fluka), C14E0A, C16E0A y C18E0A (Sigma-Aldrich, Steinheim, Alemania) y los cidos etoxicarboxlicos: C1E1A, C1E2A, C1E3A y C2E1A (Fluka). Tambin se emple la mezcla comercial FINDET 10/18 (una
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mezcla de oligmeros C10Em con un EO promedio nominal de 6, y que contiene aproximadamente un 20% de agua, Molins-Kao, Barcelona). Los cosmticos y productos para el cuidado corporal fueron adquiridos en las tiendas locales. Las muestras de agua de mar se recogieron en contenedores de polietileno, en una playa cerca a la zona urbana de La Pobla de Farnals (Valencia, Espaa).
III.1.2.2. Reactivos de uso general Los diferentes disolventes empleados fueron de grado HPLC. Se utiliz HAcO, MeOH, acetona, acetato de etilo, ACN, HCl y H2SO4 (Scharlab), butilamina (Fluka), TMBA (como patrn interno, Sigma-Aldrich), sulfito de sodio anhidro, y el xido de cromo(VI) (Panreac, Barcelona), todos de grado analtico. Tambin se emple agua desionizada (vase apartado III.1.1.2).
III.1.2.3. Instrumentacin y condiciones de trabajo Se emplearon un espectrmetro de masas y una bomba de jeringa para la infusin directa de las muestras (vase apartado III.1.1.3). El intervalo de barrido de masas fue de m/z 50800, en los modos NI y PI. El voltaje del capilar fue de 4 kV, el voltaje de la segunda mscara se fij en 6 V, mientras que el voltaje de la primera mscara se selecciona automticamente en funcin de la masa objetivo. La nebulizacin se efectu a 15 psi y 3 L min1, y la temperatura del gas de secado fue de 250C. En los estudios de cuantificacin, la intensidad mxima (tanto de los analitos como del patrn interno) se determinaron utilizando la masa de cada pico como masa objetivo para reducir al mnimo la influencia de ste parmetro en la sensibilidad. Otros parmetros o mtodos de trabajo son los indicados en el apartado III.1.1.3. Para estudiar el efecto de la temperatura sobre el rendimiento de la reaccin, se utiliz un bao de agua equipado con una sonda de temperatura (RTC+ETS-D4, IKA, Staufen, Alemania).
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III.1.2.4. Preparacin de estndares y muestras A menos que se indique lo contrario, el reactivo de Jones se prepar con 6,7 g de CrO3 y 6 mL de cido sulfrico, completando el volumen hasta 50 mL con agua
[Burke, 1999].
Las disoluciones madre de los estndares se prepararon
disolviendo los alcoholes (50 mg de cada uno) en acetona (25 mL). Alcuotas de 250 L de estas disoluciones se introdujeron en tubos de centrfuga provistos de tapn de rosca, aadindose 3 mL de acetona y 1 mL del reactivo de Jones, este ltimo fue aadido gota a gota con agitacin constante a temperatura ambiente. Tras 5 minutos, se adiciona 0,5 mL de HCl 2M, obtenindose una disolucin de sales de cromo(III). El exceso de reactivo de Jones, indicado por el color amarillo de la disolucin, se elimin aadiendo, con agitacin, unos 150 mg de Na2SO3. Posteriormente, se aadi 4 mL de acetato de etilo. La mezcla se agit para favorecer la extraccin y luego se centrifug para acelerar la separacin en dos fases. La fase superior transparente e incolora (con un volumen aproximado de 7 mL) contiene los cidos carboxlicos y etoxicarboxilicos en un medio de acetato de etilo/acetona (4:3; v/v). Las sales de cromo(III) permanecen en la fase acuosa (capa inferior) con un volumen de aproximadamente 1 mL. A alcuotas de 2 mL de la fase orgnica se les aadi 0,2 mL de una disolucin acuosa de butilamina (0,33 M) que contena TMBA (5,5 mM), este ltimo incorporado como patrn interno. Esta mezcla se introdujo directamente en el equipo MS. Para la preparacin de la mezcla industrial FINDET 10/18, y las muestras de productos cosmticos y de cuidado personal, se tom 1 y 2 g, respectivamente, se aadi 10 mL de acetona, la mezcla se agit magnticamente durante 10 min, se centrifug y se tomaron alcuotas del sobrenadante (3 mL). Como se estudia en la seccin IV.2.1, la reduccin de la cantidad de agua antes de la adicin del reactivo de Jones es un factor importante para obtener unos altos rendimientos de la oxidacin. Por este motivo, para muestras con un alto
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contenido de agua se tomaron volmenes ms pequeos del sobrenadante (0,25 1 mL); estas alcuotas fueron diluidas con acetona hasta un volumen final de 3 mL. El anlisis de agua de mar se realiz tomando dos muestras de 5 L cada una. Un volumen de 2 L, tomado de la parte superior de cada contenedor, se pas a travs de cartuchos de SPE (C18, 500 mg/ 6 mL, 55 m, 140 , Phenomenex, Torrance, CA, USA) a un flujo de 5 mL min-1. Estos cartuchos se eluyeron con tres porciones de 2 mL de acetona. El volumen de los eluatos se redujo de 6 a unos 3 mL por evaporacin en corriente de nitrgeno a temperatura ambiente. Tras la evaporacin, se efectu la oxidacin y extraccin, tal como se ha descrito antes. Para construir un blanco de referencia, se tom un tercer volumen de 2 L de agua de mar, y se procedi a extraccin (SPE) y reduccin del eluato a 3 mL, como anteriormente se ha indicado. En el procedimiento de oxidacin de este eluato, el reactivo de Jones fue sustituido por cido sulfrico diluido, siendo el resto del procedimiento de oxidacin y de extraccin el indicado previamente. Los espectros MS de los extractos de acetato de etilo/acetona se registraron en modo NI, y se midieron las abundancias de los iones [M-H]- de los cidos carboxlicos y etoxicarboxlicos. En los estudios de rendimiento de oxidacin-extraccin, los espectros tambin se obtuvieron en modo PI, y los picos de los iones [M+H]+ correspondientes a los alcoholes etoxilados se compararon con los espectros de los correspondientes cidos etoxicarboxlicos obtenidos en modo NI. Para obtener espectros en modo PI, alcuotas de 1 mL de los extractos de acetato de etilo/acetona se acidificaron con 0,1 mL de HCl 2 M (en lugar de la disolucin de butilamina), esta disolucin se diluy con 1 mL de ACN, y se infundi en la interfaz ESI del MS. Cuando fue necesario, se emple el TMBA como patrn interno, ya que proporciona los picos [M-H]- y [M+H]+ en los modos NI y PI, respectivamente.
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III.1.3. AESs
III.1.3.1. Estndares, materias primas y otras muestras Como estndares de FAEs se emplearon: C8E0, C12E0, C14E0 (SigmaAldrich, Steinheim, Alemania) y Dehydol LT-7 (mezcla comercial industrial de FAEs, 12 n 18, m = 7, Cognis, Monheim, Alemania). Como estndares de AESs se emplearon: C8E0S, C12E0S (SDS) (Fluka, Buchs, Suiza) y una mezcla industrial comercial nominalmente lauril sulfato, si bien otros residuos de alcohol graso estn tambin presentes en cantidades importantes (LES, 12 n 18,
m =3, suministrado por Qumicas Oro). Con el objetivo de estudiar posibles
interferencias con otras clases de surfactantes comnmente empleados en las formulaciones, se utilizaron las mezclas comerciales de LAS (mezcla industrial de oligmeros con 10 n 13) y SAS (mezcla industrial de oligmeros con 14 n 16) (suministrados por Qumicas Oro). Otras muestras industriales de AESs fueron suministradas por Qumicas Oro e Industria Jabonera Lina (Torras de Cotillas, Murcia, Espaa). Los detergentes comerciales fueron adquiridos en las tiendas locales. Las muestras de agua de mar se recogieron en contenedores de polietileno, en una playa cerca a la zona urbana de La Pobla de Farnals (Valencia, Espaa).
III.1.3.2. Reactivos de uso general Se utilizaron los siguientes disolventes (grado HPLC) y reactivos (grado analtico): MeOH, HAcO, ACN, NH3, DMSO (Panreac, Barcelona, Espaa), anhdrido ftlico ( 99%), urea (99,5%) (Fluka), HCl, anhdrido difnico (98%) y 1,4-dioxano (99,8%, Sigma-Aldrich). Tambin se emple agua desionizada (vase apartado III.1.1.2).
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III.1.3.3. Instrumentacin y condiciones de trabajo Se emple un cromatgrafo de lquidos (HP 1100, Agilent, Waldbronn, Alemania), constituido por una bomba cuaternaria, un desgasificador en lnea, un compartimento termostatizado de columnas, un muestreador automtico y un DAD. Cuando fue necesario, el cromatgrafo se acopl a un espectrmetro de masas 1100 Series VL IT-MS, provisto de una fuente ESI (vase apartado III.1.1.3). La columna empleada fue una C8 del tipo ncleo fundido (AscentisExpress, 2,7 m, 15 cm x 4,6 mm de ID, Supelco, Bellefonte, PA, USA). La elucin se llev a cabo mediante la mezcla de dos disoluciones que contenan ACN/agua, con un 50% (A) y un 100% de ACN (B), en presencia de un 0,1% de HAcO en ambas mezclas. El flujo fue de 1 mL min-1, y a no ser que se indique lo contrario, la fase mvil se vari de forma lineal de A a B en 50 minutos. Los volmenes de inyeccin fueron de 20 L, siendo las alcuotas filtradas previamente mediante un filtro de nylon (Albet, Barcelona) de 0,45 micras de tamao de poro. La deteccin se realiz a 230 10 nm con una referencia fijada a 360 60 nm. Las reas de los picos se midieron con el software ChemStation para LC v.10.02 (Agilent). Cuando fue necesario, se utiliz HPLC-MS, con un rango de barrido de masas de m/z 100900 en modo NI. El voltaje del capilar fue de 4 kV, el voltaje de la segunda mscara se fij en 6 V, mientras que el voltaje de la primera mscara se seleccion automticamente en funcin de la masa objetivo. La masa objetivo se fij a un valor de m/z de 400. La nebulizacin se efectu a 35 psi y 7 L min1, y la temperatura del gas de secado fue de 300 C. Otros parmetros o condiciones de trabajo son las indicadas en el apartado III.1.1.3. Tambin se emplearon los siguientes cartuchos de SPE (Phenomenex, CA, EE.UU.): Strata C18-E (500 mg/ 6 mL, 55 m, 140 ) y Strata SAX (1000 mg/ 6 mL, 55 m, 70 ). Cuando fue necesario, parte de los disolventes fueron evaporados en una centrfuga a vaco (miVac, Genevac, Ipswich, Reino Unido).
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III.1.3.4. Preparacin de estndares y muestras Para los estudios de calibracin, se prepararon disoluciones madre de C8E0 y C12E0 (2 mg mL-1) en 1,4-dioxano. Tambin, se utilizaron disoluciones madre de C8E0S y C12E0S (2 mg mL-1) en 1,4-dioxano con un 7% de DMSO para facilitar su disolucin. Por otro lado, en los estudios de separacin con cartuchos SAX, se prepararon disoluciones madre de C14E0 y C12E0S (5 mg mL-1) en MeOH. Estas disoluciones madre se utilizaron para preparar las mezclas de 0,5 mg mL-1 de cada estndar, en medios con diferentes proporciones de MeOH/agua (20:80, 50:50 y 80:20, todos como v/v). Para los estudios de separacin en SAX, tambin se prepararon disoluciones concentradas (40 mg mL-1) de mezclas industriales de FAEs (Dehydol LT-7) y AES (LES), as como mezclas de las mismas (aproximadamente 20 mg mL-1 de cada clase de surfactante) en 50:50 MeOH/ agua. Alcuotas de 1 mL de estas disoluciones se diluyeron a 5 mL con las cantidades adecuadas de MeOH y agua, para obtener mezclas con diferentes proporciones MeOH/ agua (20:80, 50:50 y 80:20). En el anlisis de productos comerciales (detergentes), se llev a cabo una etapa de limpieza, previa a la separacin con los cartuchos SAX. Para ello, se pesaron 0,1 g del detergente y se diluy hasta 25 mL con agua. Esta disolucin se pas por un cartucho de SPE C18, previamente acondicionado con agua. La elucin se efectu con dos alcuotas, primero una de 2,5 mL de MeOH/ agua 50:50, seguida de otra de 2,5 mL de MeOH/ agua 80:20. Tras la elucin, se aadi la cantidad de agua necesaria para reducir la concentracin de MeOH al 50% antes de la separacin de las dos clases de surfactantes el cartucho SAX. El anlisis del agua de mar se realiz tomando dos muestras de 5 L cada una. Un volumen de 2 L, tomado de la parte superior de cada contenedor, se pas a travs de cartucho de SPE C18, a un flujo de unos 5 mL min-1. La elucin del cartucho
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SPE C18, se efectu como se ha indicado anteriormente en la elucin de los detergentes. El procedimiento optimizado de la separacin con el cartucho SAX para SPE se indica en el esquema de la Figura III.1. En primer lugar, el cartucho SAX se acondicion con una mezcla 50:50 MeOH/ agua. Luego, la muestra que contiene FAEs y AESs en un medio MeOH/ agua 50:50, se pas a travs del mismo. Con esta operacin los AESs se quedan retenidos en el cartucho, mientras que la mayora de los FAEs son eluidos (fraccin 1). El cartucho se lav con el mismo medio MeOH/ agua 50:50, para eliminar as los restos de cationes (fraccin 2), quedando los AES retenidos fuertemente sobre los sitios activos del cartucho catinico. Una pequea parte de los FAEs ms hidroflicos tambin eluyen con la fraccin 2. Seguidamente, el cartucho se lav con una mezcla MeOH/ agua (80:20); el mayor contenido de MeOH de esta mezcla facilita a la elucin de los oligmeros ms hidrofbicos de los FAEs, constituyendo la fraccin 3. Las fracciones de FAEs 1 + 2 + 3 se combinan en un nico tubo con tapn de rosca de 15 mL. La elucin de los AES de los cartuchos SAX se realiz con mezclas MeOH:HCl, en diferentes proporciones, utilizando HCl acuoso concentrado. Para ello, la fraccin 4 formada por los AESs hidroflicos se eluye con una mezcla MeOH:HCl 80:20 (concentracin de cido en la mezcla, 2,4 M). Los AESs ms hidrofbicos (fraccin 5) se eluyen con una mezcla MeOH:HCl 95:5 (concentracin de cido en la mezcla, 0,6 M). Las fracciones de AESs 4 + 5 se combinan en un segundo tubo con tapn de rosca de 15 mL. Seguidamente esta ltima fraccin combinada se neutraliza gota a gota con NH3 acuoso concentrado en presencia de una gota de fenolftalena al 1% en MeOH, , hasta viraje del indicador. Las disoluciones de FAEs y AESs recogidas fueron evaporadas bajo corriente de nitrgeno. La eliminacin de los ltimos restos de disolvente se realiz en una centrfuga evaporadora a vaco.
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FAEs + AESs (50:50 MeOH:H2O) Fraccin 1 (FAEs hidroflicos) Fraccin 2 Fraccin 3 50:50 MeOH:H2O (eliminacin de cationes) 80:20 MeOH:H2O (FAEs hidroflicos) 80:20 MeOH:HCl (AESs hidroflicos) 95:5 MeOH:HCl (AESs hidrofbicos) Eluatos de FAEs: Fracciones 1 + 2 + 3 combinadas Neutralizacin Eluatos de AESs: Fracciones 4 + 5 combinadas Evaporacin de disolventes Evaporacin de disolventes
Fig. III.1. Esquema de separacin de los FAEs y AESs, empleando un cartucho de SPE tipo SAX. Fracciones eluidas: FAEs (1), disolucin de lavado para eliminacin de cationes (2), FAEs hidrofbicos (3), AESs hidroflicos (4) y AESs hidrofbicos (5).
Se utilizaron los procedimientos de la bibliografa para la derivatizacin de los FAEs con anhdridos ftlico [Mic-Tormos, 2008-B] y difnico [Mic-Tormos, 2009]. En este trabajo, se evalu la aplicacin de estos mismos procedimientos a la derivatizacin de AESs que tienen lugar, mediante una reaccin de transesterificacin, de acuerdo con los esquemas de reaccin de la Figura III.2. Tras la evaporacin de los disolventes, se llevaron a cabo las derivatizaciones de FAEs y AESs en sus respectivos tubos. Para ello, a cada uno de los tubos se aadi 0,25 g de urea finamente molida, 2 mL de 1,4-dioxano, y 1 g del anhdrido ftlico o 0,5 g del difnico. El anhdrido ftlico se emple para las muestras industriales o comerciales, mientras que el difnico se utiliz para derivatizar las muestras ambientales. Los tubos con la mezcla de reaccin, se agitaron e introdujeron en un bao termosttico de aceite de silicona a 105 C durante 90 min. Tras enfriar a temperatura ambiente, los residuos fueron disueltos por adicin de 10 mL de una mezcla MeOH/ agua 2:1, que contiene 0,1 M de NH3, sin embargo, slo 2 mL de dicha mezcla se aadieron a los extractos
SAX
Fraccin 4 Fraccin 5
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de agua de mar derivatizados. Las disoluciones se inyectaron inmediatamente o se almacenaron a -20 C hasta su inyeccin.
O O R R O OH o SO3
-
COOH2O o SO3
+ O O O
COO-
Fig. III.2. Esquemas de reaccin para la esterificacin de FAEs y transesterificacin de AESs con los anhdridos ftlico y difnico.
III.2. Polmeros sintticos
III.2.1. PVP-NO
III.2.1.1. Estndares, materias primas y otras muestras Se utilizaron muestras de PVP-NO (Chromabond S-403E, 9-17 kDa, con polmeros constituidos por unos 74 - 140 monmeros, International Specialty Products, Colonia, Alemania). Con el objetivo de estudiar posibles interferencias con diferentes clases de surfactantes comnmente empleados en las formulaciones, se utilizaron las mezclas comerciales Dehydol LT-7 (mezcla de FAEs, Cognis, Dsseldorf, Alemania), LAS, LES y p-isopropil sulfonato de sodio (cumeno sulfonato de sodio, sustancia incorporada a las formulaciones por sus propiedades como hidrtopo). Estos tres ltimos componentes fueron suministrados por Qumicas Oro.
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III.2.1.2. Reactivos de uso general Se emplearon los siguientes reactivos H3PO4 (Panreac, Barcelona), NaH2PO4, Na2HPO4, NaOH, HAcO, NH4AcO (Probus, Badalona, Espaa), acetona, MeOH (Scharlab, Barcelona), PEA, xido de mesitilo (Fluka), SDS (Merck, Darmstadt, Alemania). Tambin se emple agua desionizada (vase apartado III.1.1.2).
III.2.1.3. Instrumentacin y condiciones de trabajo Se emple un sistema de electroforesis capilar HP3D (Agilent, Waldbronn, Alemania) provisto de detector espectrofotomtrico de fila de diodos, y capilares de slice fundida (Polymicro, Phoenix, AZ, USA) de 33,5 cm (25 cm de longitud efectiva) 50 m ID (375 m OD). Los capilares nuevos fueron acondicionados a 60 C con NaOH 1 M, NaOH 0,1 M y agua durante 10 minutos cada uno. Posteriormente, la temperatura del capilar se redujo a 25 C, y se pas el BGE durante 10 min ms. Diariamente, antes de iniciar la sesin de trabajo, el capilar se lav sucesivamente con disoluciones de cido fosfrico 1 M y 20 mM durante 15 minutos cada vez, seguido del BGE durante 10 min ms. Entre inyecciones, el capilar se lav con el BGE durante 10 min. La inyeccin fue hidrodinmica, aplicando 50 mbar 3 s. Las separaciones se llevaron a cabo a +20 y -20 kV en ausencia y presencia de PEA, respectivamente. La deteccin se realiz a 214 y 276 nm. El BGE se prepar semanalmente, conservndolo a 4 C. Antes de las inyecciones, las disoluciones se pasaron a travs de un filtro de nylon de 0,45 am de dimetro de poro (Albet). Para las medidas de tensin superficial se emple un mtodo basado en la masa de un nmero fijo de gotas
[Mukherjee, 1971].
Las gotas se generaron
mediante un capilar de vidrio de extremo plano y de 3 mm de dimetro externo, sostenido en posicin vertical por un soporte colocado sobre la mesa
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antivibratoria de la balanza. Para cada medida se tom el peso de 40 gotas de disolucin (por triplicado), a temperatura ambiente (22 C). La calibracin se realiz con agua (tensin superficial, 72,4 mN m-1 a 22 C) [Lide, 2003].
III.2.1.4. Preparacin de estndares y muestras Se prepar una disolucin madre de PVP-NO de 10 mg mL-1 en una mezcla de 50:50 (v/v) MeOH/ agua. Los productos comerciales de limpieza se diluyeron en proporcin 1:100 con una mezcla 50:50 (v/v) de MeOH/ agua y se inyectaron en el sistema de CE.
III.2.2. PVP
III.2.2.1. Estndares, materias primas y otras muestras Los estndares de PVP (Fig. I.9), con unas masas moleculares promedio de MW = 10, 27,9, 40, 160 y 360 kDa se obtuvieron de Fluka (Buchs, Suiza), mientras que aquellos con MW = 60 y 400 kDa fueron suministradas por International Specialty Products (Colonia, Alemania). Los productos de limpieza y farmacuticos, se obtuvieron en los comercios locales.
III.2.2.2. Reactivos de uso general Se emplearon los colorantes azoicos RC (Panreac, Barcelona, Espaa) y AB (Sigma-Aldrich, Steinheim, Alemania); cuya estructura se muestra en la Fig. VI.9. Tambin se utilizaron tetraborato de sodio decahidratado, acetona (SigmaAldrich) como marcador del EOF, as como otros reactivos de grado analtico, y agua desionizada (vase apartado III.1.1.2).
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III.2.2.3. Instrumentacin y condiciones de trabajo Para el sistema de electroforesis capilar HP3D, capilares y acondicionado de los capilares nuevos, vase el apartado III.2.1.3. Diariamente, antes de iniciar la sesin de trabajo, el capilar se lav sucesivamente con disoluciones de NaOH 0,1 M y agua durante 10 minutos en cada caso, seguido del BGE durante 10 min ms. Entre inyecciones, el capilar se lav con NaOH 0,1 M y agua, durante 2 y 3 minutos, respectivamente, seguido por el BGE durante 5 minutos ms. El BGE estaba compuesto por Na2B4O710H2O 25 mM, con el pH ajustado a 9,0 por adicin de HCl 0,1 M. Todas las disoluciones se pasaron a travs de filtros de nylon de 0,45 m de tamao de poro (Albet, Barcelona, Espaa). La inyeccin fue hidrodinmica, aplicando 50 mbar 2 s. Las separaciones se llevaron a cabo a 15 kV (polaridad positiva) a una temperatura de 25 C. La deteccin se realiz a 215 nm, as como a una longitud de onda cercana al mximo de absorcin para los colorantes, tanto en presencia como en ausencia de un exceso de PVP. Las longitudes de onda seleccionadas para CR y AB fueron 500 y 565 nm, respectivamente. Los espectros de absorcin UV-Vis se registraron empleando un espectrofotmetro UV-Vis modelo 8453 (Agilent Technologies), utilizando una celda de cuarzo de 1 mm de camino ptico (Hellma, Mllheim, Alemania). Los espectros de los colorantes se obtuvieron con disoluciones compuestas por 0,1 mM del colorante y 25 mM de tetraborato sdico en agua. Los espectros de los complejos de PVP-colorante se obtuvieron en el mismo medio, despus de la adicin de 100 g mL-1 (0,9 mM en monmeros) del PVP de 60 kDa. El espectro de esta mezcla se registr cada 10 minutos durante 5 horas, con el fin de estudiar el grado de formacin de los complejos PVP-CR con el tiempo. A menos que se indique lo contrario, las mezclas de PVP-colorante se mantuvieron a temperatura ambiente durante un mnimo de 5 horas antes de la inyeccin en el capilar de CE.
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III.2.2.4. Preparacin de estndares y muestras Se prepararon disoluciones madre acuosas de los colorantes azoicos (5 mM) y PVP de distintas MW (40 mg mL-1), y a partir de ellas se prepararon disoluciones ms diludas, tambin en agua. Se construyeron curvas de calibrado externas para la determinacin de PVP mediante la representacin del rea de la banda del complejo PVP-colorante (vase el mtodo de medida de la misma en la seccin VI.2.1.1) frente a la concentracin de monmeros de PVP. A tal efecto, las concentraciones de PVP utilizadas fueron 100, 250, 500, 1000 y 1500 g mL1
. Las curvas de calibrado interno se obtuvieron mediante la adicin de 250 y 500
g mL-1 de PVP a las disoluciones de las muestras. Dos bases de detergente, cuya composicin viene dada en la Tabla III.1, se fortificaron con PVP 60 kDa, obtenindose una concentracin final de 0,99 y 0,97% (en peso) para las bases I y II, respectivamente.
Tabla III.1. Composicin de las bases de detergente empleadas en este trabajo (porcentajes en peso). Componente AS AESa Oleina FAEb SDS Cumeno sulfonato sdico Agua y otrosc
a b c
Base I (%) 5 7 11 1,5 75,5
Base II (%) 8 3,5 4 4 80,5
Cadenas entre 12 y 18 tomos de carbono y nmero medio de EO 3. Cadenas entre 12 y 18 tomos de carbono y nmero medio de EO 7. Componentes minoritarios como KOH, trietanolamina, bactericidas y perfume.
A continuacin, se pesaron 0,5 g de cada mezcla y se aadi 4 mL de una disolucin 5 mM de CR, completndose el volumen a 5 mL con agua. Alcuotas de esta disolucin se inyectaron en el equipo de CE. Con los productos de
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limpieza y farmacuticos se sigui el mismo procedimiento que el descrito con las bases de detergente. La cuantificacin se realiz a partir de una curva de calibracin externa obtenida con el CR y el PVP de 60 kDa. Todas las inyecciones se realizaron por triplicado.
III.2.3. PVA
III.2.3.1. Estndares, materias primas y otras muestras Los estndares de PVA (Fig. I.10) utilizados presentan las siguientes MW y porcentajes de monmeros residuales acetilados (nac 100%, valores entre parntesis): 15 (0,0%), 49 (0,1%) y 100 (12,5%) kDa (Fluka, Buchs, Suiza), y 31 (10,8%), 61 (1,5%), 130 (10,8%), 145 (0,6%) y 205 (10,8%) kDa (Sigma Aldrich, Steinheim, Alemania).
III.2.3.2. Reactivos de uso general Se emple el colorante azoico RC (Panreac, Barcelona, Espaa), cuya estructura puede verse en la Fig. VI.9. Se utilizaron tetraborato de sodio decahidratado, acetona (Sigma-Aldrich) como marcador del EOF, dimetil sulfxido deuterado (DMSO-d6, SigmaAldrich) as como otros reactivos de grado analtico. Tambin se emple agua desionizada (vase apartado III.1.1.2).
III.2.3.3. Instrumentacin y condiciones de trabajo Para el sistema de electroforesis capilar HP3D, capilares y acondicionado de los capilares nuevos, vase el apartado III.2.1.3. Diariamente, antes de iniciar la sesin de trabajo, el capilar se lav sucesivamente con disoluciones NaOH 0,1 M y agua durante 5 minutos cada uno, seguido del BGE durante 10 min ms. Entre inyecciones, el capilar se lav con NaOH 0,1 M y agua durante 2 y 3
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minutos, respectivamente, seguido por el BGE durante 5 minutos ms. Al finalizar cada sesin de trabajo, el capilar se lav con agua y HCl 0,1 M durante 10 min en cada caso, con agua 5 minutos ms, y con NaOH 1 M seguido de ms agua durante 20 min ms cada uno. El BGE estaba compuesto por Na2B4O710H2O 25 mM, ajustndose a pH 9,0 con HCl 0,1 M. Todas las disoluciones se pasaron a travs de filtros de nylon 0,45 m de tamao de poro (Albet, Barcelona, Espaa). Las disoluciones con PVA no se filtraron para evitar que el polmero se quedara retenido en el filtro. La inyeccin fue hidrodinmica, aplicando 50 mbar 3 s. Las separaciones se llevaron a cabo a 15 kV (polaridad positiva) y a 25 C. La deteccin se realiz a 500 nm, longitud de onda cercana al mximo de absorcin de los colorantes en ausencia de PVA. Los espectros UV-Vis se obtuvieron tanto con el equipo de CE, como con un espectrofotmetro (indicado en la seccin III.2.2.3). Para obtener los espectros en CE, el capilar se rellen con una disolucin de brax 20 mM que contena CR 4 mM y concentraciones crecientes de PVA (muestra de MW = 49 kDa). En el espectrofotmetro, para no saturar la seal, se emplearon las disoluciones anteriores diluidas en un factor 1:40 (0,1 mM CR). El tiempo de pre-equilibrado de los complejos PVA-CR se estudi tanto por CZE como por espectrofotometra. La tacticidad (estereorregularidad) de las muestras de PVA se estableci registrando los espectros de NMR 13C con un espectrmetro de NMR Ultrashield DRX 400 (Bruker, Silberstreifen, Alemania). Para ello, las muestras de PVA se deshidrataron en una centrfuga evaporadora a vaco durante 8 horas a 60 C (miVac, Genevac, Ipswich, Reino Unido) y se prepararon disoluciones de PVA al 1% con DMSO-d6. Se acumularon 2 104 espectros en todos los casos. Las estructuras de las tres posibles tradas en una cadena de PVA (mm, rm y rr) se muestran en la Figura. III.3.
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HO H HO H HO H
HO H
H OH H OH
HO H
H OH HO H
Fig. III.3. Estructuras de las tres posibles tradas en una cadena de PVA: (A) mm, (B) rm y (C) rr.
Los porcentajes de cada trada se estimaron a partir de las reas relativas de los tres multipletes que se encuentran dentro del rango de 64-69 ppm [Moritani, 1972;
Wu, 1973; Wu, 1977; Fukae, 2000].
En la Figura III.4, se representan los
porcentajes de las triadas rr encontrados en las muestras, frente el porcentaje de las tradas mm. En la misma figura, se representaron tambin datos obtenidos de diversas publicaciones [Moritani, 1972; Wu, 1973; Wu, 1977; Fukae, 2000].
40 61 (1.72) 30
H
31 (1.67) 15 (1.39)
100 (1.09) M 49 (1.05) 130 (0.85)
rr %
145 (1.04)
20
205 (0.71)
6 2 15 20 25 70
mm %
Fig. III.4. Porcentajes de las triadas rr y mm, estimados por 13C NMR en las muestras de PVA empleadas en este trabajo (). Los nmeros que se encuentran junto a los smbolos (), son la MW (kDa) y la relacin rr/mm (valores entre parntesis). Los otros smbolos corresponden a datos bibliogrficos
[Moritani, 1972; Wu, 1973; Wu, 1977; Fukae, 2000]
para muestras
de PVA altamente sindiotctico (), atctico () y altamente isotctico ().
Como se observa en esta figura, las muestras de PVA utilizadas en este trabajo resultaron agrupadas como sigue: un grupo de tres muestras con proporciones
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rr/mm dentro del rango 1,72 - 1,39, otro grupo de tres muestras dentro del rango 1,09 - 1,04 y finalmente, las dos muestras con mayor MW mostraron los menores valores de la relacin rr/mm, en concreto 0,85 y 0,71. Para facilitar la discusin en la seccin IV.3, estos grupos se denominaron en funcin de la relacin rr/mm como H (alto), M (medio) y L (bajo).
III.2.3.4. Preparacin de estndares y muestras Se prepararon disoluciones madre acuosas de RC (5 mM) y PVAs de distinta MW (10 mg mL-1, 227 mM en monmeros), preparndose diluciones y mezclas de los mismos con los tampones adecuados.
III.3. Enzimas
III.3.1. CZE
III.3.1.1. Estndares, materias primas y otras muestras Los concentrados de enzimas industriales utilizados en esta seccin se muestran en la Tabla III.2. Estos fueron suministrados en forma lquida o como slidos granulares. Para el estudio de posibles interferencias y efectos de la matriz se utilizaron las siguientes mezclas industriales comerciales: Dehydol LT7 (mezcla de FAEs, Cognis, Dsseldorf, Alemania), LAS y LES (donados por Qumicas Oro).
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Tabla III.2. Clase, nombre comercial, fabricante, seccin de esta memoria en la que se utilizan las diferentes enzimas estudiadas, as como el conjunto asignado para la construccin de modelos de LDA en las secciones indicadas.
Clase Proteasa
Amilasa
Celulasa
Lipasa
Nombre comercial Alcalase 2,5 L Savinase 16L, Type EX Everlase 16 L, Type EX Esperase 8.0 L Polarzyme 12Ta Bioproteasa L 450 Bioproteasa L 800 Enziprot 450L Deterzyme L 660 Deterzyme Apy L 560 Properase 1600L Purafect Prime HA Properase 4000Da Excellase 2250Da Purafect OX 8000Da Termamyl Ultra 300L Duramyl 300 L, Type DX Stainzyme 12L Enziamilasa Purastar ST 15000L Purastar ST 6000Da Endolase 5000L Carezyme 4500L Celluzyme 0,7Ta Deterzyme CL-5 Puradax HA 400Ea Puradax EG 7000L Lipolase 100L, Type EX Lipex 100L Lipolase 100Ta Lipex 100Ta
Fabricantee Novozymes Novozymes Novozymes Novozymes Novozymes Biocon Biocon ChemWorld Enmex Enmex Genencor Genencor Genencor Genencor Genencor Novozymes Novozymes Novozymes ChemWorld Genencor Genencor Novozymes Novozymes Novozymes Enmex Genencor Genencor Novozymes Novozymes Novozymes Novozymes
Seccin VII.1; VII.2c; VII.3b,c; VII.4 VII.1; VII.2d; VII.3d VII.1; VII.2d; VII.3d; VII.4 VII.1; VII.2d; VII.3d VII.3d VII.2d; VII.3c VII.2d; VII.3c VII.2c; VII.3d VII.2c; VII.3d VII.2d; VII.3d VII.3d VII.2d; VII.3d VII.3d VII.3d VII.3d VII.1; VII.2c; VII.3b,c VII.1; VII.2c; VII.3d; VII.4 VII.1; VII.2d; VII.3c VII.2c; VII.3c VII.2d; VII.3d; VII.4 VII.3d VII.1; VII.2c; VII.3d VII.1; VII.2c; VII.3b,c VII.1; VII.2d; VII.3d VII.2c; VII.3c; VII.4 VII.3d; VII.4 VII.2d; VII.3c VII.1; VII.2c; VII.3c; VII.4 VII.1; VII.2c; VII.3b,c; VII.4 VII.2c; VII.3c VII.2d; VII.3d
a b
Muestras suministradas como slidos granulados (el resto de muestras son lquidas). Se emplean tanto como materia prima industrial como para aditivar las bases de detergente (Tabla III.3).
c d e
Empleado en el conjunto de entrenamiento. Empleado en el conjunto de evaluacin. Donadas por las correspondientes casas comerciales: Novozymes (Bagsvaerd, Dinamarca), Biocon (Bangalore, India), ChemWorld (Barcelona, Espaa), Enmex (Tlalnepantla, Mxico), Genencor (Rochester, NY, USA)
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III.3.1.2. Reactivos de uso general Se emple IDA, urea, PVA de MW media de 70 a 100 kDa (Sigma, St. Louis, MO, EE.UU.), y HEC con una MW media de 27 kDa (Polysciences, Warrington, PA, EE.UU.). Tambin se utilizaron NaOH y sodio tetraborato decahidrato (Probus, Badalona, Espaa), acetona (Scharlau, Barcelona, Espaa) y sdio dihidrgeno fosfato (Panreac, Barcelona, Espaa). El contenido de protena de las muestras se calcul por el mtodo de Bradford
[Bradford, 1976]
utilizando
el Protein Quantification Kit Rapid (Fluka, Buchs, Suiza) y la BSA (Fluka) como estndar. Tambin se emple agua desionizada (desionizador Barnstead, Sybron, Boston, MA, USA)
III.3.1.3. Instrumentacin y condiciones de trabajo Para el sistema de electroforesis capilar HP3D, capilares y acondicionado de los capilares nuevos, vase apartado III.2.1.3. Para trabajar en medio bsico, se utiliz un BGE compuesto por NaH2PO4 50 mM, Na2B4O7 25 mM y PVA al 0,1%, ajustado a pH 9,0 con NaOH 0,1 M. Al comienzo de cada sesin de trabajo, el capilar se lav con NaOH 0,1 M y agua durante 5 minutos cada uno, y 10 minutos ms con el BGE. Entre inyecciones el capilar se lav con el BGE durante 10 min. Las separaciones con el BGE bsico se realizaron a 25 C a 15 kV (polaridad positiva). Para trabajar en medio cido, se emple un BGE compuesto por 50 mM de IDA (pH = 2,30 a 25 C), HEC al 0,5% y urea 6 M (pHaparente = 3,1). Diariamente, antes de iniciar la sesin de trabajo, el capilar se lav con el BGE cido durante 30 minutos y entre inyecciones el capilar se lav durante 5 minutos con el mismo BGE. La temperatura del capilar se fij a 25 C y separaciones se realizaron a 25 kV (polaridad positiva), lo que dio lugar a una corriente tpica de 30 mA. Para ambos BGEs, las disoluciones de las posiciones andica y catdica se renovaron tras cada serie de cinco inyecciones [Bossi, 1999].
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Los BGEs se pasaron a travs de filtros de nylon de 0,45 micras de tamao de poro (Albet, Barcelona, Espaa). Para las inyecciones hidrodinmicas se aplicaron 50 mbar 2 s. La seal se monitoriz a 214 nm. Para medir la absorbancia a 600 nm en el ensayo de Bradford, se emple un espectrofotmetro UV-Vis modelo 8453 (Agilent Technologies), utilizando una celda de cuarzo de 1 cm de camino ptico (Hellma, Mllheim, Alemania). El contenido proteico en las distintas muestras se obtuvo siguiendo el protocolo descrito en el Protein Quantification Kit Rapid (Fluka). Los resultados obtenidos con las diferentes enzimas, expresados como porcentaje de BSA se indican en la Tabla VII.1.
III.3.1.4. Preparacin de estndares y muestras Los concentrados de enzimas industriales lquidas con contenidos entre 110% de protena, fueron diluidos con agua para obtener disoluciones de aproximadamente un 0,01% de protena. A partir de estas disoluciones, se tomaron alcuotas de 1 mL y se les aadi 4 mL de acetona. Tras agitacin, se centrifug a 5500 rpm 4 min, se descart el sobrenadante, y los precipitados se disolvieron en 1 mL de agua o de urea 3 M, en funcin de que la muestra se fuese a inyectar en el BGE bsico o cido, respectivamente. Para las enzimas comercializadas granuladas, se pes aproximadamente 1 g y se aadi 5 mL de agua. La suspensin resultante se agit vigorosamente, se sonic durante 15 minutos, y se centrifug a 5500 rpm durante 4 minutos, tomndose una alcuota del sobrenadante. A continuacin, se adopt el procedimiento anteriormente descrito para concentrados lquidos de enzima. Para establecer el contenido de protena segn el ensayo de Bradford
[Bradford, 1976],
se prepar una disolucin de BSA de 4000 g mL-1 (0,4%) en
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urea 3 M. A partir de esta disolucin se prepar una curva de calibrado de nueve puntos en un intervalo de 10 a 2000 g mL-1 (0,001 a 0,2%). Tambin se estudiaron las interferencias ocasionadas por los surfactantes ms utilizados en la formulacin de productos de limpieza. Para ello, a disoluciones que contenan un 5% de LAS, LES o Dehydol LT 7, se les aadi un 0,01% de enzima. La separacin de las enzimas de estas disoluciones se realiz tomando alcuotas de 5 mL y aadiendo 20 mL de acetona. Tras agitacin, se centrifug a 5500 rpm durante 4 min, se descart el sobrenadante y el precipitado resultante se diluy en 1 mL de agua o urea 3 M, segn se fuese a inyectar en el BGE bsico o cido, respectivamente.
III.3.2. Aminocidos por infusin directa en MS
III.3.2.1. Estndares, materias primas y otras muestras Los concentrados de enzimas industriales utilizados en este trabajo se muestran en la Tabla III.2, siendo suministrados en forma lquida o como slidos granulares. Tambin, se emple Dehydol LT-7 (mezcla de FAEs, Cognis, Dsseldorf, Alemania), LES, oleina y cumeno sulfonato sdico (donados por Qumicas Oro). Dos detergentes para lavado de textiles que contenan proteasa (segn declaraba el fabricante) se obtuvieron en los comercios locales.
III.3.2.2. Reactivos de uso general Se emple acetona, etanol (Scharlau, Barcelona, Espaa) y HCl (37%) (Panreac, Barcelona, Espaa). Tambin se emple agua desionizada (vase apartado III.1.1.2).
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III.3.2.3. Instrumentacin y condiciones de trabajo Se emplearon un espectrmetro de masas (otros detalles en III.1.1.3) y una bomba de jeringa para la infusin directa de las muestras (vase apartado III.1.1.3). Las condiciones de trabajo en el espectrmetro de masas se adaptaron de trabajos de la bibliografa [Peris-Vicente, 2005; Lerma-Garca, 2007]. El rango de barrido de masas en el modo PI fue de m/z 50300. El voltaje del capilar fue de 3,5 kV, el voltaje de la primera y segunda mscara fue de 19.6 y 6 V, respectivamente. La nebulizacin se efectu a 25 psi y 8 L min1, y la temperatura del gas de secado fue de 250 C. La masa objetivo se fij a m/z 122 ([M+H]+ para la cistena). Otros parmetros o mtodos de trabajo son los indicados en el apartado III.1.1.3. El tratamiento estadstico de los datos espectrales se realiz con el paquete estadstico SPSS (v. 12.0.1; Statistical Package for the Social Sciences Inc., Chicago, IL, USA).
III.3.2.4. Preparacin de estndares y muestras Para los concentrados de enzimas industriales, tanto lquidos como slidos, se siguieron dos procedimientos adaptados de la bibliografa
Adelantado, 2002; Peris-Vicente, 2005]. [Gimeno-
Para los concentrados industriales lquidos
de enzimas se pes alrededor de 1 g en un tubo con tapn de rosca, se aadieron 4 mL de acetona, y se recogi el precipitado por centrifugacin. Se desech el sobrenadante y se aadieron 200 l de HCl 12 M, mantenindose este tubo bien cerrado a 110 C durante 24 h. Para las enzimas suministradas como slidos granulares, se pes alrededor de 1 g, se aadieron 5 mL de agua, y la suspensin se someti a sonicacin durante 15 min. Tras centrifugacin se tom 1 mL del sobrenadante, llevndose a cabo la precipitacin e hidrlisis enzimtica como la descrita para los concentrados lquidos de enzima.
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Para el estudio de las posibles interferencias causadas por la matriz, se prepararon dos bases de detergente cuya composicin viene dada en la Tabla III.3. Unos 5 g de las bases de detergente fueron aditivadas con 50 L o 50 mg, de concentrado lquido o granulado industrial de enzimas, respectivamente. Para hacer las extracciones de enzimas a partir de las bases de detergente, o de los detergentes comerciales, se tomaron porciones de unos 5 g, se aadieron 20 mL de acetona y se agit, separndose el precipitado por centrifugacin. El residuo se disolvi en 1 mL de agua, y se efectu una reprecipitacin con 4 mL de acetona. El precipitado se hidroliz tal y como como se ha indicado anteriormente para los concentrados lquidos de enzimas industriales.
Tabla III.3. Composicin de las bases de detergente empleadas en este trabajo (% en peso). Componente AES Olena FAE Sodio Cumeno sulfonato Agua Base I (%) 5 10 10 5 70 Base II (%) 10 5 15 5 65
Tras la hidrlisis cida de la muestra, el residuo se agita con 5 mL de una mezcla EtOH/HCl (0,1 M) 1:1 (v:v). La suspensin se hace pasar por un filtro de nylon de 0,45 m y se infunde directamente en el espectrmetro de masas, o se conserva a -20 C hasta su uso. Se hidrolizaron tres alcuotas de todos los concentrados industriales de enzimas, las bases de detergentes aditivadas y los productos de limpieza, realizndose asimismo un mnimo de tres infusiones por alcuota.
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III.3.2.5. Construccin de las matrices de entrenamiento y evaluacin Como se indica en la Tabla III.2, se utilizaron tres concentrados industriales diferentes, dentro de cada una de las cuatro clases de enzimas, para construir el conjunto de entrenamiento. Por lo tanto, cada categora contena el mismo nmero de muestras (y los espectros correspondientes) que las dems categoras, para evitar que un peso excesivo en una de las categoras conduzca a un modelo incorrecto. Cada enzima se precipit e hidroliz tres veces de forma independiente, y cada hidrolizado se infundi tambin por triplicado, pero en el conjunto de entrenamiento slo se incluye la media de los datos obtenidos en las tres infusiones. De esta forma, la variacin interna de las diferentes categoras se redujo, lo que es importante para reducir el nmero de variables seleccionadas por el algoritmo que utiliza el SPSS para la construccin del modelo. Por lo tanto, el conjunto de entrenamiento I estaba constituido por los datos de 36 objetos (4 clases de enzimas 3 enzimas de cada clase 3 hidrolizados de cada enzima un promedio de las tres infusiones de cada hidrolizado). Para evaluar la posible influencia de las matrices de la muestra, generalmente productos de limpieza, las dos bases de detergente de la Tabla III.3, fueron aditivadas con tres concentrados industriales de enzimas de cada categora, y se procesaron como se ha indicado anteriormente. As pues, el conjunto de datos II contena 72 objetos (2 bases de detergente 4 clases de enzimas 3 enzimas de cada clase 3 hidrolizados de cada enzima un promedio de las tres infusiones de cada hidrolizado). Como se comentar posteriormente en la seccin VII.2, el conjunto de datos II, fue utilizado para evaluar los modelos construidos con el conjunto de datos I, sin embargo, posteriormente los datos de los conjuntos I y II fueron utilizados conjuntamente para mejorar la capacidad de prediccin de los modelos.
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Por ltimo, el conjunto de datos III fue construido a partir de todos los datos espectrales de los concentrados industriales de enzimas y productos de limpieza que no se incluyeron en la construccin de los conjuntos anteriores (Tabla III.2). Este tercer conjunto contena 42 objetos (12 concentrados industriales de enzimas ms 2 detergentes comerciales 3 hidrolizados de cada enzima un promedio de las tres infusiones de cada hidrolizado).
III.3.2.6. Seleccin de variables predictoras para la construccin de un modelo de LDA El LDA, es una tcnica de clasificacin supervisada, siendo una excelente herramienta para obtener los vectores que muestran la resolucin mxima entre las diferentes clases o categoras. En un LDA se obtienen los vectores que hacen mnima la lambda de Wilks, W
[Vandeginste, 1998].
La W es una funcin de las
distancias eucldeas entre los puntos medidos en la direccin de los vectores (funciones discriminantes) que constituyen el modelo. Usando el SPSS, las distancias eucldeas se miden en un espacio normalizado, donde todas las variables de entrada tienen una media de cero y uno de desviacin estndar. Para obtener W, la suma de los cuadrados de las distancias eucldeas entre todos los puntos pertenecientes a la misma categora se divide por la suma total de cuadrados. Los valores de W cercanos a cero se obtienen con categoras bien separadas, mientras que la presencia de por lo menos un par de categoras superpuestas conduce a valores de W prximos a uno. Mediante el uso de LDA se construyen hasta N-1 funciones discriminantes, donde N es el valor ms bajo, entre el nmero de variables predictoras o el nmero de categoras. Para seleccionar las variables predictoras a incluir en los modelos, se utiliz el algoritmo paso a paso del SPSS. El valor de W disminuye cada vez que una variable predictora con un poder discriminante significativo (de acuerdo
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a las categoras establecidas) se incluye en el modelo. De acuerdo con el algoritmo paso a paso, una variable predictora se incorpora al modelo, cuando tras su inclusin en el mismo, se supera un umbral, Fin. El criterio Fin es un ensayo F que compara la reduccin de varianza residual producida por la entrada de la variable con la varianza residual que queda. Sin embargo, la entrada de un nuevo predictor modifica la reduccin de varianza residual debida a las dems predictoras presentes en el modelo. Por esta razn, tras la inclusin de una nueva predictora, se aplica un criterio de rechazo, Fout, para decidir si la predictora debe ser eliminada del modelo. El proceso termina cuando no hay predictoras que puedan entrar o ser eliminadas del modelo. Inicialmente se tomaron los valores de probabilidad que emplea el SPSS por defecto, siendo 0,05 y 0,10 para los criterios Fin y Fout, respectivamente.
III.3.3. Aminocidos derivatizados con OPA-NAC
III.3.3.1. Estndares, materias primas y otras muestras La Gly y los siguientes levo-aminocidos fueron utilizados como estndares: Ala, Arg, Asp, Glu, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Tyr, Val y Cys (Sigma). Otros aminocidos como el Trp, Asn y Gln, no se incluyeron en este estudio, ya que el primero se destruye completamente durante la hidrlisis cida, y la Asn y la Gln, se convierten en Asp y Glu
[Hurst, 2002].
Los
concentrados de enzimas industriales utilizados en este trabajo se muestran en la Tabla III.2. Dehydol LT-7 (mezcla de FAEs, Cognis, Dsseldorf, Alemania), LES, olena y sodio cumeno sulfonato (donados por Qumicas Oro). Dos detergentes para lavado de textiles que contenan proteasa (segn declaracin del fabricante) se obtuvieron en los comercios locales.
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III.3.3.2. Reactivos de uso general Se emple acetona, etanol (Scharlau, Barcelona, Espaa) y HCl (37%) (Panreac, Barcelona, Espaa). Tambin se emplearon otros reactivos como OPA, NAC (Fluka, Buchs, Switzerland), cido brico (Panreac, Barcelona, Espaa) y cido ctrico anhdrido (Sigma, St. Louis, MO, USA). Tambin se emple agua desionizada (desionizador Barnstead, Sybron, Boston, MA, USA)
III.3.3.3. Instrumentacin y condiciones de trabajo
Se emple un cromatgrafo de lquidos (HP 1100, Agilent, Waldbronn, Alemania), constituido por una bomba cuaternaria, un desgasificador en lnea, un termostato para la columna, un muestreador automtico y un detector UV-Vis de longitud de onda variable. La columna empleada fue una Kromasil C18 (5 m, 25 cm x 4 mm de ID, Anlisis Vnicos, Tomelloso, Espaa). Los gradientes de elucin, incluyendo el gradiente ptimo multi-segmentado (Tabla VII.3), se obtuvieron mezclando dos disoluciones que contenan agua con ACN al 5% y 50% (mezclas A y B, respectivamente), ambas tamponadas con cido ctrico/citrato sdico 5 mM a pH 6,5. El flujo de la fase mvil fue de 1 mL min-1, y los volmenes de inyeccin fueron de 5 L. La deteccin se realiz a 335 nm. Las reas de los picos se midieron con el software ChemStation para LC v.10.02 (Agilent). Para la normalizacin de las variables, as como para transferir los datos al paquete estadstico SPSS (v. 12.0.1; Statistical Package for the Social Sciences Inc., Chicago, IL, USA) se emple el programa Excel (Microsoft). El SPSS fue empleado tambin para el tratamiento estadstico de los datos espectrales. Para la seleccin de las variables predictoras que se incluyeron en los modelos, se utiliz el algoritmo paso a paso (seccin III.3.2.6) del paquete
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estadstico SPSS, donde se adoptaron inicialmente los valores de Fin y Fout, 3,84 y 2,71, respectivamente.
III.3.3.4. Preparacin de estndares y muestras Se siguieron los mismos procedimientos de hidrlisis y extraccin de enzimas comentados en la seccin III.3.2.4. En este caso, se hidrolizaron dos alcuotas de cada concentrado de enzima industrial, base de detergente aditivada o muestra comercial. Las dos alcuotas hidrolizadas se diluyeron y derivatizaron como se indica a continuacin, realizndose una inyeccin de cada alcuota. El reactivo de derivatizacin estaba compuesto por una mezcla que contena OPA 1,25 10-2 M y NAC 2,5 10-2 M, tamponada con una disolucin de cido brico/sodio borato 1 M a pH 9,5. El reactivo de derivatizacin se protegi con papel de aluminio y fue conservado a 4 C, renovndose semanalmente. La derivatizacin se realiz directamente por la adicin de 1 mL de la disolucin de OPA-NAC a una alcuota de 100 L de los hidrolizados. Para identificar los picos correspondientes a cada aminocido a lo largo del cromatograma, se prepararon disoluciones de un aminocido o tambin mezclas de dos o tres aminocidos (1000 g mL-1 de cada) que fueron derivatizados como anteriormente se ha explicado. Tras la derivatizacin, estas disoluciones fueron usadas tanto para inyectar directamente o para aditivar los hidrolizados cuando fue necesario. En la Figura III.5 se muestra la reaccin de derivatizacin de los aminocidos.
O R1 H2N H Aminocido COOH O O H H O OPA NAC Isoindol OH HN HO O S N OH HN HO COOH R1 O
+ HS
Fig. III.5. Reaccin de formacin de isoindoles.
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III.3.4. Digestos con tripsina
III.3.4.1. Estndares, materias primas y otras muestras Se utiliz tripsina de pncreas bovino y BSA (Fluka, Buchs, Suiza). Los concentrados de enzimas industriales utilizados en esta seccin estn indicados en la Tabla III.2. Tambin se emple Dehydol LT-7 (mezcla de FAEs, Cognis, Dsseldorf, Alemania), LES, oleina y sodio cumeno sulfonato (donados por Qumicas Oro), as como otros componentes empleados para preparar las bases de detergentes empledas en este trabajo (Tabla III.1). Dos detergentes comerciales que contenan proteasa (segn declaracin del fabricante) se adquirieron en los comercios locales.
III.3.4.2. Reactivos de uso general Se emple TFA ( 99,5%), DTT y (NH4)HCO3 (Sigma-Aldrich, Viena, Austria), cido frmico (98-100%, Riedel de Haen, Seelze, Alemania), as como ACN grado HPLC y acetona (VWR, Viena).
III.3.4.3. Instrumentacin y condiciones de trabajo Se emple un cromatgrafo de lquidos (HP 1100, Agilent, Waldbronn, Alemania), constituido por una bomba cuaternaria, un desgasificador en lnea, un termostato para la columna, un muestreador automtico y DAD. El sistema HPLC estaba equipado con un inyector automtico termostatizado, que permit que las muestras se mantuviesen a 5 C hasta su inyeccin. Las principales caractersticas de las columnas estudiadas se presentan en la Tabla III.4. La elucin se llev a cabo usando mezclas de dos disoluciones que contenan agua y ACN (fases A y B, respectivamente), ambas en presencia de un 0,1% de TFA. A menos que se indique lo contrario, la elucin se efectu en dos pasos, un primer
126
paso isocrtico con 1% de B durante 5 minutos, seguido de un gradiente lineal del 1 al 40% de B durante los siguientes 58 minutos. Los volmenes de inyeccin fueron de 5 L y los flujos se modificaron para obtener la misma velocidad lineal (1,3 mm/s) en todas las columnas. La deteccin se realiz a 214 y 280 nm.
Tabla III.4. Columnas empleadas en este trabajo. Caractersticas Tipo Tamao de partcula (m) Dimensiones, longitud ID (mm) Fabricante
a
ProSwift Monoltica polimricaa 50 4,6 Dionex
Chromolith Monoltica slice (C18) 100 4,6 Merck
Gemini 5 m Particulada (C18) 5 150 4,6 Phenomenex
Gemini 3 m Particulada (C18) 3 150 3 Phenomenex
Kinetex Particulada (C18) 2,6 100 3 Phenomenex
Columna monoltica de fenil-poliestireno.
III.3.4.4. Preparacin de estndares y muestras Los concentrados de enzimas industriales lquidas fueron diluidos en un factor 1:10 con una disolucin de (NH4)HCO3 40 mM (pH = 8,5). Para las enzimas suministradas como slidos granulares, se pesaron 0,2 g, se aadi 1 mL de agua, y se sonic la suspensin durante 15 minutos. Tras centrifugar, se tom una alcuota del sobrenadante y se trat como se ha indicado para los concentrados lquidos de enzima. Se prepar una disolucin de BSA de 3,5 mg mL-1 en el tampn de (NH4)HCO3. La digestin se efectu de la siguiente manera: se tomaron alcuotas de 100 L de cada enzima diluida y de la disolucin de BSA, y se aadieron 5 L de una disolucin de DTT 200 mM. Esta mezcla se dej reaccionar bajo agitacin a 100 C para reducir los grupos disulfuro. Tras dejar enfriar a temperatura ambiente, se aadieron 25 L de una disolucin de tripsina de 0,5 mg mL-1 preparada en el mismo tampn. La mezcla se digiere durante toda la noche a 37 C, utilizando un mezclador termosttico con agitacin. La digestin se detiene por adicin de 5 L de cido frmico al
127
10%. Los digestos resultantes se filtraron a travs de un filtro de 0,2 m Phenex RC (Phenomenex, Torrance, IL, USA) y se inyectaron en el cromatgrafo. Tambin se prepar un blanco de muestra siguiendo el mismo protocolo, pero en esta ocasin la alcuota de 100 L de enzima diluida fue sustituida por el mismo volumen de tampn. Se pesaron alrededor de 2 g de las bases de detergente y de los detergentes comerciales. Las bases de detergente se aditivaron con 20 L de una enzima concentrada industrial. Las enzimas fueron precipitadas por adicin de 10 mL de acetona con agitacin. Tras eliminar el sobrenadante, el precipitado se aisl por centrifugacin a 4000 rpm 10 minutos, y se disolvi en 200 L de agua. Esta disolucin se re-precipit con 1 mL de acetona y el precipitado se aisl por centrifugacin a 13400 rpm 10 min. El residuo se disolvi en 100 L del tampn de (NH4)HCO3 y se llev a cabo la digestin como se ha indicado anteriormente.
III.4. Columnas monolticas
III.4.1. Monmeros, agentes entrelazantes, iniciadores, estndares y reactivos de uso general Para la preparacin de columnas monolticas se emplearon los siguientes reactivos: LMA, EDMA, META (al 75% en agua), 1,4-butanodiol, metacrilato de 3-(trimetoxisilil)propilo (como reactivo enlazante o silano binding), LPO y DMPA (Aldrich, Milwaukee, WI, USA), 1-propanol, ACN y MeOH (Scharlau, Barcelona, Espaa); AIBN, BPO y Tris fueron suministrados por Fluka (Buchs, Suiza). Se utiliz tiourea como marcador del EOF, una mezcla de PAHs conteniendo pireno, antraceno, naftaleno, fluoreno, benzo[a]antraceno y benzo[k]fluoranteno (Riedel de Han, Seelze, Alemania), y una mezcla de
128
alquilbencenos
conteniendo
tolueno,
etilbenceno,
n-propilbenceno,
n-
butilbenceno, n-pentilbenceno y n-hexilbenceno. Como estndares de prueba se utilizaron los siguientes solutos bsicos: anilina, 4-nitroanilina, N,N-
dimetilanilina, 2,4,6-trimetilanilina y piridina (Aldrich). Se utiliz agua desionizada obtenida con un desionizador Barnstead (Sybron, Boston, MA, USA).
III.4.2. Tratamiento de columnas
III.4.2.1. Acondicionado de columnas Antes de rellenar las columnas con las disoluciones que contienen las mezclas para la sntesis de monolitos, se procede a modificar la superficie interna del capilar de slice. Esta operacin tiene como objeto favorecer el anclaje covalente del monolito a dicha superficie interna. Para ello se sigui el procedimiento descrito por Frchet y col. [Peters, 1997]: Los capilares que se emplearon eran de slice fundida con dimensiones 375 m OD 100 m ID, con la capa externa transparente a la radiacin UV (Polymicro Technologies, Phoenix, Arizona, USA). Por una seccin de capilar de 4 m de longitud se hicieron pasar sucesivamente, mediante la ayuda de una jeringa Hamilton conectada a una bomba de jeringa (kd Scientific, Holliston, MA, USA), las siguientes disoluciones a un caudal de 200 L min-1 (salvo que se especifique lo contrario): 1. Acetona, hasta ver aparecer algunas gotas a la salida del capilar, para asegurar la limpieza de la pared interna. 2. Agua nanopura, hasta la eliminacin completa de la acetona. 3. NaOH 0,2 M, hasta observar pH bsico a la salida del capilar. 4. Agua nanopura, hasta eliminar el NaOH, para evitar cambios bruscos del
129
pH. 5. HCl 0,2 M, hasta observar pH cido a la salida del capilar. 6. Agua nanopura, hasta pH neutro a la salida del capilar, para eliminar los restos de cido. 7. EtOH hasta olor persistente, con el fin de eliminar el agua y evitar la hidrlisis del reactivo enlazante (silano-binding) que se adiciona en la siguiente etapa. 8. Disolucin de reactivo enlazante (silano-binding) al 20% (m/v) en EtOH, acidulado con cido actico hasta pH 5; el enlazante se pasa a un caudal de 0,25 L min-1 durante 60 min. 9. Acetona, para eliminar el exceso de reactivo enlazante. Para finalizar, se aplic una corriente de nitrgeno para secar el capilar, dejndose en estas condiciones durante 24 h hasta completar la reaccin de condensacin de los grupos silanol con el reactivo enlazante (silano-binding). Tras este periodo, se retir la fuente de nitrgeno y se sellaron los extremos del capilar con sendos tapones, con el fin de evitar la hidrlisis de los enlaces siloxano.
III.4.2.2. Preparacin de columnas monolticas Los monolitos se preparan mediante polimerizacin de mezclas de un monmero base (LMA), un agente entrelazante (EDMA), un monmero con carga para la generacin del EOF (META), y disolventes porognicos, siendo los empleados aqu el 1,4-butanodiol y el 1-propanol. Estas mezclas se preparan mediante la pesada de cada uno de sus componentes en una balanza analtica. Las mezclas se polimerizan mediante fotoionizacin. Los iniciadores empleados fueron AIBN, DMPA, BPO y LPO. Antes de iniciar la polimerizacin, y con el fin de eliminar el oxgeno disuelto de las disoluciones,
130
stas se sonican durante 10 minutos y se purgan con nitrgeno durante 10 min ms. Los capilares, previamente pre-acondicionados, fueron cortados en trozos de 33,5 cm de longitud, rellenndose un segmento de 8,5 cm de longitud con las mezclas de polimerizacin. Una vez rellenos los segmentos, se sellan los extremos del capilar mediante tapones y se procede a la polimerizacin. En el caso de la fotopolimerizacin los capilares se sometieron a una irradiacin de 0,9 J/cm2 durante 10 minutos dentro de una cmara UV equipada con cinco lmparas UV (5 8 W, 254 o 365 nm). La lmpara UV de 254 nm fue empleada para polimerizar con todos los iniciadores, a excepcin del AIBN para el que se utiliz radiacin de 365 nm [Peters, 1998-A; Eeltink, 2005; Geiser, 2007; Ngola, 2001]. Una vez terminada la polimerizacin, se cortaron ligeramente los extremos de las columnas resultantes para liberar restos adheridos a los tapones, y garantizar la homogeneidad del relleno. A continuacin, las columnas se desobstruyeron con MeOH y la ayuda de una bomba de HPLC, eliminando tanto los disolventes porognicos como los posibles monmeros sin reaccionar, y los oligmeros no incorporados a la estructura del monolito. A continuacin, se cortaron las porciones de capilar necesarias para ajustar la posicin de la ventana ptica a 8,5 cm de uno de los extremos, y tambin para que la longitud total del capilar fuera de 33,5 cm. Cortar un extremo tambin garantiza una seccin transversal del monolito perpendicular al eje longitudinal del capilar en dicho extremo. Finalmente, antes de la inyeccin de estndares o muestras, se hace pasar fase mvil a travs del capilar durante 30 min.
131
III.4.3. Instrumentacin y condiciones de trabajo Los ensayos de CEC se realizaron con un equipo de electroforesis capilar Agilent, modelo HP3D (Agilent Technologies, Waldbronn, Alemania), dotado de un DAD, y de un sistema auxiliar capaz de suministrar hasta 10 bar de presin externa de nitrgeno sobre ambos extremos del capilar simultneamente. La presurizacin del capilar es importante para evitar la aparicin de burbujas que cortaran el paso de corriente elctrica por el mismo. Para la adquisicin de los datos se utiliz el software ChemStation (Rev. A.10.01, Agilent). Con el fin de proceder a su acondicionado con la fase mvil, la columna se coloc en el equipo de CEC y se equilibr con la fase mvil a 25 C, incrementndose el voltaje progresivamente en el rango de 5 a 25 kV, presurizando en todo momento los viales de entrada y salida a 10 bares, hasta observar una seal analtica y una corriente estables. Esta etapa de equilibrado tuvo una duracin de 45-60 min, dependiendo de las caractersticas de flujo de cada columna. Para los ensayos de CEC se emple, cuando fue necesario, una muestra de tiourea como marcador del EOF. Las separaciones se realizaron a 25 C y a varios voltajes. En todos los casos se utiliz nitrgeno para presurizar ambos viales a 1 MPa. Se prepar una disolucin de Tris en agua a pH 8.0 ajustado con HCl 1 M. Las fases mviles fueron preparadas mezclando el tampn de Tris con diferentes proporciones de ACN y agua para obtener disoluciones con una concentracin constante de 5 mM de Tris. Se prepar una mezcla de seis PAHs (pireno, antraceno, naftaleno, fluoreno, benzo[a]antraceno, benzo[k]fluoranteno) y tiourea (100 200 g mL-1, de cada sustancia) para evaluar el funcionamiento de las columnas. La inyeccin de la mezcla de PAHs se hizo de forma electrocintica (5 kV 3 s), y la deteccin se fij a 214 y 254 nm.
132
La morfologa de los materiales monolticos se estudi mediante el empleo de un microscopio electrnico de barrido Hitachi modelo S-4100 (Ibaraki, Japn), provisto de un sistema de captacin de imgenes EMIP 3.0. Previamente, se metaliz la superficie expuesta de la seccin de las columnas con un depsito de oro y paladio. Para ello, se utiliz un recubridor por pulverizacin BIORAD modelo SC-500 (Hemel, Hempstead, Reino Unido).
133
RESULTS
CHAPTER IV
NON IONIC SURFACTANTS IN CLEANING PRODUCTS
Chapter IV. No ionic surfactants in cleaning products
IV.1. APGs
IV.1.1. APGs by HPLC-MS The APGs are non-ionic surfactants produced from renewable materials (glucose and fatty alcohols)
[Koch, 1993; Balzer, 1996; Balzer, 2000; Hill, 1997;
Rybinski, 1998; Biermann, 1993].
Owing to their favourable properties in terms of
foaming performance, synergism with other surfactants, and environmental and skin compatibility, they are widely used in cleaning and personal care products
[Balzer, 1996; Rybinski, 1998; Biermann, 1993; Garca, 1997; Uppgard, 2000],
and in
the oil and gas industry
[McGregor, 2004].
APGs are industrially obtained as
complex mixtures of alkylpoliglucosides. The oligomers are distinguished by the alkyl chain length, the number of glucose units and the isomerism (Fig. I.5). Three types of isomerism are present: stereoisomerism with - and -epimers, ring isomerism (pyranoside and furanoside forms), and positional isomers, with predominant 1,4-and 1,6-interglucosidic linkages between glucose units. The total amount of APGs can be determined by hydrolysis followed by derivatisation with anthrone and colorimetry [Buschmann, 1995; Buschmann, 1996B],
potentiometric titration after sulfonation with the SO3DMF complex near-infrared spectrometry
[Kim, 2001]
[Buchmann, 1996-A],
and enzymatic
hydrolysis
[Kroh, 1999].
APGs can be determined in environmental samples after
hydrolysis, followed by distillation of the alcohols and fluorogenic derivatisation

[Meissner, 1999].
The separation by thin layer chromatography on C8 and C18

[Buchmann, 1996-A; Buschmann, 1996-B; Buschmann,
phases has been described
1996-C; Spilker, 1996; Klaffke, 1998].
Underivatised alkylmonoglycosides can be

[Rybinski, 1998; [Buchmann,
separated with isomer resolution using high-temperature GC

Spilker, 1996].
APGs have been also separated by GC after silylation
1996-A; Spilker, 1996; Billian, 1998].
The pyranoside and furanoside forms can be
139
distinguished by the different fragments obtained by GCMS
[Billian, 1998].
Micellar electrokinetic chromatography with electrochemical detection has been applied to the determination of APGs in shampoo
[Hbner, 2006].
To implement
the photometric detection of APGs after HPLC separation, several post-column chromogenic derivatisation procedures have been described
[Kramer, 1992].
Alternatively, refractive index detection [Klaffke, 1998], ELSD [Buchmann, 1996-A;

Buschmann, 1996-B; Lafosse, 1992; Czichocki, 2002; Armari, 2003] [Czichocki, 2002; Eichhorn, 1999; Klaffke, 1999; Khn, 2004],
or MS detection
can be used. The
chromatographic behaviour of alkylglucopyranoside standards using C8, C18, phenyl and polyvinylalcohol columns has been described
[Lafosse, 1992],
and the
separation of APGs with silica, C18 and polyvinylalcohol columns have been compared
[Czichocki, 2002].
Eichhorn and Knepper
[Eichhorn, 1999]
have used
HPLCMS with an ESI to determine APGs in spiked river and waste waters. Using a C18 column and gradient elution with ACN/water, the - and -epimers and the ring isomers were resolved. Alkylglucopyranosides can be further distinguished by their higher affinity to form [M+NH4]+ adducts with respect to alkylglucofuranosides. Kuhn and Neubert
[Khn, 2004]
have used HPLCESI-
QTOFMS with a C18 column, a MeOH/water mobile phase and gradient elution, to characterize industrial mixtures of APGs; however, isomers were not resolved. In this section, the HPLC separation of APG mixtures using either an alkylamide or a cyanopropyl column, with ACN/water mixtures as mobile phases, was studied. The alkylamide column in isocratic conditions was adequate to separate alkylmonoglucosides with full resolution between epimers and ring isomers. Retention was lower on the cyanopropyl column, but equilibration time was much shorter. Using the cyanopropyl column with gradient elution the ring isomers were also resolved. The influence of the alkyl chain length on the
140
response factors was studied. The procedures were applied to the characterization and determination of APGs in toiletries.
IV.1.1.1. Optimization of working conditions using continuous infusion MS Using continuous infusion MS in the NI mode, peaks of the [MH] ions of the standards and the components of Glucopone and Plantacare were obtained. In the PI mode, the corresponding [M+H]+ and [M+Na]+ peaks were observed. The [M+ Na]+ peaks largely predominated when NaAcO was added to the infused solution. Maximal intensity was obtained with 20 mM NaAcO. The PI mode, which gave peak intensities ca. 20 times larger than those obtained with the NI mode, was selected. Glucopone and Plantacare showed several groups of [M+Na]+ peaks, each of them corresponding to the oligomers with a given number of glucose units, up to m = 6. Within each group, two large peaks corresponding to the C8Gm and C10Gm oligomers, and a small one due to the C12Gm oligomer, were observed. The intensity also decreased as m increased. Using infusion of the Glucopone solution, the dry gas flow, spray temperature and spray pressure were optimized.
IV.1.1.2. Separation of APGs by HPLCMS in isocratic conditions The alkylamide column and a mixture containing 20:80 ACN/water in the presence of the HAcO/NaAcO buffer were first used. The EICs at the m/z values observed in the infusion spectra were examined. The AMGp standards gave single chromatographic peaks, and complex chromatograms were obtained for the technical grade mixtures. As shown in Fig. IV.1 for Glucopone, a complete separation of the four C8G1 isomers (- and -epimers, and ring isomers, on the EIC at m/z 315) was achieved.
141
-C8G1p 1.5
Abundance x 107
-C8G1p 1.0
-C8G1f 0.5 C8G2 -C8G1f m/z 315 m/z 477 0 10 20 30 40 50 Time (min)
Fig. IV.1. EIC of Glucopone. The alkylamide column was used. The mobile phase contained water with 20% ACN in the presence of 40 mM HAcO/NaAcO buffer of pH 4.7. The flow rate was 0.4 mL min1.
The epimers and ring isomers of C10G1 and C12G1 appeared also resolved at longer retention times (not shown). Also, a partial separation of the isomers of the alkyldiglucosides (C8G2) was evidenced (EIC at m/z 477). The identification of - and -C8G1p was made by injecting solutions of the corresponding standards. As far as we know, standards of APGf are not available; however, the two peaks of low intensity which appeared at longer retention times than the and -C8G1p peaks on the m/z 315 trace, were attributed to the corresponding and -C8G1f isomers. Retention should be higher for these isomers as a consequence of the longer CH(OH)CH2OH chain bound to the
alkylglucofuranoside ring in comparison with the CH2OH chain of the alkylglucopyranosides. Next, to reduce the analysis time of the C10Gn and C12Gn isomers, the ACN concentration in the mobile phase was increased. With 30% ACN (Fig.
142
IV.2), the - and -epimer pairs of C8G1 were not resolved, but the four isomers of C10G1 were resolved within 20 min. As also shown in Fig. IV.2, the presence of alkyldiglycosides (C8G2 and C10G2) and alkyltriglycosides (C8G3) with partial isomer resolution was evidenced.
1.5 C8G1p
C10G2 Abundance x 106 1.0 C8G3 -C10G1p m/z 639 C8G2 0.5 C8G1f m/z 315 m/z 477 0 2 4 6 8 Time (min) 12 -C10G1f -C10G1f m/z 343 14 16 18 Time (min) 20 m/z 505 -C10G1p
Fig. IV.2. EIC of Glucopone. The alkylamide column was used. The mobile phase contained water with 30% ACN in the presence of 40 mM HAcO/NaAcO buffer of pH 4.7. The flow rate was 0.2 mL min1.
The chromatograms of Plantacare were similar to those of Glucopone, but with higher proportions of the C10Gm and C12Gm oligomers with respect to those of C8Gm (not shown). A chromatogram of a baby shampoo, eluted with 50% acetonitrile, is shown in Fig. IV.3. The ring isomers of the AMGs of C8G1, C10G1, C12G1 and C14G1 were resolved. The - and -epimers of C12G1f, C14G1p and C14G1f were also resolved. The isomers of the alkyldiglycosides C8G2, C10G2 and C12G2 were partially resolved.
143
C8G2 2.5 C10G2 m/z 505 2

Abundance x 107
m/z 477
C12G2 m/z 533
C12G1p -C14G1f -C14G1f
1.5
C8G1p
C10G1p m/z 399
1 C8G1f 0.5 m/z 315 0 C10G1f -C14G1p m/z 343 -C14G1p -C12G1f -C12G1f m/z 371 0 2 4 6 8 10 m/z 399 14 16
18 20 Time (min)
18 Time (min)
Fig. IV.3. EIC of a baby shampoo. The alkylamide column was used. The mobile phase contained water with 50% ACN in the presence of 40 mM HAcO/NaAcO buffer of pH 4.7. The flow rate was 0.2 mL min1. The inset shows the m/z 399 trace with the intensity axis multiplied by 50.
Retention was much weaker with the cyanopropyl column; for instance, using 20% ACN, the C8G1 isomers were eluted within 6 min (not shown). Using 95 and 100% water in the mobile phase, the C8G1 isomers appeared within 30 and 40 min, respectively. In all cases, the ring isomers were well resolved, but the epimer pairs were poorly resolved with this column.
IV.1.1.3. Separation of APGs by HPLCMS with gradient elution The alkylamide column and mobile phases containing 60:40 (A) or 90:10 (B) ACN/water (v/v) in the presence of the HAcO/NaAcO buffer were used. Mobile phase A was first pumped until stabilization of the pressure, thus the Plantacare solution was injected and the composition of the mobile phase was
144
linearly varied from A to B in 20 min. The AMGs were separated with partial resolution between the epimers, and with excellent resolution between the ring isomers, within 12 min (not shown). However, the re-equilibration of this column was observed to be rather slow. Pumping of mobile phase A during several hours was required to achieve reproducibility between successive injections.
C12G1p 4
Abundance x 107
C16G1p
C8G1p m/z 427 C16G1f C10G1p C12G1f C14G1p C14G1f 13 14 Time (min)
C8G1f m/z 343 4 8
C10G1f
m/z 427 m/z 399 m/z 371
m/z 315 0
Time (min)
12
Fig. IV.4. EIC of Plantacare. The cyanopropyl column was used. The composition of the ACN/water mobile phase (also containing 40 mM of the HAcO/NaAcO buffer of pH 4.7) was linearly varied from 25 to 90% acetonitrile in 15 min. The flow rate was 0.2 mL min1. The inset shows the m/z 427 trace with the intensity axis multiplied by 20.
Using the cyanopropyl column, mobile phases containing 25:75 (A) and 90:10 (B) ACN/water (v/v) in presence of the acetic acid/sodium acetate buffer, were used. The mobile phase was linearly varied from A to B in 15 min. As shown in Fig. IV.4 for Plantacare, all the AMGs, from C8G1 up to C16G1, were separated within 14 min. The epimer pairs were not resolved, but a good resolution between the ring isomers was achieved. Successive injections of the
145
sample were performed after pressure stabilisation (15 min). Excellent reproducibility of the chromatograms was obtained. The chromatogram of a hand cream, which has a rather complex matrix, showed the resolved peaks of the ring isomers of C12G1 and C14G1 (Fig. IV.5).
C12G1p
Abundance x 105
6 C14G1p 4
2 C14G1f C12G1f m/z 371 0 10 12 Time (min) 14 m/z 399
Fig. IV.5. EIC of a hand cream. The cyanopropyl column was used. The composition of the ACN/water mobile phase (also containing 40 mM of the HAcO/NaAcO buffer of pH 4.7) was linearly varied from 25 to 90% ACN in 15 min. The flow rate was 0.2 mL min1.
IV.1.1.4. Quantitation studies The calibration curves were obtained using the cyanopropyl column in the optimized gradient elution conditions. Solutions containing a mixture of the standards (-pyranosides C1G1, C6G1, C8G1, C10G1 and C12G1), in the presence of 50 g mL1 -pyranoside C9G1 as internal standard, were injected. Mixtures of the -pyranosides of C1G1 and C8G1 were also injected. The
146
concentration of the standards was varied from 10 to 120 g mL1 (from ca. 60 600 M for C1G1 to 30360 M for C12G1). In all cases, the calibration curves gave a good linearity, with r2 > 0.99 (6 points per curve). As shown in Fig. IV.6, the relative sensitivities of the -epimers (or response factors, calculated by using -C1G1p as reference) increased almost linearly from C1G1 to C8G1, reached a maximum at C10G1 and decreased for C12G1. Relative rather than absolute sensitivities were plotted in this figure to enhance the differences between the standards, independently from detector sensitivity and some working conditions. A range of relative sensitivities for C14G1 was also plotted on Fig. IV.6. This range was obtained by injecting Plantacare, and using the sum of the areas of the C14G1p and C14G1f peaks, and the composition given by the manufacturer (6.19.5% total C14G1 for a 5153% pure product), to calculate the relative sensitivity range. As deduced from Fig. IV.6, this range indicated a further decrease of the relative sensitivity for alkyl chains longer than 12 carbon atoms. Since a range of concentrations was not declared (Table IV.1), the relative sensitivity for C16G1 could not be calculated. The influence of the mobile phase composition on the relative sensitivities was also studied. For this purpose, a 20:80 (A) or a 75:25 (B) ACN/water (v/v) mixtures, containing 20 mM NaAcO and 20 mM HAcO, were used. Dilutions of the stock solutions of the standards were prepared using both A and B. The dilution factor was 1:20, but 1:10 was used for C12G1. All the diluted solutions also contained 50 g mL1 of the internal standard, -C9G1p. The autosampler was directly connected with the ESI source with a PEEK tube in the absence of the separation column, and the solutions were injected successively using A and B as mobile phases. No significant differences between the peak areas obtained with mobile phases A and B were observed. Thus, the differences among the response factors observed in Fig. IV.6 should be attributed to the length of the
147
alkyl chains rather than to the increase of the acetonitrile concentration along the elution gradient.
16
90
12
70
Relative sensitivity
50 8
30 4 10 0 0 4 8 n 12
Fig. IV.6. Relative sensitivities (left axis and dashed line) of - () and alkylglycopyranosides () with respect to -C1G1, and LODs (, right axis and dotted line) vs. the number of carbon atoms in the alkyl chain, n (-C9G1p used as internal standard). All values were obtained from pure standards, but for C14G1 the two points delimit the range of relative sensitivity, which was estimated from its concentrations in Plantacare (6.1 9.5% C14G1 for a 5153% pure product, as declared by the manufacturer).
Taking into account that Na+ is bound to the same oxygen as the alkyl chain
[Klaffke, 1999],
the increase of the relative sensitivity from C1G1 up to
C10G1 can be due to stabilization of the adduct by the higher electronic density provided by the longer alkyl chain. The smaller volatility, the formation of ionpairs or micellization are possible explanations for the sensitivity decrease when the alkyl chain has 12 and 14 carbon atoms. Thus, the response factor can be approximately predicted by linear interpolation within the C1G1C10G1 range, but not when n > 10. Owing to the large differences, large systematic errors
148
LOD (M)
should be expected if calibration is not performed by using all the appropriate CnG1 standards. The relative sensitivities of the -epimers of C1G1p and C8G1p with respect to -C1G1p (used as a reference compound) were also plotted on Fig. IV.6. The -epimer/-epimer sensitivity ratio was 0.995 for C8G1, but 0.43 for C1G1. Thus, the response factor of the -epimer was less than half that of the epimer for C1G1, approaching to the sensitivity of the corresponding -epimer for longer alkyl chains. This can be explained by a lower ability of the -epimer of C1G1 to bind Na+ with respect to the -epimer. Further, the higher electronic density on the oxygen atom bound to the chain could explain both the sensitivity increase when the alkyl chain becomes longer, and the small sensitivity differences between the C8G1 epimers. Small or negligible systematic errors should be expected by using the calibration curve of a CnG1 epimer (n 6) to predict concentrations of the other epimer, or the sum of the concentrations of the two epimers. The LODs were obtained as the concentrations giving a peak area equal to three times the standard deviation of the calibration points with respect to the regression straight-line (standard deviation of the residuals). As observed in Fig. IV.6, the LODs were 85 M for C1G1 and of ca. 25 M from C6G1 up to C12G1, which agrees with the higher response factors of the latter. Also, from the standard deviation of the residuals, the limits of quantitation (as the concentrations giving a 10% precision in the determination of the analytes) were 280 M for C1G1 and 80 M from C6G1 up to C12G1. When the industrial products were analyzed, significant differences between the peak area ratios of the respective furanoside/pyranoside forms were observed. Thus, integration of the areas for the C10G1 and C12G1 isomer pairs showed that this ratio was 0.12 0.13 for Glucopone and the baby shampoo, 0.26
149
0.30 for Plantacare and ca. 0.04 for the hand cream. Thus, the furanoside/pyranoside concentration ratio can be used to characterize industrial samples. However, owing to the lack of standards, only the total concentration of each CnG1 group of isomers was obtained. For this purpose, the sum of the peak areas of the pyranoside and furanoside forms, and the calibration curve of the corresponding -pyranoside standard, were used. For Plantacare, the calibration curves predicted concentrations within the ranges declared by the manufacturer (Table IV.1). The analysis of a baby shampoo gave 0.5% C8G1, 0.2% C10G1, and 0.6% C12G1. The peaks of the C14G1 isomers were also observed in the chromatograms of this sample.
Table IV.1. Declared and found concentrations of alkylmonoglucosides in Plantacarea Compound Declared (%) Found (%) C6G1 C8G1 C10G1 C12G1 C14G1 C16G1
a
Max. 0.26 12.2 15.9 7.6 11.7 18.9 22.3 6.1 9.5 Max. 2.12
0.06 13.0 9.7 18.9
Range from minimal up to maximal declared concentrations multiplied by
minimal (51%) and maximal (53%) declared purity, respectively.
IV.1.2. Fragmentation of D-Glucose and AMGs CID fragmentation constitutes a powerful tool for the structural elucidation of oligosaccharides. Alkaline ions and ammonium
[Harvey, 2000-A;
Harvey, 2000-B; Chiarelli, 1987; Cancilla, 1999; Harvey, 1994; Harvey, 1997; Kovik, 1995; Penn, 1996; Asam, 1997], 2005],
divalent cations
[Harvey, 2001; Madhusudanan,
and iron salts
[Carlesso, 2000; Carlesso, 2001]
have been used; however, the
150
most informative fragmentation with maximal sensitivities has been obtained with sodium and calcium ions
[Harvey, 2000-A; Harvey, 2000-B; Harvey, 2001].
The
fragmentation of oligosaccharides using IT-MS [Zaia, 2004] and the application of MALDI-MS to the analysis of carbohydrates and glycoconjugates have been reviewed. When using low fragmentation energies, and according to the Domon Costello nomenclature [Zaia, 2004; Domon, 1988] (see Fig. I.6), the cleavage of the glucosidic bonds produces mainly B and Y fragments [Asam, 1997; Creaser, 2002]. Entire linkage sequences of linear and branched polysaccharides can be elucidated by analyzing the fragments
[Asam, 1997; Harvey, 2001; Zaia, 2004]. [Harvey, 2006]
Using higher fragmentation energies, cross-ring cleavages, which provide additional linkage information, are also produced [Harvey, 2000-A; Harvey, 2000-B;
Harvey, 2001; Cancilla, 1999; Asam, 1997; Zaia, 2004; Creaser, 2002].
Less attention has been paid to the CID spectra of monosaccharides and their derivatives. The stereochemical differentiation of monosaccharides in the presence of Zn2+ demonstrated.
[Gaucher, 1998]
and iron chloride isomers and
[Carlesso, 2000]
has been sulfated
The
positional
diastereomers
of
monosaccharides have been distinguished [Minamisawa, 2005]. The CID spectra of D-glucose and 2-fluorodeoxyglucose have been compared [Macek, 2003]. Using CID spectra, several pentoses and hexoses, including stereoisomers, were differentiated by the ratio of competing fragmentation processes
[March, 2005].
Multivariate analysis of TOF-SIMS has been used to distinguish among several furanoses, as well as among several pyranoses
[Berman, 2006].
However, studies
addressed to distinguish five- from six-membered ring isomers using CID fragmentation are rather scarce. The ring size of the last unit at the non-reducing end of oligosaccharides has been related to the abundance ratio of the [M+Na90]+ and [M+Na-104]+ ions of the CID spectrum
[Kovik, 2001].
Using TOF-
151
SIMS, five-membered ring monosaccharides were more stable than the corresponding six-membered ring isomers, also giving different peak profiles upon fragmentation [Berman, 2006]. APGs, which are industrially produced as complex mixtures from glucose and fatty alcohols, constitute an important class of nonionic surfactants
1997; Rybinski, 1998; Balzer, 2000]. [Hill,
Because of their foaming performance,
synergism with other surfactants, and environmental and skin compatibilities, APGs are widely used in cleaning and personal care products
Garca, 1997; Uppgard, 2000] [Rybinski, 1998;
and in the oil and gas industry [McGregor, 2004]. The
aim of this work was to study the ion-trap stepwise fragmentation of AMGs, which are the major components of industrial APGs. Also, the industrial production of APGs leads to mixtures containing major amounts of AMGp, and minor but significant concentrations of their five-membered ring isomers, AMGf. Thus, in this section we describe the CID spectra of both AMGp and AMGf, using several isotopic forms of D-glucose to assist interpretation. The successive MS2, MS3 and pseudo-MS4 spectra of D-glucose, D-glucose-13C6, D-glucose-6,6d2, D-glucose-1-13C and AMGp having up to 12 carbon atoms in the alkyl chain were obtained, at increasing fragmentation energies, with an ESI-IT-MS. Standards of the corresponding AMGf were not available; however, previous separation of a commercial mixture of APGs by HPLC, and their MS and MS2 spectra were obtained on-line.
IV.1.2.1. Optimization of the IT-MS detection conditions Using infusion in the IT-MS, both [M+H]+ and [M+Na]+ ions were observed to predominate in the PI spectra of D-glucose, the AMGp standards and Glucopone. The spectrum of Glucopone showed the predominant peaks of the octyl- and decyl-monoglucosides, plus the peaks of the corresponding
152
diglucosides and triglucosides with lower abundances. Concerning to the alkyl chain length, the octyl- and decylglucopyranosides predominated, the corresponding octyland decylglucofuranosides showing much lower
concentrations. Optimization of the IT-MS detection conditions was performed using Glucopone solutions. The abundance of the [M+Na]+ ions largely increased when sodium acetate was added to the MeOH/water solutions. The abundance of the [M+Na]+ ions increased with the Na+ concentration, and a plateau was reached within the 10 20 mM range. Then, the drying gas flow, spray temperature and spray pressure were optimised in the presence of 20 mM NaACO for a maximal intensity of the [M+Na]+ peaks of the AMGs of Glucopone. The rules to nomenclature of fragmentation are given in section I.2.3.
IV.1.2.2. Fragmentation of D-Glucose and their isotopic forms using MSn Using continuous infusion of the standard solutions in the optimized detection conditions, the MS2 spectra of D-glucose, D-glucose-13C6, D-glucose6,6-d2 and D-glucose-1-13C were obtained at increasing fragmentation energies. In Fig. IV.7, the MS2 spectra of the [M+Na]+ ions of D-glucose and D-glucose13
C6, both obtained at an energy of e = 0.45, are given. Using this fragmentation
energy, the peak of the parent ion was still observed together with the peaks of next ion generation. By comparing the MS2 spectra of D-glucose and D-glucose13
C6, it is deduced that the m/z 185 peak of D-glucose corresponds to an ion with
six carbon atoms. This peak was interpreted as due to a loss of a water molecule from the [M+Na]+ parent ion (m/z 203). At lower m/z values, the MS2 spectra of D-glucose showed an abundant peak at m/z 143 and a weak one at m/z 113, which according to MS2 spectrum of D-glucose-13C6 (Fig. IV.7, part B), corresponded to ions with four and three carbon atoms, respectively.
153
143
A
413
Intensity x 103
1.5 1 0.5 113 0 1.5 100 147 200 300 400 500
[M+Na]+
593 571 557 m/z 600
185 203
B
413 599 577 563 300 400 500 m/z 600
Intensity x 103
0.5 116 0 100
191 209 200
Fig. IV.7. MS2 spectra of the [M+Na]+ parent ion of (A) D-glucose (m/z 203) and (B) D-glucose-13C6 (m/z 209), both obtained at e = 0.45 (, parent ions).
By comprehensively computing all the possible cross-ring cleavages leading to an m/z 143 positive ion with four carbon atoms, the following possibilities were found:
0,2
A,
1,3
X,
2,4
[M+Na]+
X and
0,4
X; however, by using the corresponding [M+Na]+
peak as parent ion, the MS2 spectrum of D-glucose-6,6-d2 (not shown) yielded a peak at m/z 145. This indicated that carbon atom 6 was retained by the m/z 143 ion fragment of D-glucose. This excluded the
0,4
X fragmentation. Further, the
MS2 spectrum of D-glucose-1-13C (not shown) presented a peak at m/z 143, which evidenced that carbon atom 1 was not present in this ion fragment. This definitely excluded all the X fragmentations. Therefore, the m/z 143 ion of the MS2 spectrum of D-glucose should be exclusively produced by a
0,2
fragmentation. The two possible structures of this fragment ion are shown in Fig. IV.8.
154
Similarly, the
0,3
A,
1,4
A,
0,3
X and
1,4
X fragmentations, plus a
3,5
fragmentation followed by loss of a water molecule, are possible routes leading to an m/z 113 ion. However, the MS2 spectrum of D-glucose-6,6-d2 showed a peak at m/z 115, indicating that carbon atom 6 should be retained by the m/z 113 ion of D-glucose. Further, the spectrum of D-glucose-1-13C showed a peak at m/z 113, indicating that carbon atom 1 should be excluded from the composition of this ion. Thus, according to the spectra, only the two fragments obtained by a 0,3A fragmentation (Fig. IV.8) are possible to explain the formation of the m/z 113 ion.
Cross-ring cleavage OH
0,2A
Possible structures Na+ 5 4 HO 3 OH OH Na+ 5 4 O OH 6 5 4 HO H O OH HO O Na+ O 6 OH 5 4 3 OH H Na+
m/z 143
0,3A m/z 113
Fig. IV.8. The two possible structures of the m/z 143 and m/z 113 ions of the MS2 spectrum of the [M+Na]+ parent ion of D-glucose, produced by
0,2
A and 0,3A cross-ring cleavages, respectively.
Concerning to the abundant m/z 413 peak which was present in the MS2 spectrum of the [M+Na]+ parent ion of D-glucose, no shifts with respect to the equivalent spectra obtained with either D-glucose-13C (Fig. IV.7, part B), Dglucose-6,6-d2 and D-glucose-1-13C (not shown) were observed. This indicated the absence of fragments of glucose in the composition of the m/z 413 peak. However, this abundant peak was also present in the MS2 spectra of all the AMGs, and peaks showing the mass of the unfragmented molecules plus 413 m/z
155
units also appeared in the spectra of both D-glucose and all the AMGs. For these reasons, further efforts to interpret the m/z 413 peak were performed. First, MS2 spectra of the [M+Na]+ parent ion of D-glucose were obtained both in the absence of MeOH, and also using 20 mM NaHCO3 instead of 20 mM NaAcO. In both cases, an m/z 413 ion, with a similar abundance as that observed using 50% MeOH and 20 mM NaAcO, was obtained. Therefore, the presence of MeOH, AcO- or HAcO in the composition of the m/z 413 ion was also discarded. On the other hand, the m/z 413 peak was not observed when a 20 mM NaAcO solution in the absence of D-glucose was infused. Thus, the m/z 413 peak was formed only in the presence of D-glucose or an AMG, and could contain Na+, OH- and H2O, but not carbon atoms. Thus, fragmentation of the m/z 413 ion yielded peaks at m/z 301 and 189, which corresponded to two successive losses of 112 Da, in the MS3 spectrum. These losses could correspond to the uncharged fragment NaOH4H2O. A peak at m/z 171 which was attributed to a loss of water with respect to the m/z 189 ion was also observed. Further fragmentation of the m/z 301 ion was achieved by setting the compound stability parameter at 300%. Again, the m/z 189 and 171 ions were exclusively observed on this pseudo-MS4 spectrum. Then, all the possible combinations of Na+, OH- and H2O (up to 20 units of each, respectively) matching m/z 413, with the restriction of having a single positive charge, were computed. The following single combination was found: 4 Na+ + 3 OH- + 15 H2O. However, the exact mass of this combination, 413.1257 Da, did not match with the m/z values of the peaks observed in the high-resolution spectra of Dglucose (413.2341 Da) and the AMGp (413.2349 - 413.2406 Da). Thus, no more efforts to interpret the m/z 413 peak were done. As deduced from the comparison of the MS2 spectra of Fig. IV.7, parts A and B, the ion of D-glucose at m/z 593 had six carbon atoms. A likely
156
explanation is the formation of an adduct containing a molecule of D-glucose plus the m/z 413 ion (m/z = 413 + 180 = 593). The MS2 spectra obtained from the [M+Na]+ parent ions of D-glucose-6,6-d2 and D-glucose-1-13C (not shown) gave equivalent peaks at m/z 595 and 594, respectively, which agreed with this composition. Similarly, the D-glucose peaks at m/z 571 and 557 (Fig. IV.7, part A) were interpreted as coming from the m/z 593 adduct by gain of a molecule of water and loss of NaOH, and by loss of two molecules of water, respectively. These m/z 571 and 557 ions also showed shifts of +6, +2 and +1 Da when the MS2 spectra were obtained with D-glucose-13C6, D-glucose-6,6-d2 and Dglucose-1-13C, respectively, which agrees with the proposed compositions.
IV.1.2.3. Fragmentation of AMGp using MSn As summarized in Table IV.2, and as showed in Fig. IV.9 for octyl-GP, the MS2 spectra of the [M+Na]+ parent ions of the AMGp exhibited peak patterns similar to those of D-glucose, but with a few significant differences. Thus, the m/z 185 ion, which was present in the MS2 spectra of D-glucose and all the AMGp, and which was interpreted as a loss of water in D-glucose, should correspond to the loss of the alkyl alcohol in the AMGp (B1 fragmentation). Differently from D-glucose, which can undergo water losses at several locations, in the AMGp the alkyl alcohol chain is exclusively bound to carbon atom 1. The m/z 143 ion, which was present in the MS2 spectra of all the AMGp, can be exclusively formed by a
0,2
A fragmentation. Owing to the presence of the alkyl
chain, this m/z value did not matched with the possible fragment ions obtained by other cross-ring cleavages. This also agrees with the
0,2
A cross-ring cleavage
deduced above for D-glucose. The MS2 spectra of all the AMGp also showed an m/z 129 ion which was not present in the MS2 spectra of D-glucose. This m/z 129 ion was weak for methyl-MGp and abundant for the other AMGp. This suggested
157
that the presence of the hydrocarbon chain was necessary to stabilize the resulting non-ionic fragment. By computing all the possible combinations of C, H, O and Na giving rise to an m/z 129 ion with a positive charge, the two following possibilities were found: C4H10O3Na+ and C3H6O4Na+; however, a 2,5A fragmentation is the only way to produce the former ion in a single step, whereas the less likely concurrence of at least two fragmentations, required to obtain the latter.
Table IV.2. Ions observed on the MS2 spectra of the AMGp (m/z values). Ion \ n
2,5 0,2
3,5
X and C1, are
10
12
A A
B1 [M+Na]+ Unknown [M+413-2H2O]+ [M+413+H2O-NaOH]+ [M+413]+ Others
129 143 185 217 413 571 585 607 235
129 143 185 287 413 641 655 677 209 249
129 143 185 315 413 669 683 705 209 235 275
129 143 185 343 413 697 711 733 235 309
129 143 185 371 413 725 739 761 291 315
Concerning to the peaks at large m/z values, and as also occurred with Dglucose, they matched with the following compositions: [M+413]+,
[M+413+H2ONaOH]+ and [M+4132H2O]+, where M = 180 + n 14 (n = carbon atoms in the alkyl chain). Thus, analogously to that proposed for D-glucose, they were attributed to the formation of adducts of the m/z 413 ion with a molecule of the corresponding alkyl-GP, and to related ions produced by further gains and losses of water and NaOH. As also indicated in Table IV.2, in comparison to the MS2 spectrum of Dglucose, a few additional ions which depended on the length of the alkyl chain were also observed in the MS2 spectra of the AMGp. The m/z 235 ion of methylMGp can be attributed to the gain of water to yield [M+Na+H2O]+. For octyl-
158
MGp, the abundant m/z 275 ion (Fig. IV.9) could be due to a loss of NaOH to yield [M-OH]+. Finally, significant differences between the MS2 spectra of the and -epimer pairs of methyl- and octyl-MGp were not observed.
275
3
Intensity x 104
129 1 235 143 185209 0 100 200 300 400 500 600 315 413 669 683 705
m/z
700
Fig. IV.9. MS2 spectrum of the [M+Na]+ parent ion () of octyl-MGp obtained at e = 0.80.
IV.1.2.4. Fragmentation of AMGf using MSn Because of the lack of standards, the MSn spectra of AMGf could not be obtained by continuous infusion in the IT-MS. Instead of this, injections of Glucopone using HPLC with MS and MS2 detection were performed. Isocratic elution with mobile phases constituted by ACN/water mixtures, also containing the sodium acetate buffer, was employed. Using 40 % ACN, the ring isomers of the octyl- and decyl-MGp were well resolved in a short time. The extracted ion chromatograms at the m/z values of the [M+Na]+ ions of the octyl- and decylMGs are shown in Fig. IV.10. Then, HPLC with MS2 detection, with an increase of the fragmentation energy between injections, was used. The MS2 total ion chromatograms (MS2-TIC), and the MS2 spectra at the retention times of the
159
octyl-MGp and octyl-MGf peaks, are shown in Fig. IV.11. At low fragmentation energies (e < 0.6), the peak area of octyl-MGp was much higher than that of octyl-MGf (see the MS-TIC chromatograms of Fig. IV.10, which correspond to MS2-TIC with e = 0); however, at increasing energies, the peak area of the octylMGp decreased at a higher rate than that of the octyl-MGf.
octyl-MGp 1.5
Intensity x 108
1.0 decyl-MGp
0.5 octyl-MGf decyl-MGf m/z 315 0 1 2 3 m/z 343 4 5 Time (min) 6 7
Fig. IV.10. Chromatogram of Glucopone obtained using MS detection. The traces are the extracted ion chromatograms at the m/z values of the [M+Na]+ ions of the octyl- and decyl-monoglucosides. Isocratic elution with a 40:60 ACN/water mobile phase containing 20 mM NaAcO and 20 mM HAcO at 0.3 mL min-1 was performed.
As shown in Fig. IV.11, left part, the area of the octyl-MGp peak was even smaller than that of the octyl-MGf peak when e = 0.80. Also, as observed in Fig. IV.11, right part, the MS2 spectra of the alkyl-MGf differed ostensibly from those of the corresponding alkyl-MGp. Thus, in comparison with the octyl-MGf, the m/z 413 ion and its adducts with unfragmented molecules were more abundantly formed by the octyl-MGp. The differences between the MS2-TICs indicated an easier fragmentation of the alkyl-MGp compared to the alkyl-MGf.
160
e = 0.7 octyl-MGp 150 100 50 octyl-MGf 80
315
octyl-MGp e = 0.7
315
octyl-MGf e = 0.7
20
143 413 413 octyl-MGp e = 0.8 683 669 octyl-MGp e = 0.9 683 705 143 315 octyl-MGf e = 0.8
Intensity x 104
e = 0.8 octyl-MGp 4 2 octyl-MGf 1 0.5 316 185 315
705
185
413
705 octyl-MGf e = 0.9
413 octyl-MGp e = 0.9 octyl-MGf 0.6
143
2 1 1
0.2 100
185
315 300 500
669 700 100
185 315 300
413 500
705 m/z 700
2 3 Time (min)
m/z
Fig. IV.11. Total ion HPLC-MS2 chromatograms of Glucopone recorded at the indicated fragmentation energies (left), and corresponding MS2 spectra at the retention times of the peaks (right). The [M+Na]+ ions of octyl-MGp and octyl-MGf (m/z 315) were used as parent ions (). Other conditions as in Fig. IV.10.
On the other hand, as also deduced from the MS2 spectra of Fig. IV.11, the cross-ring cleavage at
0,2
A to give the m/z 143 ion was much more likely in
the alkyl-GFs than in the alkyl-GPs. This could be due to a lower stability of the five-membered furanoside rings in comparison to the six-membered pyranoside rings. Finally, as also observed in Fig. IV.11, the alkyl-GPs and alkyl-GFs showed a similar trend to lose the alkyl chain giving rise to the m/z 185 ion.
161
IV.2. Non-ethoxylated and ethoxylated alcohols
IV.2.1. Chromium(VI) oxide oxidation of non-ethoxylated and ethoxylated alcohols for determination by ESI-MS Primary alcohols are common components of many biological and industrial samples, in addition to being present at trace levels in the aquatic environment. Alcohols in the C10C18 range (fatty alcohols) are potent pheromones while those with higher molecular masses are essential components of plant waxes [Bianchi, 1995]. The industrial importance of alcohols derives from their widespread use as solvents and fillers in industrial and household products, while fatty alcohols are common ingredients of body-care and cosmetic products
[Farm, 2006].
Fatty alcohols also serve as the raw materials in the production of
important surfactant classes, including alkyl ether sulfates and sulfonates, and FAEs
[Farm, 2006.].
FAEs are mostly obtained from renewable resources as
complex mixtures of oligomers with the structure: CH3(CH2)n1(OCH2CH2)mOH. A special class of ethoxylated alcohols comprises those of low molecular mass (both n and m <4). These compounds, known as cellosolves, are used, for example, as anti-icing additives in aviation fuels and as solvents in cleaning solutions
[Cheremisinoff, 2003].
GC has been successfully employed for the
determination of cellosolves and nonethoxylated alcohols with 26 carbon atoms

[Nichols, 2006],
but not FAEs with m >4, due to the limited volatility of these
compounds [Rudewicz, 1986; Crescenzi, 1995; Battersby, 2001]. The major drawback of HPLC methods for FAEs has been the lack of an adequate detector [Rudewicz,
1986; Petrovic, 2001].
Although this problem was partially resolved through the

[Kudoh, 1984; Mengerink, 1991; Cho, 2003; [Mengerink, 1991; Heinig, 1998; Bear, 1988;
introduction of refractive index

Trathnigg, 2002]
and ELSD methods
Miszkiewicz, 2000; Miszkiewicz, 1996-B; Kamiusuki, 2000],
the LODs are high for the
162
former, while the sensitivity of ELSD is poor for volatile compounds such as non-ethoxylated alcohols (m = 0) and FAE oligomers with a low degree of ethoxylation (m <3) [Miszkiewicz, 1996-A; Bernab-Zafn, 2006]. Finally, low LODs are achieved with chromogenic and fluorogenic pre-column derivatization procedures [Marcomini, 1996; Schmitt, 1990; Kiewiet, 1995; Lemr, 1994; Zanette, 1996;
Lemr, 1996; Sun, 1997; Hoffman, 2004-A; Lemr, 2003; Okada, 1991; Okada, 1992; Hoffman, 2004-B; Bachus, 2003; Desbne, 2005; Heinig, 1996-A; Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Zu, 2010].
In the determination of FAEs by PI-MS with ESI or APCI interfaces, problems arise because the sensitivity of these methods decreases with decreasing m; thus, sensitivity is low when m <4, and non-ethoxylated alcohols (m = 0) are not detected
[Rudewicz, 1986; Crescenzi, 1995; Petrovic, 2001; Zu, 2010;
Chiron, 2000; Sherrard, 1994; Dunphy, 2001; Cassani, 2004; Sparham, 2005].
To
overcome this problem, derivatization procedures, in which a chromophore or a permanent charge is added to the oligomers, have been described
[Lemr, 1994;
Lemr, 1996; Lemr, 2003; Desbne, 2005; Heinig, 1996; Zu, 2010; Dunphy, 2001; Cassani, 2004; Sparham, 2005].
However, the derivatization of FAEs is difficult because it requires an anhydrous medium [Okada, 1992; Zu, 2010; Dunphy, 2001; Barry, 2003], and there is an increased risk of interference from both the reagent excess and the byproducts of the reaction
[Sun, 1997; Hoffman, 2004-A; Mic-Tormos, 2008-A; Mic-
Tormos, 2008-B; Dunphy, 2001].
Furthermore, owing to the high risk of losing
oligomers with low m values by volatilization, the water content of the samples must be reduced with particular care, which increases analysis time
2001]. [Dunphy,
Derivatization with cyclic anhydrides is, by contrast, tolerant to the

[Mic-Tormos, 2008-A; Mic-
presence of small amounts of water in the samples

Tormos, 2008-B; Mic-Tormos, 2009; Zu, 2010].
163
Chromium(VI) oxide (CrO3) in aqueous media containing sulfuric or acetic acid (Jones reagent) is a well-known reagent used in the oxidation of primary alcohols to carboxylic acids [Hudlik, 1990; Burke, 1999]. The carboxylate group has a low molar absorptivity and is therefore of little interest for UV-Vis detection, but can be detected with high sensitivity by MS, particularly when the alkyl chain contains a large number of carbon atoms
[Holapek, 2005].
In this
section, the determination of non-ethoxylated primary alcohols by MS, previous oxidation to carboxylic acids with the Jones reagent, was studied. In the proposed procedure, the reagent excess was removed with sodium sulfite, and the oxidized alcohols were separated from the resulting chromium(III) salts by extraction in ethyl acetate-acetone. After addition of an amine to increase pH, and water to further enhance ionization of the carboxylate groups, the extracts were directly infused in the ESI source of the MS. The study was also extended to FAEs and cellosolves, which yielded the corresponding ethoxy-carboxylic acids. Application to the characterization and determination of fatty alcohols in complex samples, such as cosmetics and body care products, and in environmental samples as seawater, was also demonstrated.
IV.2.1.1. Optimization of the infusion medium In order to increase ionization of the carboxylate groups, and therefore to enhance sensitivity in the NI mode, the influence of the addition of butylamine, water and ACN to the ethyl acetate-acetone extracts, was studied. A stock solution of four standards (C12E0A, C14E0A, C16E0A and C18E0A, 200 g mL-1 each) in a 4:3 (v/v) ethyl acetate-acetone mixture was prepared. Aliquots of this solution were diluted in a 1:1 ratio to obtain the following media: (a) ethyl acetate and acetone in a 4:3 ratio; (b) as in a but in the presence of 30 mM butylamine; (c) as in b but the medium contained also 10% water; and (d) as in b
164
but the medium contained also both 10% water and 40% ACN. These solutions were infused in the MS, and the abundances of the [M-H]- ions were measured. As shown in Fig. IV.12, sensitivity of the fatty acids increased as n increased, and also varied largely with the nature of the infusion medium. Thus, sensitivity increased ca. 5 times when butylamine was added to the extracts (Fig. IV.12, from a to b). This was probably due to ionization of the acids in a medium with a higher pH. Sensitivity further increased ca. 360 times when water was also added (Fig. IV.12, from b to c).
1.2
C12E0A C14E0A C16E0A
Abundance x 107
0.8
C18E0A
100 x
0.4
0.0 a b c d
Fig. IV.12. Negative-ion MS relative sensitivities for four fatty acids (n = 12, 14, 16 and 18, 100 g mL-1 each) in several media: (a) ethyl acetate and acetone in a 4:3 ratio; (b) as in a but in the presence of 30 mM butylamine; (c) as in b but containing also 10% water; and (d) as in b but containing both 10% water and 40% acetonitrile. In a and b, the bar lengths were multiplied by 100.
This should be mainly attributed to ionization enhancement upon increasing the permittivity (dielectric constant) of the alkalinized medium. Remarkably, the addition of water produced a much larger sensitivity increase than that observed by solely increasing the pH of the extracts. This suggests that water was rather scarce in the ethyl acetate-acetone extracts. Retention of most water into the
165
water-rich layer during extraction could be due to the large concentrations of both mineral acids and chromium(III) and sodium salts, which should made ionic strength to be very large. In a different series of experiments, up to 13% water was added to 4:3 ethyl acetate-acetone mixtures before observing two-phase separation. Then, as indicated in the recommended procedures, an aqueous butylamine solution up to a final concentration of 10% was added to the extracts before obtaining NI mode spectra. Finally, sensitivities were only slightly higher in the presence of 40% ACN.
IV.2.1.2. Relative sensitivity of carboxylic and ethoxy-carboxylic acids as a function of n and m Relative sensitivities are useful in the qualitative interpretation of MS spectral profiles of unknown samples. They are also necessary to avoid bias in predicting alcohol concentrations after calibration with a standard different from the addressed alcohol. To estimate the relative sensitivities of carboxylic and ethoxy-carboxylic acids at increasing values of both n and m, the [M-H]- peaks of the standards in ethyl acetate-acetone containing butylamine and water, and at several concentrations of each acid (0, 100, 200 and 400 g mL-1), were measured in the NI mode. Relative sensitivities with reference to the internal standard were calculated as follows:
fi = Ii I s [M ]i
(E.IV.1)
where Ii and Is are the abundances of the [M-H]- ion of the compound of interest and the internal standard, respectively, and [M]i is the molar concentration of the compound. As shown in Fig. IV.13, part A, relative sensitivities increased as the alkyl chain increased. This agreed with the reported APCI-MS response factors of fatty acids [Holapek, 2005]. Standards of ethoxy-carboxylic acids having large values of both n and m are not commercially available; then, only the relative
166
sensitivities of a few ethoxy-carboxylic acids with low values of both n and m (cellosolves) could be studied. As also shown in Fig. IV.13, parts A and B, relative sensitivity of ethoxy-carboxylic acids was higher than that of the corresponding non-ethoxylated carboxylic acids, and increased when either n or m increased.

log fi
m=3 m=2 m=1
-1 -2

m=0
A
4 8 12 n 16

-3 0
0 -1 -2 n=2
log fi
n=1
B
-3 0 1 2 m 3
Fig. IV.13. NI MS relative sensitivities of carboxylic and ethoxycarboxylic acids versus the number of carbon atoms in the alkyl chain, n, and at increasing values of the number of EO units, m (A), and vice versa (B). Relative sensitivities in logarithmic scales, log fi, are given with reference to the internal standard (TMBA).
167
IV.2.1.3. Oxidation yields of primary non-ethoxylated alcohols and extraction recoveries of the corresponding carboxylic acids Recovery of carboxylic acids with short and large chain lengths during the extraction step, independently from the yield of the previous oxidation step, was first evaluated. For this purpose, the proposed procedure was applied to solutions of C3E0A and C14E0A in acetone, and the NI mode spectra were measured. The abundance of the [M-H]- ions was compared to that obtained by using standard solutions of the acids in the ethyl acetate-acetone extraction medium. Recoveries were 100% (duplicated extractions). Then, in order to study oxidation yields, the proposed procedure was applied to a series of standard solutions containing increasing amounts of C10E0 in acetone. The concentrations used corresponded to 0, 100, 200 and 400 g mL-1 in the infused solutions (duplicated points). Using the NI mode, the abundance of the [M-H]- ions was measured. A regression straight-line with an excellent linearity (r2 > 0.999, calibration curve A) was obtained. The standard deviation of the residuals was used to calculate the standard deviation of the slope
2000]. [Miller,
Assuming a zero intercept, and in relative terms, this standard deviation
was 1.3% of the slope, which corresponded to a confidence limit of 3.0% (95% confidence level, two-sided). Using increasing concentrations of the
corresponding carboxylic acid, C10E0A, a linear relationship (r2 > 0.999, calibration curve B) was also obtained. The ratio of the slopes of the two calibration curves (as A/B) was 1.016. Thus, the oxidation yield of C10E0 was not statistically different from 100%. The influence of the addition rate of the Jones reagent was also studied. For this purpose, aliquots of the C10E0 solution in acetone were oxidized according to the proposed procedure, but the manual dropwise addition of the Jones reagent was substituted by manual quick addition, and also by slow
168
addition with a syringe pump under stirring during 40 min. The abundance of the [M-H]- ions, measured in the NI mode, was compared to that obtained using calibration curve B (expected concentration in the infused solutions, ca. 200 g mL-1). Either, dropwise or slow addition of the Jones reagent equally led to a 100% yield. The influence of the reaction temperature during oxidation was also investigated. Alcohols with a short (C3E0) and a large (C12E0) chain length were used. A water bath was used to set the initial temperature of the reaction mixture. Then, the Jones reagent was added, and after extraction the abundance of the [M-H]- ions was measured. The ion abundances did not increase nor decrease by regulating the initial temperature of the reaction mixture to 25, 35 and 55 C.
IV.2.1.4. Influence of water concentration on the oxidation yield To study the influence of water concentration on the oxidation yield of non-ethoxylated alcohols, two series of experiments were done. In all cases, a C10E0 stock solution in acetone was used, and the proposed procedure was applied, but with the modifications which are next indicated. First, the Jones reagent was prepared in the presence of decreasing water concentrations; for this purpose, water was substituted by increasing concentrations of acetic acid
[Hudlik, 1990; Burke, 1999].
Aliquots of the C10E0 stock solution were further
diluted using acetone, and the proposed procedure was applied; however, oxidation was performed with the series of Jones reagent prepared with increasing acetic acid concentrations (decreasing water concentrations). Second, aliquots of the C10E0 stock solution were diluted with acetone-water mixtures containing increasing amounts of water, instead of using pure acetone. Jones reagent prepared in aqueous sulfuric acid, as indicated in the proposed procedure,
169
was added to these solutions. In all cases, the NI spectra were obtained, and internal standard correction using TMBA was applied. As shown in Fig. IV.14, the yield was maximal when the reagent was prepared as indicated in the proposed procedure, and decreased moderately when water was partially substituted by acetic acid (Fig. IV.14, part A).
Oxidation yield, %
100 80 60 40 20 100 80 60 40 0 25 50 75 100 Initial H2O % in the sample solution
40 60 80 H2O % in the Jones reagent
Oxidation yield, %
Fig. IV.14. Reaction yield of C10E0 in the presence of different water concentrations during oxidation. The Jones reagent was prepared by substituting water by increasing concentrations of HAcO (A), and the analyte was dissolved in acetone-water mixtures with increasing amounts of water instead of using pure acetone (B).
Oxidation yield also decreased when the Jones reagent was added to C10E0 solutions containing increasing amounts of water (Fig. IV.14, part B). Therefore, in the recommended procedure, industrial samples containing water (i.e. cosmetics and body care products) were diluted with acetone. In addition,
170
after centrifugation, a low-volume aliquot of the supernatant was further diluted with acetone before Jones reagent was added.
IV.2.1.5. Oxidation yields of ethoxylated alcohols The oxidation yield of a short-chain ethoxylated alcohol, C2E1, was first studied. For this purpose, the C2E1-C2E1A pair of standards was used. The proposed procedure was first applied to a series of solutions containing increasing C2E1 concentrations, and the abundance of the [M-H]- ions were measured in the NI mode. As performed above for C10E0, the slope of this calibration curve was compared with that obtained using increasing C2E1A concentrations. These two straight-lines were highly linear (r2 > 0.999), but the slopes differed, indicating an oxidation yield of ca. 65%. As also observed for C10E0, this yield was not modified by increasing or decreasing the addition rate of the Jones reagent (both sudden manual addition and slow addition with a syringe pump under stirring during 40 min). The yield was not modified either by increasing the reaction time to more than 2 hours after addition of the Jones reagent (before removing the reagent excess with sodium sulfite). The influence of the sample temperature before addition of the Jones reagent was also studied. Using a C4E2 solution, a constant 65% yield was obtained at 25, 35 and 55 C. The oxidation yield of fatty alcohols with an increasing degree of ethoxylation was studied. For this purpose, standards of the C12Em series were used. Standards of the corresponding ethoxylated fatty acids were not available, which hindered the comparison of the abundances of the [M-H]- ions obtained in the NI mode. Instead, abundances of the [M+H]+ ions obtained in the PI mode were measured. The proposed procedure was applied, and the abundance of the [M+H]+ ion of the remaining non-oxidized FAEs was compared to that of the corresponding ethoxy-carboxylic acid. As shown in Fig. IV.15, two intense
171
peaks corresponding to the C12E3 - C12E3A pair were observed in the spectrum obtained after oxidation of C12E3. The peak of the remaining non-oxidized C12E3 was higher than that of C12E3A; however, taking into account that the C12E3/C12E3A sensitivity ratio in the PI mode was close to 1.8, an oxidation yield of ca. 60% was calculated. Two other low intensity peaks, corresponding to the [M+H]+ ions of the C12E2 - C12E2A pair, were also observed. These two peaks were attributed to loss of an EO unit during oxidation. From the peak abundances, this side-reaction amounted to less than 4% of the initial C12E3 concentration. Along the C12Em series, oxidation yield, which was 100% for C12E0, decreased to ca. 65 and 60% when m = 2 and 4, respectively. The oligomers with m = 5 and 6 also gave a ca. 60% yield.
[C12E3+H]+ [C12E3A+H]+ 3
Abundance x 106
1 [C12E2+H]+ [C12E2A+H]+ 0 300 m/z 350
Fig. IV.15. PI MS spectrum of C12E3 after oxidation with Jones reagent showing the [M+H]+ peaks of the C12E3 C12E3A and C12E2 C12E2A pairs. Target mass of the ion trap was set at m/z 275 to enhance the abundances of the diethoxylated species.
172
Finally, ethoxylated alcohols of the CnE1 and CnE2 series were used to study the influence of n from n = 2 to 18. In all cases, ca. 65% yields were obtained. In addition to the expected peaks, two additional peaks due to the FAE with loss of an EO unit, and its corresponding ethoxy-carboxylic acid, were observed. However, yields for this side reaction were small (4-8% of the initial amount of the FAE).
IV.2.1.6. Application to industrial and environmental samples
A) Industrial raw materials The NI mode spectrum of industrial raw material FINDET 10/18 showed the intense peaks of the oxidized oligomers of the C10Em series, including the nonethoxylated alcohol, C10E0 (Fig. IV.16). All these peaks were not observed on the NI mode spectrum obtained without previous oxidation of the sample (not shown). Also without oxidation, peaks of the [M+H]+ ions of ethoxylated alcohols were observed on the PI mode spectrum, but sensitivity was small for m = 1 and 2, and a peak for C10E0 was not observed (spectrum not shown). Thus, the proposed procedure is particularly useful for the quick identification of nonethoxylated fatty alcohols at low concentrations. However, owing to ion suppression effects, previous separation by HPLC or CE should be recommended for quantitative purposes. Quantitative evaluation of mixtures of FAEs by the proposed procedure is also burdened by bias due to the 4-8% breakdown of the EO chain during oxidation (with loss of an EO unit), as well as by lack of available standards of ethoxylated fatty acids, which hinders the determination of response factors of the oligomers.
173
[C10E4A-H]-
[C10E5A-H]-
[C10E6A-H]-
[C10E3A-H]-
[C10E7A-H]-
Abundance x 106
[C10E1A-H]-
[C10E2A-H]-
[C10E8A-H]-
[C10E9A-H]-
[C10E0A-H]-
[C10E10A-H]-
[C10E11A-H]-
300
500
m/z
700
Fig. IV.16. NI MS spectrum of the industrial surfactant FINDET 10/18 after oxidation with Jones reagent. The peaks of the [M-H]- ions of the oxidized oligomers of the C10Em series are labelled (IS = internal standard).
B) Body care and cosmetic products The proposed procedure was also applied to the body care and cosmetic products of Table IV.3. In the NI mode, all these samples showed the intense [M-H]- peaks of C16E0A and C18E0A (cetyl and stearic acids), as well as those of other fatty acids. By applying again the proposed procedure but with addition of diluted sulfuric acid instead of Jones reagent (CrO3 was not added), the peaks of these fatty acids were absent, or present with much lower abundances. The spectrum of a varicose vein cream is shown in Fig. IV.17. In this sample, the abundances of the peaks of C16E0A and C18E0A increased ca. 30 and 10 times, respectively, upon oxidation with the Jones reagent. The procedure was then applied to the determination of C16E0 and C18E0 in the commercial products of
174
[C10E12A-H]-
[C10E13A-H]-
IS
Table IV.3. Solutions of C16E0A and C18E0A were used to construct external calibration curves (6 duplicated points). To check possible matrix effects, internal calibration by the standard addition method was also applied; for this purpose, two duplicated calibration points per sample were performed (with addition of alcohol standards to the samples before the oxidation step).
[C18E0H-H][C16E0H-H]-
Abundance x 106
IS
0 200 250 m/z 300
Fig. IV.17. NI MS spectrum of a commercial varicose vein cream after oxidation with Jones reagent showing the peaks of the cetyl and stearic acids resulting from the oxidation of the cetyl and stearic alcohols, respectively.
As shown in Table IV.3, most samples showed satisfactory agreement between the alcohol contents found by external and internal calibration. Limits of detection (LODs) for C16E0 and C18E0 in the varicose vein cream, calculated as the concentration corresponding to a net signal equal to three times the standard deviation of the blank, were 0.1 and 0.03%, respectively. However, taken into account that the extract of this sample was diluted in a 1:40 ratio before infusion
175
in the MS, LODs of 25 and 7.5 g per gram of sample, respectively, were calculated.
Table IV.3. Mass percentages of cetyl and stearic alcohols found in several commercial samples by external and internal (standard addition) calibration (ND = not detected). C16E0 (%) C18E0 (%)
Sample Shampoo Hair conditioner Lip protector stick Deodorant stick Body-care oil Varicose-vein cream
External 0.71 0.55 0.66 ND 0.10 3.8
Internal 0.80 0.35 0.69 ND 0.16 3.3
External ND 0.25 0.34 5.0 0.15 2.0
Internal ND 0.24 0.34 5.6 0.15 2.6
C) Seawater The presence of saturated and unsaturated fatty alcohols at the g L-1 level has been reported in seawater samples taken from coastal and high seas
1992; Tolosa, 2003; Belanger, 2009]. [Parrish,
As shown in Fig. IV.18, peaks which could
correspond to linear fatty alcohols with zero, one and two double bonds, and to phytol (a common diterpenoid), were observed in the spectra of the oxidized extracts obtained with seawater. These peaks were present with much lower abundances in the spectrum of the seawater extract which was not oxidized with the Jones reagent. In Fig. IV.18, ratios calculated by dividing the ion abundances obtained after application of the proposed procedure to the seawater extract, by those obtained without oxidation with the Jones reagent (reference sample), are indicated between parentheses.
176

1.5
C12:2 (600) C18:1 (10)
Abundance x 106
1.0
C20:2 (92) C20:1 + phytol (13) C20:0 (4)
C12:1 (140) C12:0 (13)
C16:1 (16) C16:0 (30)
C18:0 (15)
C22:2 (25) C22:1 (4)
C22:0 (2)
C24:2 (10)
0 200 300 m/z 400
Fig. IV.18. NI MS spectrum of a seawater extract after oxidation with Jones reagent. Several peaks could be ascribed to common saturated and unsaturated fatty acids and to phytol, is indicated (e.g., C18:1 is octadecenoic acid). Numbers within parentheses are abundance ratios between the addressed peak in this spectrum and in the spectrum of the seawater extract not oxidized with the Jones reagent.
C14:0 (8)
177
C26:1 (12)
0.5
C26:2 (30)
CHAPTER V
ANIONIC SURFACTANTS
Chapter V. Anionic surfactants
V.1. AESs
V.1.1. Determination of FAEs and AESs by anionic exchange separation, derivatization with a cyclic anhydride and HPLC FAE and AES are two important surfactant classes, widely used in cleaners and body care products
[Fiedler, 1989].
FAE are industrially obtained as
complex mixtures of oligomers with the following structure (shortened below as CnEm): CH3(CH2)n1(OCH2CH2)mOH where n is the number of carbon atoms in the alkyl moiety of the molecule, and m is the number of EO groups. FAE mixtures obtained from vegetal oils contain linear hydrocarbon chains with even values of n, whereas both linear and branched chains, with even and odd values of n, can be found in FAE obtained from mineral oils [Sparham, 2005; Marcomini, 1996]. On the other hand, AES are obtained by esterification of FAE with either sulfur trioxide or chlorosulfonic acid. Then, FAE and AES have essentially the same molecular structure, with a hydrocarbon chain attached to an EO chain, but AES oligomers end with a sulfate group in substitution of the OH group of FAE oligomers [Strain, 1959; Suter, 1944; Arthur, 1991]: Na+CH3(CH2)n1(OCH2CH2)mOSO3Accordingly, AES oligomers are shortened below as CnEmS. Important characteristics of both FAE and AES, including viscosity of their aqueous solutions, detergency, foam formation and skin compatibility, as well as their environmental impact, depend on the variable distributions of both the alkyl and EO chains
[Marcomini, 1996; Arthur, 1991; Tadros, 2005; Rudewicz, 1986; Eadsforth,
2006; Belanger, 2006; Van Compernolle, 2006; Ribosa, 2007; Jurado, 2007].
Both the
hydrocarbon cut (range of n for the predominant hydrocarbon series) and m
181
(average number of EO units) are important in industrial quality control. Thus, methods for their characterization and determination are required; however, owing to the complexity of the sample matrices, lack of chromophore, and wide ranges of polarity and volatility of the oligomers, the analysis of FAE and AES, which are usually found in complex mixtures with other surfactant classes, is not an easy task. In addition, the unavailability of commercial standards constitutes an added difficulty of AES analysis. In determination of FAE, the low volatility and thermal instability of long EO chains limit the use of GC to the oligomers with m < 4
Crescenzi, 1995], [Rudewicz, 1986;
and owing to the high volatility of the oligomers with short EO
chains, the employ of HPLC with ELSD provides biased distributions

[Miszkiewicz, 1996-A; Bernab-Zafn, 2006].
In addition, the PI MS response factors
for underivatized FAE oligomers decrease ca. two orders of magnitude when m decreases from 4 to 1
2001]. [Sparham, 2005; Rudewicz, 1986; Crescenzi, 1995; Dunphy,
Further, non-ethoxylated alcohols (m = 0) are not detected in a mass
spectrometer [Sparham, 2005; Rudewicz, 1986; Crescenzi, 1995; Bernab-Zafn, 2006;

Dunphy, 2001; Sherrard, 1994].
Underivatized FAE can be also characterized and

[Trathnigg,
determined using isocratic elution and a refractive index detector

1993; Trathnigg, 1994; Trathnigg, 2004; Trathnigg, 2005],
but selectivity is poor and
the limits of detection are large. Derivatization procedures designed to increase volatility of FAE oligomers, followed by GC analysis have been proposed
[Marcomini, 1996; Kiewiet, 1996; Hbner, 2007].
Several derivatization procedures
for FAE, addressed to add a chromophore or a charge to the oligomers, followed by HPLC
[Marcomini, 1996; Dunphy, 2001; Hoffman, 2004-B; Sun, 1997; Zanette,
1996; Lemr, 2003; Desbne, 2005]
or CE
[Sparham, 2005; Bernab-Zafn, 2006;
Dunphy, 2001; Lemr, 2003; Desbne, 2005; Wallingford, 1996; Heinig, 1998],
have
been also described. FAE derivatives have been separated by either NP- or RP-
182
HPLC using UV-Vis
[Marcomini, 1996; Kiewiet, 1996; Zanette, 1996; Bachus, 2003;
Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009]
or MS detection
[Crescenzi, 1995; Bernab-Zafn, 2006; Levine, 2005; Jandera, 1998; Krogh, 2002].
On the other hand, nonspecific determination of AES and other anionic surfactants can be jointly performed by the methylene blue active substance method
[Llenado, 1983].
Non-ethoxylated alkyl sulfates have been studied using

[Boiani, 1987; Shamsi, 1995], [Pan, 1995; Nair, 1998]
ion pair chromatography with indirect UV detection ionic chromatography with conductimetric detection
and
HPLC with post-column ion-pair formation followed by membrane phase separation and fluorimetry
[Smedes, 1982-B].
Also, conversion of AES to the

[Neubecker, 1985],
corresponding alkyl bromides followed by GC-FID HPLC coupled to ESI-MS
as well as have
[Popenoe, 1994; Bruno, 2002; Lara-Martn, 2005],
been applied to the determination of AES in waters, sewage sludge and marine sediments. In former works of research group, we have developed procedures for the RP-HPLC-UV determination of FAE previous derivatization with a cyclic anhydride
[Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Mic-
Tormos, 2010].
However, we have observed that cyclic anhydrides also derivatize
AES to yield exactly the same derivatives as FAE, i.e. the hemiesters of the alkyl- or alkyl-ethoxy residues. Thus, the peaks corresponding to the sum of both FAE and AES oligomers are obtained on the chromatograms if the two surfactant classes are present in the samples. Therefore, in this work, a procedure for the separation of these two surfactant classes, followed by the independent derivatization of each class with a cyclic aromatic anhydride, and RP-HPLC-UV determination of the derivatized oligomers, was developed. Separation of the two surfactant classes was achieved by SPE on a SAX cartridge. Then, using either phthalic or diphenic anhydride, FAE are esterified and AES are transesterified.
183
Separation of the derivatized oligomers was achieved by RP-HPLC using gradient elution with ACN/ water in the presence of 0.1% acetic acid. The proposed method was applied to the analysis of FAE and AES in commercial products (liquid cleaners) and environmental samples (seawater extracts).
V.1.1.1. Derivatization and HPLC separation of the derivatives As shown in Fig. V.I, an industrial mixture of AES (LES) was quantitatively derivatized at 105 C in about 70 and 50 min using phthalic and diphenic anhydrides, respectively. Similar results were reported for the esterification of FAE
[Mic-Tormos, 2008-B; Mic-Tormos, 2009].
Thus, to assure
derivatization of the two surfactant classes, 90 min was selected. Chromatograms of Dehydol LT-7 and LES, obtained by previous derivatization with phthalic anhydride, are shown in Fig. V.2.
100 Relative sum of peak areas 80 60 40 20 0 0 40 80 Reaction time (min) Formation of phthalates
Formation of diphenates
Fig. V.1. Relative sum of the chromatographic peak areas of the derivatives of all the oligomers given by an industrial AES (LES) sample versus reaction time using phthalic (rhombus and dashed line) and diphenic anhydrides (squares and dotted line). Each point represents an independent derivatization performed at 105 C in 1,4-dioxane.
184
Both chromatograms showed the successive hydrocarbon series at increasing values of n, from n = 10 to 18. Hydrocarbon series having exclusively even values of n were observed with Dehydol LT-7, but significant amounts of the n = 13 and 15 odd series were also present in LES. The large difference between the peak profiles of the same hydrocarbon series for Dehydol LT-7 and LES is due to the different average EO number, namely m = 7 for Dehydol LT-7 and m = 3 for LES. The elution order of the oligomers within the series, which is indicated in the Fig. V.2 for the n = 12 series, was established with UV-Vis detection, by injecting standards of derivatized oligomers, and was also confirmed by HPLCMS using extracted ion chromatograms (EICs, not shown).
C12E0 C12E1 + + C12E5 C12E4 C12E6 C12E7 C12E8 C12E3 C12E9 C12E2
n = 12
300 200 100 0
Absorbance at 230 nm (mAU)
n = 14 n = 16 n = 18
n = 12
300 200 100 0
C12E1S + C12E6S C12E4S C12E3S C12E7S C12E8S C12E2S C12E9S
C12E0S + C12E5S
n = 14 n = 13 n = 15 n = 16 n = 18
10
20
30
Time (min)
40
Fig. V.2. Chromatograms obtained after derivatization of Dehydol LT-7 (A) and LES (B) with phthalic anhydride. In both cases, ca. 40 mg were derivatized and final volume before injection was 12 mL. Elution with a linear gradient from 50 to 100% ACN in 50 min at 25C. The insets show peak identifications for the n = 12 series.
185
Except for m = 0 and 1, the oligomers within the series eluted by following the order of decreasing m. The consecutive pairs of oligomers were also fairly well resolved; however, the peaks of the oligomers with m = 1 and 0 overlapped with other oligomers within their respective hydrocarbon series. At 25 C and for the n = 12 and 14 series, overlapping of the pairs m = 1 and 4, and m = 0 and 5, was produced. Reversion of the elution order for m = 1 and 0 within the hydrocarbon series has been explained as due to the rigidity of the short hydrophilic moiety of these oligomers of the EO chain, which hinders intramolecular solvation, thus making their hydrophobicity to decrease with respect to the oligomers with m 2
[Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Mic-Tormos, 2010].
V.1.1.2. Optimization of the SAX separation of the surfactant classes Since derivatization of both FAE and AES leads to the same derivatives, a previous separation of both surfactant classes was implemented. For this purpose, the method proposed by Fendinger et al. for alkylsulfate analysis in water was modified
[Fendinger, 1992].
The SAX separation procedure was optimized to
achieve quantitative isolation of the two surfactant classes, including both hydrophilic (low n and high m values) and hydrophobic (high n and low m values) oligomers. Conditioning of both sample and SAX cartridge with 80:20 MeOH/H2O led to the coelution of part of the AES jointly with the FAE. Thus, according to the optimized scheme of Fig. III.1 and section III.1.3.4, conditioning was performed with 50:50 MeOH/H2O. In this medium, partial elution of FAE, but without coelution of AES oligomers, was achieved. Solubilization of the most hydrophobic oligomers, together with disruption of FAE-AES mixed micelles, was also procured with 50% MeOH. At this point of the procedure, an increase of the MeOH concentration to 80% to complete FAE elution led to the partial coelution of AES. Coelution was avoided by washing
186
the cartridge with additional portions of 50% MeOH. In this way, residual cations from the sample (mostly, Na+) were washed away, thus strongly fixing AES on the SAX cartridge before increasing the hydrophobicity of the medium. Experiments performed with LES in 50% MeOH showed the absence of AES oligomers in the chromatograms obtained by increasing MeOH concentration to 80% after washing the cartridge first with more 50% MeOH.
300
A
200 Absorbance at 230 nm (mAU)
100
40
20
0 10 20 30 Time (min) 40
Fig. V.3. Chromatograms of Dehydol LT-7 derivatized with phthalic anhydride: (A) FAE oligomers eluted with 50:50 MeOH:H2O (combined fractions 1 and 2 of Fig. 1); (B) FAE oligomers eluted with 80:20 MeOH:H2O (fraction 3 of Fig. III.1). Chromatographic conditions as in Fig. V.2; other details as indicated in section III.1.3.3.
As shown in Fig. V.3, application of the optimized procedure to a Dehydol LT-7 solution led to the elution of an 82% and an 18% FAE with 50% (fractions 1 + 2 of Fig. III.1) and 80% MeOH (fraction 3 of Fig. III.1), respectively. Further, in comparison to the expected oligomer distribution for Dehydol LT-7, the 80%
187
MeOH fraction showed a higher proportion of the hydrophobic oligomers, including both oligomers with large values of n, and oligomers with low values of m within the series. A residual amount of 0.4% of the total FAE was obtained after washing the cartridges with two additional 4-mL volumes of 80:20 MeOH/H2O. Next, AES were eluted using HCl solutions in MeOH as a means to achieve both high concentrations of Cl- and a highly hydrophobic medium. According to the chromatograms shown in Fig. V.4, which were obtained using LES, a 62% of the AES oligomers was eluted with 80:20 MeOH:HCl (Fig. V.4, part A, fraction 4 of Fig. III.1). An additional 35%, containing a higher proportion of hydrophobic oligomers than the former fraction, was eluted by increasing MeOH to 95% (Fig. V.4, part B, fraction 5 of Fig. III.1). Total elution of LES oligomers was checked by washing the cartridge with two additional 4-mL volumes of both 80:20 and 95:5 MeOH:HCl; in this way, a residual 3% AES was recovered (chromatogram not shown). As indicated in the optimized procedure (Fig. III.1 and section III.1.3.4), the combined 4 + 5 fractions containing the AES were neutralized with concentrated ammonia to prevent the release of acid fumes during solvent evaporation. Finally, the isolated FAE and AES fractions were derivatized and injected. Application of this procedure to mixtures of Dehydol LT-7 and LES (ca. 20 mg each) according to the scheme of Fig. III.1, gave rise to chromatograms of the FAE and AES fractions closely resembling the chromatograms given in Fig. V.2, which were obtained by directly derivatizing and injecting Dehydol LT-7 and LES solutions. In addition, the optimized procedure, including the SAX separation into two fractions, was also independently applied to Dehydol LT-7 and LES solutions. For Dehydol LT-7 (ca. 40 mg), the chromatogram obtained from the combined fractions 1 + 2 + 3 (see Fig. III.1) was closely similar to that observed
188
in Fig. V.2, part A, whereas the chromatogram obtained with the combined fractions 4 + 5 showed no significant peaks. Therefore, FAE were quantitatively retained in the combined fractions 1 + 2 + 3.
A
100 Absorbance at 230 nm (mAU)
50
B
20
0 20 30 Time (min) 40
Fig. V.4. Chromatograms of LES derivatized with phthalic anhydride: (A) AES oligomers eluted with 80:20 MeOH:HCl (fraction 4 of Fig. III.1); (B) AES oligomers eluted with 95:5 MeOH:HCl (fraction 5 of Fig. III.1). Chromatographic conditions as in Fig. V.2; other details as indicated in section III.1.3.3.
Similarly, using LES, the chromatogram obtained for the combined fractions 4 + 5 was closely similar to that observed in Fig. V.2, part B. On the other hand, the chromatogram obtained with the combined fractions 1 + 2 + 3 showed a series of small peaks which followed the same pattern as that observed in Fig. V.2, part B, but with much smaller peak areas. This later chromatogram was attributed to the FAE impurities which are always present in industrial AES, due to the nonquantitative sulfatation of FAE during AES manufacture. The total peak area
189
obtained from fractions 4 + 5 of LES, divided by the total peak area obtained from fractions 1 + 2 + 3 indicated a molar percentage of ca. 1.2 % FAE in the LES sample.
V.1.1.3. Calibration studies Standard solutions of C8E0, C12E0, C8E0S and C12E0S were independently used to construct independent calibration curves for FAE and AES. For each standard, the proposed derivatization procedure was applied to series of solutions containing increasing concentrations, ranging from 0.06 up to 1.7 mM in the injected solutions (six solutions per standard). Plotting the areas, an excellent linearity was obtained for all the standards (r2 > 0.999). Further, the slopes of the calibration plots obtained with the four standards did not differ significantly from each other, i.e. the maximal slope difference was 1%. Therefore, sensitivity was the same for non-ethoxylated alcohols and alkylsulphates, independently from the length of the alkyl chain. The similarity of the slopes indicated both a high degree of derivatization, presumably close to 100%, for the two surfactant classes. Further, since AES are quantitatively converted to the same derivatives as those obtained with FAE, then, the UV-Vis response factors of the FAE oligomers, which were established in previous work for the derivatives obtained with the phthalic anhydrides
[Mic-Tormos, 2009], [Mic-Tormos, 2008-B]
and diphenic
should be also valid for the corresponding
derivatized AES oligomers. This is of practical interest, because standards of non-ethoxylated fatty alcohols are widely available, whereas non-ethoxylated alkylsulfates are rare and expensive. Further, FAE oligomers with m > 0 are commercially available, which does not occur with the corresponding AES oligomers. Thus, in sections V.1.4 and V.1.5, the unexpensive and widely available dodecyl alcohol (C12E0) was exclusively used as a standard to evaluate
190
without bias total surfactant class concentrations, hydrocarbon series and distribution and average EO numbers ( m ) of the complex mixtures of FAE and AES oligomers found in industrial and environmental samples. For this purpose, six duplicated calibration points, up to 1.7 mM C12E0 were obtained using the peak areas. Then, the proposed procedure was applied to the samples of Table V.1. A pair of chromatograms per sample, corresponding to the FAE and AES fractions, was obtained in duplicate, and all the peak areas were measured.
Table V.1. Declared and found composition for industrial samples.
In mass percentages; the average EO number, m number found for each AES sample is indicated between parentheses.
Overlapping of the m = 0 and 1 peaks with the m = 5 and 4 peaks, respectively, within their respective hydrocarbon series, made necessary to establish an indirect way of estimating the individual areas corresponding to these oligomers. To estimate first the area of the m = 5 and m = 4 peaks by interpolation is straightforward and sufficiently accurate for most applications. Interpolation is possible at 25 C due to the perfect overlapping of the peaks by pairs of oligomers (m = 0 with m = 5, and m = 1 with m = 4)
[Mic-Tormos, 2008-B].
As
shown in Fig. V.5, the peak areas of the m = 5 and 4 oligomers were estimated by non-linear interpolation of the peak areas of the 2 m 3 and m 6 oligomers of their respective hydrocarbon series. Thus, the peak areas of the
191
Dehydol LT-7 ( m = 7) and LES ( m = 3) fitted well to the cubic equation, and to exponential equations of the form A=be-am, respectively.
Relative area
FAE, m = 7 n = 12
AES, m = 3 n = 12

n = 14

0 2
n = 14
10 Number of EOs
12
Fig. V.5. Estimation of the uncorrected peak areas of the m = 4 and 5 oligomers of the n = 12 and 14 series in: Dehydol LT-7 (empty symbols, solid lines by cubic interpolation); LES (full symbols, dashed lines by exponential interpolation). Experimental (rhombus) and interpolated peak areas (squares).
For simplicity, when the peak areas of the m = 4 and 5 oligomers of the n = 12 and 14 series of Dehydol LT-7 were estimated, the contribution of the oligomers with m > 13 of the n = 14 and 16 series (see Fig. V.2), respectively, was neglected. Then, the peak areas of the m = 0 and 1 oligomers were obtained by difference. All the peak areas were next divided by the tabulated response factors of the respective oligomers
[Mic-Tormos, 2008-B; Mic-Tormos, 2009],
and the
molar concentrations of the oligomers were obtained by dividing the corrected peak areas by the slope of the C12E0 calibration curve. Then, the average EO number, m , was calculated from the molar distribution of the oligomers. Total
192
mass concentrations of the surfactant classes, and the mass distribution of hydrocarbon series, were finally calculated by taking into account the molar masses of the oligomers and the dilution factor in the injected solutions.
V.1.1.4. Application to industrial raw materials and commercial products As indicated in Table V.1, a 70% generic LES and liquid laundry products containing mixtures of several surfactant classes were analyzed. The common anionic surfactants SAS and LAS were studied as potential interferences. These anionic surfactants should be retained together with the AES in the SAX cartridge; however, application of the procedure to a SAS sample gave a chromatogram with no additional peaks with respect to that of the reagent blank. Also, a 1:1 mixture of LES and a SAS sample (ca. 20 mg each) gave rise to a chromatogram which was undistinguishable from that of LES. Then, SAS showed no reaction with the anhydrides and did not cause interference either. On the other hand, LAS are aromatic surfactants which absorb in UV-Vis. The HPLC separation of a LAS sample in the conditions used to separate FAE and AES derivatives lead to a series of large and wide bands, close to the dead volume, on the chromatogram of the AES fraction. Further, this pattern was not modified by application of the derivatization procedure to a LAS sample; then, LAS did not react either with phthalic anhydride. Application of the procedure to a mixture of LAS and LES gave rise to a chromatogram showing the bands of LAS followed by the expected peak pattern of the derivatives of the LES oligomers; however, the absorbance due to the LAS bands decreased largely before the elution time corresponding to the AES hydrocarbon series with n = 8 (chromatogram not shown). Therefore, LAS did not cause interference either in the evaluation of the AES series of industrial interest (n 8). The determination of residual FAE in industrial AES is important to control both the sulfation
193
process and the quality of the final product. As indicated in Table V.1, application of the proposed procedure to the 70% generic LES sample showed a 65% AES and a residual 1.2% FAE. According to the data given in Table V.1, the proposed procedure is also useful for the characterization and determination of both FAE and AES in commercial cleaners.
V.1.1.5. Seawater analysis Application of the proposed procedure using diphenic anhydride to seawater extracts led to the chromatograms shown in Fig. V.6.
12 8 4 0 4 3 Intensity 105 6 4
Absorbance (mAU)
A
FAE n = 12
40 20 0
B
AES n = 12
20
C
AES n = 14
0 0 4 0
2 0 1
4 2 2 5 3 4 3 5 1 26 0 30 32 2 1
0 22 24 26 Time (min)
0 22 24 Time (min)
34
Time (min)
Fig. V.6. Chromatograms of a seawater extract obtained by derivatization with diphenic anhydride and using UV-Vis (upper parts) and MS detection (EICs, lower parts): (A) FAE, n = 12 series; (B) AES, n = 12 series; (C) AES, n = 14 series. The numbers at the peaks are m values. Elution conditions: linear gradient from 60 to 90% ACN in 50 min at 25C.
Using both UV-Vis and MS detection, the seawater samples showed measurable amounts of several FAE oligomers of the n = 12 series, and several AES
194
oligomers of the n = 12 and n = 14 series. According to the C12E0 calibration curve, 0.25, 2.3 and 0.99 g L-1 were found in seawater for C12E0, C12E0S and C14E0S, respectively (calculated as 1500 times less than in the injected solutions). Then, the proposed method was capable of distinguishing the oligomers of the two surfactant classes in the seawater, AES being present at higher concentrations than FAE. This could be due to the faster biodegradation of FAE in comparison to AES. This also indicates that previously reported data for FAE in environmental samples, obtained by methods based on derivatization with anhydrides, actually informed about the sum of FAE and AES. Due to the labitity of the ester bond of AES, the same problem could bias the FAE concentrations reported by using other derivatization reagents. Therefore, where appropriate, the reported data should be revised.
195
CHAPTER VI
SYNTHETIC POLYMERS
Chapter VI. Synthetic polymers
VI.1. PVP-NO
VI.1.1. Characterization of PVP-NO by FSCE and MEKC Several water-soluble polymers capable of partially inhibiting the unwanted dye transfer from colored to white fabrics are used as additives in laundry products
[Bertleff, 1998; Oakes, 2003].
These polymers, which are called
DTIs, act as dye scavengers, keeping the washed-off dyestuffs in solution, thus helping in the prevention of redeposition on the fabrics. For years, PVP and their copolymers have been mainly used; however, the dye scavenger efficiency of these polymers is reduced in the presence of anionic surfactants [Oakes, 2003]. For this reason, new generations of DTI, with superior complexing properties and a higher tolerance to anionic surfactants, have begun to be employed. A relatively common DTI of the new generation is PVP-NO (see structure in Fig. VI.1).Molecular masses between 9 and 36 kDa, which correspond to polymer chains constituted by ca. 75 and 300 monomers, respectively, are found in different types of commercial PVP-NO for laundry
Industrial and Institutional Cleaning, 2006]. [Oakes, 2003; Household
In spite of the use of increasing amounts of polymers in laundry and other applications, analytical methodologies for quality control of these compounds and for the evaluation of their environmental impact have been scarcely reported. In addition, the study of the interaction of polymers with surfactants in aqueous solution is important for both fundamental research and industrial application. In this work, the migration characteristics of PVP-NO in both FSCE and MEKC have been studied. The determination of the polymer in commercial additives for laundry is also demonstrated. CE has been widely applied to the separation of biopolymers and to the characterization and determination of synthetic polymers
[Gallardo, 1999; Cottet,
199

2005].
Mainly, FSCE and CGE have been employed to separate polyelectrolytes
(polymers with either positive or negative charges) and polyampholytes (polymers with both positive and negative charges), and MEKC has been used to separate non-charged polymers
[Gallardo, 1999].
The application of FSCE, CGE
and MEKC to the analysis of synthetic polymers has been reviewed [Cottet, 2005]. Both FSCE and MEKC can provide useful information about the behavior of polymers in solution, as well as quantitative information MEKC, Gallardo et al.
[Gallardo, 1999] [Bohrisch, 2000].Using
were able to separate two different
components of a non-ionic copolymer, i.e. a fraction rich in methacrylate and another one rich in vinylpyrrolidone. MEKC has been also used to obtain information about the synthesis progress, nature and composition of ionic copolymers
[Aguilar, 2002].
On the other hand, in CGE, molecular mass
discrimination of ionic polymers is achieved by sieving through a BGE which contains a non-ionic polymer, such as a PEG derivative, dextran or other polysaccharides
Starkweather, 2000; Welch, 2001]. [Grosche, 2000], [Bohrisch, 2000;
a cellulose
Clos, 1998;
In FSCE, and for short-chain polyelectrolytes, the electrophoretic mobility is a function of the chain length. Thus, the mobility increases as more charged monomers are added to the chain; however, the mobility reaches a maximum and begins to decrease when the polyelectrolyte changes from the rod-like to the coil conformation. Finally, for longer polyelectrolytes the charge-to-mass ratio become constant, and the polymer migrates with the so-called free draining mobility [Cottet, 2000]. Therefore, over a given chain length, and independently of the chain length range, a polyelectrolyte gives a single peak in FSCE
2000; Grosche, 2000]. [Bohrisch,
However, due to additional effects, some polymers can
actually give two or more signals at different migration times rather than a single peak.
200
Thus, copolymers composed by a long polyanionic chain bound to a short hydrophobic chain have shown to form micelles in aqueous solutions
2001]. [Cottet,
Using FSCE, two peaks, which were attributed to the very slow rate of
exchange between the free and aggregated or micellized states of the polymer in comparison to the time scale of electrophoresis, were observed. The free chains of the polymer (the unimers) exhibited a larger mobility (in absolute values) than the micellized polymer. Further, the peak of the micellized polymer was broader than that of the free polymer, which was attributed to scatter in the aggregation numbers. Using FSCE and a PVP-NO/ maleic acid anionic copolymer, Gyrffy et al.
[Gyrffy, 1998]
also found a narrow peak followed by a wider one. The
presence of two components in the polymer was proposed, but the possible nature of the components was no further discussed. Using both SEC and FSCE, tpnek et al.
[tpnek, 2001]
have shown that some copolymers form micelles
in solution, and that the micelles coexist in a mobile equilibrium with the unimers. Further, the unimer-micelle equilibrium was shown to be kinetically frozen in aqueous media, the micelles behaving as independent nanoparticles. In this work, a narrow peak was obtained for PVP-NO solutions using both FSCE and MEKC; however, at least an additional broad band was also present in most electropherograms. The nature of the signals obtained is discussed at the sight of both the studies reported in the literature and the new results. In addition, low-molecular-mass zwitterions, including trimethylglycine (betaine) and sarcosine, have been used for a long time to reduce adsorption in the FSCE separation of proteins
[Bushey, 1989];
however, betaine and sarcosine
have carboxylate groups, which hinders their application below pH 56. Thus, to separate proteins in more acidic media (pH 25), PEA has been used [Chen, 1992].
201
In this work, PEA is used to reduce adsorption of PVP-NO in phosphate buffers of acidic pH.
VI.1.1.1. FSCE studies in the absence and presence of PEA Separation of PVP-NO was first tried in the absence of PEA. As shown in Fig. VI.1, using positive polarity and an acid BGE (10 mM HAcO, pH 4), a large peak located after the EOF marker peak was observed. Two narrow peaks plus a broader band in the middle were observed at pH 7 (10 mM NH4AcO) (electropherograms not shown). All the peaks and band at both pH 4 and 7 showed the spectrum of the polymer (see the insets in Fig. VI.1).
CH2 - CH
40
Abs. (mAU)
40 20 0 8 4 0 200 300 (nm) 400
N+ O20
EOF
0 0 2 4 6 8 Time (min) 10
Fig. VI.1. Electropherograms of an EOF marker (mesityl oxide) and PVPNO (2000 g/mL) obtained using positive polarity and a BGE containing 10 mM HAcO (pH 4). The insets show the chemical structure of PVP-NO, and the UV spectra at both the peak maximum and at the baseline location indicated by an arrow.
Taking into account the large molecular masses of this polymer (917 kDa), and its nominally nonionic nature, a single peak at the EOF migration time was actually expected. However, as discussed below in Section VI.1.1.2, the
202
electropherograms and associated spectra indicated the presence of at least two PVP-NO components or forms in the solutions, these forms also exhibiting nonzero net electrophoretic mobilitites. The reproducibility of the migration time and area of the PVP-NO peak at pH 4 was 1.8 and 7.2%, respectively, and similar values were obtained for the peaks and band at pH 7 (n = 5). However, as revealed by recording the UV spectra along the baseline after the PVP-NO peaks, a drawback was the presence of severe peak tailing due to adsorption (see the insets in Fig. VI.1). Then, FSCE in the presence of large concentrations of PEA was tried. The program SPARC
[Hilal, 1995]
output the following pKa values for the
ionization of PEA: 1.07, 5.75 and 8.91; thus, the PEA species without a net charge predominates, at least on a 9:1 basis, within the 2.074.75 pH range. Therefore, a BGE containing 20 mM H3PO4 (pH 2.2) was used to investigate the effect of the PEA concentration on the migration behavior of PVP-NO and the EOF. As shown in Fig. VI.2, using this BGE in the presence of increasing PEA concentrations, PVP-NO gave a large narrow peak (after an initial small peak) within the anionic migration region. A broader band at the beginning of the cationic migration region (immediately after the peak of the EOF marker) was also observed. Both the large narrow peak and the band exhibited the spectrum of the polymer. Except when otherwise indicated, adsorption along the baseline after the peak locations was not detected by using 150 mM or higher PEA concentrations. Contrary to what occurred in Fig. VI.1, negative polarity was required in Fig. VI.2 to observe the PVP-NO peaks. As revealed by using acetone as a marker, the EOF was reversed in the presence of large PEA concentrations. The negative EOF could be due to the formation of esters between the phosphate groups of PEA and the silanols. This would lead to the predominance of the
203
positively charged amino groups of PEA covering the capillary walls, thus reversing the EOF.
A
40
B
20
0 0 4 8 12 Time (min)
Fig. VI.2. Electropherograms of 1000 g/mL PVP-NO, obtained using negative polarity and a BGE containing 20 mM H3PO4 (pH 2.2) and the following PEA concentrations: 150 (A), 250 (B) and 350 mM (C). The arrow shows the location of the peak of the EOF marker (acetone).
The electroosmotic mobility increased (in absolute values) rapidly up to 150 mM PEA, reaching a plateau between 225350 mM PEA (triplicate points, not shown). The net electrophoretic mobility of PVP-NO (according to the large narrow peak) progressively increased (in absolute values) when the PEA concentration was increased from 150 to 200 mM. At PEA concentrations higher than 225 mM and up to 300 mM, PVP-NO showed an almost constant mobility. Baseline disturbances were produced with PEA concentrations over 300 mM; accordingly, further studies were made using 250 mM PEA. The contribution of PEA to the capillary current was negligible.
204
Then, the influence of pH in the presence of 250 mM PEA was studied. First, H3PO4 (pH 2.2) was substituted by NaH2PO4 (pH 3.0). The net mobility of the large PVP-NO peak increased, which suggested an intensification of the anionic character of the polymer. As shown by comparing the electropherograms of Fig. VI.2, part B and Fig. VI.3, the intensity of the broad band near the EOF migration time also increased at rising pHs, and it was partially resolved in several bands. Some of these bands were clearly located within the cationic migration region. Both the migration time and area of the large narrow peak of PVP-NO were highly reproducible (0.3 and 2.4%, respectively). On the contrary, as also shown in Fig. VI.3, along successive replicated injections on the same capillary the broad band showed a progressive shifting towards shorter migration times. As discussed below in Section VI.1.1.2, the large narrow peak and the broader band were attributed to two forms of PVP-NO in solution. The progressive shifting of the broad band along successive injections could be due to residual adsorption of the corresponding form of the polymer. In fact, adsorption was not observed in Fig. VI.3, parts A and B, but the spectrum of the polymer was observed at migration times longer than that of the band in Fig. VI.3, parts C to E. Significant differences regarding pH 3.0 were not observed at pH 4.3 (obtained using Na2HPO4). The concentration of NaH2PO4 (pH 3.0) was also varied within the 540 mM range; however, significant differences regarding 20 mM were not observed either. Significant variations of the shapes of the peak and band were not observed at increasing capillary temperatures, from 25 to 45C
(electropherograms not shown); however, the large narrow peak appeared at slightly shorter migration times. Thus, the net electrophoretic mobility of this
205
peak varied from -17.6 to -18.2 and -19.7 (in 109 m2s1V1) when the capillary temperature was increased from 25 to 35 and 45C, respectively.
200
A
160
B
120
C
80
D
40
E
0 0 4 8 12 16
Time (min)
Fig. VI.3. Electropherograms of 1000 g/mL PVP-NO, obtained using negative polarity and a BGE containing 250 mM PEA and 20 mM NaH2PO4 (pH 3.0). From (A) to (E), successive replicated injections on the same capillary. The arrow shows the location of the peak of the EOF marker (acetone).
VI.1.1.2. Discussion on the results obtained by FSCE PVP-NO has large molecular masses and does not contain ionizable groups, thus, a single narrow peak at the EOF migration time should be expected using FSCE. Instead of this, a peak with a well-defined anionic behavior was observed in Figs. VI.1-VI.3, and at least an additional broad band with a weak cationic behavior was observed in Figs. VI.2 and VI.3. As next discussed, and as
206
supported by other reports from literature, the anionic character of PVP-NO can be explained by the separation of charges along the N-O bond, and the subsequent formation of a ionizable complex with water. A possible explanation for the additional broad band is the presence of a micellized form of the polymer. The nitrogen and oxygen atoms of pyridinium oxides exhibit a remarkable separation of the respective positive and negative charges
[Ochiai, 1953].
This
charge separation has been also demonstrated in PVP-NO
[Okamoto, 1998].
According to viscosity and light scattering measurements in solution, PVP-NO has been shown to behave like a polyelectrolyte, rather than as a noncharged polymer
[Okamoto, 1998; Lee, 1996].
This behavior has been attributed to the
presence of strong interparticle forces, which result from the charge separation across the N-O bond
[Lee, 1996].
In addition, PVP-NO has been observed to
exhibit a remarkable electrodonating character, which has been explained by means of resonant structures
[Yamamoto, 1996].
Further, PVP-NO solutions in
water have a non-zero electrical conductivity (2.81073.4106 S/cm), which contrasts with the electrically insulating properties of the corresponding nonoxidated poly(4-vinylpyridine) (<1014 S/cm)
[Yamamoto, 1996].
Finally, purified
PVP-NO fractions gave solutions with pHs as low as 3.44.5 when solved in water, which was attributed to the formation and subsequent ionization of a PVPNO/water complex [Okamoto, 1998]. The formation and subsequent ionization of a PVP-NO/water complex fully agrees with the net anionic electrophoretic mobility of the large narrow PVP-NO peak observed in this work. Further, the acidity of some N-O bonds along the polymer chain can be enhanced by the presence of neighboring N-O bonds, as occurs with polycarboxylic acids [Gyrffy, 1998], which would lead to a range of pKa values along the polymer. This would also explain the increase of
207
the net anionic mobility showed by this PVP-NO peak (in absolute terms) at rising pH values from 2.2 to 3.0 (Figs. VI.2, part B and VI.3). Another point to be explained is the presence of the broad bands near the EOF migration time. At the sight of the previous studies
1998; tpnek, 2001], [Cottet, 2001; Gyrffy,
a possible explanation is the presence of associated forms
of the polymer. These associated or aggregated forms could coexist with the free form (the unimers) in a very slow or frozen equilibrium between them, as reported for some copolymers
[tpnek, 2001].
Since PVP-NO has not well-
defined hydrophobic and hydrophilic regions, association cannot be produced by the action of the hydrophobic forces. However, in the case of PVP-NO, the likely alternative is association through strong dipole-dipole interactions.
VI.1.1.3. MEKC studies in the absence and presence of PEA The MEKC behavior of PVP-NO using 60 mM SDS was first studied in the absence of PEA. As shown in Fig. VI.4, a small peak, followed by a large peak with a shoulder and by a second large peak at longer migration times, was observed in an acid medium (20 mM H3PO4, pH 2.2). These peaks were observed using negative polarity. As discussed above for FSCE, the presence of two peaks can be explained by the coexistence of a free and an aggregated form of PVPNO. The EOF should be small at this low pH, thus the PVP-NO peaks along the anionic migration time region can be explained as due to association of the two PVP-NO forms with the SDS micelles. The SDS concentration was varied within the 20100 mM range. Electropherograms similar to that shown in Fig. VI.4 were obtained; however, a major drawback was the poor reproducibility of the migration time of the second large peak. Along replicated injections of the same solution, this peak appeared at progressively longer migration times. This
208
problem was not overcome by conditioning the capillary between injections with hot NaOH solutions, as indicated in section III.2.1.3 for new capillaries.
Abs. at 214 nm ( mAU)
100 50 0 0 4 Time (min) 8
Fig. VI.4. Electropherogram of 2000 g/mL PVP-NO, obtained using negative polarity and a BGE containing 20 mM H3PO4 and 60 mM SDS. The two large peaks and the shoulder showed the spectrum of the polymer.
Lack of reproducibility was attributed to adsorption, then, the behavior of PVPNO at increasing SDS concentrations was studied in the presence of 250 mM PEA. Under these conditions, and using pH 3.0 (20 mM NaH2PO4), PVP-NO showed the reproducible but complex behavior which is outlined in Fig. VI.5. Between 1 and 3 mM SDS (Figs. VI.5, parts A and B), a single peak at increasingly longer migration times was observed. With 3 mM SDS, a new broad band at a short migration time, and a new narrow peak, began to appear. The band increased with 5 mM SDS, and reached a constant intensity within the 20 60 mM SDS range (Figs. VI.5, parts C-F). The narrow peak was large at 520 mM SDS (Figs. VI.5, parts C and D), and appeared at longer migration times and became broader at higher SDS concentrations (Figs. VI.5, parts E and F). Using 20 mM SDS and 35C (instead of 25C), an electropherogram similar to that shown in Fig. VI.5, part D was obtained, but with the large narrow peak located at a longer migration time (ca. 10 min). A possible explanation of the results summarized in Fig. VI.5 is next given and discussed.
209
200
A B
C
100
D E F
0 0 10 Time (min) 20 30
Fig. VI.5. Electropherograms of 1000 g/mL PVP-NO, obtained using negative polarity and a BGE containing 250 mM PEA, 20 mM NaH2PO4 (pH 3.0) and increasing SDS concentrations. From A to F: 1, 3, 5, 20, 30, 60 mM SDS, respectively.
VI.1.1.4. Discussion on the results obtained by MEKC The association between SDS micelles and non-ionic polymers, such as PEG and PVP, has been studied by measuring the decrease of the surface tension as the concentration of the SDS increases, and by other techniques [Cabane, 1977;
Arai, 1971].
According to Cabane [Cabane, 1977], PEG forms mixed micelles with
SDS. Mixed PEG-SDS micelles have a definite composition, with the excess material, whether polymer or SDS, being present as free polymer molecules or regular SDS micelles, respectively. When SDS is added to a solution of the polymer, two concentrations, x1 and x2, mark the beginning and the completion of the association of SDS with PEG, respectively. The association process starts abruptly above a certain surfactant concentration, x1, which is lower than the CMC, and saturates abruptly above another surfactant concentration, x2, where
210
the coverage of the polymer by adsorption of surfactant molecules along its chain is completed up. Beyond x2 the surface tension of the solution is close to that of a solution containing regular surfactant micelles. The difference, x2 - x1, measures the amount of surfactant bound to the polymer. According to NMR studies
[Cabane, 1977],
PEG is attached to the
hydrocarbon/water interface of the mixed PEG-SDS micelles, with some monomers of the polymer replacing water molecules in the vicinity of the head of the SDS molecules. About 10% of the monomers of the polymer are thought to be directly bound to the micelles, while the others form loops in the surrounding water. At the saturation point, x2, depending on the polymer concentration, and independently from the polymer molecular mass, the composition corresponded to 3.34.1 PEG monomers for one SDS molecule. Arai et al.
[Arai, 1971]
also found two transition points on the surface
tension vs. SDS concentration curves when recorded in the presence of PVP. Association of SDS with PVP to form mixed micelles began at an SDS concentration 40% lower than the CMC. The PVP/SDS weight ratio at the point where adsorption was completed, x2, was 1:2.3, regardless of the PVP concentration. This corresponded to 1.1 PVP monomers for one SDS molecule. According to literature, PVP-NO also interacts strongly with the SDS micelles. Thus, the inhibition of the dye scavenger capability of PVP-NO in the presence of SDS and other anionic surfactants has been attributed to displacement of the dye from the polymer/dye complex to form a polymer/surfactant complex
[Gyrffy, 1998] [Oakes, 2003-A].
Using MEKC, Gyorffy et al.
also found a strong interaction between SDS micelles and an
anionic PVP-NO/maleic acid copolymer. In a UV-Vis spectroscopic study, Oakes et al. [Oakes, 2003-C] have shown that PVP-NO binds SDS micelles. Since the hydrophobic region of PVP-NO (the hydrocarbon skeleton) is not well
211
separated from the hydrophilic regions, the interaction was explained by the formation of mixed polymer/SDS micelles, in which short segments of the polymer occupied substantial parts of the micelles. As illustrated in Fig. VI.6, in these parts of the micelles, a segment of the hydrocarbon skeleton was proposed to be located inside the micelle, and the polar pyridine N-oxide groups were assumed to be positioned among the SDS sulfate groups. Since a single polymer molecule can bind several micelles, mixed aggregates containing a number of SDS micelles was proposed to be formed
C].
OOSO2 O N+
[Cabane, 1977; Arai, 1971; Oakes, 2003-
Fig. VI.6. Scheme adapted from ref.
[Oakes, 2003-C],
showing the likely
structure of a free PVP-NO/SDS mixed micelle.
Curves of the surface tension against the SDS concentration, obtained both in the absence and in presence of 1000 g mL-1 PVP-NO, are shown in Fig. VI.7. These measurements were made in the conditions of Fig. VI.5, that is, in the presence of both 20 mM NaH2PO4 and 250 mM PEA. In the absence of the polymer, the CMC of SDS was 1.8 mM. This parameter, which is 8.3 mM in pure water, decreases in the presence of salts
[Phillips, 1955; Thvenot, 2005],
which is consistent with the value obtained. Two transition points, indicating the
212
beginning and end of the association process of PVP-NO with the SDS micelles, were observed in the presence of 1000 g mL PVP-NO.
70
Surface tension (mN m-1)
60 50 40 30 0 0.5 1 log ([SDS]+1)

Fig. VI.7. Surface tension plotted against log([SDS]+1), where [SDS] is the SDS concentration in mM (the 1 was added to plot the [SDS] = 0 point). Data obtained both in the absence (, dotted lines) and in the presence of 1000 g mL-1 PVP-NO (, continuous lines).
1.5
The first point, x1, was located at 0.48 mM SDS, which corresponds to a concentration a ca. 70% lower than the CMC. This indicates a strong interaction between the polymer and SDS. The second point, x2, was placed over the CMC, at 9.7 mM SDS. The difference (x2-x1) corresponded to 0.9 PVP-NO monomers for one SDS molecule taking part in the mixed micelles. This value is close to that reported for the formation of PVP/SDS mixed micelles, 1.1
[Arai, 1971].
Assuming that PVP-NO would behave in a similar way as PEG, a mixed micelle would have about 70 SDS molecules with only a 10% of the polymer taking part of the micelles
[Cabane, 1977].
If this were true, 0.9 PVP-NO monomers for one
SDS molecule would correspond to 6.3 PVP-NO monomers per mixed micelle, which is quite realistic. This value was used to draw the scheme of Fig. VI.6.
213
On the other hand, according to the FSCE discussion of above, and in agreement with the results of tpnek et al.
[tpnek, 2001],
two forms of the
polymer, namely, the free polymer and the pure polymer aggregates, probably in a very slow-rate equilibrium between them, could be present in the aqueous solutions of PVP-NO, in the absence of SDS. Further, in agreement with the study of Oakes et al.
[Oakes, 2003-C],
both the free and the micellized polymer
forms could bind SDS micelles. Thus, it seems reasonably to assign the narrower peaks of the electropherograms of Figs. VI.4, part A, VI.5, part C and D to the association between the free polymer and SDS micelles, and the broader band at a shorter migration time to the association between the aggregated polymer and SDS micelles. In this latter, band broadening could be explained by scatter of the aggregation number. The presence of two types of PVP-NO/SDS aggregates could explain the persistent presence of a peak and a band (or group of bands) in most electropherograms, being also compatible with the presence of the two transition points observed in Figs. VI.7. The EOF should be almost zero at the low pH used; however, as indicated above, an anionic (reversed) EOF was observed in the presence of large PEA concentrations. Thus, the reduction of this cathodic EOF produced by the presence of increasing SDS concentrations could be enough to explain the broadening and shifting effects on the large narrow peak observed in Fig. VI.5, parts A and B. The first transition point of Fig. VI.7, x1, approximately coincided with the appearing of the new peak and band between Fig. VI.5, parts A and B. Thus, the sudden formation of two new peaks between 3 and 5 mM SDS could be attributed to the simultaneous formation of both free polymer/SDS and aggregated polymer/SDS mixed micelles. Since the EOF was very low in the conditions of Fig. VI.5, parts B to F, the short migration times of the band and peak indicated that the polymer species
214
had high anionic mobilities. These can be explained by both the negative charge of the free polymer, and the association of the free and aggregated polymer forms with the SDS micelles. As shown in Fig. VI.5, parts E to F, the band assigned to the associated PVP-NO/SDS mixed micelles showed no further changes of shape and location at higher SDS concentrations. On the contrary, the peak assigned to the free polymer/SDS mixed micelles was progressively broader, and appeared at longer migration times as the SDS concentration increased. An explanation for this behavior was not found.
VI.1.1.5. Quantitation studies and application to real samples using FSCE Intra- and inter-day repeatabilities of the large and narrow polymer peak in the presence of 20 mM NaH2PO4 and 250 mM PEA were measured by performing successive injections of a 1000 g mL-1 PVP-NO solution. The results are given in Table VI.1. A calibration curve was constructed by injecting seven standard solutions within the 502000 g mL-1 range, and by measuring the peak area. Excellent linearity (r > 0.993) and an LOD of 23 g mL-1 (S/N = 3) were obtained (Table VI.1). The FSCE method was applied to the determination of PVP-NO in two different commercial DTI concentrates for laundry. A representative
electropherogram is shown in Fig. VI.8. This electropherogram was closely similar to those given in Figs. VI.2, part B and VI.3, which were obtained in the same conditions, but it showed an additional intense peak (marked with an asterisk in the figure). This peak increased upon spiking the sample with cumene sulfonate, which is a common hydrotropic additive of cleaning products. The other narrow peak and the broader bands at longer migration times showed the spectrum of the polymer, and increased upon spiking the sample with PVP-NO.
215
According to the calibration curve, the area of the narrow PVP-NO peak corresponded to 161 g mL-1 in the injected solution, and to 1.53% in the sample.
Table VI.1. Migration time and peak area repeatabilities, efficiency and LOD for the large narrow peak of PVP-NO. Parameter Migration time repeatabilities Peak area repeatabilities at 1000 g/mL N, m1 LOD, g/mL (S/N = 3)
a) b)
Value 0.30a), 1.0b) 2.4a), 5.5b) 80 000 23
Intraday as RSD % (n = 5). Interday as RSD % (n = 3 5).
20
12
16
20 Time (min)
Fig. VI.8. Electropherogram of a commercial additive for laundry obtained using negative polarity and a BGE containing 20 mM NaH2PO4 and 250 mM PEA. The sample was 1:100 diluted with 50:50 v/v MeOH/water. The arrow shows the location of the peak of the EOF marker (acetone), and the asterisk identifies the peak of cumene sulfonate.
Attempts of detecting 500 g mL-1 PVP-NO in the presence of non-ionic and anionic surfactants were also made. Thus, the PVP-NO narrow peak was not disturbed by the presence of a 5% of FAEs (Dehydol LT-7); however, it was not
216
detected when the sample solution contained a 5% of an anionic surfactant (both LAS and LES were tried).
VI.2. PVP
VI.2.1. Characterization and determination of PVP by complexation with an anionic azo-dye and NECEEM A variety of nonaqueous and aqueous CE methods for the characterization and determination of synthetic polyelectrolytes, including CZE, CGE and CIEF methods, have been described
2004; Cottet, 2005]. [Clos, 1998; Grosche, 2000; Borisch, 2000; Engelhardt,
In CGE, solutions of nonionic polymers are used as sieving
media to separate polyelectrolytes [Welch, 2001]. However, as far as we know, CE methods for the characterization and determination of nonionic polymers have not been reported. To analyze PEGs by CZE, previous derivatization with an anhydride, which provides both a chromophore and charge, has been used
[Wallingford, 1996; Barry, 1998];
however, most nonionic synthetic polymers
cannot be easily derivatized. PVP is a versatile hydrophilic polymer which is widely used for a variety of purposes, including cosmetics and toiletries,
1974;
textiles
Sheth,
and
dyes, In the
pharmaceuticals and adhesives
[Frauenfelder,
1985].
formulation of cleaning products, PVP is used as a dye scavenger, keeping the washed-off dyestuffs in solution, thus inhibiting the unwanted dye transfer from coloured to white fabrics [Bertleff, 1998; Oakes, 2003-A; Oakes, 2003-B; Oakes, 2005]. The determination of PVP has been carried out in urine and blood serum by precipitation with iodine and titration with thiosulfate infrared absorptiometry
[Behen, 1964]. [Dwyer, 1964],
and by
PVP has been determined in foods,
cosmetics and laundry products by preconcentration on silicagel followed by
217
complex formation with an anionic azo-dye and colorimetry [Frauenfelder, 1974]. A spectrophotometric procedure for the determination of PVP in waste water of pharmaceutical plants, based on formation of a coloured complex with a dye, has been also reported
[Chmilenko, 2001-A].
PVP can be also determined in
pharmaceutical preparations by direct injection of the sample in a RP-HPLC column

[Jones, 2004];
however, this entails a high risk of getting PVP
permanently retained by the stationary phase. Over half a century, studies concerning to the complexation of anionic organic dyes by PVP, with special reference to total number of binding sites available for the interaction, free energy and heat of interaction, and changes in the shape of polymer molecule, have been carried out
[Sheth, 1953; Hansen, 1954;
Barkin, 1955; Frank, 1957; Luck, 1958; Molineux, 1961; Runge, 1996; Chmilenko, 2001B; Oakes, 2003-C].
The aim of this work was to develop a CE method for the
determination of PVP in cleaning products. Since the electrophoretic mobility of nonionic PVP is almost zero, mixtures containing PVP and an anionic azo-dye were prepared and injected in the capillary. A band due to the PVPdye complexes followed by a peak due to free dye was observed. The electropherograms were interpreted in the light of the theory developed by Krylov and co-workers
[Berezovski, 2002; Krylov, 2003; Drabovich, 2006; Lin, 2008;
Krylov, 2006; Krylov, 2007],
which described the method of nonequilibrium CE of
equilibrium mixtures (NECEEM) for the study of proteinprobe and DNA protein interactions. In NECEEM, non-covalently bound fluorescent probes are equilibrated with a target protein or DNA, and the mixture is injected in the capillary. In this work, application of NECEEM to the study of the interaction between PVP and an azo-dye is demonstrated. Information about the average molecular mass of the polymer, and the maximal stoichiometry, average stability constant and dissociation rate of the polymerdye complexes, can be obtained. In addition, the method was applied to the characterization and determination of
218
PVP in commercial cleaners and pharmaceutical preparations. The proposed method should also be useful to characterize and determine other synthetic and natural nonionic polymers by using the appropriate probes.
VI.2.1.1. Electropherograms of PVP in the presence of anionic azo-dyes Two anionic bisazo-dyes (Fig. VI.9), which form with PVP stronger complexes than single azo-dyes, were selected. The electropherogram of a CR solution is shown in Fig. VI.10, part A. In the same figure, parts BF, a series of electropherograms showing the effect of the addition of increasing CR concentrations to a PVP solution, are given. In the absence of CR, PVP absorbed only at low wavelengths, and its electrophoretic mobility was close to zero (Fig. VI.10, part B, trace obtained at 215 nm).
PVP
C H C H2 N O n
NH2 O N S O Na+ OO N N N S
O- Na+ O
Congo Red (CR)
H2N
N O S O- Na+ O
NH S
O O
Acid Blue 113 (AB)
Na+ O-
Fig. VI.9. Molecular structures of PVP and the azo-dyes used in this work.
219
A
60
C
6
Absorbance (mAU)
40
D
4.5
E
20
3.0
F 2.25
0 0
G 1.12
2 4
10
Time (min)
Fig. VI.10. Electropherogram of 0.5 mM CR (A) and electropherograms of 500 g mL1 PVP of 60 kDa (4.5 mM in monomers) without an azo-dye (B), and in the presence of the following CR concentrations: 0.75 (C), 1 (D), 1.5 (E) and 2 mM (F). Trace G was obtained with 4 mM AB. The numbers on the traces are the values of q (monomer/dye molar ratio). The traces were recorded at 215 (B), 500 (A, CF) and 565 nm (G). On each trace, the arrow indicates the location of the EOF marker peak. The area at the left half of the peak of the PVPdye complexes, a, was used to estimate the total area of this band (a = APVPD/2).
A significant parameter in all the experiments along this work was the monomer/dye molar ratio, which will be indicated by q throughout the article. At increasing CR concentrations (decreasing values of q), the band appeared at increasing migration time values after the EOF time. The band was also
220
progressively divided into two peaks which showed a large absorptivity in the visible region, with their respective maxima at ca. 505 and 485 nm, respectively. At q < 4, the peak with a lower migration time was Gaussian shaped, whereas the other peak was asymmetric (Fig. VI.10, parts E to F). Resolution between the two peaks, and their areas at 500 nm, increased as q decreased; however, the area of the Gaussian shaped peak remained constant when q < 4. The asymmetric peak continued increasing, approaching the migration time of CR at q <4 (Fig. VI.10, parts E and F). Interpretation of the electropherograms was made in the light of both the UVVis spectra of the free dye and the PVPCR complexes, which were obtained with a conventional spectrophotometer, and the NECEEM theory (see Section VI.2.1.3). Using a conventional spectrophotometer, the absorption spectrum of CR showed two maxima located at 340 and 487nm ( = 16,845 and 21,400 M1 cm1, respectively). Upon addition of an excess PVP (0.91 mM in monomer giving q = 9.1), the band at 340 nm was only slightly modified, but the maximum of the other band shifted from 487 to 505 nm. The molar absorptivity of this band also increased from 21,400 to 24,000 M1 cm1. Batochromic shifts between 7 and 19 nm have been reported for the 500650 nm band of azo-dyes upon complexation with PVP
[Scholtan, 1953; Runge, 1996],
which agrees with the 18 nm shift found
in this work for the formation of the PVPCR complexes. Accordingly, the Gaussian shaped peak of the electropherograms of PVPdye mixtures (Fig. VI.10, parts B to F), was attributed to the corresponding PVPdye complexes, and the asymmetric peak at a longer migration time to the free dye. As also shown in Fig. VI.10, when q < 4, the asymmetric peak was close to the location of the free dye peak obtained in the absence of the polymer (trace A). There are a large number of potential binding sites along a PVP molecule. Thus, the increase in the peak area of the complexes at rising dye concentrations was attributed to
221
an increased number of bound sites along the PVP molecules. The increasing mobility of the complexes at increasing dye concentrations (at decreasing q values up to q = 4) was also attributed to the same cause. On the other hand, at q > 4 (Fig. VI.10, parts C and D), the peak of the free dye ions was located at a migration time which was only slightly higher than that of the PVPCR complexes. Further, a similar behaviour was observed by injecting PVPAB mixtures. The peak due to the free dye was expected at longer migration times, namely, at 6.4 and 9.5 min for CR and AB, respectively; however, a peak close to these locations was obtained only when q < 4. As further discussed in Section VI.2.1.3, this, as well as the presence of an intermediate exponential region when q < 4, were explained by applying NECEEM concepts.
VI.2.1.2. Formation rate of the PVPCR complexes The formation rate of the PVPCR complexes was first studied spectrophotometrically. For this purpose, a mixture containing 0.1 mg mL1 60 kDa PVP (0.91 mM in monomers) and 0.1 mM CR (q = 9.1) was prepared, and a spectrum every 10 min was obtained. The band at 487 nm shifted progressively to 505 nm, and the molar absorptivity also increased in a process which lasted ca. 40 min. No further modifications of the spectrum were observed during the following 5 h. Then, another mixture containing 1 mg mL1 60 kDa PVP (9.1 mM in monomers) and 4 mM CR (q = 2.25) was prepared, and aliquots were injected in the capillary at increasing times after preparation. In these experiments the peak area of the PVPCR complexes reached some stability 3 h after preparation of the mixtures. At the same time, the mobility of the complexes increased, also reaching a plateau between 3 and 5 h after preparation. The slower formation of the complexes when monitored using CE instead of the
222
spectrophotometer could be due to the different values of q (9.1 vs. 2.25). However, conformational changes of the complexes, leading to an increase in their stability, could take place during several hours after preparation of the mixture. As deduced from application of NECEEM principles, conformational changes which could be blind to a spectrophotometer, could be revealed by the electropherograms. Thus, on the electropherograms, an increase of the peak area of the PVPCR complexes at increasing time values after preparation could be due not to an increase of the concentration of the complexes, but to a slower dissociation rate. This point is further commented upon in the next section. Except when otherwise indicated, in all the experiments presented in this work, the vials containing all the solutions were maintained in the carrousel of the CE instrument at ca. 25 C during a minimum of 5 h before injection.
VI.2.1.3. Application of NECEEM principles to the interpretation of the electropherograms of PVPdye mixtures According to Krylov and co-workers
[Berezovski, 2002; Krylov, 2003;
Drabovich, 2006; Lin, 2008; Krylov, 2006; Krylov, 2007],
when a targetprobe
complex is injected in the capillary, due to the violation of the equilibrium conditions during migration, at least two peaks and an intermediate exponential region should be obtained. The two peaks are due to the remaining targetprobe complex at the time of detection, and to the equilibrium concentration of the free probe. The exponential region is due to the probe which is liberated from the complex during migration. A third peak and its corresponding exponential region, due to the equilibrium concentration of the free target and to the target liberated during migration, respectively, will also appear if detection conditions sensitive to the target are used. The peaks of the targetprobe complex, free probe and free target, should appear at the migration time values corresponding
223
to their respective electrophoretic mobilities. Since both target and probe are carried away from the complex during migration, the complex dissociation rate depends exclusively on its concentration. For this reason, a first-order kinetics, which gives rise to an exponential liberation of both target and probe, is followed. The stability constant of the targetprobe complex can be established by measuring the peak area of the free probe, and the sum of the peak areas of the remaining complex and the exponential region. Correction of the areas is necessary if the complex and the probe have different sensitivities (e.g. different fluorescence quantum yields or different molar absortivities). Finally, kinetic information related to the complex dissociation rate can be obtained from the exponential profile of the intermediate region. The PVPdye mixtures used in this work gave rise to electropherograms which closely resembled to those described for proteinprobe and DNAprotein complexes when injected in NECEEM conditions. As shown in Fig. VI.10, parts C to G, the Gaussian peak attributed to the PVPdye complexes was followed by an exponential region which ended abruptly at the long migration time side; however, a peak due to the equilibrium concentration of free dye could not be distinguished in the electropherograms of Fig. VI.10. This was attributed to the use of q values not sufficiently close to the maximal stoichiometry of the complexes, which as shown later in Section VI.2.1.5, was q = 4. In fact, in Fig. VI.10, this ratio was 4.5 and 3.0 for traces D and E, respectively. At large q values, the excess PVP should reduce the equilibrium concentration of the free dye below its detection limit. On the other hand, at too low values of q, the large equilibrium dye concentration could not be distinguished from the much lower amount of dye liberated by the complexes. However, as shown in Fig. VI.11, a peak of the free dye, separated from the beginning of the exponential region, was
224
clearly observed in all the electropherograms when mixtures with q values ranging from 3.1 to 4.4 were injected.
A
Absorbance (mAU)
6 20 3.1 4 2 4.0 0 5 4 3 40 3.1 2 1 5 6 Time (min) Ae AD 6 7 Time (min) Ae AD
B
Absorbance (mAU)
4.0 0
Absorbance (mAU)
C
80 3.1 10 AD Ae 0 4.0 2 4 6 8 0 7.0 7.5 Time (min)
Time (min)
Fig. VI.11. Electropherograms of PVP of 10 (A), 60 (B) and 360 kDa (C) with 4 mM CR at the values of q (monomer/dye molar ratios) indicated on the traces. The insets show how the areas corresponding to AD and Ae were established. The electropherograms were recorded at 500 nm. Other details as in Fig. VI.10.
In order to characterize the PVPdye complexes, three areas should be measured: (i) the area of the remaining PVPdye complexes, APVPD; (ii) the area
225
due to the equilibrium concentration of the free dye, AD; and (iii) the area due to the dye liberated from the complexes during separation, Ae. The values of these three areas were measured as next explained. As indicated in Fig. VI.10, the maximum of the PVPdye peak was first located, and the area of the left half of this peak was taken as APVPD/2. Then, as indicated in Fig. VI.11 for the experiments performed at 3.1 q 4.4, the area at the right of the small dip of the asymmetric peak was assigned to the equilibrium concentration of the free dye, AD. Finally, the area of the exponential region, Ae, was calculated as the total area minus (APVPD + AD). These areas were used below in sections VI.2.1.5 and VI.2.1.7 to estimate the maximal stoichiometry and average stability constant of the PVPdye complexes, respectively.
VI.2.1.4. Influence of MW on the location and shape of the peak of the PVP dye complexes As deduced from the electropherograms of Figs. VI.10 and VI.11, when q < 4 (the dye was in excess in relation to the saturation point), the mobility of the PVPdye complexes was independent of the molecular mass of the polymer. This agreed with the model of repeating units of polymer binding individual ions. A single peak, at a migration time independent from the polymer molecular mass should be obtained for sufficiently long polyelectrolytes; further, differences in the mobility of large ionic polymers due to different MW values should be obtained only upon addition of a sieving medium to the BGE [Cottet, 2005]. As shown below in section VI.2.1.8, the peak areas of the complexes increased linearly with the monomer concentration, but were essentially independent of MW. However, as observed in Figs. VI.11, the shape of the peak of the PVPdye complexes varied with MW. In fact, the height/width ratio of the peaks increased with both MW and the PVP concentration. Thus, a method to
226
estimate MW was immediately derived. In Fig. VI.12, part B, the logarithm of the height/width ratio (in mAU min1, and estimated as indicated in Fig. VI.12, part A) divided by the PVP concentration (CPVP in g mL1) was plotted against log MW.
40
A
Absorbance (mAU)
0 2 -0.5
w1/2 4 Time (min)
B
log (h/w) - log CPVP
-1.5
1.2 2.2 3.1 3.6 4
-2.5 4 4.5 5 log MW 5.5
Fig. VI.12. Logarithmic plot of (h/w)/CPVP against MW; h and w are the height and base width of the peak of the PVPCR complexes, which were measured as indicated in part A (w =2w1/2). The units used were mAU, min and mg mL1 for h, w and CPVP, respectively. The dashed lines indicate confidence limits for a significance of 0.05. The legend in part B indicates the q values of the series. Other details as in Fig. VI.11.
This plot is useful to predict MW using electropherograms obtained at q < 4 (excess dye in relation to the saturation point). These predictions should be particularly accurate at low MW values, where both a high slope of the curve and
227
a reduced dispersion of the points are observed. This relationship between peak shape and MW could be due to the increase in the stability of the complexes as MW increased. However, another possible reason could be an increase in the polydispersity of the PVPdye complexes as MW decreased.
VI.2.1.5. Determination of the maximal stoichiometry of the PVPdye complexes To estimate the maximal stoichiometry of the PVPdye complexes, the dye concentration was increased while the PVP concentration was maintained constant. Then, the APVPD values and the remaining area, (Ae + AD), were plotted against the dye/monomer molar ratio (1/q). The plot corresponding to CR and 60 kDa PVP is shown in Fig. VI.13, part A. The peak area of the PVPCR complexes increased until reaching an approximately constant value at which saturation of the polymer with the dye was produced. On the other hand, the remaining area, (Ae + AD), increased only slightly at low values of 1/q, and increased steeply after saturation. This agreed with the complexation of all the available dye by the polymer at large q values, whereas the dye added to the solution over the saturation point remained free. As shown in Fig. VI.13, part B, the absolute mobility of the PVPCR complexes increased as the dye concentration in the complexes increased, reaching a constant value immediately after the saturation point. A higher absolute mobility of the complexes should be attributed to the increase of their charge density as a result of the increasing dye polymer ratios. Mixtures of CR with PVP, and AB with PVP, at all the available values of MW, gave plots which were closely similar to those shown in Fig. VI.13, parts A and B. In all cases, the increase in both the peak area and mobility of the PVPCR and PVPAB complexes pointed out to q 4.0 0.5 at the saturation point (1/q 0.25 0.04 in Fig. VI.13).
228

300 Relative A PVP-CR values 1000 Relative Ae + AD values
A
200 500 100 60 kDa PVP 0 3 0
B
(-108) (m2 s-1 V-1) 2 27.9 kDa PVP 60 kDa PVP 400 kDa PVP
0 0 0.2 0.4 0.6 1/q (reverse of the monomer/ dye ratio)
Fig. VI.13. Area (APVPD, rhombus and continuous line) (A) and electrophoretic mobility (B) of the peak of the PVPCR complexes at increasing CR concentrations, keeping a constant monomer concentration of 4.5 mM. In A, the squares and dashed lines correspond to Ae + AD (right scale). The MW values are indicated on the plots. The electropherograms were recorded at 500 nm.
Next, the PVP concentration was increased, while the dye concentration was maintained constant. As shown in Fig. VI.14, part A for the CR complexes with 60 kDa PVP, the peak area of the complexes increased at increasing q values. This plot, and similar plots obtained with PVP having other MW values, as well as by using AB, also indicated the formation of a complex with a maximal monomer: dye molar ratio of q 4. A difference with respect to Fig. VI.13, part A, was the linearity of the relationship between APVPD and the PVP concentration, at least up to q = 3.5. Linearity was attributed to saturation of the
229
polymer in the presence of an excess dye in this region of the curves. As shown later in this work, this was of interest for the CE determination of the polymer. Another feature of Fig. VI.14, part A, which was not observed in Fig. VI.13, part A, was an abrupt increase of the peak area of the PVPCR complexes immediately before reaching the saturation point.
1200
Relative APVP-CR values Relative Ae + AD values
800 1000
A
400 60 kDa PVP 0 3.0 0
(-108) (m2 s-1 V-1)
2.5
27.9 kDa PVP 60 kDa PVP 400 kDa PVP
2.0 0 2 4 6 q (monomer/ dye molar ratio)

Fig. VI.14. Area (APVPD, rhombus and continuous lines) (A) and electrophoretic mobility (B) of the peak of the PVPCR complexes at increasing PVP concentrations, and at a constant CR concentration of 4 mM. Other details as in Fig. VI.13.
This could be due to a higher stability of the complexes in the vicinity of the saturation point, where a more favourable conformation could exist; however, a slower dissociation rate of the complexes could also contribute. As also observed in Fig. VI.14, part A, the remaining area, (Ae + AD), decreased at increasing PVP
230
concentrations, approaching zero when q > 4, as expected from the increasing amount of complexed dye. In Fig. VI.14, part B, the variation in the electrophoretic mobility of the PVPCR complexes at increasing PVP concentrations while maintaining a constant CR concentration (increasing q values), was plotted. At q values well below the saturation point, the mobility of the PVPCR complexes did not vary, showing a perturbation (a local decrease of the absolute mobility) when the saturation point was approached. Finally, the absolute mobility of the complexes decreased steadily in the presence of an excess PVP. Therefore, Fig. VI.14, part B also suggested a conformation change of the PVPCR complexes in the vicinity of the saturation point. Extrapolation of the approximately linear regions at low and high values of q pointed out to values of the saturation point close to q = 4. As also shown in Fig. VI.14, part B, the absolute mobility of the PVPCR complexes when q > 4 decreased with a higher absolute slope as greater was MW. This agreed with the curves of Fig. VI.13, part B, where at 1/q values approaching zero, lower absolute slopes as higher was MW were observed. Series of experiments performed with either CR or AB and PVP samples at all the available values of MW, led to plots closely similar to those shown in Fig. VI.14, parts A and B. Both the peak areas and the mobility of the PVPAB complexes also indicated a saturation point close to q = 4 for this dye.
VI.2.1.6. Influence of MW on the mobility of the PVPCR complexes at low q values The relationship between the initial slopes of the curves in Fig. VI.13, part B (variation of the absolute mobility of the complexes vs. increasing values of 1/q) and MW was studied. To obtain accurate values of the initial slopes of the curves, series of experiments constituted by five triplicated points per series at
231
5002000 g mL1 PVP, and at 1/q values equal to zero, 0.05 and 0.1 were performed. These series were monitored at 215 nm. The curves were fitted to the cubic equation, and for each curve, the initial slope was obtained as the regression coefficient of the first grade term. A plot of the initial slopes against log MW is shown in Fig. VI.15. Although with some dispersion, this plot showed a decrease in the initial slopes when log MW increased. The possible reasons of this behaviour are discussed next.
/ (1/q) at (1/q) 0
200
100
0 4 5 log MW 6
Fig. VI.15. Initial slopes of the curves in Fig. VI.13, part B (variation in the mobility of the complexes vs. 1/q) plotted against log MW. Data obtained as indicated in the text.
First, at a constant q value, and at increasing values of MW, a lower absolute mobility of the complexes could be produced by a lower amount of dye complexed by the polymer. However, attending to the peak area of the PVPdye complexes, the opposite behaviour was actually observed when
electropherograms at constant 1/q values were compared. In fact, the APVPD values indicated an increase in the degree of complex formation as MW increased, which agreed with the increased stability of the complexes at increasing MW values (section VI.2.1.7). Second, the surface area: volume ratio of the PVPCR
232
complexes should decrease as MW increases; however, this should lead to an increase in the absolute mobility of the complexes at increasing values of MW. Again, the opposite behaviour was actually observed in Figs. VI.13, part B and VI.15, thus suggesting that the absolute mobility of the complexes at low values of 1/q should be influenced by another factor. This factor could be the location of the bound dye ions closer to the surface of the complexes, which would increase their zeta potential. A higher zeta potential should be more important at low molecular masses, since the surface area:volume ratio of the complexes probably decreases as MW increases.
VI.2.1.7. Determination of PVPdye stability constants Although the NECEEM theory was developed to be applied to protein and DNA complexes with fluorescent probes, to adapt it to the CE of polymerdye mixtures with spectrophotometric detection is straightforward. Thus, the equilibrium concentration of free dye is proportional to AD:
[ D ] eq =
AD b D
(E.VI.1)
where bD is the optical path multiplied by the molar absorptivity of the dye. The equilibrium molar concentration of the complexes is dependent on APVPD and Ae as follows:
[ PVP D ] eq =
APVP D A + e b PVP D b D
(E.VI.2)
where PVPD is the average molar absorptivity of the complexes, which should be calculated with reference to the molar concentration of the complexed dye. This is equivalent to considering that a dye ion is complexed on a 1:1 basis by a group formed by a fixed number of monomers, complexing polymer units, or binding sites. In this way, the possible influence of a stoichiometry different from 1:1 is ruled out. The ratio of the two equilibrium molar concentrations is:
233
R=
[ PVP D ] eq [ D ] eq
APVP D ( D / PVP D ) + Ae AD
(E.VI.3)
The stability constant, Ka, is given by:

Ka = 1+ R [C ] 0 (1 + 1 R) [ D ] 0
(E.VI.4)
where [C]0 and [D]0 are the analytical molar concentrations of the complexing polymer units and the dye, respectively. Since the maximal stoichiometry of the PVP complexes with CR and AB is 4:1, we have:
[C ] 0 = [ PVP ] 0
N 4
(E.VI.5)
where N is the average number of monomers and [PVP]0 is the analytical concentration of the polymer in mol L1. To estimate Ka values, series of electropherograms obtained at q values ranging from 3.1 to 4.5, and at increasing MW values, were used. The values of APVPD, AD and Ae were measured as indicated in sections III.2.2.3 and VI.2.1.2. The D/PVPD ratio was measured at 500 nm; for this purpose, a conventional spectrophotometer was used (Section III.2.2.3). The values of log Ka are given in Table VI.2. From top to bottom in Table VI.2, it is deduced that log Ka varied with q, showing a maximum value at the saturation point (q = 4) and decreasing slightly at higher q values. It should be indicated that this variation could be partially due to systematic errors associated to the difficulty in estimating AD independently from Ae; however, the presence of the perturbations observed in Fig. VI.14, parts A and B, suggests that more stable complexes were actually formed in the vicinity of the saturation point than at other values of q.
234

Table VI.2. Stability constants, log Ka, for the PVPCR complexes using PVP with different molecular masses and at increasing q values. MW (kDa) q 3.1 3.6 4.0 4.5
*
10 4.350.05 5.010.05 5.040.03 4.670.08
27.9 4.290.01 4.940.12 4.350.01
40 4.330.01 5.040.05 5.23* 5.09*
60 4.300.01 5.220.03 5.430.12 4.990.11
360 4.300.01 4.170.71 4.730.14 4.820.15
400 4.450.02 5.460.07 5.660.06 5.230.11
n = 2; the other values are means with n = 3.
In addition, the log Ka values given in Table VI.2 also showed that the stability of the complexes when q = 4 increased with MW. Finally, it should be noted that owing to the Joule heating, the values in Table VI.2 were obtained at a capillary temperature which was actually higher than 25C [Evenhuis, 2009].
VI.2.1.8. Analytical applications The increase in the peak area of the PVPCR complexes at increasing PVP concentrations, when an excess dye is present (q < 4), was used to quantify the polymer. Calibration curves were constructed using either CR or AB and PVP standards at all the available MW values. As indicated in section III.2.2.4, the maximal PVP concentrations used were 1500 g mL1, which corresponded to a maximal value of q = 3.4 (at a safe distance from the saturation ratio). Linear calibrations were obtained in all cases (r2 > 0.98). As shown in Table VI.3, PVP samples with different MW values gave similar sensitivities, although for unknown reasons the 160 and 400 kDa PVP standards gave sensitivities higher than the other standards. Therefore, systematic errors can be occasionally produced by applying the proposed procedure when a PVP standard different from the PVP contained in the sample is used for calibration. In addition, the possible interference of an anionic surfactant was studied. For this purpose,
235
mixtures containing 1000 g mL1 60 kDa PVP and 4 mM of either CR or AB were injected both in the absence and presence of a 5% SDS. When SDS was present, the peak shape and area of the PVPCR complexes were not modified, but the peak area of the PVPAB complexes was reduced largely. In addition to electrostatic repulsion between the PVPdye complexes and SDS, the polar amino groups of CR could also contribute to the lack of matrix effect observed when SDS was present in the sample solution.
Table VI.3. Relative slopes of the external calibration curves obtained with PVP solutions of different MW values. MW (kDa) 10 27.9 40 60a 160 360 400
a)
CR 0.94 0.95 1.07 1.00 1.54 0.92 1.28
AB 0.87 0.96 1.01 1.00 1.57 0.93 2.00
Taken as reference.
PVP of 60 kDa is commonly used in the formulation of colour care cleaning products, thus the calibration curve constructed with this PVP and CR (r2 = 0.997) was used in the quantitation studies that followed. First, two detergent bases with the composition given in Table III.1 were spiked with 60 kDa PVP, mixed with CR as indicated in section III.2.2.4, and injected. An electropherogram of spiked detergent base II, recorded at 215 and 500 nm, is shown in Fig. VI.16. The interference of the peaks due to several sample components was removed by using 500 nm. Using external calibration, the found/expected PVP concentrations were 1.67/0.99% and 1.19/0.97% for detergent bases I and II (Table III.1), respectively. Using internal calibration
236
with two standard additions, the found/expected concentrations were 1.18/1.17% and 0.62/0.97%, respectively. Thus, the matrix effect due to the detergent components was partially reduced using internal calibration. Then, the curve of Fig. VI.12, part B was used to predict MW in the spiked samples. The values MW = 51 and 60 kDa were obtained for detergent bases I and II, respectively.
150 Abs. 500 nm (mAU) 20
Absorbance (mAU)
10
100
0 3.0 Time (min) 4.0
A
50
215 nm
B 500 nm
0 0 2 4
Time (min)
Fig. VI.16. Electropherograms of detergent base II spiked with ca. 1% 60 kDa PVP. Data obtained at 215 (A) and 500 nm (B). The inset shows the peak of the PVPCR complexes for a series of calibration points.
Several commercial cleaning products and pharmaceutical preparations were also analyzed. To reduce iodine, drops of an ascorbic acid aqueous solution were added to the topical antiseptic until decolouration. Then, aliquots of the samples were mixed with 4 mM CR and injected. The PVP concentrations, predicted by using external calibration with 60 kDa PVP as standard, are given in Table VI.4. The found values agree with the expected concentrations for colour care liquid cleaners, which may contain up to 1% PVP
[Prudhomme de Lodder,
237

2006].
The concentrations found for the cough syrup and the antibacterial tablet
were well within the usual ranges, namely 0.55% as binders in tablets and <5% for dispersing agents in syrups [Rowe, 2005]. Finally, the declared amount of PVP in the topical antiseptic was 10%. Then, the plot of Fig. VI.12, part B was used to predict the MW values of Table VI.4. Values below 35 kDa were predicted for the laundry cleaners. Owing to the interference of the surfactants, these values were probably lower than the actual values. A large value (MW = 400 kDa) was obtained for the cough syrup, which was attributed to the presence of the anionic azo-dye amaranth (E123). This dye could form also complexes with PVP with a mobility similar to that of the PVPCR complexes, thus contributing to the peak area. Finally, MW = 22 and 16 kDa were predicted for the antibacterial tablet and topical antiseptic, respectively. The actual MW values in these products are to be expected along wide ranges [Rowe, 2005].
Table VI.4. PVP concentrations predicted using external calibration with 60 kDa PVP and MW values estimated by applying the plot of Fig. VI.12, part B. MW (kDa) Laundry cleaner I Laundry cleaner II Laundry cleaner III Cough syrup Antibacterial tablet Topical antiseptic Concentration (%wt) 0.89 0.31 0.45 0.95 2.42 12.4 MW (kDa) 35 10 <10 400 22 16
238
VI.3. PVA
VI.3.1. Evaluation of MW and tacticity of PVA by NECEEM of a polymer and a dye A variety of nonaqueous and aqueous CE methods for the characterization of synthetic polyelectrolytes, including CZE, CGE and CIEF, have been described
[Clos, 1998; Grosche, 2000; Borisch, 2000; Engelhardt, 2004; Cottet, 2005].
Using CE, information about size, shape, surface charge and formation of intramolecular associates can be gained
[Engelhardt, 2004].
Further,
polyelectrolytes have been separated by CGE using solutions of nonionic polymers as sieving media
[Borisch, 2000; Engelhardt, 2004; Cottet, 2005; Welch,
2001; Starkweather, 2000; Cottet, 1997].
Models describing the electrophoretic

[Long,
mobility of polyampholytes in free solution CZE have been developed

1998],
and CZE has been also used to study both the polymerization degree and
the sulfonation rate of polyestyrenesulfonates [Cottet, 2000]. MECK has been used to characterize highly charged polysaccharides (heparins) polyacrylic acids
[Collet, 1996], [Stefansson, 1994],
and
as well as to study the synthesis progress and
composition of ionic copolymers [Aguilar, 2002]. However, the characterization of non-charged polymers using CE has been scarcely investigated. In this connection, polyethylene glycols have been analyzed by CZE previous derivatization with an anhydride, which provides chromophore groups and electrical charges at both polymer ends previous work
[Beneito-Cambra, 2009-A], [Wallingford, 1996; Barry, 1998].
In a
we described a CZE method to
characterize and evaluate PVP; for this purpose, we used the azo-dye CR which forms a charged and colored PVPCR complex. The electropherograms of PVP CR mixtures were interpreted at the light of the NECEEM theory, which was developed by Krylov et al.
[Berezovski, 2002; Krylov, 2003; Drabovich, 2006; Lin,
239

2008; Krylov, 2006; Krylov, 2007]
to obtain information about proteins and DNA
fragments using fluorescent markers. PVA is a synthetic polymer which is widely used for a variety of purposes within the fields of cosmetic, pharmaceutical and food technologies. Dependingonthe route of synthesis, PVA with a different tacticity is obtained. Tacticity or stereoregularity is a characteristic feature of those polymers which have repeating adjacent chiral centers along the main chain. Tacticity depends on the class percentages of pairs of adjacent monomers or diads. The two possible classes of diads are: m or meso (with the same orientation) and r or racemic (with opposite orientation). Accordingly, three classes of units constituted by three adjacent monomers or triads can exist: mm, mr and rr. The relative percentages of mm, mr and rr triads, established by 1H NMR, or preferably by 13C NMR, are normally used to evaluate PVA tacticity
Fukae, 2000; Wu, 1973]. [Snchez, 2000; Moritani, 1972; Wu, 1977;
As far as we know, CE methods for PVA characterization have not been reported, and the possibility of evaluating the tacticity of polymers using CE methods has not been investigated either. In this work, we have applied the NECEEM principles to the study of the electropherograms obtained by injecting PVACR mixtures. The formation of complexes between PVA and azo-dyes has been known for decades
Tsujimoto, 2002]. [Fraunenfelder, 1974; Ikkai, 1996; Atkin, 2001; Ikkai, 1994;
When excess borate was added to both the injected PVACR
mixtures and the BGE, the expected NECEEM pattern for a mixture of a noncharged macromolecule and a charged marker was obtained. Commercial PVA samples with different molecular masses, also differing in tacticity, were studied. The electropherograms of PVACR mixtures provided information about the electrophoretic mobility, maximal stoichiometry, thermodynamic stability constant and pseudo first-order dissociation rate constant of the PVACR
240
complex. These parameters were observed to depend on both molecular mass and tacticity of PVA.
VI.3.1.1. Effect of PVA on the UVVis absorption spectrum of CR The formation of PVACR complexes has been described
1974; Ikkai, 1996; Atkin, 2001; Ikkai, 1994; Tsujimoto, 2002]. [Fraunenfelder,
As indicated in section
III.2.3.3, the formation of the complexes was first studied by filling the capillary with PVACR mixtures containing 20 mM borax. As illustrated in Fig. VI.17 for the 49 kDa PVA sample, the UVVis spectrum of a 4 mM CR solution was largely modified when the mixture also contained increasing PVA
concentrations.
q=6 q=9 600 Absorbance (mAU)
400 q = 12 200 q=3 q=0
0 300 400 500 600 Wavelength (nm)

Fig. VI.17. UVVis absorption spectra obtained by filling the capillary with solutions containing 20 mM borax, 4 mM CR and the following PVA concentrations (49 kDa): 0, 12, 24, 36 and 48 mM (the resulting q values are indicated on the traces).
Thus, from q = [monomer]/[dye] = 0 to 6, the molar absorptivity at the maximum of the main absorption band increased in a ca. 30%, and a large bathochromic
241
shift of about 40 nm was also observed (from 479 to 519 nm). Similarly, an intensity increase of ca. 55% and a bathochromic shift of ca. 50 nm (from 489 to 539 nm) were observed with a conventional spectrophotometer when 0.4 mM PVA was added to a 0.1 mM CR solution (spectra not shown). This implies a major change in the physico-chemical environment of CR [Olsen, 1975]. The large modification of the CR spectrum could be partially due to replacing of water molecules attached to the polar locations of CR by the polar groups of the polymer; however, as discussed below in section VI.3.1.4, another possible reason is the stacking of dye ions in proximity to each other within the structure of the PVACR complex. An absorptivity decrease at all wavelengths was also observed when q > 6. This could be due to an increase of the refraction index of the solution or to the optical screening of the dye in the presence of a large polymer excess.
VI.3.1.2. Selection of working conditions In Fig. VI.18, traces A to D, electropherograms of mixtures of CR (4mM) and PVA (15 kDa, increasing concentrations) obtained in a BGE containing 25 mM borax are given. The injected mixtures were also equilibrated with borax; however, 20 mM borax was used to preserve sample stacking. Pre-equilibration with borax improved peak repeatability. In Fig. VI.18, traces E and F, electropherograms obtained by injecting 2.5 and 4 mM CR solutions, respectively, in the absence of PVA, are also shown. Truncation of the peak for the 4 mM CR solution was attributed to the local concentration increase produced by stacking, followed by precipitation of the dye. Truncation of this peak was not observed when PVA was present in the injected solution with q 1. According to the NECEEM theory, when a solution containing an uncolored macromolecule and a colored charged marker is injected into the capillary, two
242
peaks with a superimposed exponential decay region in the middle should be observed
[Berezovski, 2002; Krylov, 2003; Drabovich, 2006; Lin, 2008; Krylov, 2006;
Krylov, 2007].
Thus, in Fig. VI.18, traces A to D, the first band was attributed to
the PVACR complex, and the peak that followed was assumed to be contributed by both the initial CR excess in free form and the dye released by the complex during migration (contributing mainly to the left half of the peak). As q increased (from traces A to D), the area of the band due to the PVACR complex increased, and the area of the peak due to the free dye decreased. Above q 4 the electropherograms showed the single band of the complex, which progressively widened when q further increased (trace D). In addition, a BGE containing 20 mM Na2HPO4 (pH 10) instead of borax was tried. Mixtures containing 4 mM CR and 16 mM PVA (q = 4) were injected; however, a low intensity wide band due to the PVACR complex, and a large peak and exponential decay due to the free dye, were observed (electropherograms not shown). Thus, owing to the better shape and higher intensity of the complex band, the BGE containing a borax excess was preferred. The time required to equilibrate the mixtures before injection was studied. For this purpose, aliquots of a solution containing 20 mM borax, 4 mM CR and 16 mM PVA monomers (49 kDa, q = 4) were injected at regular time intervals after mixing the reagents. The area due to the remaining complex, APVA-D, was estimated as indicated in Fig. VI.18, traces A to D, by doubling the area of the left half of the complex band. The rest of the area, due to both the initial free dye and the dye released by the complex during migration, was measured as ATotalAPVA-D. A slow increase of APVA-D (ca. 8%), a decrease of the rest of the area (ca. 20%), and a small increase of the electrophoretic mobility of the complex (ca. 4%), were observed during the first 2 h after mixing the reagents (sequence of electropherograms with time, not shown). This suggested a slow
243
reorganization of the complex involving an increase of complex stability and compactness, or a decrease in its dissociation rate, or both processes at a time. Thus, to obtain reproducible electropherograms in the experiments that followed, sample injection was performed with a delay of ca. 23 h after mixing the reagents.
300 Absorbance at 500 nm (mAU) APVA-D / 2
A
200
EOF
q=1 q=2 q=3 q=9
B C
100
D E F
0 0 2
4 Time (min)
Fig. VI.18. Electropherograms of mixtures of CR (4 mM) and PVA (15 kDa, increasing concentrations) (A to D). The q values are monomer/dye molar ratios. The procedure used to calculate APVA-D (used to construct Fig. VI.19) is indicated on the traces. Traces E and F are electropherograms of 2.5 and 4 mM CR, respectively. The BGE contained 25 mM borax (pH 9), and all the injected solutions contained 20 mM borax.
VI.3.1.3. Maximal stoichiometry of the PVACR complex and its relationship with log MW and tacticity Series of electropherograms also obtained with 4 mM CR and increasing PVA concentrations (increasing q values) were used to estimate the saturation point or maximal stoichiometry of the PVACR complexes, qsat, for all the PVA
244
samples. As shown in Fig. VI.19 for the 15 kDa PVA sample, when q was increased the area of the PVACR complex, APVA-D, increased linearly up to q 4, and the rest of the total area decreased proportionally. When q 5, that is, above the saturation point, the peak due to the excess dye was not observed any longer, and APVA-D decreased steadily. This agreed with the decrease in the absorptivity of the complex which was observed by recording spectra at large q values (Fig. VI.17).
100 Atotal APVA-D
APVA-D
0 0 2 4 6 q = [monomer] / [CR] 8 10
Fig. VI.19. Relative areas obtained from electropherograms of a series of solutions containing 4 mM CR and increasing PVA (15 kDa) concentrations in the presence of 20 mM borax. Other conditions as in Fig. VI.18. The continuous and dashed lines join points corresponding to the area assigned to the PVACR complex (APVA-D, estimated as indicated in Fig. VI.18) and the rest of the area under the peaks, respectively.
The other PVA samples behaved similarly, although with differences in the location of the saturation point indicating the maximal stoichiometry of the complex. In order to establish the saturation point of PVA complexes, qsat, as accurately as possible, mixtures with q values close to the expected qsat values
245
were injected; however, repeatability of the electropherograms was poor when q qsat. Therefore, the qsat values used in the discussion that follows were established for the different PVA samples as the point where the linear extrapolation of the decrease of ATotalAPVA-D crossed zero (Fig. VI.19, dashed line). In Fig. VI.20 (full symbols), the qsat values obtained in this way for the PVA samples were plotted against log MW. As observed, H samples formed a clearly resolved group with respect to the M+L samples (see section III.2.3.3). Also, within each tacticity group, qsat decreased as log MW increased. Further, the upper and lower limits of the qsat range were similar for the H and M+L sample groups, starting at qsat 4.9 at a low molecular mass, and decreasing down to qsat 3.5 at a large molecular mass. The two L samples, which had very large molecular masses, gave both qsat values close to 3.5. Thus, qsat decreased from ca. 4.9 to ca. 3.5 at increasing molecular masses, but this variation took place at different log MW values depending on tacticity.
5.0
4.5 qsat and qsat,c
4.0
3.5 1.5 Log MW

Fig. VI.20. Plots of the maximal stoichiometry of the complex against log MW without (qsat, full symbols) and with correction for the acetyl percentage (qsat,c, empty symbols). Symbols indicating tacticity groups: H (diamonds), M (circles) and L (triangles).
2.0
246
Next, a correction of the qsat values, thus to take into account the different proportions of residual acetyl groups in the PVA samples was tried. The molar proportions of residual acetyl groups per mol of monomers, nac, for the PVA samples used in this work, were given in section III.2.3.1. In a PVA molecule, acetyl groups could reduce the number of OH groups which are actually available to link the dye ions. Thus, in nac 100% acetylated PVA, only (1nac)100% of the monomers would be available for bonding; however, this should not reduce the complexing capacity of PVA in an equivalent (1nac)100% factor, since acetylated monomers can also perform as nonbonding bridges between bonded monomers. Nevertheless, we studied next the effect which would have on qsat the extreme situation in which the complexing capacity of PVA would be reduced in a full (1nac)100% factor. Thus, the correction applied was: qsat,c = qsat/(1nac). As shown in Fig. VI.20 (empty symbols), full correction for the acetyl percentage essentially confirmed the conclusions obtained above using uncorrected qsat values concerning both the decrease of qsat at increasing molecular masses and the noticeable difference between the H and M+L tacticity groups.
VI.3.1.4. Structure of the complex When qsat 4.9, each dye ion could be bonded by a maximum of four and probably a minimum of two monomers (since CR is a symmetrical structure), leaving an average of 0.92.9 non-bonded monomers per dye ion, respectively. Similarly, when qsat 3.5, each dye ion could be bonded by a maximum of three and a minimum of two monomers, leaving an average of 0.51.5 non-bonded monomers per dye ion, respectively. In both cases, a small number of nonbonded monomers are available to perform as bridges between adjacent dye ions. Thus, in all the possible scenarios, the number of non-bonded monomers per dye
247
ion is very low, which suggests a proximity between the dye ions. As depicted in the temptative structure of Fig. VI.21, stacks of dye ions by pairs, or by groups constituted by a higher number of dye ions, could be formed. As proposed in this figure, it seems reasonably to stack the dye ions by alternating the sulfonate and amino groups, rather than putting together all the sulfonate groups at one side and all the amino groups at the other side of the stack. Also, from a geometrical point of view, it seems reasonably to link the PVA monomers with the alternated sulfonate and amino groups though hydrogen bonds. As indicated in the literature, the azo groups are also possible sites for hydrogen bonding
2001; Olsen, 1975.]; [Atkin,
however, below the saturation point, and owing to the low
stoichiometry, the number of available monomers per dye ion is rather short. Then, hydrogen bonding of the PVA monomers with both amino and azo groups, which are located in outer and inner sites of the dye ion, respectively, seems to be less likely than the structure proposed in Fig. VI.21 However, the probability of bonding the OH groups with azo groups should increase after the saturation point, when an excess of monomers are available. Dye stacking, resulting in a large absorptivity increase and blue shifting of the absorption maximum, has been described in solutions of plant pigments when certain ligands and Mg2+ are present [Ellestad, 2006]. Thus, dye stacking could explain both the low qsat values of Fig. VI.20 and the very large modification of the absorption spectrum when PVA is added to CR solutions (Fig. VI.17). Further, the distance between donoracceptor atoms in adjacent stacked dye ions should be constricted by the distances between pairs of adjacent OH groups along the PVA chain, and these later are longer for r than for m diads. Thus, in PVA samples having a larger proportion of r diads, adjacent dye ions could be stacked at longer distances from each other than in samples with larger proportions of m diads. As discussed below, this could also explain the
248
correlations found between other electrophoretic parameters and PVA tacticity as established by 13C NMR.
O O O O H H H O N N N S N H H O O S O OH H O H OS O N N N N H O H N H H O OO S N O N N H H N
H OH O S O
O H H O
O OO H
O H O-
O N H H H
H O
Fig. VI.21. Temptative structure for two adjacent PVACR complex units (with two stacked dye ions).
The complex is probably compelled to adopt a given structure when an excess of either dye ions or unbound monomers is present. This could explain the excellent reproducibility of the electropherograms when q < qsat or q > qsat, respectively. Therefore, poor reproducibility of the electropherograms when q qsat could be due to the different ways the dye ions could be arranged within the complex when the available monomers are either in a small defect or a small excess with respect to qsat.
249
VI.3.1.5. Influence of molecular mass and tacticity on the electrophoretic mobility of the complex Electropherograms obtained with PVA samples of increasing molecular mass, also corresponding to different tacticities, are shown in Fig. VI.22. These electropherograms were obtained with a small CR excess with respect to the saturation point. Thus, traces A and C, which correspond to complexes with qsat = 4.51 were obtained at q = 4, and the other traces, which correspond to complexes with qsat = 3.51 were obtained at q = 3. As further commented in section VI.3.1.6, this was important to distinguish the AD area from that of the exponential decay contribution, Ae. As observed in Fig. VI.22, the electropherograms of M+L samples (traces CE) showed a sharper complex band than that of the H samples (traces A and B). In addition, within each tacticity group, the electrophoretic mobility of the complex was reduced at increasing molecular mass (AB and CD pairs). The relationships among electrophoretic mobility of the complex formed in the presence of an excess dye, molecular mass and tacticity are better recognized in Fig. VI.23 (full symbols). Within each tacticity group, the absolute electrophoretic mobility decreased slightly at increasing log MW values. Therefore, electrophoretic mobilities indicated that charge density of the complexes decreased at increasing molecular masses. In addition, absolute mobility increased when the rr/mm ratio decreased between the H and M sample groups. Therefore, absolute mobility increased when the average distances between the OH groups of adjacent monomers decreased as a result of the reduction of the proportion of r diads, probably giving rise to an increase of complex compactness. In free solution, the electrophoretic mobility of polyelectrolytes with the same linear structure but with different molecular masses is very similar [Grosche,
2000; Long, 1998; Cottet, 2000].
This is due to the almost identical charge-to-
250
volume ratio which is achieved when units with a given charge-to-volume ratio are increasingly added to the polyelectrolyte. This electrophoretic behavior of polyelectrolytes has been called the free draining regime [Cottet, 2000].
APVA-D / 2 200
AD
EOF
4; 4.5 AD
Absorbance at 500 (mAU)
150
3; 3.5 A
D
C
100
4; 4.5
D
50
3; 3.5
AD
E
0
0 2
3; 3.5 4 6 Time (min) 8 10
Fig. VI.22. Electropherograms of PVACR mixtures containing 20 mM borax, 4 mM CR and the following PVA monomer concentrations: (A, C) 16 mM and (B, D, E) 12 mM. The molecular masses were: (A) 15, (B) 31, (C) 49, (D) 100 and (E) 205 kDa. Sample tacticities: H (A, B), M (C, D) and L (E). The numbers on the traces are q and qsat values (in italics). The expanded parts show how the AD areas were estimated; APVA-D values were estimated as indicated in trace A. Other conditions as in Fig. VI.18, traces AD.
251
60 - 105 (cm 2 V -1 s -1) 30
50
40 25 1.0 1.5 Log MW 2.0
Fig. VI.23. Electrophoretic mobility of the PVACR complex against log MW, without (full symbols, dotted lines and axis at the left) and with correction for the complex stoichiometry (empty symbols, dashed lines and axis at the right); correction was performed with both VD/Vm = 15 and 10. Symbols indicating tacticity groups: H (diamonds), M (circles) and L (triangles). Other conditions as in Fig. VI.22.
As occurs with polyelectrolytes, the mobility of the PVACR complexes could also reach a constant value at increasing molecular masses, not further dependent on the molecular mass of PVA. However, in the case of the PVACR complexes formed in the presence of an excess CR, to increase the molecular mass of the polymer is not the only factor to be taken into account for a free draining regime to be reached. At increasing molecular masses, both the stoichiometry and the apparent charge density of the saturated complex, qsat, should be also maintained constant. As discussed above in section VI.3.1.3, stoichiometry of the complex formed in an excess dye varied with both molecular mass and tacticity; however, to show if a free draining regime is approached at high molecular masses, a model taking into account the stoichiometry variations can be constructed. For this purpose, the concept of average electrophoretic mobility per complex unit, unit, can be used. This parameter was defined as the mobility due to a unit constituted by a single dye ion and the average number of monomers directly
252
- [qsat + (VD / Vm)] 104 (cm 2 V -1 s -1)
attached to that ion, plus the average share of monomers which are necessary to link the complexed dye ion to the neighboring complex units. The mobility per complex unit, uni, should be directly proportional to the charge of a dye ion, 2, and inversely proportional to the volume of the complex unit:
unit =
2f VD + qsatVm (E.VI.6)
where f is a coefficient of proportionality, VD is the volume of a dye ion bound to the polymer chain, and Vm is the average volume of a monomer in the complex. If both sides of equation E.VI.6 are multiplied by the electrophoretic mobility of the complex, , and the equation is reorganized, we have:
2f = [ qsat + (VD / Vm) ] Vm unit (E.VI.7)
Therefore, if a free draining regime would be reached at large molecular masses, the product [qsat+ (VD/Vm)] would also reach a constant value. Since the VD/Vm ratio was unknown, two approximations were used: first, VD/Vm was taken as the ratio of the molecular masses of the dye ion and the monomers, 651/44 15, and second, VD/Vm was taken as the ratio of the total number of C, H, O and N atoms involved, 68/7 10. These two values of VD/Vm are rough approximations; however, as shown below, the use of any of them led to essentially the same conclusions. In Fig. VI.23 (empty symbols), product [qsat+ (VD/Vm)] was plotted against log MW. As observed in this figure, the differences among tacticity groups were enhanced upon correction of the electrophoretic mobility of the complex by the effects of complex stoichiometry. This correction confirmed the independent reduction of the absolute mobility at increasing log MW values for the H and M+L tacticity groups. Therefore, both a higher molecular mass and a higher rr/mm ratio implied a larger volume of the monomerdye complex units, that is, a smaller packing density of the complex. This agreed with the longer distances between the OH groups in r diads compared to m diads. On the other hand, the
253
corrected mobilities of Fig. VI.23 also indicated that the complex did not reach a free draining regime at increasing molecular masses. This meant that coefficient f in equation E.VI.7 was not constant within any molecular mass range, that is, packing density of the complex decreased at increasing molecular masses for all the PVA samples used in this work.
VI.3.1.6. Stability and dissociation rate constants of the complex In Fig. VI.22, it can be also observed that the area of the peak due the free dye was larger for the H (A and B traces) than for the M+L (CE traces) tacticity groups. This indicated either a higher complex stability or a slower dissociation rate, or both features at a time, for the H group in comparison to the M+L groups. Thus, next the stability and dissociation rate constants of PVACR complexes were estimated. For this purpose, three areas are needed: (i) APVA-D, due to the PVACR complex; (ii) AD, due to the free dye present in the initial equilibrium conditions; and (iii) Ae, generated by the dye released by the complex during migration. In the electropherograms of Fig. VI.18, AD could not be distinguished from Ae. This was attributed to the use of q values far from the maximal stoichiometry of the complexes, qsat. Thus, the free dye concentration should be very low at large values of q, and conversely, at low q values, the large initial concentration of the free dye made also difficult to distinguish its contribution from that due to the dye released during complex migration. However, as shown in the expanded regions of the electropherograms of Fig. VI.22, at q values slightly lower than qsat, it was possible to distinguish between these two contributions. Therefore, the maximum of the PVACR complex peak was located, and as indicated in the same figure (trace A), the area of the left half of the band was taken as APVA-D/2. In this band half, the contribution to the area due to the dye released during migration was assumed to be negligible (since the free
254
dye migrates towards increasing migration times). Then, the area at the right of the first depression of the free dye peak (see the expanded parts in Fig. VI.22) was assigned to AD, and Ae was calculated as the total area minus the contributions of the complex and initially free dye: Ae = ATotal (APVA-D + AD). These assignments probably implied a systematic error in the estimation of AD, and therefore also in the calculation of the stability constants; however, this made possible to compare the constants obtained with the different PVA samples. To estimate stability constants, the equilibrium molar concentration of free dye, [D]eq, was obtained as:
[ D ] eq =
AD b D
(E.VI.8)
where bD is the optical path multiplied by the molar absorptivity of the dye. The equilibrium molar concentration of the complex is given by:
[ PVA D ] eq =
APVA D A + e b PVA D b D
(E.VI.9)
where PVA-D is the molar absorptivity of the complex. Instead of reasoning about complex formation in terms of qsat monomers per dye ion, calculations are much simpler if the formation of a 1:1 complex is assumed, being the ligand the complexing units formed by groups of qsat monomers. The stability constant, Ks, is then given by:
Ks = 1+ R [C ] 0 (1 + (1/ R)) [ D ] 0 (E.VI.10)
where [C]0 and [D]0 are the total analytical molar concentrations of the complexing polymer units and the dye, respectively, and where R is given by:
R=
[ PVA D ] eq [ D ] eq
APVA D ( D / PVA D ) + A e AD
(E.VI.11)
255
The value of [C]0 can be calculated as:
[C ] 0 =
n [ PVA] 0 q sat
(E.VI.12)
where n is the average number of monomers along the polymer chains, and where [PVA]0 is the total analytical molar concentration of the polymer in mol L1. The molar absorptivity ratio D/PVA-D = 0.77 was obtained with a spectrophotometer at 500 nm by measuring several PVACR mixtures with q values sligthly lower than the respective qsat values; in these conditions, the formation of a predominant complex with a 1:1 stoichiometry, in which the ligand was constituted by qsat monomers, was assumed. In Fig. VI.24, part A, the resulting values of log Ks for q = 2 and 3 (full and empty symbols, respectively) were plotted against log MW. Contrary to that observed for other CZE parameters, log Ks values did not show any significant trend concerning to either molecular mass or tacticity. The electropherograms with q = 2 used to obtain log Ks were further processed to estimate pseudo-first-order rate constants for the dissociation of the PVACR complexes. For this purpose, half-life measurements were made, and rate constants were estimated as k = ln2/t1/2, where t1/2 is the half-life. The exponential decay curve within the region close to the peak of the excess dye, where the contribution of the remaining PVACR complex was small, was used. Five measurements of the half-life were made by selecting 5 different starting points on the decay curve of each electropherogram of series of triplicated injections. The starting points were evenly spaced from each other in about 5 s. Estimations of k were obtained with low uncertainties (ca. 4.3%). In Fig. VI.24, part B, the average values of k were plotted against log MW. Within each tacticity group, the dissociation rate constant of the complex was reduced at increasing molecular masses; in addition, H samples showed differences with respect to the M+L samples.
256
5.0 4.0 3.0 2.0 1.0 5.0 4.0 3.0 2.0 1.0 1.0 1.5 Log MW 2.0
Fig. VI.24. Plots of (A) log Ks and (B) k vs. log MW. Data were calculated from electropherograms obtained with (A) q = 3 and 2 (full and empty symbols, respectively) and (B) q = 2. Symbols indicating tacticity groups: H (diamonds), M (circles) and L (triangles). Other conditions as in Fig. VI.22.
257
CHAPTER VII
ENZYMES
Chapter VII. Enzymes
VII.1. Intact enzymes by CE
VII.1.1. Identification of enzymes for the detergent industry by CZE of the intact proteins Today enzymes constitute important components of laundry cleaners, dishwashers and other cleaning products
[Novozymes, 2002].
Since the
introduction of Alcalase, an alkali-tolerant bacterial protease in 1963 [Novozymes,

2002],
the role of enzymes in cleaning products has changed from that of a minor
[Olsen, 1998].
additive to becoming a key ingredient
Enzymes for cleaning
products constitute the largest segment of the world market for industrial enzymes
[Novozymes, 2002].
Cleaning power enhancement by enzymes leads to a
reduction of washing times and temperatures, with the subsequent savings of water and energy. Further environmental advantages arise from the lower consumption of surfactants, hypochlorite and alkalis, with the additional benefit of a better care of fabrics during washing [Olsen, 1998]. In spite of their interest in the detergent industry, and in environmental and toxicological studies
[www.heraproject.com.],
identification and quantification
methods for enzymes in cleaning products have been scarcely investigated. Enzymes are commonly detected and quantified by monitoring the hydrolysis of a substrate, or by precipitation with an antiserum
Dunn, 1971; Kulkarni, 1999; Lorentz, 2000]. [Novozymes, 2002; Olsen, 1998;
Polyacrylamide gel electrophoresis has

[Deschreider, 1972].
been used to separate proteases in cleaners
Protein
characterization and quantification is frequently carried out by hydrolysis followed by chromatographic determination of the resulting amino acids [Phillips,
1983].
Amino acid profiles can be established by a variety of separation and
detection techniques, including GC previous derivatization with ethyl chloroformate

[Gimeno-Adelantado, 2002; De la Cruz-Caizares, 2004],
or with a
261
silylating reagent [Rampazzi, 2002], and a wide variety of HPLC methods [MolnrPerl, 2000; Molnr-Perl, 2001; Tcherkas, 2001-A; Tcherkas, 2001-B; Concha-Herrera, 2007; Hanczk, 2007; Chernobrovkin, 2007; Pereira, 2008].
The resulting amino acid

[Beneito-Cambra, 2008;
profiles have been used to predict the enzyme class

Beneito-Cambra, 2009-B].
However, amino acid analysis using hydrolysis followed
by either GC or HPLC requires long analysis times. Capillary electroseparation techniques have the advantages of being simple, fast and highly efficient. The application of these techniques to the analysis of peptides and proteins has been periodically reviewed
Dolnk, 2006; Dolnk, 2008; Cifuentes, 1997]. [Jmeian, 2009;
MECK has been used to analyze
Savinase (a protease) and various analogues in cultivation broth [Vinther, 1992-B], and both CZE and MEKC have been applied to the identification of serine protease analogues [Eriksen, 1996]. The aim of this work was to examine CZE of intact proteins as a means of classifying and identifying enzymes in industrial raw materials of the detergent industry. The four main enzyme classes used in cleaners were included in this study, i.e. proteases, amylases, lipases and cellulases. Charge and structural changes of proteins are largely dependent on pH; thus, in order to gather more information for enzyme identification, both a basic and an acid BGEs were used. An alkaline borate buffer of pH 9, also containing PVA as dynamic coating to hinder protein adsorption
[Ruiz-ngel, 2002],
was selected. On the other hand, an
acidic BGE containing urea, iminodiacetic acid as low conductive isoelectric buffer
[Righetti, 1997; Bossi, 1997; Righetti, 1998; Capelli, 1998; Stoyanov, 1997]
and
HEC to inhibit adsorption, was also used. Due to their much reduced conductivities, large concentrations of isoelectric buffers are compatible with high voltage gradients, thus further reducing adsorption and favouring high resolution within short migration times
Capelli, 1998]. [Righetti, 1997; Bossi, 1997; Righetti, 1998;
Further, the total amount of protein in the samples was established

262
by the Bradfords method. This made possible to use total peak areas of the electropherograms obtained with both BGEs to establish relative sensitivities, which were also useful for enzyme identification. The enzymes used in this work are indicated in Table III.2.
VII.1.1.1. CZE with a basic BGE With the basic BGE, most enzymes gave rise to a single predominant peak or a group of partially resolved intense peaks, which was frequently followed by several small peaks (Fig. VII.1, parts A to D).
A
Esp
B
400 Sta EOF 120
EOF
800
EOF
80 Lx 80
Cel
Eve 200 400 Dur Sav
40 40 EOF Ls Alc Ter 0 2 4 2 4 Time (min) 0 2 4 0 2 4
Car
End
Fig. VII.1. Electropherograms of enzyme brands in the basic BGE (pH 9.0): (A) proteases, (B) amylases, (C) lipases and (D) cellulases. Experimental conditions in text (sections III.3.1.3 and III.3.1.4).
For all proteases (Fig.VII.1, part A), the main peak was located within the cationic migration region in the vicinity of the electroosmotic flow (EOF) time (ca. 2.14 min). At pH = 9.0 charge density should be low for proteases (8.4 < pI < 11), which agrees with the cationic behaviour of the main peak. The other
263
enzymes (Fig.VII.1, parts B to D), with the exception of Sta, gave also an intense peak or a group of peaks within the anionic migration region. Sta gave two partially resolved peaks close to the EOF time, but within the cationic region. An amylase, Ter, and a lipase, Lx, showed two peaks of similar intensity, with baseline resolution, instead of a single predominant peak (Fig.VII.1, parts B and C). The presence of a second intense peak close to the EOF time made Ter to be easily distinguishable from its engineered variant Dur (Fig.VII.1, part B). Similarly, the sharp intense peak within the cationic region made Lx to be easily distinguishable from the other lipase, Ls (Fig.VII.1, part C). Also, some cellulases as Cel and End (Fig.VII.1, part D) showed a division of the main band into several partially resolved intense peaks. In addition to the main peaks, the electrophoretic profiles of most enzymes also provided several small peaks which can be useful as fingerprints for identification. Intra- and inter-day repeatabilities in this BGE were obtained by injecting a 0.01% Alc aqueous solution. Five injections per day during three consecutive days were performed. Intra- and inter-day migration times showed RSDs lower than 3.5 and 7.5%, respectively. More than 100 injections were performed without the need of replacing the capillary.
VII.1.1.2. CZE with an acid BGE In agreement with literature, an excellent current stability was observed in the acid BGE [Piergiovanni, 2005]. The EOF was also very low in this medium. As shown in Fig. VII.2, parts A to D, the electropherograms also showed several distinctive features according to both enzyme class and the nature of the particular enzyme. Thus, proteases gave a predominant peak together with several small peaks within the 5.5 6.5 migration time region (Fig. VII.2, part A). Further, Eve and Sav also gave an additional and quite characteristic intense
264
sharp peak at ca. 3.5 min. The presence of two large peaks in these two enzymes could be due to the occurrence of two predominant protein fractions in the raw material. It should be noted that Esp and Sav are two closely related proteases with well known three-dimensional structures, and a high degree of structural similarity
[Georgieva, 2001].
However, in spite of this structural similarity, these
two enzymes can be clearly distinguished by CZE in the acid BGE.
A
120 200 Esp
Sta
C
40
60
D
Cel
Dur Eve 80
Lx
40 Car
100
Sav 40
20 20 Ls Ter
End 0
Alc 0 4 6 0 4 6 Time (min) 0 4 6 4 6
Fig. VII.2. Electropherograms of enzyme brands in the acid BGE (apparent pH 3.1): (A) proteases, (B) amylases, (C) lipases and (D) cellulases. Experimental conditions in text (sections III.3.1.3 and III.3.1.4).
Concerning to amylases (Fig. VII.2, part B), Ter showed a single predominant peak, rather than the two large peaks which were observed in the acid BGE (Fig. VII.1, part B). As observed in the basic BGE (Fig. VII.1, part C), lipases also gave rather characteristic patterns in the acid BGE, with several partially resolved sharp peaks (Fig. VII.2, part C). Thus, both Lx and Ls were clearly distinguishable by CZE using either the basic or acid BGEs. Concerning to cellulases (Fig. VII.2, part D), Car gave two peaks, whereas Cel and End
265
gave a single asymmetric peak. Using Alc, migration time intra- and inter-day repeatabilities were below 2.7 and 5.3%, respectively. At least 120 injections were performed without the need of replacing the capillary. Therefore, both in the basic and acid BGEs, most enzymes gave characteristic patterns which allowed class identification, and in many cases the electropherograms also showed enough distinctive details to allow the safe identification of the individual enzyme within its class.
VII.1.1.3. Relative sensitivities and application to enzyme identification In order to establish relative sensitivities, the protein contents in the raw enzymes were previously measured by the Bradfords method. The protein concentrations found using BSA as reference are given in Table VII.1.
Table VII.1. Class, commercial name, protein content and relative sensitivities of the enzyme industrial concentrates used in this work. Relative sensitivityc) Basic Acid BGE BGE 1.0 1.0 1.8 2.8 1.5 1.9 0.8 1.0 1.2 0.8 1.1 1.5 4.9 1.7 1.0 1.4 1.1 0.3 0.9 1.2 0.7 1.0 1.4 0.4
Class
Commercial name Alcalase 2.5 L
Abbreviation Alc Sav Eve Esp Ter Dur Sta Ls Lx End Car Cel
Protein content (% BSA)b) 4.03 0.03 1.81 0.07 1.24 0.03 2.45 0.05 2.95 0.02 4.87 0.03 2.69 0.01 1.79 0.01 2.90 0.01 2.56 0.01 0.74 0.08 1.31 0.06
Protease
Savinase 16L, Type EX Everlase 16L, Type EX Esperase 8.0L Termamyl Ultra 300L
Amylase
Duramyl 300L, Type DX Stainzyme 12L
Lipase
Lipolase 100L, Type EX Lipex 100L Endolase 5000L
Cellulase
a)
Carezyme 4500L Celluzyme 0,7Ta)
Sample supplied as granular solid (the other samples were liquid concentrates) Estimated using the Bradfords assay (calibration curve with 9 points) c) Relative sensitivities for the total area of the electrophoretic peaks (respect to Alcalase 2.5L)
b)
266
Then, for each industrial enzyme a calibration curve was constructed. For this purpose, industrial enzymes were diluted with water at several concentration levels within the 0.2-20% v/v range, and aliquots were taken. Precipitation with acetone, re-dissolution and injection in the capillary using both the basic and acid BGEs were then performed. The plots of total peak area versus protein concentration were linear (r > 0.992). From these plots, relative sensitivities were established as follows:
S r = S x S Alc
(E.VII.1)
where Sx and SAlc are the slopes for the assayed enzyme and Alc which was used as reference, respectively. As shown in Table VII.1, the ranges were 0.8-4.9 and 0.3-1.7 for the basic and acid BGEs, respectively. The differences between relative sensitivities were small in a number of cases, but several enzymes gave rather characteristic values. Thus, Eve and Sav gave high sensitivities in the basic and acid media, respectively, Dur gave low sensitivities in both media, and Cel exhibited a very high sensitivity in the basic BGE but a very low one in the acid BGE. As also deduced from Table VII.1, the differences among relative sensitivities were predominantly due to the nature of the individual enzymes rather than to their respective classes.
VII.1.1.4. Influence of surfactants commonly used in the formulation of cleaning products The influence of some common surfactants, widely used in the formulation of cleaning products, on the migration times and areas of the electrophoretic peaks was examined. For this purpose, solutions of surfactants spiked with Alc were treated as indicated in section III.3.1.4. Using the basic BGE, the electrophoretic profile of the enzyme (Fig. VII.3, part A) was slightly modified in the presence of fatty alcohol ethoxylates (Dehydol LT-7) (Fig. VII.3,
267
part B); however, rather different profiles were obtained when the sample solution contained anionic surfactants (Fig. VII.3, parts C and D). Similar experiments were performed in the acid BGE. The influence of Dehydol LT-7 gave electropherograms similar to that of Fig. VII.2, part A for Alc; however, in the presence of anionic surfactants, a significant increase in the retention time of the enzyme peaks was observed (data not shown). Similar results were obtained for selected enzymes of the other three classes. Consequently, the proposed method for enzyme identification can be safely applied in the presence of nonionic surfactants, but will fail probably when anionic surfactants would be present at large concentrations. Work addressed to reduce the interference of anionic surfactants is in progress.
80
80
40
40
0 20
0 20
C
10 10
0 0 4 8
0 0 Time (min) 4 8
Fig. VII.3. Influence of surfactants on the electrophoretic profile of Alc (0.01%) in the basic BGE: (A) absence of surfactant, (B) 5% Dehydol LT7, (C) 5% LAS and (D) 5% LES. Other conditions as in Fig. VII.1.
268
VII.2. Classification of enzymes by MS
VII.2.1. Rapid classification of enzymes in cleaning products by hydrolysis, MS and LDA The first enzyme-containing detergent was commercialized in 1913; however, enzymes were little used for cleaning purposes until the introduction of Alcalase, an alkali-tolerant bacterial protease, in 1963
[Novozymes, 2002].
Since
then, the role of enzymes in cleaning products has changed from one of a minor additive to becoming a key ingredient [Olsen, 1998]. Cleaning power enhancement by enzymes leads to a reduction of washing times and temperatures, with the subsequent savings of water and energy. Further environmental advantages arise from the lower consumption of surfactants, hypochlorite and alkalis, with the additional benefit of a better care of fabrics during washing [Olsen, 1998]. Mainly proteases, amylases, lypases and cellulases are used in the formulation of modern cleaning products. Proteases are used to remove proteinrich soil stains, including blood and grass. Amylases remove starch-containing stains, and prevent starch from adhering to fabrics and dishes. Lypases help in removing fat and edible oil stains. Finally, cellulases are used to remove fuzz and pills (small balls of fabric) from cotton. In spite of the importance of these enzymes in the detergent industry, and in environmental and toxicological studies
[www.heraproject. com],
identification and quantification methods for enzymes in
cleaning products have scarcely been investigated. Enzymes are commonly detected and quantified by monitoring the hydrolysis of a substrate, or by precipitation with an antiserum
Kulkarni, 1999; Lorentz, 2000]. [Novozymes, 2002; Olsen, 1998; Dunn, 1971;
Enzymes of different classes differ in molecular structure, and they also have rather dissimilar amino acid profiles, which can be useful for enzyme
269
classification. After hydrolysis, the amino acid profile of the enzyme can be established by a variety of separation and detection techniques, including GC following previous derivatization with ethyl chloroformate
2002; De la Cruz-Caizares, 2004], [Gimeno-Adelantado, [Rampazzi, 2002],
or with a silylating reagent
and a variety of HPLC methods
[Molnr-Perl, 2000; Molnr-Perl, 2001; Tcherkas,
2001-A; Tcherkas, 2001-B; Garca Alvarez-Coque, 1989; Concha-Herrera, 2007; Hanczk, 2007; Chernobrovkin, 2007; Pereira, 2008].
Useful sample classification methods without previous chromatographic separation have been described by using MS. For this purpose, the samples have been directly infused into the mass spectrometer using an ESI
[Goodacre, 2002;
Gama-Melo, 2006; Ng, 2004; Peris-Vicente, 2005; Catharino, 2005] [Gmez-Ariza, 2006]
or an APPI
ion source. Amino acid profiles obtained by protein
hydrolysis have been used as fingerprints to classify protein-binding media used in works of art according to their biological sources
2003], [Peris-Vicente, 2005; Llet, [Lerma-Garca, 2007],
vegetable oils in accordance to their botanical origin

[Wang, 1998],
rice cultivars
and tea varieties

[Erbe, 2000].
[Alczar, 2007],
as well as to
authenticate high quality beer principal component analysis analysis

2007], [Poulli, 2005],
Multivariate algorithms including hierarchical cluster
[Goodacre, 2002; Poulli, 2005],
LDA
[Peris-Vicente, 2005; Gmez-Ariza, 2006; Lerma-Garca, [Yang, 2002]
partial least-squares
and ANN
[Garca-Gonzlez, 2004],
have
been used to treat the spectral data. The aim of this work was to develop a quick and straightforward method for enzyme identification and classification in cleaning products. For this purpose, the enzymes were isolated by precipitation with acetone, hydrolyzed, and the hydrolysates were directly infused in the ESI ion source of a mass spectrometer. After normalization, the ion abundances of the amino acids were used as predictors to construct LDA models for enzyme classification. The
270
procedure was validated by analyzing enzyme industrial concentrates and laundry products. The enzymes used in this work are indicated in Table III.2.
VII.2.1.1. Mass spectra and normalization of the variables After protein hydrolysis, the mass spectra of the enzyme industrial concentrates showed the [M+H]+ ions of the following amino acids: Gly (m/z 76.1), Ala (m/z 90.1), Ser (m/z 106.1), Pro (m/z 116.1), Val (m/z 118.1), Thr (m/z 120.1), Cys (m/z 122.2), Ile/Leu (single common ion at m/z 132.2), Asn (m/z 133.1), Asp (m/z 134.1), Lys (m/z 147.2), Glu (m/z 148.2), Met (m/z 150.2), His (m/z 156.2), Phe (m/z 166.2), Arg (m/z 175.2), Tyr (m/z 182.2) and Trp (m/z 205.5). A typical mass spectrum obtained from the hydrolysate of a protease is shown in Fig. VII.4.
1.2
Leu+Ile
Val
0.8
Relative abundance x 107
Gly Ala
Pro
Asn Asp Lys Glu Met His
Thr Cys
Tyr
0
Met
Ser
0.05
Gly Ala
0 80 120 160 m/z 200

Fig. VII.4. Typical ESI mass spectrum of the hydrolysate of a protease. The [M+H]+ ions of the amino acids used as variables in this study are indicated.
Ser
Cys
271
Trp
0.4
Phe
Arg
In order to reduce the variability associated with the total amount of protein recovered from the samples, normalized rather than absolute ion abundances were used. For this purpose, two normalization procedures were tried. In procedure A, the ion abundance of each amino acid was divided by the sum of the ion abundances of all the amino acids in the corresponding spectrum. In procedure B, the ion abundance of each amino acid was divided by each of the ion abundances of the other amino acids in the corresponding spectrum. In this way, and taking into account that 18 peaks were measured on each spectrum and that a pair of peaks should be considered only once, (1817)/2=153 nonredundant ion abundance ratios were obtained. The ion abundances of these 18 amino acids were either intermediate or low, but in all cases signal-to-noise ratios adequate for data analysis were obtained. Significant differences between the amino acid profiles given by different enzyme classes were observed. After normalization according to procedures A and B, the significance of several variables was checked using analysis of variance (ANOVA). Using 3 enzymes of each class 3 infusions of each, all the variables checked showed significances better than 0.05.
VII.2.1.2. Construction of LDA models Using the normalized variables, LDA models capable of classifying the samples according to the respective enzyme classes were constructed. Thus, training data set 1 was used in combination with normalization procedures A and B to constitute matrices M1A and M1B. Taking into account the 18 and 153 predictors generated by normalization procedures A and B, the dimensions of the M1A and M1B matrices were 36 18 and 36 153, respectively. Similarly, data set 2 was used to constitute matrices,M2A and M2B, whose dimensions were 72 18 and 72 153, respectively, and evaluation data set 3 was used to constitute
272
matrices, M3A and M3B, whose dimensions were 42 18 and 42 153, respectively. Using training matrix M1A, an LDA model with 12 variables (selected out of 18), which showed an excellent resolution between all the possible pairs of categories, was obtained. Using training matrix M1B, an LDA model with 15 variables (selected out of 153) was obtained. This latter model showed a slightly better W value (0.035) than the previous one (0.043). Then, matrices M2A and M2B, which were obtained by using detergent bases spiked with enzymes, were used to evaluate the models. With some exceptions, all the objects of matrices M2A and M2B were erroneously assigned to the protease category. This revealed a relevant sample matrix effect, which could be due to the modification of the ion suppression effect in the mass spectrometer. Ion suppression may depend on both the concentrations of the enzymes, which was much smaller in the spiked detergent bases than in the enzyme industrial concentrates, and the saline contents of the infused solutions, which could be higher in those obtained by enzyme precipitation from spiked detergent bases. Therefore, to take into account the sample matrix effect in the design of the training set, matrices M1A and M2A were jointly used. Analogously, matrices M1B and M2B were also jointly used to construct another LDA model. Both models yielded excellent resolution between all the pairs of categories; however, in comparison with the model obtained using normalization procedure A, procedure B led to a smaller number of predictors (18 instead of 20), and to a much lower value of W (0.019 instead of 0.047). Thus, model construction using normalization by procedure B was selected for further studies. The effect of requiring an entrance threshold of Fin=0.01 instead of 0.05 in the stepwise algorithm for variable selection was studied. With Fin=0.01, the four categories were also very well resolved, W increased only slightly (from 0.019 to
273
0.022), and the number of predictors selected from the 153 variables initially established decreased from 18 to 13. The predictors and their standardized coefficients for the three discriminant functions obtained by using Fin=0.05 and 0.01 are shown in Table VII.2. Using normalization procedure B, the ion abundance ratios of pairs of amino acids rather than the ion abundances of individual amino acids are used as predictors.
Table VII.2. Predictors selected and standardized coefficients of the LDA models constructed by jointly using training matrices M1B (enzyme industrial concentrates) and M2B (detergent bases spiked with enzyme industrial concentrates) with two different entrance thresholds. Selected variable Val/Met Val/Thr Leu+Ile/Met Phe/His Phe/Lys Phe/Arg Phe /Ser Phe/Trp Phe/Cys Phe/Pro Glu/His His/Arg Lys/Arg Lys/Trp Gly/Trp Ser/Thr Thr/Trp Tyr/Pro Phe/Thr f1 3.44 -2.78 -2.12 3.24 -6.78 -6.43 2.06 3.08 -1.63 1.78 -1.07 0.95 10.6 -8.26 -1.21 0.513 5.75 -0.158 Fin=0.05 f2 f3 6.34 -0.800 0.715 -0.744 0.805 12.1 4.47 0.849 -3.20 -4.76 -1.34 -1.70 -2.95 -17.0 15.5 0.846 -1.19 -4.06 -3.63 5.71 -0.22 0.92 3.74 -2.02 0.070 -0.261 -0.424 -2.07 3.69 -0.831 -5.19 2.02 0.426 0.088 -0.255 0.022 f1 1.92 0.295 -4.10 3.02 6.41 -1.67 -3.43 -0.927 1.85 -2.47 -5.17 2.42 2.14 Fin=0.01 f2 0.51 1.70 0.839 2.57 0.172 -0.247 -2.16 0.698 0.275 -0.545 -2.41 2.11 0.290 f3 5.82 -0.326 1.04 3.75 -2.72 -0.237 -0.572 -1.22 4.21 -0.254 -3.99 0.646 -0.002
This prevented us from assigning any particular relevance to the discriminant capability of any individual amino acid; however, the coefficients of Table VII.2
274
suggested that Phe was important in distinguishing enzyme classes, and that the pairs Lys/Arg, Lys/Trp, Phe/Lys and Phe/Arg, among others, were also particularly important.
30
Second discriminant function Third discriminant function
10
A
20 lipases
proteases lipases 0 cellulases
10 cellulases proteases amylases -10 -60 -40 -20 0 20 First discriminant function
-10 amylases
-20 -10
0 10 20 30 Second discriminant function
C
First discriminant function
20 0 -20 -40
lipases
proteases
amylases cellulases
30 20 10 0 -10 0
10
Fig. VII.5. Score plots on the planes of the first and second (A), and second and third discriminant functions (B), and on an oblique plane of the 3-D space defined by the three discriminant functions (C). Matrices M1B (industrial concentrates of enzymes selected for training) and M2B (two detergent bases spiked with the same industrial concentrates of enzymes) were jointly used to construct the LDA model. The predictors were selected using Fin=0.01.
275
As observed in Fig. VII.5, part A, the variance gathered by discriminant function 1 was mainly associated with the resolution between cellulases and the other categories, whereas discriminant function 2 was associated with the resolution between lipases and the rest of the categories. According to Fig. VII.5, part B, amylases were resolved with respect to the other categories along discriminant function 3. As illustrated in Fig. VII.5, part C by using a plane oblique to the three discriminant functions, all the possible pair of categories were very well resolved from each other.
VII.2.1.3. Evaluation of the prediction capability of the optimal LDA model The LDA model obtained by jointly using matrices M1B and M2B for training (with Fin=0.01) was used to predict the enzyme categories of the objects of the evaluation set (M3B matrix). The enzymes of all the samples (42 objects), including the two commercial laundry cleaners (6 objects), were correctly classified, with assignment probabilities higher than 98%.
VII.3. Classification of enzymes by HPLC-UV-Vis
VII.3.1. Enzyme class identification in cleaning products by hydrolysis followed by derivatization with o-phthaldialdehyde, HPLC and LDA Today, enzymes are important components ofmost laundry and dishwasher cleaners, spot removers and other household products [Olsen, 1998]. In fact, enzymes for cleaning products constitute the largest division of the world market for industrial enzymes
[Novozymes, 2002].
Using enzymes, substantial
reductions of washing times and temperatures, with the subsequent savings of water and energy are achieved. Further, the concentrations of surfactants and harsh chemicals as alkalis and strong oxidants are reduced
[Olsen, 1998],
with the
276
additional benefit of a better care of fabrics duringwashing. Mainly proteases, amylases, lypases and cellulases are used in the formulations. Proteases are used to remove protein-rich soil stains (as blood and grass), amylases are addressed to solubilise starch-containing stains, also preventing starch from adhering to fabrics and dishes, lypases help in removing fat and edible oil stains, and cellulases are used to remove fuzz and little balls of fibers from the surface of cotton fabrics. In spite of their interest in the detergent industry, and in environmental and toxicological studies
[www.heraproject.com],
identification and
quantificationmethods for enzymes in cleaning products have been scarcely investigated. Enzymes are commonly detected and quantified bymonitoring the hydrolysis of a substrate, or by precipitation with an antiserum
Novozymes, 2002; Dunn, 1971; Kulkarni, 1999; Lorentz, 2000]. [Olsen, 1998;
Enzymes of different classes largely differ in molecular structure, also having rather dissimilar amino acid profiles, which can be useful for enzyme class identification. After hydrolysis, the amino acid concentration profile of the enzyme can be established by a variety of analytical techniques, including gas chromatography previous derivatization with ethyl chloroformate
Adelantado, 2002; De la Cruz-Caizares, 2004], [Rampazzi, 2002], [Phillips, 1983], [Gimeno-
or with a silylating reagent
automated ion exchange chromatography previous hydrolysis

[Molnr-Perl, 2000; Molnr-Perl,
and a variety of HPLC methods
2001; Tcherkas, 2001-A; Tcherkas, 2001-B; Garca-lvarez-Coque, 1989; ConchaHerrera, 2007; Hanczk, 2007; Chernobrovkin, 2007; Pereira, 2008].
Among these,
RP-HPLC with pre-column derivatization using either phenylisothiocyanate or OPA, in the presence of a reagent containing an SH group, is most frequently used [Molnr-Perl, 2000]. Using OPA, isoindoles, which can be detected by either UVVis spectrophotometry
[Concha-Herrera, 2007],
fluorimetry
[Molnr-Perl, [Tcherkas,
2001; Tcherkas, 2001-B; Pereira, 2008] 2001-A],
or electrochemical techniques
are quickly and easily obtained. Among the SH-group-containing

277
additives, 3-mercaptopropionic acid, NAC

2004; Concha-Herrera, 2007]
[Molnr-Perl, 2001; Concha-Herrera, [Hanczk, 2007],
and ethanethiol
which yield rather
stable isoindoles, have been recommended. Amino acid concentration profiles of protein hydrolyzates have been used to classify protein-binding media used in works of art
[Llet, 2003; Peris-Vicente, 2005], [Wang, 1998].
vegetable oils
[Lerma-Garca, 2007],
and rice cultivars
The contents of free amino acids have been also and to authenticate high quality beer
[Llet, 2003]
used to classify tea varieties

[Erbe, 2000].
[Alczar, 2007]
Multivariate data treatment techniques, including ANN
and LDA
[Peris-Vicente, 2005; Lerma-Garca, 2007],
have been used to construct
the models for class prediction. In this work, we describe a method for the identification of the enzyme class in raw materials of the cleaning industry and household cleaners by HPLCUV-Vis detection. The enzymes are first precipitated with acetone and hydrolyzed with HCl, the resulting amino acids are derivatized with OPA in the presence of NAC, and their concentration profiles are established by RP-HPLC with UVVis detection. Then, either the normalized peak areas (divided by the sum of the peak areas of the chromatogram), or ratios of pairs of peak areas, are used as predictors in the construction of LDA models for enzyme class prediction. The enzymes used in this work are indicated in Table III.2.
VII.3.1.1. Optimization of OPANAC derivatization and chromatographic conditions The conditions for OPANAC derivatization, initially taken from literature
[Concha-Herrera, 2006],
were further optimized using hydrolyzates of
Alc. First, the OPA concentration was increased, while both an OPA/NAC molar ratio of 1:2 and an OPANAC/hydrolyzate ratio (v/v) of 10:1 were maintained at fixed values. The peak areas increased when the OPANAC concentration
278
increased from 2.5104M
[Concha-Herrera, 2006]
to 1.25102 M. Then, was
1.25102 M, close to the OPA solubility in water
[www.cas.org/SCIFINDER],
selected. Owing to the increase of the dilution factor, the peak areas were reduced to ca. 50% when the OPANAC volume was increased from 1 to 2mL, while maintaining an hydrolyzate volume of 100 L. The amino acid peak areas also decreased when the hydrolyzate volume was increased to 0.5 mL, which could be due to a reduction of the reaction yield at the decreasing pH of the mixture. Thus, further studies were performed with an OPANAC/hydrolyzate ratio of 10:1 (v/v). Under these conditions, the other enzymes of Table III.2, section III.3.1.1 also provided satisfactory peak intensities. To optimize the chromatographic separation of the amino acids, the multisegmented gradient of Concha-Herrera et al.
[Concha-Herrera, 2006]
was initially
used. This consisted of three linear steps where the ACN concentration was increased as follows: 518.5% (30 min), 18.522% (40 min) and 2227.5% (10 min). However, with this gradient a few peaks overlapped at long retention times. Then, careful trial-and-error optimization of the multi-segmented gradient was carried out in order to improve resolution between the critical peak pairs. With the gradient described in Table VII.3, and as illustrated in Fig. VII.6, all the amino acid peak pairs, except the Phe/Leu pair, were baseline resolved.
Table VII.3. Optimal multi-segmented gradient accomplished by mixing 5% (A) and 50% (B) ACN/water solutions, both buffered at pH 6.5 with 5mM sodium citrate/citric acid. Time (min) 0 28 36 42 50 55 A(%) 100 72.5 70 44.4 42.4 0 B(%) 0 27.5 30 55.6 57.6 100
279
Thr 120
Phe + Leu 30 Ala
Asp 80 Val Glu 40 Ser His 0 0 10 20 30 40 Time (min) 50 Arg Tyr Gly Ile Met 0 Lys 10 20
ACN %
Fig. VII.6. Chromatogram of a hydrolyzate of Alc showing the peaks of the isoindoles of the amino acids and the optimal multi-segmented gradient (dashed line and Table VII.3). Other conditions as indicated in section VII.3.1.1.
Also, the Ser peak, which was well resolved in the chromatogram of Fig. VII.6,was only partially resolved from an unidentified peak (see Fig. VII.6) in the chromatograms of a few other enzymes. Thus, to construct LDA models, the Ser peak was not used, and the Phe/Leu peak pair was jointly measured, 13 peak areas corresponding to 14 amino acids being then selected.
VII.3.1.2. Data matrices and construction and evaluation of the LDA models In order to reduce the variability associated to the total amount of protein recovered from the samples and to their hydrolysis, normalized rather than absolute values of the peak areas were used. For this purpose, two normalization procedures were tried. In procedure A, the area of each amino acid peak was divided by the sum of the areas of all the amino acid peaks of the chromatogram. In procedure B, the area of each amino acid peak was divided by each one of the
280
areas of the other 12 amino acid peaks; in this way, and taking into account that a pair of peaks should be considered only once, (1312)/2=78 non-redundant ratios of peak areas were obtained. To construct LDA models for enzyme class prediction, the training set was first constituted by the enzyme industrial concentrates indicated in Table III.2, section III.3.1.1. Three enzymes from each one of the four enzyme classes were selected. As indicated in section III.3.3.4, two aliquots of each enzyme were hydrolyzed and injected; however, only the average of the peak areas of the two injections was included in the training matrix. In this way, the internal variance of the categories was reduced, which was important to also reduce the number of variables selected by the stepwise algorithm during model construction. Therefore, the training matrices were initially constituted by 12 objects each (3 enzymes 4 enzyme classes 1 average of two hydrolyzates) and either 13 or 78 variables obtained according to normalization procedures A and B, respectively. The resulting LDA models were used to predict the enzyme class in the two detergent bases spiked with the four enzymes indicated in Table III.2. Data obtained from a total of 16 chromatograms were evaluated (4 enzyme industrial concentrates 2 detergent bases 2 hydrolyzed aliquots of each mixture). However, the two models showed a poor prediction capability (2530% of correct assignments for a 95% probability level) which was attributed to the matrix effect produced by the anionic surfactants and other components present in large concentrations in the detergent bases. Thus, in order to increase the prediction capability of the models, data obtained with the spiked detergent baseswere also included in the training set. Thus, the expanded training matrices had 20 objects (3 enzyme industrial concentrates plus 2 spiked detergent bases 4 enzyme classes 1 average of two hydrolyzates), and either 13 or 78 predictors as indicated. Then, the duplicate hydrolyzates of the enzyme industrial
281
concentrates of Table III.2 which were not included in the training set, plus the duplicated hydrolyzates of the spiked detergent bases and the two commercial cleaners, were used to construct the evaluation matrices. Thus, the two evaluation matrices had 58 objects each (19 industrial concentrates of enzymes plus 8 spiked detergent bases and 2 cleaning products 2 hydrolyzates each). Using normalization procedure A, an LDA model with 9 variables, showing an excellent resolution between all the possible pairs of categories (W = 0.032), was obtained. However, only a 30% of the samples of the evaluation setwere correctly classified by this model. On the other hand, using normalization procedure B, an LDA model with 8 variables, showing a slightly better value of W (0.019) than the previous model (0.032) was obtained. Further, all the 58 samples of the evaluation set were correctly classified, with assignment probabilities higher than 99%. The predictors and respective standardized coefficients of the three discriminant functions of this model are shown in Table VII.4. The use of ratios of areas of peak pairs as predictors, rather than individual peak areas, prevented from reliably assigning any particular relevance to the discriminant capability of individual amino acids.
Table VII.4. Predictors and their corresponding standardized coefficients of the optimal LDA model. Selected variables f1 f2 f3
Asp/Val Glu/His Glu/Ala His/Thr Thr/(Leu+Phe) Ala/Tyr Ala/(Leu+Phe) Gly/Ile
0.92 0.63 0.07 0.20 1.09 -0.45 -1.26 0.97
0.56 -0.76 1.04 -3.77 0.25 2.35 0.15 1.47
-0.55 1.76 0.82 2.29 1.46 -1.70 0.15 0.81
282
As observed in Fig. VII.7, part A, the variance gathered by discriminant function f1 was mainly associated to the resolution between the protease class and the rest of the classes (amylase+lipase+cellulase), whereas discriminant function f2 explained variance associated to the resolution of the amylase class with respect to the rest of the classes (protease+cellulase+lipase).
10 4 cellulases 2
A
f3
cellulases
f2
0 0 proteases -4 amylases -10 -8 -6 -4 -2 0 2 4 6 8 10 -6 lipases -2
amylases proteases
lipases 0 10
-8 -10
f1
f2
C
10
f2
0 proteases
cellulases lipases
amylases 4 2 0 -2 -4 -6 f
3
10 0
f1
Fig. VII.7. Score plots on the planes of the first and second (A), and second and third discriminant functions (B), and on an oblique plane of the 3D space defined by the three discriminant functions (C) of the LDA model of Table VII.4.
283
Finally, according to Fig. VII.7, part B, the lipase class was resolved with respect to the other classes (protease+cellulase+amylase) mainly along discriminant function f3. As illustrated in Fig. VII.7, part C by using a plane oblique to the three discriminant functions, all the possible pairs of classes were very well resolved. Then, the peak areas of the most stable amino acids (Ala, Arg, Asp, Glu, Gly, His, Leu, Lys and Phe) were exclusively used as predictors to construct an LDA model. In this case, the following four peak area ratios were selected by the stepwise algorithm: Glu/His, Glu/Ala, His/Ala, Ala/(Leu+Phe). This model gave W = 0.387, and the score plots along the discriminant functions showed well resolved classes. The resolution between classes was only slightly lower than that observed in Fig. VII.7 for the model obtained with the eight predictors of Table VII.4.
VII.4. Tryptic digests of enzymes, columns comparation
VII.4.1. Comparison of microparticulate and monolithic columns for RP-LC of tryptic digests of industrial enzymes in cleaning products Today enzymes are commonly used in cleaning product formulations, particularly in developed countries, with over half of all detergents presently available containing enzymes
[Olsen, 1998].
In fact, the detergent industry is the
largest single market for enzymes, constituting 25 - 30% of total sales in the enzyme market
[Novozymes, 2002].
Enzymes allow a reduction of washing times
and temperatures, jointly with lower consumptions of aggressive chemicals, which translates into additional environmental benefits and better care of fabrics during washing [Olsen, 1998; Novozymes, 2002]. An active research area within this field is the development of enzymes capable of maintaining their activity in
284
extreme temperatures and high pH values, and in the presence of chelate agents for calcium ions. For this purpose, several techniques (i.e. DNA technology) are used [Olsen, 1998; Novozymes, 2002]. Enzymes are used in small amounts (0.4 - 0.8% crude enzyme by weight) in most formulations [Novozymes, 2002], being by far proteases, lipases, amylases and cellulases the most commonly used enzyme classes. Proteases are used to remove protein-rich stains (including blood and grass), amylases are addressed to solubilize starch-containing stains, also preventing starch from adhering to fabrics and dishes, lipases help in removing fat and edible oil stains, and cellulases are used to remove fuzz and little balls of fibres from the surface of cotton fabrics. Analytical methods for enzyme identification and quantitation in raw materials and manufactured products are required in industrial quality control. Further, enzyme producers and their customers are also interested in investigating the enzyme market trends, which also requires analytical support. Finally, analytical methods for enzymes are also needed to assess their potential impact on water treatment plants, as well as to optimize plant operation. However, in spite of their interest in the detergent industry, and in environmental and toxicological studies
[www.heraproject.com],
analytical methods for enzymes
in cleaning products have rarely been investigated. Enzymes are commonly detected and quantified by monitoring the hydrolysis of a substrate, or by precipitation with an antiserum
Kulkarni, 1999; Lorentz, 2000]. [Olsen, 1998; Novozymes, 2002; Dunn, 1971;
Other methods are based on complete hydrolysis of
the protein into its constitutive amino acids followed by derivatization and separation/detection by GC [Gimeno-Adelantado, 2002; De la Cruz-Caizares, 2004;
Rampazzi, 2002]
or HPLC
[Molnr-Perl, 2000; Molnr-Perl, 2001; Tcherkas, 2001-A;
Tcherkas, 2001-B; Garca-lvarez-Coque, 1989; Concha-Herrera, 2007; Hanczk, 2007; Chernobrovkin, 2007; Pereira, 2008].
The resulting amino acid profiles have
285
been also used to predict the enzyme class [Beneito-Cambra, 2008; Beneito-Cambra,
2009-B];
however, much information about the nature of the protein is lost during
total hydrolysis. In proteomics, a common approach for protein analysis is the enzymatic digestion with trypsin. In this way, the resulting peptides, which are much more specific for protein identification than the amino acid profiles can be studied. After digestion, profiles are studied by HPLC-MS
Opiteck, 1997-B; Opiteck, 1998]. [Nilsson, 2000; Opiteck, 1997-A;
Although this routine is well-established in
proteomics, it has been not applied to industrial enzymes in detergents. Further, there is a need to develop highly efficient and/or fast analytical methods to assess the quality of peptides produced by biotechnological procedures in terms of identity, content and purity. Within this concern, several analytical strategies related to column technology have been developed in LC, including the use of monolithic supports
[Cabrera, 2004; Guiochon, 2007],
packed
columns with fused-core (or core-shell) sub-3 m particles

Marchetti, 2007-A; Marchetti, 2007-B],
[Cunliffe, 2007;
or with sub-2 m particles operating at ultra-
high pressure (UPLC) [Mazzeo, 2005; Guillarme, 2007]. In the present study, several commercial chromatographic supports (monolithic and particulate columns) are comparatively applied to the LC-UV analysis of enzymes of the different classes commonly used in the detergent industry. Using industrial concentrates of the raw enzymes, both the intact proteins and their tryptic digests were analyzed. A polymeric and a silica monolithic, and particulate columns, including classical and core-shell particle technology, were compared. For this purpose, and for each column, peak capacity, resolution and number of peaks were evaluated. The best column was also used to analyze tryptic digests of enzymes in spiked detergent bases and commercial cleaners.
286
VII.4.1.1. Study of intact enzymes and tryptic digests using a ProSwift polymeric monolithic column First, a commercial ProSwift RP polymeric monolithic column was applied to the monitoring of raw industrial concentrates of enzymes using intact proteins. This column has been employed for the analysis of proteins and peptides
[Rao, 2006; Causon, 2010-B].
The gradient elution conditions for intact
proteins were as follows: an isocratic step with 1% ACN for 2 min followed by a linear gradient up to 75 % ACN in 18 min.
120 Absorbance 80 40 0
150 100 50 0
Absorbance
40
40
20
20
0 5 10 15 20 Time (min)
0 5 10 15 20 Time (min)
Fig. VII.8. Chromatograms of intact enzymes using a ProSwift polymeric monolithic column: (A) protease (Everlase), (B) amylase (Duramyl), (C) lipase (Lipex) and (D) cellulase (Deterzyme). Elution conditions: isocratic with 1% ACN for 2 min followed by a linear gradient up to 75 % ACN in 18 min at 1 mL min-1.
As shown in Fig. VII.8, most enzymes gave rise to a single predominant peak; however, a comparison of enzymes of different classes showed a coelution of the main peaks for the following enzyme class pairs: proteases and cellulases (main
287
peak within the ca. 11-12 min range), and amylases and lipases (main peak within the ca. 13-14 min range). No improvement in the resolution between enzyme classes was achieved by modifying the elution conditions. Then, the chromatograms of the intact enzymes are useful to assess the quality of the protein for any enzyme class; however, chromatography of the intact enzymes did not provide the necessary information to unequivocally identify unknown enzymes. For this purpose, application of the tryptic digests of the enzymes was investigated.
120 Absorbance 80 40 0 80 5 10
15
B
Absorbance 40
0 10 20 30 40 Time (min)
Fig. VII.9. Chromatograms of a tryptic digest of BSA using a ProSwift polymeric monolithic column under different elution conditions: (A) as in Fig. 1, and (B) isocratic step with 1% ACN for 5 min followed by a linear gradient up to 40 % ACN in 58 min at 1 mL min-1.
50
In addition to the enzymes, BSA was also used as a reference protein to check the performance of the tryptic digestion, as well as a probe to optimize the
288
separation of the digests. Fig. VII.9, part A shows a chromatogram of a BSA digest obtained with the same elution gradient employed in Fig. VII.8 for intact proteins. A number of peptides, most of them partially resolved, were observed. Optimization of elution conditions to improve resolution was performed. Fig. VII.9, part B shows the best conditions achieved, selecting 50 min gradients as compromise between analysis time and peak resolution.
Absorbance
10
20
30
40
50 Time (min)
Fig. VII.10. Chromatograms of a tryptic digest of (A) a protease (Alcalase) and (B) a lipase (Lipolase) using the ProSwift polymeric monolithic column under elution conditions as in Fig. VII.9, part B.
According to literature, an increase in the column temperature can lead to an increase in efficiency of separations of tryptic digests
2010-A]. [Ruta, 2010; Causon,
Thus, a tryptic digest of BSA was chromatographed at several
temperatures (25, 40 and 60 C; chromatograms not shown). At 40 C, the
289
resolution between the weakly retained peptides decreased, whereas at 60 C a reduction of peak intensity, which could be due to peptide hydrolysis, was observed. Then, 25 C was selected for further studies. Under these conditions, tryptic digests of enzymes belonging to different classes were analyzed. The chromatograms of a protease and a lipase, showing rather different fingerprints are given in Fig. VII.10. A series of partially resolved peptides at retention times below 20 min, were observed for the protease, whereas, the lipase provided a wide range of satisfactorily resolved peptides. Also, acceptable separations were achieved with amylases and cellulases (chromatograms not shown); however, as evidenced, low efficiencies were obtained for the tryptic digests of the four classes of enzymes. Changes in gradient elution conditions did not lead to significant improvements in efficiency and resolution, then, other stationary phases were investigated.
VII.4.1.2. Study of tryptic digests of enzymes using microparticulate and silica monolithic columns Chromatographic supports with C18 particulate packings and a silica monolithic bed (see Table III.4) were investigated. Fig. VII.11 shows the chromatograms of a tryptic digest of a protease obtained with different columns and using the gradient elution conditions found in the previous section. Since the columns differed in their cross section, the flow rate was adapted to maintain a constant value of the average linear flow velocity. As evidenced, a satisfactory separation of the tryptic digest of the protease was achieved for all these columns, in contrast with that obtained for the same enzyme by using the ProSwift column (Fig. VII.10, part A).
290
*
20 Absorbance
*
20 Absorbance
* C D
0 0 20 Time (min) 40 0 20 Time (min) 40
Fig. VII.11. Chromatograms of a tryptic digest of a protease (Alcalase) obtained using different columns: (A) Chromolith, (B) Gemini 5 m, (C) Gemini 3 m and (D) Kinetex. The flow rate was 1.12, 1.5, 0.4 and 0.4 mL min-1, respectively. Other elution conditions as Fig. VII.9, part B. The asterisk indicates a blank peak i.e. a peak not originating from the enzyme analyte.
In order to evaluate the chromatographic performance of the columns, the number of resolved peaks, peak capacity (PC) and global resolution (RG) were obtained by selecting as representative probes a protease (Fig. VII.11) and a lipase. The PC was experimentally determined using the well-known equation
[Snyder, 1986.]:
PC = 1 + (t g / w)
(E.VII.1)
where w is the average peak width at 4 (13.4% of peak height) in time units (experimentally measured) and tg is the gradient time. The global resolution, RG, was also measured as the geometric mean of the resolution between the
291
consecutive peak pairs. To perform this evaluation unresolved peak pairs or with RS values 0.5 were excluded. Fig. VII.12 shows the number of peaks, PC and RG found for a protease and a lipase using the different columns. As it can be seen for the protease (Fig. VII.12 part A), the PC values increased in the following order: ProSwift < Gemini (5 m) < Chromolith ~ Gemini (3 m) < Kinetex, and a similar order was obtained for the lipase, providing in this case the Chromolith column higher PC values than the Gemini (3 m) column. A similar trend was observed for the number of peaks and RG; in both cases, the Kinetex column showed the best performance. The differences in number of peaks, PC and RG values among the columns are a consequence of differences in their morphological features. Comparing monolithic beds, the results obtained for complex tryptic digests for the ProSwift column suggest a larger globule/polymer skeleton diameter and/or bed inhomogeneity than for its silica counterpart (Chromolith Performance RP18), which translates into lower plate numbers and reduced peak capacities as well as, along with absence of mesoporous structure, into lower surface area and less retention of analytes. Silica monoliths show larger porosities than packed beds. Hence, they exhibit a higher permeability than packed columns. The silica skeleton in Chromolith is mesoporous and has mean thickness of around 1-3 m leading to a mass-transfer kinetics faster than that of packed columns with 5-m particles and comparable to those of columns packed with 34 m particles
[Guiochon, 2007],
in rough agreement with the results shown in Fig. VII.12.
The Kinetex column, packed with 2.6 m core-shell particles (porous shell 0.35 m, fused-core 1.9 m) showed a better performance than both Gemini columns, packed with conventional 5- or 3-m particles and monolithic supports.This can be explained by the particular characteristics of the Kinetex
292
packing, where the particles are constituted by porous outer layer surrounding a solid non-porous core [Snyder, 1979].
80
A
Number of peaks
60 40 20 0 400
Peak Capacity Log (Global Resolution)
300 200 100 0
C
20
10
Fig. VII.12. Evaluation of separation performance of tryptic digests of a protease (Alcalase, dotted bars) and a lipase (Lipolase, striped bars) using different columns: (A) number of peaks, (B) peak capacity and (C) log (global resolution). Elution conditions as indicated in Fig. VII.11.
293
*
Absorbance 40
0 Absorbance
*
20
Absorbance
0 40
0 0 20 40 Time (min)
Fig. VII.13. Chromatograms of tryptic digests of (A) an amylase (Purastar), (B) a lipase (Lipolase) and (C) a cellulase (Puradax) using the Kinetex column. Other details as in Fig. VII. 11, part D.
60
In comparison to conventional particle technology, efficiency is gained due to the faster mass transfer through the shorter diffusion distance provided by the porous layer. In addition, the PC values obtained for the Kinetex column were similar to those found in literature for other shell-core columns (Ascentis), and lower than those reported for columns packed with sub-2 m particles (Acquity, BEH C18) operating at much higher pressures (UHPLC technology)
[Ruta, 2010].
The
Kinetex column which showed the best chromatographic performance for tryptic digests, was chosen for further studies. Chromatograms of a protease, an amylase, a lipase and a cellulase are shown in Figs. VII.11, part D and Figs. VII.13, parts A to C, respectively.
294
VII.4.1.3. Influence of matrixes commonly used in cleaning product formulations In order to evaluate the feasibility of the proposed method to analyze enzymes in cleaning products, two detergent bases containing surfactants and other reagents commonly employed in the formulation of cleaning products (see composition in section III.3.4.4) were used. These solutions, spiked with different classes of enzymes, were treated as indicated in section III.3.4.4 and injected into the HPLC system. Fig. VII.14, part A shows a representative chromatogram of a tryptic digest resulting from detergent base II spiked with a protease (Alcalase). This profile was closely similar to those obtained for detergent base I spiked with the same enzyme (chromatogram not shown) and by directly digesting the raw enzyme (Fig. VII.11, part D). Similarly, detergent bases (I and II) spiked with other classes of enzymes also gave chromatograms closely resembling those obtained with the corresponding raw enzymes. Therefore, interference due to the matrix components used to prepare the detergent bases was not observed. The method was also tested with several commercial cleaning products containing mainly proteases, which constitute the most common enzymes used today by the detergent industry [Watson, 2006.]. Fig. VII.14, part B shows a chromatogram of a liquid detergent containing a protease (Everlase, as declared by the manufacturer Qumicas Oro, S.A.). Again, a closely similar profile was obtained for the tryptic digest of the industrial concentrate of this enzyme (chromatogram not shown). Other commercial household cleaners containing proteases of unknown origin were analyzed, and matrix interferences were not evidenced in any case.
295
*
Absorbance 40
*
Absorbance 40
0 0 20 40 Time (min)
Fig. VII.14. Chromatograms of tryptic digests of (A) detergent base II spiked with a protease (Alcalase) and (B) liquid detergent containing protease (Everlase). Other details as in Fig. VII. 11, part D.
60
296
CHAPTER VIII
MONOLITHIC COLUMNS
Chapter VIII. Monolithic Columns
VIII.1. Monolithic columns of lauryl methacrylate
VIII.1.1. Photo-polymerized LMA monolithic columns for CEC using LPO as initiator Polymer-based monolithic stationary phases have been developed over the past two decades as an alternative to particle-packed phases for CEC and HPLC. The advantages of these monolithic supports are very well documented
2005; Lmmerhofer, 2003], [Svec,
and include good efficiencies, simplicity of
manufacturing and low back-pressure in HPLC. These phases are usually prepared by in situ free-radical polymerization of a mixture containing one or more functional monomers, including a crosslinker, a porogenic solvent and an initiator. Heat
[Peters, 1997; De Vries, 2008; Peters, 1998-A; Yu, 2002; Eeltink, 2005] [Peters, 1998-A; Eeltink, 2005; Geiser, 2007; Ngola, 2001]
and UV irradiation
are the
most common ways of initiating polymerization, while other techniques, such as microwaves
[Faure, 2007],
-radiation
[Zhang, 2008; Sfrny, 2005],
electron beam
[Beiler, 2007] Mirapeix,
and chemical agents

Cant-Mirapeix,
[Bandari, 2007; Holdvendov, 2003; Cant2008-B]
2008-A;
have been scarcely employed.
Advantages of photo-initiation are speed and easy selection of polymerization regions by using masks, which is particularly important in relation to the manufacturing of microfluidic chips. Acrylate and methacrylate-based materials have been proved to be excellent as stationary phases, with outstanding chemical stability over a broad pH-range
[Peters, 1997; De Vries, 2008; Peters, 1998-A; Yu, 2002; Eeltink, 2005;
Sfrny, 2005; Beiler, 2007; Cant-Mirapeix, 2008-C; Throckmorton, 2002; DelaunayBertoncini, 2004; Augustin, 2006].
Several authors have described the preparation of

[Peters, 2008-C;
monoliths using UV irradiation, mostly in the presence of either AIBN

1998-A; Eeltink, 2005; Geiser, 2007; Ngola, 2001; Cant-Mirapeix,
299

Throckmorton, 2002; Delaunay-Bertoncini, 2004; Augustin, 2006]
or 2,2-dimethoxy-
2-phenylacetophenone [Augustin, 2008; Ro, 2004; Lee, 2004; Huo, 2007] as initiators. LPO, as other diacyl peroxides, constitutes a source of free-radicals, when decomposed by thermolysis, irradiation or activation by tertiary amines [Gu, 2007;
Redington, 1948; Sanchez, 1996; ODriscoll, 1960].
This compound has been
employed in the preparation of methyl methacrylate polymers by thermal initiation

[Denisov, 2003; Bevington, 2004],
and recently, we have described its use
in the preparation of LMA-based monolithic columns for CEC also by thermal polymerization
[Gao, 2005].
The monoliths obtained with this initiator provided
better permeability and a finer control of the pore size over the studied range of 1,4-butanediol/1-propanol ratio, than columns prepared with AIBN. This suggested that the combination of LPO and UV irradiation could also be an attractive way for the fast preparation of LMA-based monoliths. In this work, the preparation of LMA-based monolithic columns for CEC by UV irradiation using LPO as an initiator is described. In order to obtain satisfactory column performances, the composition of polymerization mixture (i.e. ratios of monomers/porogens and monomer/crosslinker, and the composition of the porogenic solvent) was optimized. SEM photographs were used to characterize the morphology of the resulting monoliths, and the CEC performance of different columns was evaluated by measuring the retention factors and efficiencies of test mixtures of non-charged solutes. Photopolymerized LMA stationary phases were compared with those prepared by thermal initiation. These columns were also compared with those prepared using the more common AIBN instead of LPO as an initiator.
300
VIII.1.1.1. Preparation and characterization of columns photo-initiated with LPO The conditions to prepare photo-polymerized LMA-based monoliths were adapted from our previous work, where CEC columns were thermally polymerized using LPO as initiator [Gao, 2005]. Initially, the selected composition of the polymerization mixture was 40 wt% monomers (59.8 wt% LMA, 39.9 wt% EDMA and 0.3 wt% META) and 60 wt% porogens (17 wt% 1,4-butanediol and 83 wt% 1-propanol) in the presence of a 0.3 wt% LPO. However, when a PAH test mixture was injected in this monolith (column C1), wide peaks of components with several overlappings (naphthalene/fluorene and
pyrene/benz[a]anthracene peak pairs) were evidenced (Fig. VIII.1). These peaks showed much lower efficiencies than those reported for a thermally polymerized LMA column [Gao, 2005].
60 1
Absorbance (mAU)
40 3 20 4 2 5+6 7 0 1 2 3 Time (min) 4
Fig. VIII.1. Electrochromatogram of a PAH test mixture obtained with an LMA-based monolithic column photo-polymerized with LPO (column C1 of Table VIII.1) and SEM photograph of the monolith (inset). CEC conditions: mobile phase, 80:20 v/v ACN/aqueous 5 mM Tris (pH = 8.0); applied voltage, 10 kV; UV detection at 254 nm. Peak identification: (1) thiourea, (2) naphthalene, (3) fluorene, (4) anthracene, (5) pyrene, (6) benz[a]anthracene and (7) benzo[k]fluoranthene.
301
It was attributed to that the photo-polymerized columns yielded monoliths with larger pores and globules compared with those initiated thermally, which is consistent with the literature
[Peters, 1998-A; Cant-Mirapeix, 2008-D].
This was
also corroborated by the SEM pictures, where the photo-polymerized column C1 showed larger flow-through pores and globule sizes (inset of Fig. VIII.1) than a thermally initiated column obtained with the same polymerization mixture
2005]. [Gao,
In order to improve the CEC properties of the photopolymerized LMA monoliths, the influence of the ratios of monomers/porogens and 1,4butanediol/1-propanol on the porosity and performance of the columns was first studied (Table VIII.1). For this purpose, SEM pictures were obtained, and a mixture of PAHs was used to measure retention and efficiency values (by giving the minimum plate height, Hmin obtained from van Deemter plots). The ratio of monomers/porogens was varied from 30:70 to 60:40 wt/wt at several 1,4butanediol percentages. For 17 wt% 1,4-butanediol and a 30:70 wt/wt ratio of monomers/porogens, the polymerization inside the capillaries was not produced. Stable monoliths were obtained with 40:60 and 50:50 ratios; however, for a 60:40 ratio and at all the studied 1,4-butanediol percentages, the bed permeability was significantly reduced, thus, leading to column blockage. At 17 wt% 1,4butanediol, when ratio of monomers/porogens was increased from 40:60 (column C1) to 50:50 (column C2), efficiency improved (see Table VIII.1); however the pyrene/benz[a]anthracene pair was still not resolved by column C2. As shown in Table VIII.1, the k-values increased from column C1 to column C2, while the flow rate (u) remained almost the same. The increase in k-values can be explained by the smaller globule structure of column C2 when compared with column C1, and therefore, the column C2 gave higher surface/column volume ratio.
302
Table VIII.1. Composition of the polymerization mixtures used for the preparation of photo-polymerized LMA-based monolithic
columns with LPO as initiator and their CEC properties
303 Chapter VIII. Monolithic Columns
Solvents: pore-forming solvents.
Weight percentages.
Flow rates and retention measured at 5 kV. Mobile phase, 80:20 v/v ACN:5 mM Tris (pH = 8.0)
On the other hand, the similar u-values suggested similar flow-through pore sizes for columns C1 and C2, which was consistent with their SEM pictures (data not shown). The similarity of the pore sizes could be due to the excessively large 1,4butanediol content employed in the preparation of these two monoliths.
Fig. VIII.2. SEM photographs of LMA monoliths photo-polymerized with LPO prepared with 10:90 wt/wt 1,4-butanediol/1-propanol, at several ratios of monomers/porogens (wt/wt): 30:70 (column C3) (A), 40:60 (column C4) (B) and 50:50 (column C5) (C). Part (D) shows a monolith prepared with a 40:60 wt/wt ratio of monomers/porogens and a 6:94 wt/wt ratio of 1,4-butanediol/1-propanol (column C7). Other details about the
composition of the columns are given in Table VIII.1.
The influence of the ratio of monomers/porogens on the CEC properties was also studied at 1,4-butanediol contents lower than 17 wt% (columns C3C8). As shown in Table VIII.1, at 10 and 6 wt% 1,4-butanediol in the porogenic mixture, and when the content of this mixture was reduced (from 30:70 to a 50:50 ratio of monomers/porogens), an increase in k- and a decrease in u-values
304
were observed. This trend was consistent with the SEM pictures of these monoliths. As a representative example, monoliths photo-polymerized with 10 wt% 1,4-butanediol showed a significant decrease in both flow-through pore and globule sizes when the ratio of monomers/porogens was increased from 30:70 to 50:50 (columns C3C5) (Fig. VIII.2, parts A to C).
A 20
Hmin (m)
10
0 0 60 B 0.5 1 1.5 2
Hmin (m)
40
20
0 0 0.5 1 u (mm s-1) 1.5
Fig. VIII.3. Van Deemter plots of LMA monolithic columns photopolymerized with LPO and using different polymerization mixtures (wt/wt ratios): (A) 40:60 monomers/porogens and 10:90 1,4-butanediol/1propanol (column C4); (B) 50:50 monomers/porogens and 6:94 1,4butanediol/1-propanol (column C8); other details about the composition of the columns are given in Table VIII.1. Compounds: () naphthalene, () anthracene, and () benzo[k]fluoranthene. CEC conditions: mobile phase, 80:20 v/v ACN: 5 mM Tris (pH=8.0); UV detection at 254 nm; injection, 5 kV for 3 s.
305
Regarding to the efficiency, at 10 and 6 wt% 1,4-butanediol, a 30:70 wt/wt ratio of monomers/porogens (columns C3 and C6) provided the worst Hmin values. The best efficiencies for 10 and 6 wt% 1,4-butanediol were achieved at ratios 40:60 (column C4) and 50:50 (column C8), respectively. Figure VIII.3 shows the van Deemter plots obtained for these columns for naphthalene, anthracene and benzo[k]-fluoranthene. The column C4 gave Hmin values comprised 8.911.1 m (at optimum u-values 0.650.67 mm/s), whereas for the column C8, the Hmin values ranged between 18.622.7 m (u-values 0.300.33 mm/s) (see Table VIII.1). Additionally, this latter monolith showed higher mass transfer contributions (C-term) (26.547 ms) than those obtained with the column C4 (6.813 ms). The separation of the PAH test mixture on columns prepared with these two monoliths was examined. In both cases, all the analytes were well resolved, giving the monolith prepared with a 40:60 ratio (column C4) the shortest analysis time (see Fig. VIII.4, parts A and B). Next, the influence of the content of 1,4-butanediol in the porogenic solvent was studied at a given ratio of monomers/porogens. At 40:60 wt/wt monomers/porogens, by decreasing the 1,4-butanediol from 17 (column C1) to 6 wt% (column C7), the flow rates did not change significantly, but the k-values progressively increased. This retention behavior was attributed to the reduction of the globule size, which was corroborated by the SEM pictures (see inset of Fig. VIII.1 and Fig. VIII.2, parts B and D for columns C1, C4 and C7, respectively). A reduction of the globule size produced by a reduction of the porogenic solvent content was also reported for LMA-based monoliths prepared by thermal polymerization in the presence of LPO as initiator
[Gao, 2005].
The
increase in retention was also observed at 50:50 wt/wt monomers/porogens, being more pronounced than 40:60 wt/wt monomers/porogens (see columns C2, C5, C8 compared with columns C1, C4, C7). At 50:50 wt/wt
306
monomers/porogens, with decreasing 1,4-butanediol, u decreased from column C2 to C5, for later did not change significantly for column C8. This behavior can be explained as follows. The reduction of 1,4-butanediol in polymerization mixtures prepared at 50:50 wt/wt monomers/porogens led to monoliths with small macropore and globule sizes, which translated in a reduction in u and increase in k-values. However, at 6 wt% 1,4-butanediol percentage (column C8), no substantial changes in u and an increase in k-values was observed. It can be explained by considering that this polymerization media produced a decrease in globule size and non-significant variations in macropore size. The influence of the 1,4-butanediol percentage in the mixture of porogenic solvents on the CEC separation performance was also evaluated. When the PAH test mixture was injected using columns prepared with a 40:60 wt/wt ratio of monomers/porogens, the decrease of the 1,4-butanediol percentage from 17 wt% (column C1, Fig. VIII.1) to 10 wt% (column C4, Fig. VIII.4, part A) led to an improvement of the resolution between all the successive analyte pairs. The column prepared with 6 wt% 1,4-butanediol (column C7, Fig. VIII.4, part C) also provided baseline resolution of analytes; however, it showed lower efficiency than the column C4. Thus, this monolithic bed, which represented the best compromise between resolution and analysis time, was selected for further studies. Changes in the monomer to crosslinker ratio have been proved to have significant effects on monolith porosity
Okay, 2000]. [Svec, 1995-A; Svec, 1995-B; Viklund, 1996;
Thus, the LMA:EDMA ratio was increased from 40:60 to 50:50,
60:40 and 70:30 wt/wt (columns C9, C4 and C10, respectively). When 40:60 was used the columns exhibited a large flow resistance, which caused column blockage.
307

30 1
Absorbance (mAU)
3 4
20 6 10 5 2 7
0 0 40
Absorbance (mAU)
2 1 4
6 B
30 20 10 0 0 60 4 8 4 40 12 16 C 2 3 5 6
Absorbance (mAU)
1 3
20 2 0 0 2 4 6 Time (min) 8 5 6 7
Fig. VIII.4. Electrochromatograms of a PAH test mixture obtained using LMA columns photo-polymerized with LPO and using different polymerization mixtures (wt/wt ratios): (A) 40:60 monomers/porogens and 10:90 1,4-butanediol/1-propanol (column C4); (B) 50:50
monomers/porogens and 6:94 1,4-butanediol/1-propanol (column C8); and (C) 40:60 monomers/porogens and 6:94 1,4-butanediol/1-propanol (column C7). Other details about the composition of the columns are given in Table VIII.1. CEC conditions and peak identification are as in Fig. VIII.1.
308
When LMA:EDMA ratio was increased from 50:50 (column C9) to 60:40 (column C4), a decrease in retention without any separation improvement was observed (see Table VIII.1). Finally, with 70:30 (column C10), overlapping of the naphthalene/fluorene and pyrene/benz[a]anthracene peak pairs was produced. This was consistent with the SEM pictures of these columns, which showed how the decrease of the crosslinker concentration from 60 to 30 wt% led to monolithic beds with larger globules (pictures not shown). This was in agreement with other studies reported in the literature, where a decrease in the crosslinker percentage was also accompanied by an increase in the average pore size [Svec, 1995-A; Svec,
1995-B; Okay, 2000].
This behavior can be explained as follows. When the
LMA/EDMA ratio was progressively increased from 50:50 to 70:30 wt/wt, the hydrophobicity of the monomer mixture was increased, which resulted in an earlier phase separation. The aggregates preferentially tended to swell with the LMA monomers than with porogenic solvent. Overall, the globules and voids formed in this system will be larger. It should translate in a reduction both in kvalues and efficiency along this series. However, similar Hmin values were observed from column C9 to C4, whereas as expected an increase in Hmin was observed for column C10. As shown in Table VIII.1, the LMA:EDMA ratio of 60:40 wt/wt (column C4) provided the best Hmin values for all the PAHs of the test mixture, thus being this ratio selected for the studies that followed. These efficiencies are comparable with the performance of microparticulate columns packed with 5 m silica particles [Vlakh, 2007; Eeltink, 2004]. Using LPO, the photo-polymerized LMA-based monolithic columns were also compared with those prepared by thermal polymerization. For this purpose, the 1,4-butanediol and porogenic solvent contents, which were found to be optimal for each polymerization processes were used. The monoliths of the two types showed similar efficiencies for naphthalene, i.e. Hmin
naphthalene
= 11.1 and
309
9.5 m for photo-polymerized and thermal initiated columns, respectively, but the former showed much lower k-values than the latter (kpyrene = 1.76 and 4.58, respectively). A comparison in terms of efficiency with monolithic columns made with either LMA or other similar bulk monomers (i.e. LA) was also performed. Regarding to LMA columns initiated thermally with AIBN, the Hmin values found were similar to the 915 m for alkyl benzenes (at optimum flow rates 0.51.0 mm/s) obtained by Buszewski et al.
[Buszewski, 2004].
Our efficiencies were also
comparable with the values of 513 m for PAH compounds (flow rates: 1.01.5 mm/s) obtained with photo-initiated LA monoliths with AIBN
[Geiser, 2007].
However, the plate heights achieved were slightly higher than those obtained for LA columns chemically initiated with LPO
[Buszewski, 2004],
where the Hmin
values for PAH compounds ranged from 2.6 to 5.3 m (flow rates: 0.51.5 mm/s).
Table VIII.2. Reproducibility of CEC properties of LMA-based monolithic columns photo-polymerized with LPOa Column-to-column (n=3) Parameter u (mm/s) kanthracene Hminb (m)
a
Batch-to-Batch (n=3) Mean 1.29 1.00 13.1 RSD (%) 5.4 6.3 6.0
Mean 1.25 1.08 11.0
RSD (%) 4.5 2.8 2.1
Polymerization mixtures prepared with 40 wt% monomers (59.8 wt% LMA, 39.9 wt% EDMA and 0.3 wt% META) and 60 wt% porogens (10 wt% 1,4-butanediol and 90 wt% 1-propanol); LPO, 0.3 wt%. Applied voltage, 15 kV; other conditions as in Fig. VIII.2.
Hmin values obtained for naphthalene.
To estimate the reproducibility of photo-polymerized columns initiated with LPO, three independent batches of three columns each were prepared. As it
310
can be observed in Table VIII.2, satisfactory reproducibilities were achieved for all the tested parameters, with RSD values comprised between 2.1 and 4.5% for column-to-column, and between 5.4 and 6.3% for batch-to-batch. These reproducibilities were similar to those reported for thermally polymerized monoliths using also LPO as initiator (<5.3%) [Gao, 2005].
VIII.1.1.2. Comparison of LPO and AIBN as initiators for photopolymerization Since AIBN is used as initiator for UV polymerization in most of the studies reported in the literature, the CEC performance of columns photopolymerized using either LPO or AIBN was next compared. The features of columns prepared by using the same ratios of monomers/porogens (40:60 wt/wt) and LMA/EDMA (60:40 wt/wt), and the same 1,4-butanediol range in the porogenic solvent (176 wt%), are shown in Tables VIII.1 and VIII.3. Using PAHs for testing, the columns photo-initiated with AIBN at 17 wt% 1,4butanediol in the porogenic solvent showed better efficiencies than the corresponding columns obtained using LPO (column C1, Table VIII.1). At 6 wt% 1,4-butanediol, the columns polymerized with AIBN gave slightly better efficiencies (see Table VIII.3 and column C7, Table VIII.1) and comparable separations were achieved for both initiators (data not shown). As deduced from Table VIII.3, the optimal efficiency for the monoliths photo-initiated with AIBN was found at 10 wt% 1,4-butanediol in the porogenic mixture. Figure VIII.5 shows the electrochromatogram obtained with the column that provided the best Hmin using AIBN. As it can be observed, the solutes of the PAH test mixture were resolved at 10 kV with similar peak efficiencies as those obtained with the optimal monolith initiated with LPO (see Fig. VIII.4, part A), but within a longer analysis time.
311

Table VIII.3. CEC properties of LMA-based monolithic columns prepared from mixtures with different 1,4-butanediol/1-propanol ratios using AIBN as initiatora)
a)
The polymerization mixtures contained 40:60 wt/wt monomers/porogens and 60:40 LMA/EDMA. b) Weight percentages. c) Flow rates and retention measured at 5 kV. Mobile phase, 80:20 v/v ACN: 5 mM Tris (pH = 8.0).
4 80
Absorbance (mAU)
40
1 3 6 2 5 7
0 2 4 6 8 Time (min)
Fig. VIII.5. Electrochromatogram of a PAH test mixture obtained with an LMA-based monolithic column photo-polymerized with AIBN, and SEM photograph of the monolith (inset). Polymerization mixture: 40 wt% monomers (59.8 wt% LMA, 39.9 wt% EDMA and 0.3 wt% META) and 60 wt% porogens (10 wt% 1,4-butanediol and 90 wt% 1-propanol). CEC conditions and peak identification are as in Fig. VIII.1.
10
As shown in Tables VIII.1 and VIII.3 for LPO and AIBN, respectively, at the optimal ratios of monomers/porogens (40:60 wt/wt) and LMA/EDMA
312
(60:40 wt/wt), when the 1,4-butanediol contents was decreased from 17 to 6 wt%, the u-values did not varied significantly. As also shown in these tables, the LPO photo-initiated monoliths gave higher u-values than those initiated with AIBN. Concerning to retention, for 10 and 17 wt% 1,4-butanediol, the monoliths photo-polymerized with AIBN gave higher k-values than those obtained with LPO. This could be explained by the presence of a higher number of micropores in the globule structure of the AIBN columns, and consequently, by a higher surface area. This was confirmed by the SEM pictures, which showed smaller globules in beds made with AIBN than in those prepared with LPO (see for instance inset of Figs. VIII.5 and VIII.2, part B for columns prepared with 10 wt% 1,4-butanediol). The performance of these monolithic stationary phases as RP packings was also evaluated using alkyl benzenes and a mixture of basic compounds (Figs. VIII.6 and VIII.7, respectively). From Fig. VIII.6, it can be seen that the column initiated with LPO showed a fast baseline separation of alkyl benzenes in less than 4 min. Similar theoretical plates (7.118.3 m) were achieved with this monolithic column than the monolithic column initiated with AIBN, which yielded 8.216.4 m for a separation under 6 min. The analysis of basic compounds in HPLC commonly undergoes of peak broadening and tailing due to the interaction of these compounds with residual silanol groups on RP columns
[Cant-Mirapeix, 2009-B].
Figure VIII.7, part A
shows the separation of five basic compounds in less than 12 min with column efficiency of up to 20 m, whereas the column initiated with AIBN (Fig. VIII.7, part B) as in Fig. VIII.6 showed longer analysis times with theoretical plates ca. 24 m. Similar efficiencies were achieved for RP columns, in particular XTerra RP18 and XBridge C18, two columns with low-silanophilic activity
2003], [McCalley,
with theoretical height plates ranged between 1625 m. In any case, it
313
can be observed that symmetric peaks were obtained in CEC due to suppressed electrostatic interaction between the neutral solutes and the positively charged monolithic surface, then; these stationary phases were suitable for the analysis of basic compounds.
3 4
Absorbance (mAU)
5 1
0 3
Absorbance (mAU)
3 2
7 5 1
1 0 0 1
5 6 Time (min)
Fig. VIII.6. Electrochromatograms of a mixture of alkylbenzenes obtained by using LMA monoliths photo-polymerized with LPO (A) and AIBN (B) as initiator. The composition of the polymerization mixtures is as in Figs. VIII.4, part A and VIII.5, respectively. CEC conditions: mobile phase, 70:30 v/v ACN: 5mM Tris (pH = 8.0); UV detection at 214 nm; applied voltage, 25 kV; injection, 5 kV for 3 s. Peak identification: (1) thiourea, (2) toluene, (3) ethylbenzene, (4) n-propylbenzene, (5) n-butylbenzene, (6) npentylbenzene and (7) n-hexylbenzene.
314
6
Absorbance (mAU)
1 4 3 2 2 4 5
A
6
0 0
Absorbance (mAU)
10
20
30
4 2 1 0 0 10 20 1 2 3 4 5
30 40 Time (min)
Fig. VIII.7. Electrochromatograms of a mixture of basic compounds obtained by using LMA monoliths photo-polymerized with LPO (A) and AIBN (B) as initiator. The composition of the polymerization mixtures is as in Figs. VIII.4, part A and VIII.5, respectively. CEC conditions: mobile phase, 40:60 v/v ACN: 5mM Tris (pH=8.0); UV detection at 254 nm; applied voltage, 10 kV; injection, 5 kV for 3 s. Peak identification: (1) thiourea, (2) pyridine, (3) aniline, (4) 4-nitroaniline, (5) 2,4,6trimethylaniline, (6) N,N-dimethylaniline.
315
VIII.2. Comparison of photo-initiators for methacrylate monolithic columns
VIII.2.1. Comparison of photo-initiators for the preparation of methacrylate monolithic columns for capillary electrochromatography In the last years, monolithic columns have attracted considerable attention due to their properties such as the ease of preparation, the good efficiencies, the speed of chromatographic separations at low back-pressure and non-requirement of frits, constituting a competitive technology to the conventional particlepacked phases used in HPLC and CEC
[Svec, 2003; Legido-Quigley, 2003].
Organic
polymer-based monoliths are prepared in situ by thermal

1998-A; Eeltink, 2005; Geiser, 2007], Delaunay-Bertoncini, 2004; Faure, 2007] Mirapeix, 2009-A]
[Peters, 1997; Peters,
photoinduced
[Geiser, 2007; Ngola, 2001;
or chemical [Cant-Mirapeix, 2008-A; Cant-
polymerization of a mixture that contains one or more
functional monomers, a cross-linking agent, a porogenic solvent and an initiator. Photo-initiation process is faster than other initiation ways and provides a spatial and temporal control of the polymerization reaction, since the radiation can be focused on a location of interest and stopped at a specified time. Thus, UV irradiation constitutes the most common way of photo-polymerization
[Geiser,
2007; Ngola, 2001; Delaunay-Bertoncini, 2004; Faure, 2007; Yu, 2002; Augustin, 2006],
although other sources of electromagnetic radiation (e.g., electron beam, -rays or microwaves [Bandari, 2007; Sfrny, 2005; Zhang, 2008]) have also been used to induce the polymerization reaction. Typically, the morphological properties of polymeric monoliths are controlled by the type of porogenic solvent selected and the amounts of both porogen and crosslinker used [Peters, 1997; Peters, 1998-A; Eeltink, 2005; Svec, 1995B; Eeltink, 2007].
In contrast, very less attention has been paid to the effects of
316
other variables such as type of free-radical initiator and its concentration, which both affect the kinetics of the free-radical polymerization. Consequently, these will affect the morphology of the resulting polymer. Most literature concerning UV-initiated polymeric-based monoliths has reported the use of AIBN
[Geiser, 2007; Ngola, 2001; Delaunay-Bertoncini, 2004;
Faure, 2007; Yu, 2002; Augustin, 2006; Augustin, 2008; Bernab-Zafn, 2009]
or
DMPA [Ro, 2004; Lee, 2004; Huo, 2007; Gu, 2007] as initiators, whereas others, such as BPO and LPO
[Chen, 2007; Du, 2007; Bevington, 2004; Cant-Mirapeix, 2008-D]
have been less employed. In this work, the preparation of LMA-based monoliths for CEC by photopolymerization using several free-radical initiators is described. Fig. VIII.8 shows the decomposition schemes for the four initiators investigated (AIBN, DMPA, BPO and LPO). Using a 1,4-butanediol/1-propanol mixture as porogenic solvent, the influence of each type of initiator and its content in the polymerization mixture was comparatively investigated. The morphology of the resulting columns was characterized by SEM images, whereas their CEC properties were evaluated by measuring retention factors, efficiencies and resolution of a test mixture of non-charged solutes. The run-to-run and columnto-column reproducibilities of CEC columns were also studied.
VIII.2.1.1. Preparation and characterization of LM columns photo-initiated with AIBN or DMPA A series of LMA-based monolithic columns photo-initiated with AIBN was prepared by modifying the percentage of this initiator in the polymerization mixture. The composition of this mixture was taken from a previous work
[Bernab-Zafn, 2009].
It contained 40 wt% monomers (59.8 wt% LMA, 39.9
wt% EDMA and 0.3 wt% META) and 60 wt% porogens (10 wt% 1,4-butanediol
317
and 90 wt% 1-propanol). The AIBN percentage was varied from 0.05 to 0.8 wt% in the polymerization mixture. As it can be seen in Table VIII.4, minor changes in efficiency (minimum theoretical plate, Hmin obtained from Van Deemter plots) were observed for the range of AIBN tested. On the other hand, small variations in u-values were observed with increasing AIBN content, while the k-values increased from 0.05 to 0.30 wt% AIBN, followed by a decrease at contents higher than 0.6 wt% (see Table VIII.4).
N N N N
N2
O C
OCH3 C OCH3 OCH3 C OCH3
O C
OCH3
C
OCH3
O C OCH3 +
CH3
O O O O O O O O CH2(CH2)9CH3
CH3(CH2)9CH2
CH3(CH2)9CH2
Fig. VIII.8. Photolytic cleavage schemes of four investigated photoinitiators: AIBN (A), DMPA (B), BPO (C) and LPO (D).
In order to understand this behaviour, SEM pictures of these monoliths were taken. Thus, from 0.05 (Fig. VIII.9, part A) to 0.30 wt% AIBN (Fig. VIII.9, part B), the characteristic dimensions of the globules are decreased, which produce an increase in surface area, and consequently, larger retention.
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Fig. VIII.9. SEM photographs of LMA monoliths photo-polymerized with AIBN at several contents: 0.05 wt% (A), 0.3 wt% (B) and 0.8 wt% (C). Part (D) shows a monolith photo-polymerized with 0.05 wt% DMPA. Polymerization conditions: 40 wt% monomers (59.8 wt% LMA, 39.9 wt% EDMA and 0.3 wt% META) and 60 wt% porogens (10 wt% 1,4butanediol and 90 wt% 1-propanol); photo-polymerization at 0.90 J/cm2 for 10 min. The bar lengths stand for 3.0 m.
However, SEM photograph of monolith photo-initiated with 0.8 wt% AIBN (Fig. VIII.9, part C) did not show significant changes in globule size compared to Fig. VIII.9, part B. It would be reasonable to assume that lower concentrations of initiator (i.e., 0.05 wt%) would lead to lower number of nuclei and thus polymers with slightly large globules (Fig. VIII.9, part A). Increasing the initiator concentration increases the polymerization rate, which would produce the formation of a larger number of free-radicals and growing nuclei, which would result in smaller globules.
319
Tabla VIII.4. Electrochromatographic properties of LMA-based monolithic columns prepared with several photo-initiators.
320
Flow rate and retention measured at 5 kV. Mobile phase, 80:20% (v:v) ACN:water (5mM Tris buffer pH 8.0). Mean and RSD values (%) obtained for run-to-run repeatability with n = 10 at optimal initiator content. c Mean and RSD values (%) obtained for column-to-column repeatability with n = 3 at optimal initiator content. d Global resolution as geometrical mean of resolution between the consecutive PAH pairs.
Table VIII.4. Continue.
321 Chapter VIII. Monolithic Columns
Flow rate and retention measured at 5 kV. Mobile phase, 80:20% (v:v) ACN:water (5mM Tris buffer pH 8.0). Mean and RSD values (%) obtained for run-to-run repeatability with n = 10 at optimal initiator content. c Mean and RSD values (%) obtained for column-to-column repeatability with n = 3 at optimal initiator content. d Global resolution as geometrical mean of resolution between the consecutive PAH pairs.
It was in agreement with Fig. VIII.9, part B and C, and with the increase in retention observed from 0.05 to 0.3 wt% AIBN. However, a reduction in kvalues was observed at larger AIBN contents (Table VIII.4). Several possible explanations could be given for this irregular retention behaviour. The effect of initiator concentration on the final globule size is likely to be dependent on the competition between the rates of nucleation and termination. With increasing AIBN content, an increase in termination step could be favoured, which leads to the formation of less chains able to grow a sufficient length either to become nuclei or to stabilize the growing nuclei. It could reduce the available free sites for interactions within the polymer, and would produce a decrease in retention of analytes
[Paine, 1990; Lai, 1997].
Another potential explanation is taking into
account the screening effect of initiator, as has been reported [Lai, 1997; Gruber,
1992; Guthrie, 1986; Hutchison, 1973; Moad, 1995].
At large initiator concentrations,
the distribution of initiating species will become less uniform, being the majority of these concentrated in a relatively narrow region close to the radiation source. Thus, this surface layer will effectively screen the deeper layer of initiating species against UV radiation. This non-uniformity will give rise to an overall reduction in the rate of polymerization, and consequently, monoliths with less binding capacity. The possibility of employing DMPA as free-radicals source by photoinitiation instead of AIBN was also evaluated. Different amounts of DMPA ranging between 0.02 and 0.2 wt% were tested (Table VIII.4). Concentrations above 0.15 wt% led to a fast polymerization of mixture at room temperature, making a proper filling of the capillaries unfeasible. Along the DMPA range, similar flow rates and Hmin values were obtained, while the k-values decreased from 0.02 to 0.05 wt% DMPA, remaining practically constant at 0.15 wt%. However, SEM pictures of these monoliths did not show significant differences in morphology. A representative example of porous structure of these monoliths is given in Fig. VIII.9, part D.
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The retention behaviour could be explained by taking into account the above considerations. Additionally, one should keep in mind that the initiation efficiency of generated radicals depends on the type of photo-initiator. In fact, each initiator will have its own specific decomposition rate, which would depend on its concentration and the selected experimental conditions [Gruber, 1992]. A comparison of CEC performance of monoliths photo-initiated with AIBN and DMPA was also accomplished. As it can be seen in Table VIII.4, at 0.05 wt% for each initiator, slightly better efficiency values were achieved for columns photo-initiated with DMPA. The k-values were also higher in columns obtained with DMPA. This fact suggests the presence of higher number of micropores in the globule structure for monoliths photo-polymerized with DMPA, and consequently, larger retention, which is confirmed by SEM pictures (see Fig. VIII.9, parts A and D). Rohr et al.
[Rohr, 2001]
have reported the use
of DMPA as UV initiator for polymerization 2-hydroxyethyl methacrylate monoliths providing a much faster polymerization rate than those photo-initiated with AIBN. Faster polymerization rate results in a faster initiation and therefore the formation of smaller globules. Additionally, the RG, measured as the geometrical mean of the resolution between the consecutive PAH pairs, was also evaluated. Monoliths prepared with DMPA showed slightly better resolution values than those obtained for AIBN taking into account the studied range for each initiator (Table VIII.4). Fig. VIII.10, parts A and B shows the electrochromatograms of PAHs obtained with monoliths photo-initiated at the optimal percentage of AIBN and DMPA, respectively.
323
80
Absorbance (mAU)
80 60 40 3 1 1 80 60 40 3 1
60 40 20 0 1 160 2 4 3 4 1 2 3 5 6 7
20 0
5 6 2 2 4
Absorbance (mAU)
120 80 40 0 1 2 Time (min) 3 1 2 3 5 6 7
6 5 2 1 Time (min)
20 0
Fig. VIII.10. Electrochromatograms of a PAH test mixture obtained using LMA-based monolithic column photo-polymerized with several initiators: 0.3 wt% AIBN (A), 0.05 wt% DMPA (B), 0.05 wt% BPO (C) and 0.3 wt% LPO (D). Other details about the composition of the columns are given in Fig. VIII.9. CEC conditions: mobile phase, 80:20% (v/v) ACN/aqueous 5mM Tris (pH 8.0); applied voltage, 25 kV. Peak identification: (1) thiourea, (2) naphthalene, (3) fluorene, (4) anthracene, (5) pyrene, (6) benz[a]anthracene and (7) benzo[k]fluoranthene.
VIII.2.1.2. Preparation and characterization of LMA columns photo-initiated with BPO or LPO LMA-based monolithic columns were prepared using BPO or LPO as photo-initiators and the same polymerization mixture described in the previous section was adopted. The BPO content was varied from 0.02 to 0.8 wt% in the polymerization mixture, while the content of LPO ranged from 0.05 to 0.8 wt% (see Table VIII.4). When BPO was increased from 0.02 to 0.30 wt%, a decrease in the k-values was observed, followed by an increase at 0.6 wt%, and a later
324
decrease at 0.8 wt%. The beds initiated with 0.05 (Fig. VIII.11, part A) and 0.6 wt% (Fig. VIII.11, part C) showed globule sizes smaller than those obtained with 0.3 wt% (Fig. VIII.11, part B), which would explain its lower retention. The monolith initiated at 0.8 wt% did not show significant changes in morphology compared to 0.6 wt%.
Fig. VIII.11. SEM photographs of LMA monoliths photo-polymerized with BPO at several contents: 0.05 wt% (A), 0.3 wt% (B) and 0.6 wt% (C). Other details about the composition of the columns are given in Fig. VIII.9.
As we commented in previous section, a possible explanation of this retention behaviour could be related to the competition between the rates of coagulation and nucleation along initiator concentration. LMA-based monolithic columns using LPO as initiating system were also prepared. At 0.05 wt% LPO (Fig. VIII.12, part A), large globule sizes were observed, whereas beds initiated with higher LPO contents gave similar
325
monolithic structures (see Fig. VIII.12, part B as representative example). Thus, these latter monoliths showed small variations in u- and k-values.
Fig. VIII.12. SEM photographs of LMA monoliths photo-polymerized with LPO at several contents: 0.05 wt% (A) and 0.6 wt% (B). Other details about the composition of the columns are given in Fig. VIII.9.
A comparison of CEC performance of both diacyl peroxides was also performed. Taking into account the initiator content employed, for both types of monoliths, some differences in the studied parameters could be observed. As it can be seen in Table VIII.4, at low initiator contents (i.e., 0.05 wt%), the kvalues were slightly higher with BPO than those observed for LPO, which was consistent with SEM pictures of these columns (see Figs. VIII.11, part A and
VIII.12, part A). On the other hand, for initiator contents comprising between
0.050.3 wt% BPO and 0.150.6 wt% LPO, the k-values obtained with BPO initiated monoliths were lower than those observed for LPO ones. This result was consistent with SEM pictures, which showed larger globules in beds made with BPO than those prepared with LPO (see Figs. VIII.11, part B and VIII.12, part
B). However, for BPO contents above 0.6 wt%, the use of this initiator provided
again monoliths with larger retention. As we commented above, these variations in retention could be probably ascribed to the yield of radical generation, which depends on the type and concentration of photo-initiator [Gruber, 1992].
326
As observed for LPO, similar k-values were obtained within the range 0.150.8 wt%, in contrast to their BPO counterparts. This fact would allow the possibility of a fine control of morphological and electrochromatographic properties of LMA-based monolithic columns prepared using LPO. In terms of efficiency and resolution, similar values were achieved both using BPO and LPO over the tested initiator range, except at 0.3 wt% BPO, where the lowest efficiency and resolution values (Table VIII.4) were achieved, probably due to its large globule size. Fig. VIII.10, parts C and D shows the electrochromatograms of PAHs that provided the best chromatographic performance obtained with the monoliths initiated with BPO and LPO, respectively. Finally, a comparison of four investigated photo-initiators was performed taking into account the optimal initiator concentration found for each one. As shown in Table VIII.4, the four initiating systems investigated showed in their optimum conditions similar Hmin and RG values. Fig. VIII.13, parts A to D shows the Van Deemter plots for several PAHs with the optimal monoliths found for each photoinitiator. The AIBN and LPO monoliths showed low mass transfer contributions (C-term): 5.612.1 and 6.812.5 ms, respectively, followed by DMPA (8.214.3 ms) and BPO (7.816.6 ms). At the sight of these values and Fig. VIII.13, the AIBN monoliths showed the flatter profile, which offered the possibility of increasing the speed of analysis without a significant loss in efficiency. Anyway, as shown in Fig. VIII.10, for all tested monoliths, the PAHs were satisfactorily separated at 25 kV in less than 4 min. The monoliths initiated with LPO gave the best compromise between separation performance and analysis time, followed by AIBN, DMPA and BPO. The repeatability of LMA-based monolithic columns prepared using the optimal monolithic columns found for each photoinitiator was also examined
327
(see Table VIII.4). Satisfactory run-to-run repeatabilities in the tested chromatographic parameters were found for each initiator, whereas for columnto-column repeatability, the beds photo-polymerized with BPO showed the largest RSD values (see Table VIII.4).
30
30
Hmin (m)
20
20
10
10
0 0 30 0.4 0.8 1.2 1.6
0 0 30 0.4 0.8 1.2 1.6
Hmin (m)
20
20
10
10
0 0 0.4 0.8 1.2 1.6 u (mm s-1)
0 0 0.5 1 1.5 2 u (mm s-1)
Fig. VIII.13. Van Deemter plots of LMA monolithic columns photopolymerized with 0.3 wt% AIBN (A), 0.05 wt% DMPA (B), 0.05 wt% BPO and 0.3 wt% LPO (D). Compounds: () naphthalene, () anthracene, and () benzo[k]fluoranthene. Polymerization conditions as in Fig. VIII.9 and CEC conditions as in Fig. VIII.10.
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CAPTULO IX
CONCLUSIONES GENERALES / GENERAL CONCLUSIONS
Captulo IX. Conclusiones generales
IX.1. Surfactantes
IX.1.1. APGs
IX.1.1.1. Separacin y determinacin de APGs por LC con deteccin ESI-MS Los espectros de masas de los APGs en el modo PI estn constituidos por picos de iones [M+Na]+. Utilizando una columna de alquilamida, elucin isocrtica y deteccin MS, se consigui una buena separacin de los AMGs, con resolucin entre los epmeros - y - y los ismeros de anillo. Los ismeros de los APGs (alquildiglicsidos y alquiltriglicsidos) tambin se resolvieron parcialmente. Alternativamente, los ismeros de anillo pueden resolverse en menor tiempo utilizando una columna de cianopropilo y elucin en gradiente con la ventaja de que esta columna requiere un tiempo de equilibrado mucho ms corto que la columna de alquilamida. Ambos mtodos permiten la identificacin y cuantificacin de APGs en mezclas industriales y productos de tocador. Utilizando ESI-MS como sistema de deteccin, los factores de respuesta de los AMG son independientes del contenido de ACN en la fase mvil. Los factores de respuesta aumentan en ms de un orden de magnitud al pasar del C1G1 al C10G1, y nuevamente disminuyen para cadenas de alquilo ms largas; por lo tanto, para evitar un error sistemtico en la determinacin de los APGs de mayor inters industrial (C10G1-C16G1) es necesario utilizar estndares de todos los oligmeros.
The mass spectra of the APGs in the positive-ion mode were constituted by the single peaks of the [M+Na] + ions. Using an alkylamide column, isocratic elution and MS detection, the alkylmonoglycosides can be separated with resolution between the - and -epimers and ring isomers. The isomers of the
331
alkyldiglucosides and alkyltriglucosides are also partially resolved. The ring isomers can be also resolved in a shorter time using a cyanopropyl column with gradient elution. Also in comparison with the alkylamide column, the cyanopropyl column requires a much shorter equilibration time. The APGs present in industrial mixtures and products (toiletries) can be quickly identified and quantified by any of these two methods. Using ESIMS, the response factors of alkylmonoglycosides are independent from the acetonitrile contents of the mobile phase, but increase more than one order of magnitude from C1G1 up to C10G1, and decrease again for longer alkyl chains; thus, standards of all the oligomers are required to avoid systematic error in the determination of the alkylpolyglycosides of most industrial interest (C10G1C16G1).
Main points: The ESI-MS spectra of the APGs in PI mode consist mainly of [M+Na] + peaks. A column of silica-alkylamide allows the separation of the AMGs, with resolution between epimers and ring isomers, as well as the partial resolution of the APGs (alkyldiglicosides and alkyltriglicosides). Its main drawback is the long equilibration time, thus, isocratic elution should be used. Using a cyanopropyl column and gradient elution, the AMG ring isomers are resolved in shorter analysis times than using the alkylamide column. The two methods can be applied to the identification and quantification of APGs in industrial mixtures and toiletries. In ESI-MS, the AMG response factors are independent of the content of ACN in the mobile phase, increase from C1G1 to C10G1, and decrease for longer alkyl chains.
332
IX.1.1.2. Estudio de fragmentacin de la D-glucosa y los AMG en presencia de iones de sodio en IT-MS En presencia de iones Na+, los espectros MS en modo PI correspondientes a la D-glucosa y a los APGs con cadenas alqulicas de hasta 12 tomos de carbono presentaron, predominantemente, iones [M+Na]+. Los espectros de MS2 obtenidos a partir de los iones progenitores [M+Na]+ mostraron un patrn de fragmentacin comn con las siguientes caractersticas: (i) un ion a m/z 185, cuya obtencin se debe a una deshidratacin en la D-glucosa, o a la prdida de la cadena alqulica en forma de alcohol en los APGs; (ii) un ion a m/z 143 producido por una rotura 0,2A en el anillo; (iii) un ion a m/z 413, que no contiene tomos de carbono, y que se debe probablemente a un cluster o grupo constituido por Na+, OH- y H2O; (iv) aductos que contienen una molcula de D-glucosa o el correspondiente APG (se representa como M), con las estructuras [M+413]+, [M+413-2H2O]+ y [M+413+H2O-NaOH]+; y (v) unos iones adicionales, cuya estructura depende de la longitud de la cadena de alquilo. Adems, la D-glucosa tambin mostr un dbil in a m/z 113 producido por una rotura 0,3A en el anillo (Fig. IV.8), mientras que todos los APGs mostraron un ion a m/z 129 producido por una rotura transversal 2,5A del anillo. Debido a la falta de estndares comerciales de AMGf, los patrones de fragmentacin de estos ismeros de anillo se estudiaron mediante HPLC-MS y HPLC-MS2 de mezclas industriales de APGs. Estas mezclas contienen pequeas concentraciones de octil- y decil-MGf. En comparacin con los AMGf, el ion a m/z 413 y los aductos formados a partir de ste con molculas no fragmentadas se producen ms fcilmente con los AMGp. Por el contrario, el ion a m/z 143 se produce ms fcilmente con los AMGf que con los AMGp, lo que puede deberse a la menor estabilidad de los anillos de cinco miembros (furansidos) respecto a los de seis miembros (piransidos).
333
In the presence of Na+, the PI MS spectra of D-glucose and the APGs with alkyl chains up to 12 carbon atoms gave predominantly [M+Na]+ ions. The MS2 spectra obtained from the isolated [M+Na]+ parent ions showed a common fragmentation pattern with the following features: (i) an m/z 185 ion, which is due to dehydration and to loss of the alkyl chain as an alcohol in the cases of Dglucose and the AGPs, respectively; (ii) an m/z 143 ion produced by a 0,2A crossring cleavage; (iii) an m/z 413 ion, not containing carbon atoms and probably containing Na+, OH-, and H2O; (iv) adducts containing a molecule of either Dglucose or the corresponding AGP, M, with the structures [M+413]+, [M+413 2H2O] +, and [M+413+H2ONaOH] +; and (v) a few additional ions, whose structures depended on the length of the alkyl chain. In addition, D-glucose also showed a weak m/z 113 ion produced by a
0,3
A cross-ring cleavage (Fig. IV.8),

2,5
and all the AGPs exhibited an m/z 129 ion produced by a cleavage.
A cross-ring
Standards of AMGf were not available; however, the fragmentation patterns of these ring isomers were studied by using HPLC-MS and HPLC-MS2 of industrial mixtures of APGs. These mixtures contain minor concentrations of octyl- and decyl-MGf. In comparison with the AMGf, the m/z 413 ion and its adducts with unfragmented molecules were more easily formed by the AMGp. In contrast, the m/z 143 ion occurred more easily in the AMGf than in the AMGp, which can be due to the lower stability of the five-membered furanoside rings.
Main points: In the presence of Na+, the [M+Na] + peak also predominates in the ESI-MS spectrum of D-glucose in PI mode. When obtained from [M+Na] + parent ions, the ESI-MS2 spectra of D-glucose and the APGs show a common fragmentation pattern.
334
The spectra of AMGp y AMGf (ring isomers) can be separately studied by using HPLC-MS y HPLC-MS2. The corresponding spectra show differences that can be useful to identify the ring isomers.
IX.1.2. Alcoholes
IX.1.2.1. Oxidacin de alcoholes no etoxilados y etoxilados con cromo(VI) y posterior determinacin por ESI-MS Los alcoholes alifticos no pueden ser detectados por MS empleando ESI u otras interfaces de ionizacin a presin atmosfrica. Sin embargo, en el procedimiento propuesto, la oxidacin de alcoholes primarios alifticos con CrO3 (reactivo de Jones), para dar los correspondientes cidos carboxlicos de forma rpida y cuantitativa, permite su deteccin mediante ESI-MS. Una vez oxidados mediante el mtodo propuesto, los alcoholes primarios presentes en muestras industriales y ambientales pueden ser identificados y cuantificados por infusin directa en el espectrmetro de masas. Se infunde una disolucin transparente e incolora constituida por extractos en acetato de etilo/acetona. La sensibilidad en modo NI se exalta por la simple adicin de un 10% de agua alcalinizada a los extractos. Por otra parte, la sensibilidad de este mtodo es mayor al aumentar la masa molecular de los alcoholes, de manera que para alcoholes de cadena larga se consiguen LODs particularmente bajos. Los rendimientos combinados de las etapas de extraccin-oxidacin fueron de prcticamente del 100% para alcoholes no etoxilados disueltos en acetona, y disminuyeron progresivamente para muestras que presentaban cantidades crecientes de agua. Por ejemplo, con un 50% de agua se obtuvo un rendimiento del 75%. Por esta razn, las muestras industriales que contienen agua en proporciones importantes se diluyeron con acetona antes de aadir el
335
reactivo de Jones. Con ello se reduce considerablemente la concentracin final de los analitos en las muestras, lo que no constituye ningn problema en control de calidad industrial, donde las concentraciones de analito suelen ser grandes. Sin embargo, en muestras ambietales, a causa de las bajas concentraciones de analito normalmente presentes, se necesita una preconcentracin de la muestra. La preconcentracin puede hacerse por SPE, lo que permite remplazar el agua por otro disolvente (como acetona) durante la etapa de elucin. La formacin de aductos con H+ permite el anlisis por MS de los alcoholes etoxilados en modo PI, aunque la sensibilidad es baja para oligmeros con un nmero pequeo de unidades de EO. Por otra parte, los espectros en modo PI son ms complejos y con una peor relacin seal-ruido que los espectros en modo NI. Por lo tanto, la oxidacin de los FAEs a los correspondientes cidos etoxicarboxlicos, seguido por MS en el modo NI, tiene mayor inters. En el procedimiento desarrollado, los FAEs se oxidan con unos rendimientos del 65 y 60% para los oligmeros con m = 1-2 y m 2, respectivamente. La oxidacin tambin puede dar lugar a la prdida de una unidad de EO, sin embargo, el rendimiento de esta reaccin secundaria es bajo (4-8%). Por otra parte, al variar el nmero de tomos de C y de EOs, los rendimientos en la oxidacin de los FAEs fueron bastante constantes. Por otra parte, las sensibilidades en MS fueron mayores para los cidos etoxicarboxlicos que para los cidos carboxlicos con el mismo nmero de tomos de carbono (vase Fig. IV.13). Los alcoholes no etoxilados y alcoholes con un bajo grado de etoxilacin se suelen determinar por GC-FID. Las ventajas del procedimiento propuesto en este trabajo son la rapidez y las elevadas sensibilidades obtenidas para los alcoholes de cadena larga, lo que tiene inters en el anlisis de muestras con matrices complejas. En cosmticos, productos para el cuidado corporal y
336
extractos de muestras medioambientales, la presencia de muchos compuestos neutros con volatilidades relativamente altas da lugar a cromatogramas muy complejos en GC. En el procedimiento propuesto, esta complejidad se evita por la oxidacin de los alcoholes a los correspondientes cidos carboxlicos, y por el aislamiento de los mismos mediante extraccin lquido-lquido. Tras aumentar el pH, los extractos se infunden directamente en un espectrmetro de masas trabajando en modo NI. Adems, el procedimiento oxidacin-extraccin propuesto permite tambin el anlisis de alcoholes por HPLC-MS, CE-MS, y CEC-MS. Por el contrario, los alcoholes no etoxilados con altas masas moleculares no se detectan (o bien se observan con una sensibilidad muy baja) por HPLC-ELSD, mientras que la sensibilidad de alcoholes mono y dietoxilados es tambin bajas con esta tcnica
Por esta razn, otra
ventaja del procedimiento propuesto es que permite ampliar el campo de aplicaciones de los detectores evaporativos, haciendo posible la deteccin de alcoholes no etoxilados, y mejorando la respuesta de alcoholes mono y dietoxilados. Por ltimo, cuando los cidos carboxlicos correspondientes a la oxidacin de los alcoholes de inters estn inicialmente presentes en la muestra, las seales obtenidas por el mtodo propuesto corresponden a la suma de alcoholes y cidos carboxlicos. En estos casos, se puede obtener un espectro o cromatograma adicional a partir de la muestra sin la adicin del reactivo de Jones, y utilizarlo para restar la contribucin de las seales derivadas de los cidos carboxlicos inicialmente presentes en la muestra. De este modo, es posible aislar las contribuciones de los alcoholes respecto a las debidas a los cidos carboxlicos. Aunque la sustraccin de la seal aumenta el error aleatorio, la prdida de precisin es pequea cuando la concentracin de alcohol en las muestras es bastante mayor que la de los cidos carboxlicos correspondientes, lo que es frecuente en muchos casos.
337
Aliphatic alcohols cannot be detected by MS using ESI and other atmospheric-pressure ionization interfaces. However, in the proposed procedure, the oxidation of primary aliphatic alcohols with a CrO3 solution in aqueous sulphuric acid (Jones reagent) resulted in their rapid and quantitative conversion into the corresponding carboxylic acids. Primary alcohols present in industrial and environmental samples can be identified and quantitated by infusion of the transparent and uncoloured ethyl acetate-acetone extracts in a mass spectrometer. Sensitivity in the NI mode is strongly enhanced by the addition of 10% alkalinized water to the extracts. Moreover, the sensitivity of this approach further increases as the molecular mass of the alcohols increases, such that for long-chain alcohols the LODs obtained using the proposed procedure were particularly low. Oxidation-extraction yields, which were ca. 100% for nonethoxylated alcohols dissolved in acetone, decreased moderately in samples containing increasing amounts of water, e.g., a 75% yield was obtained in the presence of 50% water. For this reason, industrial samples containing large amounts of water were diluted with acetone before Jones reagent was added. While this greatly reduced the final concentration of the analytes in these samples, this should not be a problem in industrial quality control, where analyte concentrations are usually large. However, owing to the low analyte concentrations typically present in environmental samples, preconcentration of the sample, with replacement of water by another solvent, is usually required. The formation of H+ adducts enables MS analysis of ethoxylated alcohols in PI mode, although the sensitivity is low for oligomers with a small number of EO units. Furthermore, PI mode spectra are potentially more complex and noisier than NI mode spectra. Thus, oxidation of ethoxylated alcohols to the corresponding ethoxycarboxylic acids, followed by NI mode MS, is also of
338
interest. In the procedure developed in this work, ethoxylated alcohols are oxidized, with ca. 65 and 60% yields for m=12 and m 2 oligomers, respectively. Oxidation may also result in the loss of an EO unit; however, the extension of this side reaction is low (48%). Furthermore, oxidation yields of ethoxylated alcohols were fairly constant, and MS sensitivities were larger for ethoxycarboxylic acids than for carboxylic acids with the same number of carbon atoms (see Fig. IV.13).Therefore, application of the proposed procedure not only to nonethoxylated alcohols but also to ethoxylated alcohols is of much interest. Non-ethoxylated alcohols and alcohols with a low degree of ethoxylation are usually identified and determined by GC-FID. The advantages of the procedure proposed in this work are its rapidity and the prominent signals obtained from long-chain alcohols present within wide concentration ranges in samples comprising complex matrices. In cosmetics, body-care products, and extracts from environmental samples, the presence of many neutral compounds with relatively large volatilities frequently gives rise to complex gas chromatograms. In the proposed procedure, this is avoided by alcohol oxidation to carboxylic acids and isolation of the acids by liquid-liquid extraction; after the pH has been raised, the extracts can be directly infused in the mass spectrometer working in the NI mode, without the need for further purification. Moreover, the proposed oxidation-extraction procedure allows alcohol analysis by HPLC-MS, CE-MS, and CEC-MS as well. By contrast, non-ethoxylated alcohols up to high molecular masses are either not detected or are observed with low sensitivities by HPLC-ELSD, while the sensitivities for mono- and diethoxylated alcohols are likewise poor
Thus, a further advantage of the proposed
procedure is that it expands the range of applications of evaporative detectors to include non-ethoxylated alcohols and improves the response of mono- and diethoxylated alcohols. Finally, signals corresponding to the sum of alcohols and
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carboxylic acids are obtained when these acids are initially present in the sample. In this case, an additional spectrum or chromatogram, obtained without adding Jones reagent to the sample, can be used to subtract the signal contributions arising from the carboxylic acids initially present in the sample and thus to isolate the contributions of alcohols. Although signal subtraction increases the random error, the loss of precision is small when the alcohol concentration in the samples is larger than that of the corresponding carboxylic acids.
Main points: A procedure that combines the oxidation of aliphatic primary alcohols and the extraction of the resulting carboxylic acids to obtain an extract free of inorganic salts has been developed. The oxidation-extraction yields are close to 100%. Aliphatic primary alcohols are not detected in MS; however, using this procedure, the identification and quantification of aliphatic primary alcohols by ESI-MS is possible. In NI mode, sensitivity increases with pH and the molecular mass of the alcohol, being also higher when alcohol is ethoxylated. The oxidation yield decreases with increasing water content in the sample. After SPE preconcentration in SPE cartridges, followed by elution with acetone, the method can be applied to samples of the aquatic environment. Owing to the formation of adducts with H+, ethoxylated alcohol can be analyzed by MS in the PI mode; however, the sensitivity is low for oligomers with a low EO number. In addition, PI mode spectra are more complex tan Ni mode spectra. Also, the signal to noise ratio in the PI mode is lower than that usually found in NI mode spectra.
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Oxidation of FAEs to ethoxycarboxylic acids results in loss of one EO unit, but the yield of this side reaction is small (< 8%).
Ethoxylated alcohols are oxidized with lower yields (60-65%) than those obtained with non-ethoxylated alcohols (~ 100%), however, oxidation yields are independent from the number of carbon atoms and the number of EOs.
The proposed method allows the use of evaporative detectors and mass spectrometry detection in the determination of aliphatic primary alcohols. The method is direct, fast and results in lower LODs with complex samples of industrial and environmental origin.
IX.1.3. AES
IX.1.3.1. Determinacin de FAEs y AESs por separacin mediante SAX, derivatizacin con un anhdrido cclico y LC En trabajos anteriores, se han desarrollado procedimientos para la caracterizacin y la determinacin de FAEs basados en su derivatizacin con un anhdrido cclico, seguida por separacin de los oligmeros mediante RP-HPLC
[Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Mic-Tormos, 2010].
Sin embargo, cuando los FAEs se esterifican, los AESs se transesterifican, dando lugar ambas clases de surfactantes a los mismos derivados. Por lo tanto, en el caso en que las dos clases de surfactantes estn presentes en una muestra, los cromatogramas resultantes muestran, en realidad, la suma de FAEs y AESs. Por lo tanto, en este trabajo se ha desarrollado un procedimiento para la separacin de ambas clases de surfactantes, empleando SPE con un cartucho de SAX. Tras la separacin, los oligmeros se derivatizan independientemente y se determinan por RP-HPLC-UV. La separacin casi cuantitativa de las dos clases de surfactantes, incluyendo oligmeros hidroflicos e hidrofbicos, se logr
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mediante la elucin de las fracciones correspondientes a los FAEs y AESs en tres y dos pasos, respectivamente. Como ya se demostr previamente para los FAEs, la derivatizacin de los AESs tambin tiene lugar de forma cuantitativa. Para ello se puede usar anhdrido ftlico o difnico en 1,4-dioxano a 105 C. La separacin de los oligmeros derivatizados empleando RP-HPLC y elucin en gradiente con fases de ACN/agua, mostr una buena resolucin entre las sucesivas series hidrocarbonadas, y entre los oligmeros dentro de una misma serie. Tambin se ha demostrado que los FAEs pueden utilizarse como patrones de calibracin para AESs, lo que supone una ventaja, ya que los estndares de AESs son raros y caros, mientras que los estndares de alcoholes grasos son ampliamente disponibles; adems, los estndares de AES con m > 0 no se encuentran comercialmente disponibles. Por otro lado, otras clases de surfactantes aninicos de uso general en productos de limpieza, como son el SAS y el LAS, no interfieren. El mtodo propuesto se aplic satisfactoriamente a la caracterizacin y determinacin de FAEs y AESs en detergentes lquidos industriales y en extractos de agua de mar. El procedimiento propuesto es tambin til para el control de calidad industrial de los AESs, donde los FAEs suelen estar presentes como impurezas, a causa de que en el proceso de obtencin de los AESs la sulfatacin no es cuantitativa.
In previous work, procedures for FAE characterization and determination based on derivatization with a cyclic anhydride, followed by RP-HPLC with UV or MS detection, were developed [Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; MicTormos, 2009; Mic-Tormos, 2010].
However, when FAE are esterified, AES are
also transesterified, leading to the same derivatives as FAE. Thus, if the two surfactant classes are present in the samples, the resulting chromatograms show the sum of both FAE and AES oligomers. In this work, a procedure for the SAX
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separation of both surfactant classes, followed by their independent derivatization and RP-HPLC-UV, was developed. Quantitative isolation of the two surfactant classes, including both hydrophilic and hydrophobic oligomers, was achieved by eluting the FAE and AES fractions in three and two steps, respectively. As previously shown for FAE, quantitative derivatization of AES with either phthalic or diphenic anhydrides has been demonstrated. Separation of the derivatized oligomers, with good resolution between both the hydrocarbon series and the successive oligomers within the series, was achieved by RP-HPLC using gradient elution with ACN/ water. It has been also shown that FAE oligomers can be used as calibration standards for AES, which is an advantage because standards of alkylsulfates are rare and expensive, whereas standards of fatty alcohols are widely available; further, standards for AES with m > 0 are not commercially available. On the other hand, anionic surfactant classes commonly used in cleaners, including SAS and LAS, did not interfere. The proposed method was successfully applied to the characterization and determination of FAE and AES in industrial liquid cleaners and seawater extracts. The proposed procedure is also useful to control the quality of industrial AES, where FAE are always present as an impurity, due to the nonquantitative sulfatation of FAE during AES manufacture.
Main points: Reacting with a cyclic anhydride, FAEs are esterified, and AESs are transesterified, quantitatively giving rise to the same derivatives (hemiesters of the alcohol residues). Using a SAX cartridge, a procedure for the quasi quantitative separation of FAEs and AESs has been developed.
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A method for the characterization and determination of FAEs and AESs in mixtures has been developed. The method is based on the separation of the two surfactant classes with a SAX cartridge, followed by derivatization with a cyclic anhydride and RP-HPLC-UV. A good resolution between consecutive series (different hydrocarbon chain), and between the oligomers within a series is obtained.
The method allows the use of FAEs as calibration standards for AESs. The method is useful to characterize and determine FAEs and AESs in industrial liquid detergents and in seawater. Other classes of anionic surfactants such as SAS or LAS do not interfere.
IX.2. Polmeros sintticos
IX.2.1. PVP-NO
IX.2.1.1. Caracterizacin de PVP-NO por CE y MECK Los estudios mediante FSCE realizados en este trabajo han mostrado que el PVP-NO es un polmero aparentemente no inico, que se comporta como un polielectrolito con un carcter aninico bien definido en disolucin acuosa. Los resultados obtenidos por FSCE estn de acuerdo con los obtenidos previamente a travs de estudios de viscosidad, conductividad, dispersin de luz y cido-base
[Okamoto, 1998; Lee, 1996; Yamamoto, 1996].
Los resultados tambin son
consistentes con la presencia de al menos dos formas del polmero en disoluciones acuosas, probablemente una de ellas constituida por cadenas libres (unmeros) y la otra por agregados. Este enfoque estara de acuerdo con los estudios realizados por CE para otros polmeros y copolmeros
Gyrffy, 1998; tpnek, 2001]. [Cottet, 2001;
Por otro lado, los resultados obtenidos por MEKC
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indican una fuerte interaccin del PVP-NO con las micelas de SDS, y son coherentes con la formacin de micelas mixtas de dos tipos: las constituidas por polmeros libres y SDS, y las que contienen agregados de polmero y SDS
1971; Cabane, 1977]. [Arai,
Esta explicacin es consistente con los estudios

[Oakes, 2003-C].
espectroscpicos por UV-Vis de Oakes y col.
Por lo tanto, en el
presente estudio se ha demostrado la utilidad de la FSCE y la MEKC en la investigacin del comportamiento de los polmeros sintticos en disolucin. Tambin se ha demostrado la capacidad del PEA para reducir la adsorcin de polmeros sintticos sobre la pared interna de los capilares en medio cido. Por ltimo, la FSCE puede ser til en el control de calidad de materias primas y productos comerciales que contienen PVP-NO, si bien, el procedimiento se encuentra limitado a productos que no contienen surfactantes aninicos.
The FSCE studies performed in this work have shown that PVP-NO, which is a nominally non-ionic polymer, behaves as a polylectrolyte, with a welldefined anionic character in aqueous solutions. In this concern, the results obtained by FSCE agreed with those previously reported using viscosity, light scattering, conductivity and acid-base studies
Yamamoto, 1996]. [Okamoto, 1998; Lee, 1996;
The results are also consistent with the presence of at least two
forms of the polymer in aqueous solutions, likely one of them constituted by free chains (unimers) and the other one by aggregates. This would agree with the CE studies performed with other polymers and copolymers
1998; tpnek, 2001]. [Cottet, 2001; Gyrffy,
On the other hand, the results obtained by MEKC indicated
a strong interaction of PVP-NO with the SDS micelles, and are consistent with the formation of both free polymer/SDS and aggregated polymer/SDS mixed micelles [Arai, 1971; Cabane, 1977]. This explanation is consistent with the UV-Vis spectroscopic studies by Oakes et al.
[Oakes, 2003-C].
Thus, the present study
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further shows the usefulness of both FSCE and MEKC as complementary techniques in the investigation of the behavior of synthetic polymers in solution. The capability of PEA to reduce the adsorption of synthetic polymers in weak acid media has been also demonstrated. Finally, FSCE can be useful in the quality control of raw materials and commercial products containing PVP-NO, although the procedure is limited to products not containing anionic surfactants.
Main points: Using FSCE, it has been shown that PVP-NO (apparently a non-ionic polymer) behaves as a polyelectrolyte with anionic character in aqueous solution, which has been attributed to the deprotonation of water associated with the NO- group. The FSCE experiments are consistent with the presence of two forms of the polymer in solution, one made up of free chains (unimers) and the other constituted by aggregates. PVP-NO interacts strongly with SDS micelles, giving rise to mixed micelles of two types: free polymers bound to SDS, and polymer aggregates also bound to SDS. The usefulness of FSCE and MEKC in investigating the behavior of synthetic polymers in solution has been demonstrated. The ability of PEA to reduce adsorption of synthetic polymers on the inner wall of the capillaries in acid medium has been demonstrated. Although limited to samples having no anionic surfactants, FSCE is useful in the quality control of industrial products containing PVP-NO.
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IX.2.2. PVP
IX.2.2.1. Caracterizacin y determinacin de PVP por complejacin con un azo-colorante aninico seguida de NEECEM En disolucin acuosa, los colorantes aninicos estn enlazados al PVP formando complejos que muestran movilidad aninica. Durante la separacin electrofortica, estos complejos se disocian parcialmente siguiendo una cintica de primer orden. En este trabajo, la teora NECEEM, que se desarroll para estudiar las interacciones protena-marcador y protena-DNA, se aplica por primera vez al estudio de polmeros sintticos. Mediante esta teora se puede obtener informacin sobre la estabilidad de los complejos formados entre un polmero y un colorante. A pesar de la mayor polidispersidad de los polmeros sintticos en comparacin con las protenas
[Grosche, 2000; Borisch, 2000],
el
comportamiento electrofortico de las mezclas PVP-colorante ha podido ser muy bien explicado por la teora NECEEM. As, a partir de la inyeccin de mezclas con diferentes razones molares monmero/colorante, puede establecerse la estequiometra mxima de los complejos, y trabajando con razones molares monmero/colorante cercanas al punto de saturacin, puede estimarse tambin la constante de estabilidad de los complejos. El mtodo NECEEM es, por lo tanto, til para caracterizar polmeros no inicos, tanto sintticos como naturales, y estudiar su interaccin con un marcador en disolucin. Adems, trabajando a razones molares inferiores al punto de saturacin, puede obtenerse la concentracin de monmeros mediante una curva de calibracin, y puede estimarse un promedio de la masa molecular del polmero a partir de relaciones sencillas basadas en la forma del pico o banda del complejo polmero-marcador. En este trabajo se estim la estequiometra de los complejos formados por el PVP con dos azo-colorantes aninicos, CR y AB. La saturacin de PVP con
347
estos dos colorantes se produjo a una razn molar monmero/colorante prxima a q = 4. Se estimaron tambin las constantes de estabilidad de los complejos PVP-CR. Cuando se trabaja en presencia de un exceso de colorante (q < 4), la banda de los complejos PVP-CR es til para predecir la concentracin del PVP, as como su MW promedia, lo que se aplic a la caracterizacin de PVP en productos de limpieza y productos farmacuticos.
Anionic dyes are bound to PVP in aqueous solutions, forming complexes that show anionic mobility. During the electrophoretic run, the complexes partially dissociate following a first-order kinetics. On the basis of the NECEEM theory, which was formerly developed to study proteinprobe and DNAprotein interactions, information about the stability of polymerdye complexes can be obtained. In spite of the much larger polydispersity of synthetic polymers when compared to proteins [Grosche, 2000; Borisch, 2000], the electrophoretic behaviour of PVPdye mixtures is very well explained by the NECEEM theory. Thus, by injecting mixtures at several monomer/dye molar ratios, the maximal stoichiometry of the complexes can be established, and by working at monomer/dye molar ratios close to saturation, the average stability constant of the complexes can be estimated. The NECEEM method is thus useful to characterize synthetic and natural nonionic polymers, and to study their interaction with a probe in solution. In addition, at monomer/dye molar ratios lower than saturation, the monomer concentration can be obtained by using a calibration curve, and the average molecular mass of the polymer can be estimated from a simple relationship provided by the shape of the peak of the polymerprobe complexes. In this work the stoichiometry of the complexes formed by PVP with two anionic azo-dyes, CR and AB, was estimated. Saturation of PVP with these dye
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ions was produced at a monomer/dye molar ratio close to q = 4. The stability constants of the PVPCR complexes were also estimated. Upon addition of an excess dye to the sample (q < 4), the band of the PVPCR complexes was useful to predict both the concentration of PVP and the average molecular mass, Mw, in cleaning products and pharmaceutical preparations.
Main points: Anionic dyes are bound to PVP in aqueous solution, resulting in complexes with anionic mobility. During electrophoretic separation, the PVP-dye complex is partially dissociated following a first-order kinetics. It has been shown that the NECEEM theory, previously developed to characterize proteins and DNA fragments, is also useful to characterize synthetic nonionic polymers. Information on the stability of the complexes and the nature of the polymer (average molecular mass) can be obtained. Working at molar ratios below the saturation point, the stoichiometry of the complexes (number of monomers per dye ion) can be estimated. Saturation of the polymer (PVP) with CR or AB occurs at a monomer / dye molar ratio close to 4. Working in an excess dye (q < 4), the band of the PVP-CR complexes contains information which is useful to predict the PVP concentration and their average MW.
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IX.2.3. PVA
IX.2.3.1. Evaluacin de la MW y tacticidad del PVA mediante NEECEM de mezclas polmero-colorante Cuando una disolucin de PVA que contiene borato se mezcla con una disolucin que contiene CR, se forma inmediatamente un complejo PVA-CR. La formacin de este complejo se detecta por el aumento de la absortividad molar y por el notable desplazamiento batocrmico de la banda principal del espectro de absorcin UV-Vis del CR. Utilizando polaridad positiva, los electroferogramas de las mezclas PVA-CR en presencia de un exceso de CR mostraron el patrn predicho por la teora NECEEM para complejos formados por una macromolcula sin carga y un marcador aninico. Cuando la concentracin de PVA aument hasta el punto de saturacin, el rea de la banda del complejo aument tambin linealmente, y las reas debidas al exceso de colorante liberado durante la migracin disminuyeron igualmente de forma lineal. Se estim la mxima estequiometra del complejo a partir de la variacin de reas a valores crecientes de la razn molar monmero/colorante (valores de q). Esta razn oscil entre qsat ~ 4,9 y 3,5 para PVA de MW bajo y alto, respectivamente. Por otra parte, esta variacin se produjo a diferentes valores de log MW dependiendo de la tacticidad del PVA. A la vista de los valores de la qsat, se discuti la posible estructura del complejo de PVA-CR. Las correlaciones encontradas entre varios parmetros electroforticos obtenidos en las condiciones NECEEM respecto a la MW y la tacticidad del PVA, pueden explicarse suponiendo que los iones de colorante se apilan muy prximos unos a otros dentro de la estructura de los complejos PVA-CR. La correccin de la estequiometra para tener en cuenta el porcentaje de monmeros acetilados no enlazados (qsat,c) confirm las diferencias entre los grupos de muestras de distinta tacticidad. La forma de la banda de los
350
complejos PVA-CR, las reas relativas de la banda del complejo y del pico del colorante libre, la movilidad electrofortica y la constante de disociacin de pseudo-primer orden del complejo, tambin resultan estar correlacionadas con la MW y la tacticidad del PVA. Las muestras ms sindiotcticas (tipo H) dieron complejos con menor movilidad absoluta que las muestras ms atcticas (tipos M y L). Este comportamiento podra deberse a las mayores distancias entre los grupos OH adyacentes en las muestras tipo H, las cuales presentan una mayor relacin rr/mm que las muestras M y L. El producto [qsat + (VD / Vm)] disminuy para todas las MW, lo que indica que no llega a alcanzarse el rgimen de drenaje libre (donde la movilidad es constante porque la relacin carga/masa ya no aumenta al seguir creciendo la MW del polmero). La variacin de este producto, tambin confirm la dependencia entre la movilidad del complejo PVA-CR y la tacticidad. Por lo tanto, se puede obtener informacin til acerca de la MW y la tacticidad del PVA a partir de la CZE de mezclas de PVA-CR. Sin embargo, sera necesario recurrir a la calibracin multivariante, incluyendo la variacin ortogonal de las variables MW y el cociente rr/mm, para construir el correspondiente conjunto de estndares de calibracin. Sin una calibracin independiente de estas variables no es posible construir modelos capaces de hacer predicciones de la MW y la tacticidad con una precisin razonable. El conjunto de muestras comerciales de PVA recopilado y empleado en este trabajo fue suficiente para demostrar la dependencia de los parmetros de CZE obtenidos en condiciones de NECEEM sobre la MW y tacticidad, pero no es lo suficientemente bueno para hacer una calibracin multivariante. Por ltimo, aunque esto tambin debe ser estudiado, la NECEEM podra ser til para evaluar la MW y la tacticidad de otros polmeros solubles no cargados.
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A PVACR complex is immediately formed when a PVA solution containing borate and a CR solution are mixed. Complex formation produced a large increase of the molar absorptivity and a remarkable bathochromic shift of the main band of the UVVis absorption spectrum of CR. Using positive polarity, electropherograms of PVACR mixtures containing a CR excess showed the pattern predicted by the NECEEM theory for a complex formed by an uncharged macromolecule and an anionic marker. The area of the complex band increased linearly, and the areas due to the excess dye and to the dye released by the complex during migration decreased, when the PVA concentration increased up to the saturation point. The variation of the areas at increasing monomer/dye molar ratios (q values) was used to estimate the maximal stoichiometry of the complex. This ranged from qsat ~ 4.9 to 3.5 for low and high MW PVA, respectively, this variation being produced at different log Mw values depending on PVA tacticity. At the sight of the values of qsat, the possible structure of the PVACR complex was discussed. The correlations found along this work among several electrophoretic parameters obtained in NECEEM conditions with respect to both molecular mass and tacticity of PVA can be explained by assuming that the dye ions are stacked in a proximity to each other within the structure of the PVACR complex. Correction of the stoichiometry taking into account the percentage of unbonding acetylated monomers (qsat,c) confirmed the differences between tacticity groups. The shape of the PVACR complex band, the relative areas of the complex band and free dye peak, and the electrophoretic mobility and dissociation pseudo-first-order rate constant of the complex, were also related to both MW and tacticity of PVA. Thus, the H samples gave complexes with lower absolute mobilities than the M+ L samples. In comparison to M+ L samples, these differences could be due to the larger distances between adjacent OH groups in H samples, which have a higher rr/mm ratio than the M+ L
352
samples. Product [qsat + (VD / Vm)] decreased at all MW, thus indicating that a free draining regime was not reached. The variation of this product also confirmed the dependence of the mobility per complexed dye ion on tacticity. Therefore, useful information about both MW and tacticity of PVA can be gained by CZE of PVACR mixtures. However, multivariate calibration, including the orthogonal variation of the molecular mass and rr/mm ratio values along the set of standards, would be necessary to construct multivariate models capable of making predictions of these two responses with reasonable precision. The set of commercial PVA samples we collected and used in this work was adequate to demonstrate the dependence of CZE parameters obtained in NECEEM conditions on molecular mass and tacticity, but it was deficient to support multivariate calibration. Finally, although this should be also investigated, NECEEM could be useful to evaluate MW and tacticity of other soluble noncharged polymers.
Main points: The PVA-CR complex is immediately formed upon mixing a solution of PVA with a solution containing CR (in the presence of borate). Upon complex formation, an increase in the molar absorptivity of the main UV-absorption band of CR, and a large bathochromic shift, is produced. In the presence of an excess of CR, the electropherograms of PVA show the pattern predicted by the NECEEM theory. Increasing the PVA concentration up to the saturation point, the band area of the complex increases linearly, while the sum of the areas due to excess dye and the dye released during migration, also decreases linearly.
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The maximum stoichiometry of the complex ranges from qsat ~ 4.9 to 3.5 for samples of PVA of high and low MW, respectively. This variation occurs at different values of log MW, depending on the tacticity of PVA.
The correlations between MW and tacticity of PVA with various electrophoretic parameters obtained in the NECEEM conditions can be explained by assuming that in these complexes, dye ions are stacked very close together within the structure of the PVA-CR complex.
Correction of the stoichiometry of the complex to take into account the percentage of unbound acetylated monomers, confirmed the differences observed between groups of samples of different tacticity.
The shape of the band of PVA-CR complexes, the relative areas of the band of the complex and free dye peak, the electrophoretic mobility and the dissociation constant of the complex, are all correlated with MW and tacticity.
IX.3. Enzimas
IX.3.1. CZE
IX.3.1.1. Identificacin de enzimas de la industria de la detergencia por CZE de enzimas intactas Se ha demostrado que la separacin de protenas intactas por CZE, utilizando dos BGEs, uno cido y otro bsico, es potencialmente til para identificar las enzimas comnmente utilizadas en la formulacin de productos de limpieza. En particular, el BGE bsico proporciona una lnea base habitualmente plana, lo que permite distinguir una serie de pequeos picos que muestran pautas caractersticas del tipo de enzima. Adems del tiempo de migracin del pico principal, la presencia de un segundo pico de mediana intensidad tambin
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constituyen un rasgo caracterstico de algunas enzimas utilizadas en la industria de los detergentes. La cuantificacin del contenido proteico por el mtodo de Bradford, permiti calcular las sensibilidades relativas a partir de las reas totales de los picos obtenidos en los electroferogramas en los BGEs bsico y cido. Estas sensibilidades tambin pueden ser tiles para clasificar o identificar las enzimas. La aplicacin del procedimiento a muestras reales falla en presencia de surfactantes aninicos, si bien, algunas muestras que contienen surfactantes no inicos dan resultados aceptables.
It has been shown that CZE of the intact proteins using either basic or acid BGEs is potentially useful to identify the enzyme brands commonly used in the formulation of cleaning products. In particular, the basic BGE gives flat baselines, which makes possible to distinguish a number of small peaks constituting rather characteristic patterns. In addition to the migration time of the main peak, the presence of a second large well resolved peak was also a rather characteristic feature of some raw enzymes. Further, after quantitation by the Bradfords method, relative sensitivities calculated from the total peak area of the electropherograms obtained in the basic and acid BGEs can be also useful to identify the enzymes. Application of the procedure to real samples is possible in the presence of non-ionic surfactants, but not in samples containing large concentrations of anionic surfactants, more research being needed to reduce this interference.
Main points: The separation of intact proteins by CZE is potentially useful to classify or identify the enzymes used in the formulation of cleaning products.
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A basic BGE provides a flat baseline on which some small peaks are distinguished; the peaks constitute characteristic patterns which can be useful to classify or identify the enzymes.
Relative sensitivities in the acid and basic BGEs, calculated after protein quantification by the Bradfords method, may also be useful for enzyme identification.
The application of the procedure to real samples is limited to samples that do not contain anionic surfactants, being tolerated the presence of nonionic surfactants.
IX.3.2. Aminocidos por infusin directa en MS
IX.3.2.1. Clasificacin rpida de enzimas en productos de limpieza por hidrlisis aminocidos, MS y LDA Se ha desarrollado un procedimiento sencillo y rpido, capaz de clasificar las enzimas presentes en los productos de limpieza de acuerdo con su clase (proteasa, amilasa, celulasa y lipasa). Para ello, tras la precipitacin e hidrlisis de las enzimas empleadas como materia prima, los hidrolizados fueron disueltos en etanol y directamente infundidos en la interfaz ESI de un espectrmetro de masas de trampa inica. Los datos espectrales fueron utilizados para construir modelos LDA, obtenindose unas capacidades de prediccin excelentes. Los efectos de matriz de la muestra, observados al aditivar las bases de detergente con los concentrados de enzimas industriales, se redujeron en gran medida mediante la inclusin de muestras aditivadas en el conjunto de entrenamiento. El modelo de LDA resultante mostr una excelente capacidad de prediccin.
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A simple and quick procedure capable of classifying enzymes in cleaning products according to their protease, amylase, lypase and cellulase classes has been developed. For this purpose, after precipitation and hydrolysis, the hydrolysates were dissolved in ethanol and directly infused into the ESI ion source of an ion trap mass spectrometer. The spectral data were used to construct LDA models with excellent prediction capabilities. The sample matrix effects, which were observed when detergent bases spiked with enzyme industrial concentrates were used for model evaluation, were ruled out by also including the spiked samples in the training set. The resulting model showed an excellent prediction capability.
Main points: A simple and rapid procedure for enzyme classification in cleaning products has been developed; enzymes are classified according to the protease, amylase, cellulase or lipase classes. The spectral data of amino acids obtained by hydrolysis followed by ESI-MS were used in the construction of LDA models which showed excellent predictive capabilities. Sample matrix effects were reduced by including spiked samples in the LDA training set.
IX.3.3. Aminocidos derivatizados con OPA-NAC
IX.3.3.1. Clasificacin de enzimas presente en productos de limpieza mediante hidrlisis, derivatizacin con OPA-NAC, HPLC y LDA Se ha desarrollado un mtodo para la clasificacin de las enzimas presentes en productos de limpieza, basado en la hidrlisis seguida de
357
derivatizacin con OPA- NAC de los aminocidos resultantes, separacin de los derivados (isoindoles) mediante HPLC-UV-Vis. La clasificacin se efecta en funcin de las categoras ms comunes: proteasas, amilasas, lipasas y celulasas. Para la separacin se utiliz una columna C18 y elucin de los isoindoles con un gradiente multisegmentado. A partir del perfil de aminocidos se obtuvo un modelo de LDA con una excelente capacidad predictora. Se utilizaron los cocientes de las reas de pares de picos como variables predictoras para construir el conjunto de entrenamiento, adems de los concentrados industriales de las enzimas (materias primas), se incluyeron bases de detergentes reforzadas con enzimas. Todos los objetos del conjunto de evaluacin, incluyendo concentrados industriales de enzimas, bases de detergente aditivadas y productos de limpieza comerciales, se asignaron correctamente, con una probabilidad del 99%.
An HPLCUVVis method for class identification of the enzymes found in cleaning products according to the protease, amylase, lypase and cellulase classes, has been developed. For this purpose, after precipitation and hydrolysis, the amino acids were derivatized with OPANAC and aliquots were chromatographed. A C18 column and multi-segmented gradient elution with ACN/water were used to separate the isoindoles of the amino acids. An LDA model with an excellent prediction capability was obtained by using ratios of the areas of the peaks taken by pairs as predictors, and by including both enzyme industrial concentrates and spiked detergent bases in the training set. All the objects of the evaluation set, including enzyme industrial concentrates, spiked detergent bases and commercial cleaners, were correctly assigned with a 99% probability.
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Main points: A method for the classification of enzymes in cleaning products has been developed. The method is based on the total hydrolysis of the enzymes to amino acids, followed by derivatization with OPA-NAC, HPLC of the resulting isoindoles and LDA, using the chromatographic peak areas as predictors. In addition to industrial enzyme concentrates, the inclusion of detergent bases spiked with enzymes in the training set improves the prediction capability of the model when applied to real samples.
IX.3.4. Digestos con tripsina
IX.3.4.1. Comparacin de columnas microparticuladas y monolticas para RPLC de digestos trpticos de enzimas industriales en productos de limpieza Se ha realizado un estudio comparativo de las prestaciones de diversos soportes cromatogrficos comerciales, incluyendo tanto columnas monolticas (una polimrica y otra de base slice) como columnas particuladas (dos basadas en tecnologa convencional de partculas y una conteniendo partculas de ncleo fundido), para el anlisis mediante HPLC-UV de digestos trpticos de las enzimas ms comunes en la industria de los detergentes. Se desarroll tambin un procedimento para el control de calidad de las enzimas intactas en concentrados industriales, utilizando para ello la columna monoltica polimrica. Sin embargo, esta columna proporcion unas bajas eficacias y resolucin global en la separacin de los pptidos obtenidos por digestin con tripsina. Para este fin, la columna Kinetex, empaquetada con partculas de 2,6 m, con un ncleo fundido no poroso y una superficie porosa (tecnologa de partculas shell-core),
359
fue la que mostr las mejores prestaciones. La columna monoltica de slice Chromolith, y columnas convencionales empaquetadas con partculas de 5 y 3 m mostraron prestaciones intermedias. La columna Kinetex en las condiciones ptimas de elucin se utiliz para el anlisis de los digestos trpticos de los concentrados de materias primas industriales, bases de detergente aditivadas y productos de limpieza comerciales. Los cromatogramas resultantes fueron muy similares a los obtenidos directamente por la digestin de los concentrados industriales de enzimas, por lo que no se observaron interferencias ni efecto matriz. El mtodo cromatogrfico propuesto es til tambin para la clasificacin de las enzimas. Actualmente se est trabajando para mejorar este mtodo, estableciendo los pptidos que llevan a una mejor identificacin y clasificacin fiable de las enzimas a partir de datos de LC-MS.
The
chromatographic
performance
of
several
commercial
chromatographic supports, including monolithic (polymeric and silica-based) and particulate columns (with conventional and core-shell particle technology), for the HPLC-UV analysis of tryptic digests of enzymes commonly used in the detergent industry was comparatively evaluated. In addition, quality control of industrial enzymes concentrates was shown to be possible by chromatographing the intact enzymes on the polymeric monolithic column (ProSwift). However, this column gave a poor efficiency and a low global resolution for the separation of the peptides obtained by trypsin digestion. The Kinetex column, packed with supercially porous sub-3 m particles (shell-core particle technology), showed the best performance when applied to the separation of tryptic digests of the enzymes. The Chromolith silica monolithic column, and columns packed with 5 m or 3 m conventional particles showed intermediate performances. The
360
Kinetex column in the optimal elution conditions was applied to the analysis of tryptic digests from both raw industrial concentrates of the enzymes as well as to enzymes in spiked detergent bases and commercial cleaners. The resulting chromatograms were quite similar to those obtained by directly digesting the industrial enzyme concentrates; then matrix interferences were not observed. Therefore, HPLC of intact enzymes is useful in the quality control of industrial samples, as well as to discard enzyme classes; however, a more selective approach is required for a reliable classification and identification of the enzyme. For this purpose, HPLC of the tryptic digest of the enzyme using a core-shell particulate column is an excellent option. Further work on this topic, in order to establish characteristic peptides leading to the reliable identification of the individual enzymes by coupling LC with MS is in progress.
Main points: A polymeric monolithic column (ProSwift) is useful for the separation of intact enzymes, giving rise to chromatograms which show a predominant peak. These chromatograms are useful in quality control; however, they do not provide enough information to reliably classify or identify the enzymes. To separate the peptides obtained by enzyme digestion with trypsin, the polymeric monolithic column provides low efficiencies and poor overall resolution. For the separation of tryptic digests of enzymes, a Kinetex column showed an excellent performance. Columns packed with conventional 5 or 3 m particles, and a silica monolithic column (Chromolith), presented intermediate performances.
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No interference or matrix effects were observed in the separation of tryptic digests of industrial enzyme concentrates, detergent bases spiked with enzymes and commercial cleaning products, with the Kinetex column.
IX.4. Columnas monolticas
IX.4.1. Columnas monolticas de lauril metacrilato para CEC
IX.4.1.1. Columnas monolticas de LM para CEC utilizando LPO como iniciador Se ha optimizado la preparacin de columnas monolticas de LMA mediante fotopolimerizacin UV en presencia de LPO como iniciador. Se ha investigado la influencia de los cocientes monmeros/disolventes porognicos, 1,4-butanodiol/1-propanol y LMA/EDMA sobre las propiedades morfolgicas y sobre las prestaciones de las columnas en condiciones de uso en modo CEC. Utilizando LPO como iniciador, se compar la polimerizacin por va UV con la polimerizacin trmica. Ambos mtodos proporcionaron columnas con una eficacia similar, sin embargo, las columnas fotopolimerizadas mostraron menores tiempos de anlisis que las iniciadas trmicamente. Otras ventajas de la fotoiniciacin respecto a columnas obtenidas por polimerizacin trmica es el menor tiempo de fabricacin de la columna, y la mayor permeabilidad de la misma. Por otra parte, se estudi la sustitucin del LPO por AIBN como iniciador en la sntesis de columnas monolticas obtenidas por fotoionizacin. Bajo sus respectivas condiciones ptimas de polimerizacin, los dos tipos de monolitos proporcionaron eficacias semejantes. Las columnas obtenidas utilizando LPO como iniciador dieron lugar a tiempos de anlisis eran menores. Asimismo, se
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evalu el potencial de las columnas obtenidas con ambos iniciadores para separar compuestos tanto neutros como bsicos. Teniendo en cuenta los resultados obtenidos, el empleo de LPO y radiacin UV es la mejor opcin para la preparacin rpida de monolitos de LMA con buenas prestaciones
cromatogrficas.
The preparation of photo-polymerized LMA monoliths in the presence of LPO as initiator has been optimized, and the resulting columns have been characterized. The influence of ratios of monomers/porogenic solvents, 1,4butanediol/1-propanol and LMA/EDMA on the morphological and CEC properties was investigated. Under their respective optimal conditions, photoand thermal-polymerization using LPO gave rise to columns with similar efficiencies; however, photo-polymerized columns showed shorter analysis times. Other advantages of photo-initiation are shorter polymerization times and better permeabilities. Additionally, the LMA-based monolithic columns photoinitiated with either LPO or AIBN were compared. Under their respective optimal polymerization conditions, both types of monoliths provided similar efficiencies, but those prepared in the presence of LPO gave faster separations than their AIBN counterparts. The capability of columns obtained with both initiators to separate neutral and basic compounds has been also demonstrated. Thus, the employ of LPO and UV irradiation constitutes an attractive way for the fast preparation of improved LMA-based monoliths.
Main points: Using LPO as initiator, photopolymerization gives rise to better columns than thermal polymerization. Efficiencies were similar, but analysis times were
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shorter with photoinitiated columns. Also, manufacturing time was shorter, and the monoliths showed a higher permeability. Using photoinitiation in the presence of either LPO or AIBN, similar efficiencies were obtained; however, LPO led to shorter analysis times than AIBN. Excellent columns are quickly prepared using photoinitiation in the presence of LPO.
IX.4.1.2. Comparacin de fotoiniciadores para la preparacin de columnas monolticas de metacrilato para CE Se ha estudiado la preparacin y caracterizacin de columnas monolticas de LMA para CEC por fotopolimerizacin, utilizando varios iniciadores radicalarios a distintas concentraciones. El tipo y la variacin del contenido del iniciador produce cambios en la estructura monoltica. As, el tamao de los glbulos puede variar, lo que influye en las propiedades electrocromatogrficas de los monolitos. Por lo tanto, es importante encontrar una concentracin ptima para cada uno de los fotoiniciadores investigados. En las respectivas condiciones ptimas, se obtuvieron eficacias y resoluciones similares para los cuatro fotoiniciadores estudiados. Los monolitos polimerizados con AIBN y DMPA mostraron separaciones ligeramente mejores que aquellos polimerizados con BPO y LPO; sin embargo, este ltimo iniciador proporcion menores tiempos de anlisis y la posibilidad de controlar mejor las propiedades cromatogrficas del monolito. Por otro lado, la peor repetibilidad columna-columna se encontr para los lechos cromatogrficos fotoiniciados con BPO.
The preparation and characterization of LMA-based monolithic columns for CEC by photo-polymerization using several free radical initiators have been
364
described. The influence of each type of initiator and its concentration in the polymerization mixture was studied. As a result of this study, we have found that the type and variation of initiator content can produce changes in the monolithic structure, leading to an increase, a small influence, or a decrease on the final globule size, and therefore, variations in the electrochromatographic properties of monoliths. Consequently, it is important to find an optimum concentration for each photo-initiator investigated. Thus, under their respective optimum conditions, similar efficiencies and resolutions were obtained for the four investigated photo-initiators. The monoliths initiated with AIBN and DMPA showed slightly better separations than BPO and LPO, however, this latter initiator provided shorter analysis time jointly with a fine control in the retention properties. Additionally, the highest RSD values found for column-to-column repeatabilities were obtained for beds photo-initiated with BPO.
Main points: The type and concentration of the initiator may cause changes in the structure of the monoliths obtained by photopolymerization, which implies variations in the CEC properties. Each photoinitiator has its own optimal concentration. The four photoinitiators which have been studied show similar efficiencies and resolutions. The monoliths polymerized with AIBN and DMPA showed separations slightly better than those polymerized with BPO and LPO; however, LPO provided the shortest analysis time for the test compounds. On the contrary, BPO provided the worst column to column reproducibility.
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ABREVIATURAS
A
AB ABS ACN AES AIBN Ala Alc AMG AMGf AMGp ANN ANOVA AOS APCI APE APES APG APPI Arg AS Asn Azul cido / Acid blue Alquilbenceno sulfonatos / Alkylbenzenesulfonates Acetonitrilo / Acetonitrile Alquil ter sulfatos / Alkyl ether sulfates ,-azobisisobutironitrilo / ,-azobisisobutyronitrile Alanina / Alanine Alcalase 2.5 L Alquilmonoglucsido / Alkylmonoglucoside Alquilmonoglucofuransido / Alkylmonoglucofuranoside Alquilmonoglucopiransido / Alkylmonoglycopyranoside Red neuronal artificial / Artificial neuronal network Anlisis de la varianza / Analysis of variance -olefina sulfonatos / -olefin sulfonates Ionizacin qumica a presin atmosfrica / Atmospheric pressure chemical ionization Alquilfenol etoxilados / Alkylphenol ethoxylates Alquil fenol ter sulfatos / Alkyl phenol ether sulfates Alquilpoliglucsidos / Alkylpolyglucosides Fotoionizacin a presin atmosfrica / Atmospheric pressure photoionization Arginina / Arginine Alquil sulfatos / Alkyl sulfates Asparagina / Asparagine
399
Asp
cido asprtico / Aspartic acid
B
BGE BPO BSA Electrolito de fondo / Background electrolyte Perxido de dibenzoilo / Dibenzoyl peroxide Albmina srica bovina / Bovine serum albumine
C
Car CE CEC Cel CGE CI CID CIEF CMC CR Cys CZE Carezyme 4500L Electroforesis capilar / Capillary electrophoresis Electrocromatografa capilar / Capillary electrochromatography Celluzyme 0,7T Electroforesis capilar en gel / Capillary gel electrophoresis Ionizacin qumica / Chemical ionization Cationizacin seguida de colisin inducida / Cationization followed by collision-induced Isoelectroenfoque capilar / Capillary isoelectric focusing Concentracin micelar crtica / Critical micelar concentration Rojo Congo / Congo red Cistena / Cysteine Electroforesis capilar zonal / Capillary zone electrophoresis
D
DAD DMPA Detector de fila de diodos / Diode array detector 2,2-dimetoxi-2-fenilacetofenona / 2,2-dimethoxy-2-phenylacetophenone
400
DMSO DNA DP DTI Dur
Dimetilsulfxido / Dimethilsulfoxide cido desoxiribonucleico / Deoxyribonucleic acid Longitud de la cadena de poliglucsido / Degree of polymerization Inhibidor de la transferencia de color / Dye-transfer inhibitor Duramyl 300L, Type DX
E
EDMA EI EIC ELSD End EO EOF ESI Esp EtOH Eve Dimetilacrilato de etileno / Ethylene dimethacrylate Impacto electrnico / Electron impact Cromatograma de ion extrado / Extracted ion chromatogram Detector evaporativo de luz dispersada / Evaporative light scattering detection Endolase 5000L xido de etileno / Ethylene oxide Flujo electroosmtico / Electroosmotic flow Ionizacin por electronebulizacin / Electrospray ionization Esperase 8.0L Etanol / Ethanol Everlase 16L, Type EX
F
FAA FAB FAE FID Alcanolamidas / Fatty acid amide ethoxylates Bombardeo con tomos rpidos / Fast atom bombardment Alcoholes grasos etoxilados / Fatty alcohols ethoxylates Detector de ionizacin por llama / Flame ionization detector
401
FSCE FT
Electroforesis capilar en disolucin libre / Free solution capillary electrophoresis Transformada de Fourier / Fourier transform
G
GC Gf Gln Glu Gly Gp Cromatografa de gases / Gas chromatography Glucofuransido / Glucofuranoside Glutamina / Glutamine cido glutmico / Glutamic acid Glicina / Glycine Glucopiransido / Glucopiranoside
H
HAcO HEC HILIC His HPLC cido actico / Acetic acid Hidroxietilcelulosa / Hydroxyethylcellulose Cromatografa de interaccin hidroflica / Hydrophilic interaction chromatography Histidina / Histidine Cromatografa lquida de alta resolucin / High performance liquid cromatography
I
ICP ID IDA Ile IT Plasma acoplado por induccin / Inductively coupled plasma Dimetro interno / Internal diameter cido iminodiactico / Iminodiacetic acid Isoleucina / Isoleucine Trampa inica / Ion trap
402
L
LAS LC LDA LES Leu LMA LOD LPO Ls Lx Lys Alquilbenceno sulfonatos lineales / Linear alkylbenzenesulfonates Cromatografa lquida / Liquid cromatography Anlisis discriminante lineal / Linear discriminant analysis Lauril ter sulfatos / Lauryl ether sulfates Leucina / Leucine Lauril metacrilato / Lauryl methacrylate Lmite de deteccin / Limit of detection Perxido de lauroilo / Lauroyl peroxide Lipolase 100L, Type EX Lipex 100L Lisina / Lysine
M
MALDI MEKC MeOH Met META MGf MGp MS MW Desorcin-ionizacin lser asistida por matriz / Matrix-assisted laser desorption/ionization Cromatografa micelar electrocintica capilar / Micellar electrokinetic capillary chromatography Metanol / Methanol Metionina / Methionine Cloruro de [2-(metacriloiloxi)etil] trimetilamonio / [2-(methacryloyloxy)ethyl]trimethyl ammonium chloride Monoglucofuransido / Monoglucofuranoside Monoglucopiransido / Monoglucopyranoside Espectrometra de masas / Mass spectrometry Peso molecular / Molecular weight
403
N
NaAcO NAC NECEEM Acetato sdico / Sodium acetate N-acetil-cistena / N-acetyl-cysteine Electroforesis capilar en condiciones de no equilibrio de mezclas en equilibrio / Non-equilibrium capillary electrophoresis of equilibrium mixtures Acetato amnico / Amonium acetate Ion negativo / Negative ion Resonancia magntica nuclear / Nuclear magnetic resonance Fase normal / Normal phase Nonilfenoles etoxilados / Nonylphenol ethoxylates
NH4AcO NI NMR NP NPE
O
OD OPA OPE Dimetro externo / Outside diameter o-Ftaldialdehdo / o-Phthaldialdehyde Octifenoles etoxilados / Octylphenol ethoxylates
P
PAH PEA PEEK PEG Phe PI Pro PVA PVAcO Hidrocarburo poliaromtico / Polyaromatic hydrocarbon o-fosfoetanolamina / o-phosphoethanolamina Poli-ter-ter-cetona / Polyether ether ketone Polietilenglicol / Polyethylenglycol Fenilalanina / Phenylalanine Ion positivo / Positive ion Prolina / Proline Polivinil alcohol / Polyvinyl alcohol Acetato de polivinlo / Polyvinyl acetate
404
PVP PVP-NO PVP-PVI
Polivinil pirrolidona / Polyvinyl pyrrolidone Poli(1-xido-4-vinilpiridina) / Poly(4-vinylpyridine-1-oxide) Poli(1-vinilpirrolidona-co-1-vinilimidazol) / Poli(1-vinylpyrrolidona-co-1-vinylimidazole)
Q
QDA QTOF Anlisis discriminante cuadrtico / Quadratic discriminant analysis Cuadrupolo-tiempo de vuelo / Quadrupole time-of-flight
R
RG RP RSD Resolucin global / Global resolution Fase reversa o inversa / Reverse phase Desviacin estndar relativa / Relative standard deviation
S
Sav SAX SDS SEC SEM Ser SFC Savinase 16L, Type EX Intercambio aninico fuerte / Strong anionic exchange Dodecil sulfato sdico / Sodium dodecil sulfate Cromatografa de exclusin por tamao / Size exclusion chromatography Microscopa electrnica de barrido / Scanning electron microscope Serina / Serine Cromatografa de fluidos supercrticos / Supercritical fluid chromatography
SIM
Monitorizacin de iones seleccionados / Selected ion monitoring
405
SIMS SPE Sta
Espectrometra de masas de iones secundarios / Secondary Ion Mass Spectrometry Extraccin en fase slida / Solid phase extraction Stainzyme 12L
T
TAG Ter TFA Thr TIC TLC TMBA TMS Tris Trp Tyr Triacilgliceroles / Triacilgliceride Termamyl Ultra 300L cido trifluoroactico / Trifluoroacetic acid Treonina / Threonine Cromatograma de iones totales / Total ion chromatogram Cromatografa en capa fina / Thin layer chromatography cido 3,4,5-trimetoxibenzoico / 3,4,5-trimethoxibenzoic acid Trimetilsilano / Trimethylsilane Tris(hidroximetil)aminoetano / Tris(hydroxymethyl)amino ethane Triptfano / Tryptophan Tirosina / Tyrosine
U
UV Ultravioleta / Ultraviolet
V
Val Vis Valina / Valine Visible / Visible
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ANEXO I
A nexo I
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M iriam B eneito Cam bra
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ANEXO IV
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ANEXO V
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ANEXO VI
A nexo V I
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ANEXO VII
A nexo V II
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ANEXO VIII
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DEPARTAMENT DE QUIMICA ANALITICA

MIRIAM BENEITO CAMBRA

UNIVERSITAT DE VALNCIA Servei de Publicacions 2011

Copyright: Servei de Publicacions Miriam Beneito Cambra

FACULTAT DE QUMICA DEPARTAMENT DE QUMICA ANALTICA

Burjassot, Junio de 2011

Guillermo Ramis Ramos

Jos Manuel Herrero Martnez

xx1 xx3 xx3

x64 x65 x66 x68 x70

x72 x73 x73 x75 x76 x78

x79 x79 x80

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344 344 344 347

350 354 354

A partir de 1930 se empezaron a sintetizar

Miriam Beneito Cambra

Miriam Beneito Cambra

Sin embargo, en otras aplicaciones, la generacin de espuma es un

Miriam Beneito Cambra

Entre otros muchos usos, se han

Miriam Beneito Cambra

En los detergentes lquidos, especialmente en los

Miriam Beneito Cambra

Miriam Beneito Cambra

CH3(CH2)nCOO- Na+ n + 1 = 10 18 Jabones CH3(CH2)nOSO3 Na+ n + 1 = 12 18 AS

ABS CH3(CH2)n(OCH2CH2)mOSO3 Na+ AES

CH(CH2)mSO3 Na+ AOS

m + n = 9 15 m = 1 8 R: cadena alqulica APES ROOCCH CH3(CH2)nSO3 Na+ Alquil sulfonatos

ROOCCH SO3 Na+ Alquil sulfosuccinatos

Fig. I.1. Estructura y nomenclatura de las principales familias de surfactantes aninicos.

Miriam Beneito Cambra

CH3 N+ (CH2)nCH3 R1 N+ CH3 CH3 R1

n + 1 = 12 14 Sales de alquil piridinio

R1, R2: cadenas alqulicas Sales de alquil trimetil y dialquildimetil amonio

Fig. I.2. Estructura y nomenclatura de las principales familias de surfactantes catinicos.

Son de poca utilidad en procesos de limpieza, ya que al presentar

Miriam Beneito Cambra

m = 2 100 APEs O RCHN (CH2CH2O)mH R: cadena alqulica FAAs (CH2CH2O)nH

HOH2C HO HO O OH HOH2C O HO O (CH2)nCH3 OH n = 8 18 m = 0 3 APGs

Fig. I.4. Estructura y nomenclatura de las principales familias de surfactantes anfotricos.

Miriam Beneito Cambra

Se obtienen a partir de la alquilacin de

En la Fig. I.5 se muestran

los efmeros de anillo de los alquilmonoglucsidos.

Los diferentes oligmeros se pueden abreviar como CnGm,

La presencia de numerosos grupos hidroxilo en las

molculas de glucosa asegura la completa solubilidad de la molcula en agua

Los APGs con 16 o ms tomos de carbono en la cadena

Este tipo de surfactantes son buenos

Miriam Beneito Cambra

[Oldenhove de Guertechin, 1999].

Por estas propiedades favorables, as

son ampliamente utilizados