ANALYTICAL METHODS FOR THE DETERMINATION

AND ESTIMATION OF SYNTHETIC DETERGENTS

Roberto Lava, PhD

ARPA Veneto
ITALY

TAIEX Workshop on Production

Technology and Chemical Analysis
of Synthetic Detergents
Cairo (Egypt) 27-28 February 2017
 OUTLINES

-  Introduction

-  Composition and sampling

-  Physical analysis, qualitative and elemental analysis

-  Examples
surfactants, enzymes, CMC, water, solvents, fragrances

-  References and further readings

2
 INTRODUCTION I

Purpose of the analysis:

20 millions tonns of
- PRODUCER laundry detergents (2000)
composition, Quality Control

- INSPECTOR
composition, presence/absence substances,
compliance with legislation

Different typology of materials

physical state, uses, treatments

Analytical protocols
official, well recognised (ISO, ASTM, OECD)
Accreditation of the lab ISO 17025 Hautal in Handbooks of Detergents: Part C (2005)
Accurate measures - Qualitative/Quantitative
3 Investment in lab according to the purpose
 INTRODUCTION II

Different detergent market according to uses and costumes, country

4
 INTRODUCTION III

In 2016, in Egypt, laundry care recorded

overall current value growth of 18% with all
subcategories seeing positive current value
growth. Standard powder detergents
recorded the most dynamic current value
growth of 20% in 2016. Laundry care
continued to grow … driven by the
importance of laundry detergents for
consumers with different income levels….

from Euromonitor International, 2017

http://www.euromonitor.com/laundry-care-in-egypt/report
5
 INTRODUCTION IV

Regulation (EC) No 648/2004 on detergents protection of animals

in scientific research

GLP

GLP inspections
6
 COMPOSITION I

- SURFACTANTS

- BUILDERS

- BLEACHING AGENTS

- ENZYMES

- MINOR COMPONENTS

Detergents as combinations of all of them

Most contains all 4 of them, other only 2 (e.g. window cleaners)
7
 COMPOSITION II

8
 COMPOSITION III

Regulation (EC)
No 648/2004
on detergents
Annex VII

9
 SAMPLING I

A good analysis is starting always from a good sampling

Representative product samples

ISO 8212: general techniques of taking and preparing

samples of soaps/detergents during the
manufacturing process at the time of packaging

ISO 8213: general techniques of taking and preparing

samples from bigger packages or in bulk
(i.e. to assess a lot)

10 Hautal in Handbooks of Detergents: Part C (2005)

 SAMPLING II

11 Hautal in Handbooks of Detergents: Part C (2005)

 SAMPLING II

12 Hautal in Handbooks of Detergents: Part C (2005)

 PHYSICAL CHARACTERISTICS

•  pH: potentiometrically with glass electrode (1 g in 100 mL demi water od directly

•  VISCOSITY: falling ball viscometer, Ubbelohde viscometer, rotational viscometer

Tested the dynamic viscosity η [Pa s]

•  DENSITY and APPARENT DENSITY: oscillation-type densitometer for liquid/pastry,

known volume (500 mL) filled with bulk powder detergent and weighed [ g/L]

•  PARTICLE SIZE DISTRIBUTION: 100 g powder detergent sieved on a set of test

sieves. % mass for each single sieve.

•  MOISTURE or VOLATILE MATTER§: 5 ± 0.01 g of sample in porcelain dish, dry to

constant mass in oven at 105 ± 2°C (1h with a loss <0.1 %)

§: ASTM D820.93 (Reapproved 2016)

13
 QUALITATIVE TESTS I

Waldhoff in Handbooks of Detergents: Part C (2005)

-  rough idea of the chemical composition and essential ingredients

-  often sufficient to check the presence/absence of an ingredient
-  when possible run together with quantitative tests to save costs of analysis

MICROSCOPICAL CHECK: optical for manufacturing

processes (mix, extrusion, spray mix, …), SEM for
element analysis of single particles (enzymes, cellulose,
perborate, …)

IR SPECTROSCOPY: identification by characteristic functional groups,

spectra comparison with library.

NMR: 1H and 13C NMR to detect group typycal shifts,

two-dimentional NMR for more complex matrix
14 Waldhoff in Handbooks of Detergents: Part C (2005)
 QUALITATIVE TESTS II

COLORIMETRIC TESTS for surface-active agents

anionic/cationic surface-active agents detected by formation of characteristic

British Standard 3762-2

15 Waldhoff in Handbooks of Detergents: Part C (2005)

 ELEMENTAL ANALYSIS

Tawada Scientific X-Ray Fluorescence website

X-RAY FLUORESCENCE (XRF) SPECTROSCOPY

powerful, universal technique for qualitative/quantitative analysis of elements

ELEMENT SOURCE COMPONENT

Al Na Mg silicates
Si silicates, silicon oil
P phosphates, phosphonates
B borate, perborate
S surfactants, inorganic sulfate
C H O organic components
Ca calcium carbonate

Na K N cation from salts and surfactants

16
Ti V Mo
Co Mn Fe
trace components/impurities
 FRACTIONING

17 Hautal in Handbooks of Detergents: Part C (2005)

 SURFACTANTS ANALYSIS

Separation by Solid-Phase Extraction (SPE)

-  different solid phase, large selection available
-  complete retention or elution according to solvent
-  surfactants retained both by non-polar and polar
sorbents

General scheme:
-  first column non-polar sorbent, polar solvent
(non-polar surfactants retained, polar eluted)
-  second column polar sorbent, non-polar solvent

Alternative scheme:
- Buschmann and Kruse, 1992

18 Hautal in Handbooks of Detergents: Part C (2005)

 NON-IONIC SURFACTANTS I

INFRARED (IR) SPECTROSCOPY

-  simple and quick technique
-  identification and structural analysis → corresponding
functional groups
-  analysis after fractioning and surfactant separation

Ethoxylated Alcohols
-  C-O-C band at 1100 cm-1
-  CH3, CH2 at 2950-2850, 350, 960, 840 cm-1
-  no peaks at 1740 cm-1
-  no peaks between 1600-1500 cm-1

19 Hautal in Handbooks of Detergents: Part C (2005)

 lauryl alcohol ethoxylate (n=10)

octylphenol ethoxylate (n=10)

stearic acid ethoxylate (n=10)

IR SPECTRA
20 Tanaka and Igarashi in Handbooks of Detergents: Part C (2005)
 lauryl alcohol ethoxylate (n=10)

octylphenol ethoxylate (n=10)

stearic acid ethoxylate (n=10)

21 CH3 CH2 group

Tanaka and Igarashi in Handbooks of Detergents: Part C (2005)
 lauryl alcohol ethoxylate (n=10)

octylphenol ethoxylate (n=10)

stearic acid ethoxylate (n=10)

22 C-O-C group
Tanaka and Igarashi in Handbooks of Detergents: Part C (2005)
 lauryl alcohol ethoxylate (n=10)

octylphenol ethoxylate (n=10)

stearic acid ethoxylate (n=10)

23 C-O-O-C group
Tanaka and Igarashi in Handbooks of Detergents: Part C (2005)
 lauryl alcohol ethoxylate (n=10)

octylphenol ethoxylate (n=10)

stearic acid ethoxylate (n=10)

24 BENZENE group
Tanaka and Igarashi in Handbooks of Detergents: Part C (2005)
 lauryl alcohol ethoxylate (n=10)

octylphenol ethoxylate (n=10)

stearic acid ethoxylate (n=10)

25 -OH group
Tanaka and Igarashi in Handbooks of Detergents: Part C (2005)
 NON-IONIC SURFACTANTS II

EX: Quantification of 4-nonylphenol, 4n-nonylphenol, 4-tert-oclyphenol and 4-

octlyphenol in waters

Loos, Hanke, Umlaf, Eisenreich, LC-MS-MS analysis and

occurrence of octyl- and nonylphenol, their ethoxylates
and their carboxylates in Belgian and Italian textile
industry, waste water treatment plant effluents and surface waters
Chemosphere 66, 690-699 (2007)

Principle:
analytes are extracted on solid phase (SPE) and determined with LC-ESI(-)MS/MS

500 mL water
extraction: SPE automatic station (HLB Oasis cartridge 200mg, 6mL),
SPE eluent: meOH/aceton/ethylacetate (2:2:1) + formic acid 0.1%
flow 5 mL/min, 6 mL eluted, Turbovap conc
Internal standard: 4-n-nonylphenol-d8
26
 NON-IONIC SURFACTANTS III

EX: Quantification of 4-nonylphenol, 4n-nonylphenol, 4-tert-oclyphenol and 4-

octlyphenol in waters

Chromatographic conditions: gradient millli-Q water/ acetonitrile, 40 mins run

Injection 10µL, flow 0.25 mL/min
Interface: ESI, negative ionization
Product
Results: C (µg/L) = Y ± U (µg/L) Analyte
Precurs Cone
Ion (m/
Collision
or (m/z) (V) Energy
z)
Quality data: 219 45 133 32
4-nonylphenol
- 60% ≤ recovery ≤ 120 % 219 45 147 30
- R2 ≥ 0.990 calibration curve 4-n-nonylphenol 219 40 106 22
once per month, 5 standards
4-octlyphenol 205 45 106 20
- quality control every sequence
± 10% 4-tert-octlyphenol 205 40 133 20
- method blank every sequence 4-n-nonylphenol-d8
227 40 112 20
27 (IS)
 ANIONIC SURFACTANTS I

higher industrial
production

28 Spilker in Handbooks of Detergents: Part C (2005)

 ANIONIC SURFACTANTS II

Chromatographic methods

•  Gas Chromatography after derivatization in methyl esters (some cases)

•  Ionic Chromatography

•  HPLC with detector UV, Refractive Index (RI), MS

- RP-HPLC with alkyl modified silica phase columns
- separation by type and length of the hydrophobic group, but also
different hydrophilic anionic groups
- H2O/MeOH phase better than H2O/ACN (anionic surfactants poorly soluble in ACN)
- NaClO4 often added as additive on the eluent
- pH controlled with a buffer

29
 examples of HPLC
methods for
anionic surfactants

Spilker in Handbooks of Detergents: Part C (2005)

 ANIONIC SURFACTANTS III

HPLC OLD EXAMPLE…

SURFACTANTS IN TOOTHPASTES
Konig, H; Strobel, W Fresenius Z. Anal. Chem. 1988, 331,
435-438

Procedure: toothpaste suspended in the eluent,

centrifugation and filtration to eliminate insoluble matter
(1)  lauric acid taurate
(2)  lauric acid sarcosinate
(3)  lauryl sufate
(4)  lauryl sulfoacetate

Column Nucleosil C18 (25 cm, i.d. 4 mm), mobile phase

MeOH/water 80:20 + 0.25 mol/L NaClO4, no pH adjustment

31
 ANIONIC SURFACTANTS IV

HPLC MORE RECENT EXAMPLE…

ANIONIC SURFACTANTS
Liu X, Tracy M, Pohl C, commercial application note,
2010

Anionic sulfate, LOQ estimated 50 ppb

(1)  decyl sulfate
(2)  dodecil sulfate
(3)  tetradecil sulfate
(4)  hexadecyl sulfate
(5)  octadecyl sulfate

Column Acclaim PA2 (15 cm, i.d. 4.6 mm), mobile phase
ACN/Borate buffer/water in gradient + NaOH, pH 8.3,
suppressed conductivity detection

32
 CATIONIC SURFACTANTS

HPLC CATIONIC SURFACTANTS

Nakamura K, Morikawa Y, JAOCS 59, 64-68

Cationic surfactants clorides, LOQ estimated 50 ppb

(A)  alkylbenzyldimethylammonium chloride
(B)  alkylpyridinium chloride
(C)  alkylbenzyldimethylammonium chloride

Column RP 18 (15 cm, i.d. 4.6 mm), mobile phase

MeOH/water (85:15) + 1 M NaClO4, pH 2.5 (phosphoric
acid), 50°C controlled temperature of column, RI
detector (isocratic conditions)

Same chromatographic conditions for separation of fatty

acid diethanolamides, alkyldimethylaminoacetic acid
betaines and sodium alkylamino propionates in the
33 individual homologous
 SIMULTANEOUS SURFACTANTS ANALYSIS

ANIONIC SURFACTANTS BY HPLC

Liu X, Tracy M, Pohl C,
commercial application note, 2010
Detector: MS, UV (230 nm), ELSD, CAD
34
 SURFACTANTS ANALYSIS KITS

Last decade there was a development of commercial kit for the analysis of all types of
surfactants in water (cuvette tests)

•  cheap, quick methods

•  based on colorimetric methods, need a UV-VIS photometer
•  specific for anionic (MBA), cationic (CTAB) and non-ionic (TBPE, CTAS) surfactants
•  useful for surfactants in different kind of water (e.g. environmental control)
•  range of measurement: 0.2 mg/L to 20 g/L, depending by method and surfactant
•  advantage of using no glassware, no solvents and limited amount of reagents
included in the kit
•  few volume of sample (4 mL)

Procedure:
-  4 mL sample in cuvette
-  add solvent (0.4 mL) + reagent (0.2 mL)
-  60 seconds stirring
-  30 second decanting phase
35 -  photometric measure
 BUILDERS

PHOSPHATES

Lecture this afternoon

ZEOLITES

36
 ENZYMES ANALYSIS I

•  Analysis of enzymes = based on standard method of protein detection

•  The difficulty is due to perform qualitative/quantitative determinations in
complex matrix è INTERFERENCES

AMYLASE, CELLULASE, PROTEASE, LIPASE, MANNANASE, PEROXIDASE

1)  QUALITATIVE ANALYSIS

2)  QUANTITATIVE ANALYSIS, necessary to know the composition
(before a qualitative)

QUALITATIVE ANALYSIS, before because a QUANTITATIVE can often be

performed when the exact type of enzyme variant is involved (specific activity in
an active assay è the identification of the type or variant is needed in order to
correlate activity with dosage or concentration of a specific enzyme type.
To perform the identification is needed the protein isolation.

37
 ENZYMES ANALYSIS II

Process of enzyme
analysis in detergents

E + S è ES è E + P
E: enzyme
S: substrate
P: product

38 Maurer and Gabler, in Handbooks of Detergents: Part C (2005)

 ENZYMES ANALYSIS III

COLORIMETRIC (STAINING) QUALITATIVE/QUANTITATIVE METHODS

Dyed Substrate, based on cross-linked or insoluble starch linked to dyes with a
covalent bond. Spectrophotometric detection method (UV-VIS)
Many enzymes under patent (methods as well).

AMYLASE - Phadebas® Amylase Test or similar methods for the quantitative

assay of α-amylase in fluids.
Insoluble, dyed, cross-linked starch, with the dyes becoming soluble after
amylolytic degradation of the cross-linked polymer to soluble dextrins.
Mayor amylase used: Bacillus licheniformis amylase

CELLULASE - highest degree of variety among detergents enzymes. Made visible

by using activity staining methods base on CMC Congo Red or CMC Trypan Blue

LIPASE - incubation of enzyme fraction with p-nitrophenyl laurat or p-nitrophenyl

palmitate (yellow color of p-nitrophenol)

39 PROTEASE - suc-AAPF-p-nitroanilide as substrate most popular method

 ENZYMES ANALYSIS IV

GEL ELECTROPHORESIS
Methods for separation and analysis of macromolecules and their fragments,
based on size and charge.
Method used in clinical chemistry, separation of DNA/RNA. Separation due to
application of an electric field to move the negatively charged molecules in a
matrix of agarose or similar substances. By Bensaccount at English Wikipedia, CC BY 3.0, https://commons.wikimedia.org/w/index.php?curid=11497401

CHROMATOGRAPHIC METHODS
HPLC, LC-MS, size-exclusion chromatography (or gel filtration), affinity chromatography
Detector: UV, RI

40 All these methods are used for fractioning of proteins/enzymes

 CARBOXYMETHYLCELLULOSE I

Polymer additive, added 1-2 %

CMC used in detergents as antisoil redeposition agent, antygraying agent,
thickener, particle suspender, skin protector, stabilizer, colour protector agent
(cotton laundering, antiallergenic)

41
 CARBOXYMETHYLCELLULOSE II

Historical method based on hydrolysis

and dehydration of CMC to furfural
derivatives which produce a GREEN
colour with a solution of anthrone in
60% sulphuric acid.

Not specific for CMC, but most cellulose derivatives reacts (e.g. sucrose)
Green colour proportional of content.

Anthrone (9,10-dihydro-9-oxoantracene), Longman method (red dye)

UV-VIS spectroscopy, IR spectroscopy

No standards at EU level
42
 CARBOXYMETHYLCELLULOSE III

1H NMR identification

43
 WATER IN DETERGENTS I

WATER
Contained in liquid detergents as solvent, in powder as crystal water
bound in ingredients, e.g. perborate.

Methods:
azeotropic distillation in the past, now Karl-Fischer (KF) titration

KF titration
-  very precise method
-  SO2 + I2 + 2H2O - 2HI + H2SO4
-  electrometrical endpoint determination, coulometric method
recommended for low water content, volumetric system for higher
content,
-  direct and indirect method

44
 WATER IN DETERGENTS II

DIRECT (EN 13267, 2001)

An exactly weighed amount of sample or its solution in an organic
solvent introduced in the KF instrument and directly titrated

INDIRECT
when interfere substances (strong alkaline substances, oxidizing
agents). Some grams of detergent placed in a special oven
connected to an absorption vessel. Oven heated up (120-150°C).
The free water is transferred by a stream of N2 in the KF instrument
and titrated

45
 ORGANIC SOLVENTS IN DETERGENTS I

Different organic solvents: by Head Space-GC (FID or MS as detector).

Solvent type: boiling range 40-150°C

46
 ORGANIC SOLVENTS IN DETERGENTS II

Polar compound: DB-WAX,

Medium polarity:DB-624
Non-polar: DB-5

Difficulties in direct determination of very-high-boiling polyhydric alcohols

(e.g. glycerol).

2 options: - transformed in lower boiling point derivatives

- use of HPLC methods

47
 METHANOL IN DETERGENTS I

HPLC
10 ppm all analytes
Most solvents are not 100 ppm 2-propanol
UV-detectable.
Refractive Index detector,
no solvent gradient,

Weiβ (2001)
polymer-resin-based RP column
detector: pulsed amperometry

48
 METHANOL IN DETERGENTS II

49
 MUSK XYLENE I

MINOR COMPONENT
Synthetic musk fragrance (mimic natural musk)

REACH Regulation (CE) 1907/2006

Authorisation list for SVHC (Substances of Very High Concern)
Musk Xylene (MX), sunset data: August 2014
Absence: < 1.0 %
Internal method, Lab Accreditation according to ISO IEC 17025

Persistant, not biodegradable, imported 67 t/y (2000), 25 t/y (2008) in Europe

Cosmetics Regulation (CE) 1223/2009:
1% perfum, 0.4 % eau de toilette, 0.03 % other products
but not regulated in PCP and household products before REACH
February 2011 proposed for Annex XIV as vPvB substance

50
 MUSK XYLENE II

MUSK XYLENE

1,3-dimethyl-2,4,6-trinitro-5-ter-butylbenzene

•  CAS number: 81-15-2

•  EINECS number: 201-329-4 Product Mass fraction (%)
•  Molecular Mass: 297.3 Skin cream 0.0075
Deodorant 0.0075
•  Density 0.77 g/cm3 Shampoo 0.01
•  Vapour pressure: 0.00003 Pa at 20°C Household detergents 0.02
•  Water solubility: 0.15 mg/L Aftershave 0.03
Toilet soap 0.04
•  log KOW : 4.9 Air freshener 0.07
Cologne/eau de toilette 0.075
Fine fragrance 0.05–0.1
Sources: International Agency for
Research on Cancer (1996);
European Union Risk Assessment Report (2005)
51
 MUSK XYLENE III

52
 SPME - Solid Phase MicroExtraction I

2010 Sigma-Aldrich Co.

53 2010 Sigma-Aldrich Co.

 SPME - Solid Phase MicroExtraction II

Analyte adsorbed Extraction time

Factors affecting sampling recovery: type of fiber, sample

Modification, extraction time, desorption condition, GC inlet
design, GC column
54
 MUSK XYLENE III

Analytical protocol:
5 mL pure detergent sample + 100 µL MeOH + 15µL internal standard
(deuterated MX, 100 ng/µL) → final concentration 30 µg/L

55
 MUSK XYLENE IV

56
 MUSK XYLENE V

57
 CONCLUSIONS

From the analytical methods overview in this presentation:

•  Composition of SYNDET complex è clear the purpose of the analysis

•  Laboratory accredited ISO 17025, not necessary the single methods

•  Often a precise method is not necessary: QUALITATIVE/QUANTITATIVE

•  Importance of QA/QC

•  Adequate instrumental facilities and updated technology

58
 REFERENCES and USEFUL READINGS

•  Ed. H. Waldhoff, R. Spiker

Handbook of Detergents
Part C: Analysis
Vol. 123, CRC Press (2005)

•  Regulation (EC) 648/2004 of

European Parliament and of the
Council of 31 March 2004 on detergents

59
 ANY QUESTIONS….?

THANK YOU FOR

YOUR ATTENTIONS
‫شكرا‬
Contact:
LAVA ROBERTO, PhD
email: roberto.lava@arpa.veneto.it

60