BIOANA !"ICA CH#$IS"%!

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Student Name: . Registration number: . Course number (CRN): .. Instructors Name: Dr. Saravanan Rajendrasozhan

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Department of Chemistry Faculty of Science University of Hail, Hail, KSA

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PREPARED BY Dr. Saravanan Rajendraso han! "ssistant Professor of #iochemistry! Department of $hemistry! University of %ail! %ail! Saudi "rabia. OBJECTIVE The course aims to &ive students insi&ht into and an understandin& of the analytical chemical problems and procedures related to biolo&ical sample analysis. Students should &ain an understandin& of the principles of advanced analytical techni'ues as (ell as the advanta&es and disadvanta&es of the individual methods. COURSE MATERIAL ). Principles and reactions of protein e*traction! purification! and characteri ation #y %afi "hmed. $R$ Press! +,,-. .S#/0 123,3415+,542. +. Understandin& #ioanalytical $hemistry0 Principles and "pplications #y 6ictor 7ault and /eville 8c$lena&han. 9iley annotated edition! +,,1. .S#/0 ,42,,+1,:4. INDEX Experime nt Number ; ) + 5 4 : 2 3 1 ), EXPERIMENT <ab safety and .ntroduction =ualitative tests for carbohydrates =ualitative tests for proteins =ualitative tests for lipids Determination of protein concentration by #iuret method Determination of protein concentration by $oomassie #lue >#radford? method @stimation of D/" concentration by Diphenylamine method Determination of carbohydrates by phenolAsulfuric acid method @stimation of carbohydrates by dinitrosalicylic acid method Separation and identification of amino acids by paper chromato&raphy Separation and identification of D/" by a&arose &el electrophoresis PAGE 5 : 1 )+ ))2 )1 +) +5 +4 +:

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LABORATORY SA ETY RULES <aboratories are interestin& and potentially dan&erous place to (ork. <aboratory Safety is a very important aspect of science. 9ithout it! e*perimentation could result in very serious injury. To reduce the risks involved (ith e*perimentation! there are certain procedures that (e should all follo(.

Safety is the first priority in our lab

). +. 5. Be!"#e re$p%n$ib&'( The dan&ers of spilled acids! chemicals and broken &lass(are created by thou&htless actions are too &reat to be tolerated. )e"r &"b *%"t( " lab coat should be (orn durin& laboratory e*periments. .t provides protection at all times. Dre$$ pr%per&'. <on& hair and loose clothin& >such as ties and ghutra? are a ha ard in the laboratory. They may either catch fire or be chemically contaminated. <on& hair must be tied back! ba&&y clothin& must be secured. )e"r $!%e$( Shoes must completely cover the foot. Sandals are prohibited because of the ha ard from the chemical spills. )e"r e'e pr%te*t%r( 9ear appropriate eye protection >&o&&les? at all times in the laboratory and in any area (here chemicals are stored or handled. Such protection (ill protect you a&ainst chemical splashes. D% n%t +e"r *%nt"*t &en$e$ >even (ith safety &o&&les?. $ontact lenses prevent rinsin& chemical splashes from the eye. 6apors in the laboratory >e&. %$l? dissolve in the li'uids coverin& the eye and concentrate behind the lenses. BSoftC lenses are especially bad as chemicals dissolve in the lenses themselves and are released over several hours. D% n%t $m%,e( Smokin& is not just an obvious fire ha ardD it also dra(s chemicals in laboratory air >both vapors and dust? into the lun&s. D% n%t e"t %r -rin,( Do not eat food! drink bevera&es! or che( &um in the laboratory. Do not use laboratory &lass(are as containers for food or bevera&es. 9ash your hands thorou&hly (ith soap and (ater (hen leavin& the laboratory. A#%i- bre"t!in. /ume$ %/ "n' ,in-( To test the smell of a vapor! collect some in a cupped hand. Ebtain your instructorCs (ritten permission before you smell any chemical. /ever smell a chemical reaction (hile it is occurrin&. 9ork in a hood if there is the possibility that no*ious vapors may be produced.

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),. Be prep"re- for your (ork in the laboratory. $arefully read the e*periment before comin& to the laboratory. "n unprepared student is a ha ard to everyone in the room. )). 0EEP YOUR )OR0 AREA NEAT AND CLEAN( $lean up spills and broken &lass immediately. $lean up your (ork spaceD (ipe all surfaces and put a(ay all chemicals and e'uipment at the end of the laboratory period. )+. Ne#er +%r, "&%ne in the lab. There must be at least one other person present in the lab. .n addition! an instructor should be 'uickly available. )5. D% n%t per/%rm "n' un"ut!%ri1e- experiment$( Perform only those e*periments authori ed by your teacher. This includes usin& only the 'uantities instructed! no more. $arefully follo( all instructions! both (ritten and oral. $onsult your instructor if you have any doubts about instructions and laboratory manuals. )4. Be *"re/u& +!i&e !e"tin. &i2ui-$( "dd boilin& chips to avoid Bbumpin&C. Flammable li'uids such as ethers! hydrocarbons! alcohols! acetone! and carbon disulfide must never be heated over direct flame. )-. C"re/u& "b%ut $p"tter$( Spatters are common in &eneral chemistry lab. Test tubes bein& heated or containin& reaction mi*tures should never be pointed at anyone. .f you observe this practice in a nei&hbor! speak to him or the instructor. ):. A&+"'$ p%ur "*i-$ int% +"ter +!en mixin.( Ether(ise the acid can spatter! often 'uite violently. )2. Ne#er u$e m%ut! $u*ti%n in fillin& pipettes (ith chemical rea&ents. "l(ays use a suction device. )3. D% n%t /%r*e " rubber $t%pper %nt% .&"$$ tubin& or thermometers. <ubricate the tubin& and the stopper (ith &lycerol or (ater. Use paper or cloth to(elin& to protect your hands. 7rasp the &lass close to the stopper. )1. Di$p%$e %/ "&& *!emi*"& +"$te pr%per&'( Dispose of e*cess li'uid rea&ents by flushin& small 'uantities do(n the sink. $onsult the instructor about lar&e 'uantities. Dispose of solid in crocks. /ever return unused chemicals to their ori&inal container. +,. Rep%rt "n' "**i-ent >spill! breaka&e! etc.? or injury >cut! burn! etc.? to your instructor immediately. Do not panic. +). In *"$e %/ /ire %r "**i-ent ! call the instructor at once. /ote the location of fire e*tin&uisher and safety sho(ers no( so that you can use them if needed. 9et to(els can be used to smother small fires.

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.n case of chemical spill on your body or clothin&! (ash the affected area (ith lar&e 'uantities of runnin& (ater. Remove clothin& that has been (et by chemicals to prevent further reaction (ith the skin. .f a chemical should splash in your eye>s?! immediately flush (ith runnin& (ater for at least +, minutes. @*cept for superficial injuries! you (ill be re'uired to &et medical treatment for cuts! burns or fume inhalation.

++. 8ost importantly! think about (hat you are doin&. Plan ahead. .f you &ive no thou&ht to (hat you are doin&! you predispose yourself to an accident. +5. Report if you have any dia&nosed aller&ies or other special medical needs to your instructor.

INTRODUCTION Bi%"n"&'ti*"& *!emi$tr' " branch of analytical chemistry in (hich compounds of biolo&ical si&nificance! such as carbohydrates! proteins! lipids and nucleic acids! are studied. 3u"&it"ti#e "n"&'$i$ .t deals (ith the identification of elements or &roupin& of elements > e ample: #iomolecules? present in a sample. 3u"ntit"ti#e "n"&'$i$ .t deals (ith the determination of the absolute or relative abundance >often e*pressed as a concentration? of a particular substance present in a sample.

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Experiment4 5 3UALITATIVE ANALYSIS O CARBO6YDRATES 5( BENEDICT7S TEST Principle 7lucose reduces copper hydro*ide >cupric hydro*ide? in alkaline solution to red or bro(n colored cuprous o*ide. The reducin& property of &lucose is due to the presence of free aldehyde >or keto? &roup. Dependin& on the concentration! yello( to &reen color is developed. Reagents ). #enedictCs rea&ent +. Test solution0 ), &rams of &lucose in ),,, ml of distilled (ater. Procedure "dd - drops of the test solution to + ml of #enedictCs rea&ent. The solution is boiled for minutes in a (ater both. $ool the solution. Analysis Formation of red! yello( or &reen precipitate indicates the presence of &lucose. 8( E6LING7S TEST Principle 9hen the blue alkaline cupric hydro*ide present in Fehlin&Cs solution is heated (ith &lucose! it &ets reduced to yello( or red cuprous o*ide precipitate. Reagents ). Fehlin&Cs solution " and # +. Test solution0 ), &rams of &lucose in ),,, ml of distilled (ater. Procedure To ) ml of Fehlin&Cs solution "! add ) ml of Fehlin&Cs solution # and a fe( drops of test solution. #oil for a fe( minutes. Analysis Formation of bro(nish;red precipitate indicates the presence of &lucose.
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9( IODINE TEST Principle 9hen starch solution is mi*ed (ith iodine! a blue color starch;iodine comple* is formed. Reagents ). .odine solution0 + & of potassium iodide is dissolved in ),, ml of distilled (ater. To this :+ m& of iodine crystals are dissolved >or 5G potassium iodide?. +. Test solution0 ) &m of starch po(der is boiled in ),, ml of distilled (ater. Procedure Take + ml of starch solution in a test tube. To these 5 drops of iodine solution is added. Analysis Formation is deep blue color indicates the presence of starch. :( ANT6RONE TEST Principle $arbohydrates are first hydroly ed into sample su&ars usin& dilute %$l. .n hot acidic medium &lucose is dehydrated to hydro*ymethylfurfural. This compound (ith anthrone forms a &reen colored product (ith an absorption ma*imum at :5, nm. Reagents ). +.- / %ydrochloric acid. +. "nthrone rea&ent0 +,, m& of anthrone is dissolved in ),, ml of concentrated sulfuric acid. 5. Test solution0 Dissolve ) &m table su&ar or &lucose or starch in ),, ml of distilled (ater. Procedure Take about + ml of test solution in a test tube. To this + ml of anthrone rea&ent is added and mi*ed (ell. %eat for 3 min in a (ater bath. $ool the test tube. Analysis Formation is dark &reen color indicates the presence of carbohydrates.
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D"te4 REPORT

Experiment 54 3UALITATIVE ANALYSIS O CARBO6YDRATES Te$t ). #@/@D.$TCS T@ST Ob$er#"ti%n C%n*&u$i%n

+. F@%<./7CS T@ST

5. .ED./@ T@ST

4. "/T%RE/@ T@ST

Re$u&t The &iven sample contains ;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;

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Experiment4 8 3UALITATIVE ANALYSIS O PROTEINS 5( NIN6YDRIN TEST Principle /inhydrin is a po(erful o*idi in& a&ent. .t decarbo*ylate the alpha amino acids and yields an intensely colored bluish purple product. Reagents ). )G /inhydrin0 Dissolve ) & of ninhydrin in ),, ml of acetone. +. Test solution0 Dissolve ) & of peptone in ),, ml of distilled (ater >)G solution?. Procedure Take + ml of test solution is a test tube. To this add ) ml of )G freshly prepared ninhydrin solution. 8i* the contents and boil for a couple of minutes. "llo( to cool. Analysis Formation of violet or purple color indicates the presence of protein. 8( BIURET TEST Principle $ompounds (ith t(o or more peptide bonds &ive a violet color (ith alkaline copper sulphate solution. Reagents ). #iuret rea&ent +. Test solution0 Dissolve ) & of peptone in ),, ml of distilled (ater >)G solution? or separate the e&& (hite and dilute it. Procedure Take + ml of test solution. To this add + ml of biuret rea&ent. Analysis Formation of violet or pink color indicates the presence of protein.
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9( NITRIC ACID TEST Reagents ). /itric acid 5. Test solution0 Separate the e&& (hite and dilute it. Procedure Take + ml of test solution in a test tube. "dd a fe( drops of nitric acid. " precipitate appears. %eat it. Analysis Formation of lemon yello( color indicates the presence of protein.

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D"te4 REPORT

Experiment 84 3UALITATIVE ANALYSIS O PROTEINS Te$t ). /./%HDR./ T@ST Ob$er#"ti%n C%n*&u$i%n

+. #.UR@T T@ST

5. /.TR.$ "$.D T@ST

Re$u&t The &iven sample contains ;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;

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Experiment4 9 3UALITATIVE ANALYSIS O LIPIDS 5( SOLUBILITY TEST Principle <ipids dissolve in chloroform! not in (ater. Reagents ). $hloroform +. Test solution0 $oconut oil or &hee Procedure Take + drops of the sample in t(o separate test tubes. "dd + ml of distilled (ater to one tube and + ml of chloroform to another tube. Shake (ell and observe. Analysis .f the sample completely dissolved in chloroform and undissolved in (ater! it indicates the presence of lipid. 8( EMULSI ICATION TEST Reagents ). -G Sodium carbonate +. Test solution0 $oconut oil or &hee Procedure Take + drops of the sample and 4 ml of distilled (ater in a test tube. "dd 4 drops of -G sodium carbonate. Shake (ell and observe. Analysis Formation of emulsion >uniform distribution of lipid in solution? indicates the presence of lipid.

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9( IODINE TEST Reagents ). .odine solution0 Dissolve + & of potassium iodide in ),, ml of distilled (ater. To this add :+ m& of iodine crystals and dissolve. +. Test solution0 $oconut oil or &hee Procedure Take - drops of test solution in a test tube. "dd ) ml of distilled (ater and ) ml of iodine. Shake (ell. Analysis Disappearance of yello( color >due to the absorption of iodine by lipids? indicates the presence of lipid. :( SUDAN III TEST Reagents ). Sudan .... +. Test solution0 $oconut oil or &hee Procedure Take + ml of test solution and add fe( drops of sudan .... Analysis Formation of red color indicates the presence of lipid.

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D"te4 REPORT

Experiment 94 3UALITATIVE ANALYSIS O LIPIDS Te$t ). SE<U#.<.TH T@ST Ob$er#"ti%n C%n*&u$i%n

+. @8U<S.F.$"T.E / T@ST 5. .ED./@ T@ST

4. SUD"/ ... T@ST

Re$u&t The &iven sample ;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;; contains

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Experiment4 : DETERMINATION O PROTEIN CONCENTRATION BY BIURET MET6OD Prin*ip&e Under alkaline conditions substances containin& t(o or more peptide bonds >proteins? form a purple comple* (ith copper salts in the rea&ent.

E2uipment .n addition to standard li'uid handlin& supplies a visible li&ht spectrophotometer is needed! (ith ma*imum transmission in the re&ion of 4-, nm. 7lass or polystyrene cuvettes may be used. Re".ent ). #iuret rea&ent0 1 &m Sodium potassium tartrate >f.(. +3+.++?! 5 &m $opper sulfate * %+E >f.(. +41.:3?! - &m Potassium iodide >f.(.)::.,?! all dissolved in order in 4,, ml ,.+ 8 /aE% >f.(. 4,.,? before brin&in& to final volume >one liter final volume?. Discard if a black precipitate forms. A$$"' Pr%*e-ure ). 9arm up the spectrophotometer )- minutes before use. +. Prepare standards from bovine serum albumin >recommended ran&e is ,.- to +, m& protein?. 5. Prepare a reference tube (ith ) ml buffer. 4. .f possible! dilute unkno(ns to an estimated ) to ), m&Iml (ith bufferD a ran&e of dilutions should be used if the actual concentration cannot be estimated.
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-. Use ) ml sample per assay tube. :. "dd 1 ml #iuret rea&ent to each tube! vorte* immediately! and let stand +, min. 2. Read at --, nm.

An"&'$i$ Prepare a standard curve of absorbance versus micro&rams protein >or vice versa?. From the curve! determine concentrations of unkno(n samples from the amount protein! volumeIsample! and dilution factor! if any.

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Experiment4 < DETERMINATION O PROTEIN CONCENTRATION BY COOMASSIE BLUE =BRAD ORD> MET6OD Prin*ip&e The assay is based on the observation that the absorbance ma*imum for an acidic solution of $oomassie #rilliant #lue 7;+-, shifts from 4:- nm to -1- nm (hen bindin& to protein occurs. #oth hydrophobic and ionic interactions stabili e the anionic form of the dye! causin& a visible color chan&e. The assay is useful since the e*tinction coefficient of a dye;albumin comple* solution is constant over a ),;fold concentration ran&e.

E2uipment$ .n addition to standard li'uid handlin& supplies a visible li&ht spectrophotometer is needed! (ith ma*imum transmission in the re&ion of -1- nm! on the border of the visible spectrum >no special lamp or filter usually needed?. 7lass or polystyrene cuvettes may be used! ho(ever the color rea&ent stains both. Disposable cuvettes are recommended. Re".ent$ ). #radford rea&ent0 Dissolve ),, m& $oomassie #rilliant #lue 7;+-, in -, ml 1-G ethanol! add ),, ml 3-G >(Iv? phosphoric acid. Dilute to ) liter (hen the dye has completely dissolved! and filter throu&h 9hatman J) paper just before use.
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+. >Eptional? ) 8 /aE% >to be used if samples are not readily soluble in the color rea&ent?. The #radford rea&ent should be a li&ht bro(n in color. Filtration may have to be repeated to rid the rea&ent of blue components. The #io;Rad concentrate is e*pensive! but the lots of dye used have apparently been screened for ma*imum effectiveness. K%omemadeK rea&ent (orks 'uite (ell but is usually not as sensitive as the #io;Rad product. A$$"' pr%*e-ure ). 9arm up the spectrophotometer before use. +. Dilute unkno(ns if necessary to obtain bet(een - and ),, L& protein in at least one assay tube containin& ),, Ll sample. 5. "dd an e'ual volume of ) 8 /aE% to each sample and vorte*. 4. Prepare standards containin& a ran&e of - to ),, micro&rams protein >albumin or &amma &lobulin are recommended? in ),, Ll volume. -. "dd - ml dye rea&ent and incubate - min. :. 8easure the absorbance at -1- nm. An"&'$i$ Prepare a standard curve of absorbance versus micro&rams protein and determine amounts from the curve. Determine concentrations of ori&inal samples from the amount protein! volumeIsample! and dilution factor! if any.

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Experiment4 ? ESTIMATION O DNA CONCENTRATION BY DIP6ENYLAMINE MET6OD Prin*ip&e The rea&ents used in the diphenylamine reaction include acetic acid and sulfuric acid. 9hen these are heated (ith D/" they cleave the phospho;diester bonds and hydroly e the &lycosidic bonds bet(een the su&ar and purines. The free +;deo*y ribose under&oes a dehydration reaction to form M;hydro*ylevulinyl aldehyde! (hich reacts (ith diphenylamine to produce a variety of blue;colored compounds sho(in& a characteristic absorbance peak at -1- nm. Since the reaction is specific for +;deo*y ribose! the su&ar in D/"! there is no reaction (ith the ribose su&ar of R/". Thus! the presence of R/" in a sample (ill not interfere (ith the measurement of D/". E2uipment$ .n addition to standard li'uid handlin& supplies! boilin& (ater bath and visible li&ht spectrophotometer are needed! (ith ma*imum transmission in the re&ion of -1- nm! on the border of the visible spectrum >no special lamp or filter usually needed?. Re".ent$ ). Diphenylamine rea&ent0 Dissolve ) & of diphenylamine in ),, ml of &lacial acetic acid! and subse'uently add +.- ml conc. % +SE4. "l(ays use freshly prepared rea&ent and cover it (ith an aluminium foil! as the rea&ent is photosensitive. +. Stock solution0 Prepare a stock solution (ith ),, &Iml of commercially available calf thymus D/" in distilled (ater or buffered saline. A$$"' pr%*e-ure ). Prepare a series of standard D/" dilutions of , to ),, & in ) ml of each usin& stock solution. +. Set up various dilutions of unkno(n D/". 5. "dd 5 ml of diphenylamine rea&ent to all test tubes containin& the diluted D/" samples and blank. /ote0 diphenylamine rea&ent contains sulfuric and acetic acids. Use e*treme caution (hen handlin&. $lean up spills (ith plenty of (ater.
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4. 8i* the contents! and cap the tubes (ith foil! (rap (ith tape! and label each tube usin& a pencil. Place the tubes in a boilin& (ater bath for ), minutes >do not submer&e tubes in (aterN?. -. Remove the tubes from the boilin& (ater and cool them briefly in an ice bath! then allo( them to reach room temperature. :. Ence the tubes are dry! read their absorbance at :,, nm. An"&'$i$ Prepare a standard curve of absorbance versus micro&rams D/" and determine amounts from the curve. Determine concentrations of ori&inal samples from the amount of D/"! volumeIsample! and dilution factor! if any.

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Experiment4 @ DETERMINATION O CARBO6YDRATES BY P6ENOLASUL URIC ACID MET6OD "mon& many colorimetric methods for carbohydrate analysis! the phenolAsulfuric acid method is the easiest and most reliable method. .t has been used for measurin& neutral su&ars in oli&osaccharides! proteo&lycans! &lycoproteins! and &lycolipids. This method is used (idely because of its sensitivity and simplicity. Prin*ip&e This assay based on the action of concentrated sulfuric acid that causes hydrolysis of &lycosidic linka&e. The hydrolysed neutral su&ar >pentoses and he*oses? are then partially dehydrated! (ith the elimination of three molecules of (ater! to form furfural or a derivative of furfural >hydro*yl methyl furfural?.

>%e*ose? Furfural or hydro*yl methyl furfural cause condensation (ith phenol to form yello(;oran&e colored compounds. The absorbance at 4+, nm is proportional to the carbohydrate concentration initially in the sample. The sulfuric acid causes all non;reducin& su&ars to be converted to reducin& su&ars! so that this method determines the total su&ars present. Re".ent$ ). Phenol solution0 -G (Iv in (ater. A$$"' pr%*e-ure ). Set up several &lass tubes containin& - to ),, & &lucose in +,, l of (ater. Take unkno(n &lucose in +,, l of (ater. +. "dd +,, l of phenol solution. 5. "dd ) ml concentrated sulfuric acid rapidly and directly on the sample >Do not allo( sulfuric acid to touch the side of the tube?.
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4. <eave the solution undisturbed for ), minutes. -. Shake vi&orously and continue incubation for another 5, minutes. :. Read each tube at 41, nm. An"&'$i$ Prepare a standard curve of absorbance versus micro&rams carbohydrate >&lucose?. Determine the concentration of carbohydrate in the unkno(n sample from a standard plot.

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Experiment4 B ESTIMATION O CARBO6YDRATES BY DINITROSALICYLIC ACID MET6OD Prin*ip&e 5!-;Dinitrosalicylic acid >D/S? is an aromatic compound that reacts (ith reducin& su&ars and other reducin& molecules to form 5;amino;-;nitrosalicylic acid! (hich absorbs li&ht stron&ly at -4, nm.

.t (as first introduced as a method to detect reducin& substances in urine and has since been (idely used! for e*ample! for 'uantificatin& carbohydrates levels in blood. Re".ent$ ). "ssay rea&ent0 Dissolve ,.+- & 5!-;dinitrosolicylic acid and 2- & sodium potassium tartrate in -, ml +8 /aE%. Dilute to +-, ml (ith (ater. A$$"' pr%*e-ure ). Set up several &lass tubes containin& - to -,, & &lucose in ),, l (ater. Set up tube for unkno(n sample in ),, l (ater. +. "dd ) ml of assay rea&ent in to each tube. 8i* (ell. 5. .ncubate in boilin& (ater bath for ), minutes. 4. $ool to room temperature and determine absorbance at -2, nm. An"&'$i$ Prepare a standard curve of absorbance versus micro&rams carbohydrate >&lucose?. Determine the concentration of carbohydrate in the unkno(n sample from a standard plot.

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Experiment4 C SEPARATION AND IDENTI ICATION O AMINO ACIDS BY PAPER C6ROMATOGRAP6Y Prin*ip&e $hromato&raphy is a techni'ue for separatin& mi*tures into the components that they are made from. $hromato&raphy utili es the differential affinities of the components for a &as or li'uid mobile medium >mobile phase? and for a stationary adsorbin& medium >stationary phase? throu&h (hich they pass. The stationary phase holds the mi*ture until the mobile phase passes throu&h! solubili es the components! and moves them alon& at their individual rates. Ence components are separated from one another! they can be analy ed. .n paper chromato&raphy the stationary phase is the filter paper and the mobile phase is the solvent. The filter paper holds the components until the solvent dissolves them and carries them up the filter paper. The solvent travels up the filter paper by capillary action. The solventCs attraction to itself pullin& it up is &reater than the force of &ravity pullin& it do(n. The separation of components depends on their solubility (ith the solvent and their affinity to the solvent and filter paper. ObDe*ti#e$ ). To demonstrate separation of amino acids by paper chromato&raphy. +. To use chromato&raphy to identify amino acids. M"teri"&$ ). $hromato&raphy tank and lid +. $hromato&raphy paper >or filter paper?! to fit tank 5. $apillary tubes or pasteur pipettes 4. "mino acid samples >e.&. <eucine and 7lycine? in ) molar hydrochloric acid -. Solvent >#"9?0 #utan;);ol 0 "cetic acid 0 9ater :, 0 )- 0 +- >should be made up fresh on the day? :. Spray can of )G /inhydrin in butan;);ol 2. <ate* &loves Experiment"& Pr%*e-ure
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). Usin& a pencil! li&htly dra( a line ).-A+ cm above the bottom of the chromato&raphy paper. 8ake small marks at ).- cm intervals alon& the line. +. Fill a capillary tube or pipette by capillary action (ith your sample >animo acid?. Touch the tip of the capillary to the first mark and pull it a(ay. Hou should leave a small >O- mm? (et spot. <et this dry and apply the sample a&ain. 8ake a note of (hich sample is placed on the spot >you can li&htly (rite on the paper beneath the mark! usin& a pencil?. Repeat for each sample or mi*ture! on a different mark and usin& a fresh tube or pipette each time. <et the paper dry. 5. Put ) cm or less of #"9 into the chromato&raphy tank. The sample spots should not dip into the solvent. Place the tank in a cool place out of direct sunli&ht. Stand the chromato&raphy paper in the tank so that the bottom ed&e is in the solvent but the remainin& paper does not touch the tank. Place the lid on the tank and leave until the solvent reach the top. 4. 9earin& &loves! remove the damp paper and mark (here the solvent has reached. Dry the paper. -. Spray the (hole of the paper (ith )G ninhydrin. Dry the paper. :. 8ark the position of each spot that develops.

An"&'$i$ /ote the color and travel distance of each standard and hence find the amino acid composition of the unkno(n samples.
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The relative e*tent to (hich solute molecules >amino acids? move in a chromato&raphy e*periment is indicated by retardation factor >Rf? values. $alculation of Rf of the amino acids0 Rf P Distance travelled by amino acid >a or b? I Distance travelled by solvent >s?

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Experiment4 5E SEPARATION AND IDENTI ICATION O DNA BY AGAROSE GEL ELECTROP6ORESIS

"&arose &el electrophoresis is a (idely used techni'ue for the analysis of nucleic acids. D/" a&arose &els can be used to separate and visuali e D/" fra&ments of various si es. Prin*ip&e The techni'ue of D/" a&arose &el electrophoresis is based on the fact that D/" is ne&atively char&ed at neutral p% due to its phosphate backbone. For this reason! (hen an electrical potential is placed on the D/" it (ill move to(ard the positive pole. "n a&arose &el is created by suspendin& dry a&arose in a buffer solution! boilin& until the solution becomes clear! and then pourin& it into a castin& tray and allo(in& it to cool. The result is a fle*ible &elatin;like slab. Durin& electrophoresis! the &el is submersed in a chamber containin& a buffer solution and a positive and ne&ative electrode. The D/" to be analy ed is forced throu&h the pores of the &el by the electrical current. Under an electrical field! D/" (ill move to the positive electrode >red? and a(ay from the ne&ative electrode >black?. Several factors influence ho( fast the D/" moves! includin&D the stren&th of the electrical field! the concentration of a&arose in the &el and most importantly! the si e of the D/" molecules. Smaller D/" molecules move throu&h the a&arose faster than lar&er molecules. D/" in the &el (ill be visuali ed by the use of @thidium #romide! added to the &el. @thidium bromide binds to D/" and illuminates (hen e*posed to ultraviolet li&ht! causin& the D/" to B&lo(C. M"teri"&$ ). "&arose +. T"@ #uffer0 "dd 4.34 & Tris #ase to Q1,, ml %+E. "dd ).)4 ml &lacial "cetic "cid and + ml of ,.- 8 @DT" solution >p% 3? and mi*. Pour mi*ture into ) < &raduate cylinder and add %+E to a total volume of ) <. 5. :R Sample <oadin& #uffer0 $ombine ) ml of sterile % +E and ) ml of 7lycerol. "dd enou&h bromophenol blue po(der to make the solution a deep blue color >about ,.,- m&?. /ote0 #romophenol blue mi&rates at a rate e'uivalent to +,,A4,,bp D/". 4. @thidium #romide >), m&Iml?
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-. D/" ladder standard :. D/" fra&ments of different si es 2. @lectrophoresis chamber 3. Po(er supply 1. 7el castin& tray and combs ),. U6 illuminator )). 7loves and &o&&les Experiment"& pr%*e-ure Preparing the agarose gel ). 8easure ) & "&arose po(der and add it to a -,, ml flask. +. "dd ),, ml T"@ #uffer to the flask. 5. 8elt the a&arose in a micro(ave or hot (ater bath until the solution becomes clear. >if usin& a micro(ave! heat the solution for several short intervals ; only until the solution starts to boil?. 4. <et the solution cool to about -,;--S$! s(irlin& the flask occasionally so it cools evenly. -. Place the combs in the &el castin& tray. :. Pour the melted a&arose solution into the castin& tray and let cool until it is solid >it should appear milky (hite?. 2. $arefully pull out the combs. 3. Place the &el in the electrophoresis chamber. 1. "dd enou&h T"@ #uffer so that there is about +;5 mm of buffer over the &el. Loading the gel ). "dd ), l of each sample >D/" fra&ments? to + l of :R Sample <oadin& #uffer >make sure the order (hich each sample is loaded has been recorded?. +. $arefully pipette each sampleISample <oadin& #uffer mi*ture into separate (ells in the &el.
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5. Pipette - l of the D/" ladder standard into another (ell of the &el. Running the gel ). Place the lid on the &el bo*! connectin& the electrodes. +. $onnect the electrode (ires to the po(er supply! makin& sure the positive >red? and ne&ative >black? are correctly connected. 5. Turn on the po(er supply. 4. $heck to make sure the current is runnin& throu&h the buffer by lookin& for bubbles formin& on each electrode. -. $heck to make sure that the current is runnin& in the correct direction by observin& the movement of the blue loadin& dye A this (ill take a couple of minutes >it (ill run in the same direction as the D/"?. :. <et the po(er run until the blue dye approaches the end of the &el. 2. Turn off the po(er and disconnect the (ires from the po(er supply. 3. Remove the lid of the electrophoresis chamber. 1. $arefully remove the &el Staining the Gel ). 9ear &loves (hen (orkin& (ith @thidium #romide. +. Place the &el in the stainin& tray containin& )., &Im< @thidium #romide solution. "llo( the &el to stain for +,;5, minutes. 5. Remove the &el to a tray of (ater and allo( to destain for - minutes. 4. Place the &el on U6 li&ht source and observe the D/" bands. /ote0 /ever lookIobserve @thidium #romide fluorescence (ith unprotected eyesD short (ave U6 is dama&in&. -. Take a picture. An"&'$i$ <abel the identity of each sample by comparin& the si e of the bands on the D/" ladder standard.
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