The Strategy of Surfactant Analysis by HPLC Xiadong Liu, Mark Tracy, and Christopher Pohl Dionex Corporation, Sunnyvale, CA, USA

Home , Ethoxylation

INTRODUCTION DETECTION METHOD CONSIDERATION Surfactants are widely used in the consumer, industrial, agricultural, and For surfactant analysis, detection methods such as pharmaceutical markets in products as diverse as pesticides, ultraviolet/visible (UV), refractive index (RI), charged aerosol detection detergents, petroleum, and cosmetics. The separation and identification (CAD®), evaporative light scattering detection (ELSD), mass of surfactants can be difficult due to both their diversity and the spectrometry (MS), or suppressed conductivity can be used. The complexity of sample matrices. Although high-performance liquid preferred detection method for surfactants with chromophores is UV. chromatography (HPLC) is the preferred and most commonly used However, many surfactants have no chromophore and thus cannot be approach for surfactant analysis, it is always challenging to choose suit- detected by UV. MS is often used for trace analysis and in cases when able methods for specific applications. For example, the existing HPLC peak identification is required. ELSD is a universal detection method and methods do not provide optimal separation for anionic, nonionic, cationic, is compatible with gradient methods, making it suitable for surfactant and amphoteric surfactants in a single analysis. This poster provides an analysis when pursuing exploratory work or performing routine analysis overview of solutions for surfactant analysis by HPLC, including of high-concentration samples. Its limitations include poor separation columns, instrumentation, method development, and applications. reproducibility, low sensitivity, and nonlinear response. Compared to ELSD, CAD provides (1) excellent reproducibility, (2) 5 to 10 times better sensitivity than ELSD, and (3) more uniform peak response in addition to all the benefits of ELSD. Suppressed conductivity detection provides good sensitivity and excellent selectivity for ionic species, making it an economical method for ionic surfactant analysis requiring high sensitivity and selectivity. EXPERIMENTAL For UV and ELSD: Summit HPLC System (Dionex) equipped with a P680 gradient pump, ASI-100 autosampler, TCC-100 column oven, and UVD 340 detector. A Sedex 85 ELS detector (Sedere, Alfortville, France) 6 was used for detecting surfactants with weak or no chromophore. 5 10 For suppressed conductivity detection: ICS-3000 ion chromatography system (Dionex) equipped with a DP Dual Pump module, AS mV 3 autosampler, and DC Detector/Chromatography module with a 2 4 ® 7 13 conductivity detector. A CSRS ULTRA II 4 mm suppressor was used in 8 9 external water mode for detecting cationic surfactants and an 1 12 11 AMMS® III 4 mm suppressor was used in chemical mode for detecting 14 anionic surfactants. 0 5 10 15 20 25 30 35 Software: Chromeleon® 6.7 Chromatography Data System (Dionex). Minutes

HPLC-grade acetonitrile and methanol used were from Burdick and Column: Acclaim Surfactant, 5 µm Jackson (Muskegon, MI, USA). Deionized water (>18 MΩ resistivity) Dimensions: 4.6 × 150 mm Mobile Phase: (A) Acetonitrile, (B) 0.1 M NH4OAc, pH 5.4 was purified by a Milli-Q® water purification system (Millipore, Bedford, Gradient: 25% to 85% A in 25 min, then hold 85% A for 10 min MA, USA). The boric acid was obtained from EM Science (Cherry Temperature: 30 C Flow Rate: 1 mL/min Hill, NJ, USA). Ammonium acetate, acetic acid, and sulfuric acid were Inj. Volume: 25 µL purchased from Sigma-Aldrich (Milwaukee, WI, USA). All surfactant Detection: ELSD Peaks: 1. Chloride 6. Lauryldimethylbenzylammonium standards were from ChemService (West Chester, PA, USA). 2. Bromide 7. Triton® X-100 3. Nitrate 8. Cetyl betaine 4. Xylene sulfonate 9. Decyl sulfate APPLICATIONS AND DISCUSSIONS 5. Laurylpyridinium 10. Dodecyl sulfate 11-14. Dodecylbenzene sulfonate Simultaneous Analysis of a Mixture of Anionic, Cationic, Nonionic, and Amphoteric Surfactants Xylene Sulfonate Laurylpyridinium Chloride ® The Acclaim Surfactant column is the most versatile column for CH surfactant analysis in the market. Its novel chemistry provides ideal 3 CH3 selectivity for separating different types of surfactants as well as + N - - + Cl hydrotropes. Figure 1 shows the separation of a mixture of anionic, SO3 Na nonionic, cationic, and amphoteric surfactants as well as hydrotropes, with excellent resolution and peak asymmetry. The mobile phase is Triton X-100 Lauryldimethylbenzylammonium Chloride volatile, thus making it compatible with CAD, ELSD, MS, and CH UV (>230 nm) detection methods. 3 Cl-

+ C H (OCH2CH2)nOH 8 17 CH2N n ~ 10 CH3

Decyl Sulfate O Cetyl Betaine Me O O-S-O-Na+ N O- O Me

Dodecyl Sulfate Dodecylbenzene Sulfonate H O CH3 (CH2)n C (CH2)m CH3 O-S-O-Na+ m + n = 10 O

- + SO3 Na

27419

Figure 1. Simultaneous separation of inorganic anions, hydrotropes, cationic, nonionic, amphoteric, and anion surfactants.

2 The Optimization Strategy ofof Surfactantthe Separation Analysis Power by ofHPLC 1-D Nano LC Analysis of Proteomics Samples

Analysis of Cationic Surfactants Determination of the Degree of Ethoxylation and Alkyl The Acclaim Surfactant column is the column of choice for separating Chain Distribution for Nonionic Ethoxylated Surfactants cationic surfactants. Cationic surfactants are used in many formulated The Acclaim Mixed-Mode HILIC-1 column is suitable for analyzing products, such as fabric softeners, corrosion inhibitors, and nonionic ethoxylated surfactants. In HILIC mode, the degree of antimicrobial agents. Commonly used cationic surfactants include alkyl ethoxylation (EO) can be determined. In RP mode, the separation of quaternary ammonium, benzylalkylammonium, alkylpyridinium, and ethoxylated oligomers is suppressed and the alkyl chain distribution can imidazolinium salts. Silica-based, reversed-phase (RP) columns often be characterized. Figure 3 illustrates an example of analyzing Brij® exhibit peak tailing and excessive retention because of the undesirable 35 [lauryl alcohol condensed with 23 moles ethylene oxide; molecular ion-exchange interactions between residual silanols on the silica surface formula: C12H26O(C2H4)n H]. In RP mode, the surfactant is separated into and the analytes, accompanied by high hydrophobicity of cationic four single peaks, corresponding to free PEGs (early-eluting peak) and surfactants. The novel column chemistry of the Acclaim Surfactant three ethoxylates corresponding to different alkyl chain lengths. Under column effectively deactivates these interactions, resulting in excellent this condition, all EO oligomers with the same hydrophobe selectivity, high efficiency, and good peak shape (Figure 2). collapse into a single peak. Alternately, in HILIC mode, all EO oligomers are separated in addition to the hydrophobe-based separation. In this way, the degree of ethoxylation can be determined.

3 5

mV 10 6 4 9 Hydrophobe Separation 7

(RP mode, CH3CN/0.1M NH4OAc v/v 70/30) mV

0 5 10 15 20 25 EO Oligomer Separation Minutes (HILIC mode, CH3CN/0.1M NH4OAc v/v 90/10)

Column: Acclaim Surfactant, 5 µm Peaks: (100–400 µg/mL each) Dimensions: 4.6 × 150 mm 1. Lauryl pyridinium Mobile Phase: (A) Acetonitrile 2. Lauryldimethylbenzyl (B) 1% acetic acid (v/v) ammonium 0 10 20 30 40 50 Gradient: Time (min) A B 3. Octylphenoxyethoxyethyl- –12 20 80 dimethylbenzyl ammonium Minutes 0 20 80 4. Cetyltrimethylammonium 25 80 20 5. Cetylpyridinium Temperature: 30 °C 6. Diethyl heptadecyl Flow Rate: 1 mL/min imidazolinium–1 Column: Acclaim Mixed-Mode HILIC-1, 5 µm Inj. Volume: 10 µL 7. Diethyl heptadecyl Dimensions: 4.6 × 150 mm Detection: ELSD imidazolinium–2 Mobile Phase: See chromatogram for details 8. 2M2HT quat–1 Temperature: 30 °C 9. 2M2HT quat–2 Flow Rate: 1 mL/min 10. 2M2HT quat–3 Inj. Volume: 5 µL Detection: ELS detector Sample: Brij 35 (3 mg/mL)

23803 24258

Figure 2. Separation of cationic surfactants using the Acclaim Surfactant column. Figure 3. Analysis of ethoxylated fatty alcohols in both RP and HILIC modes.

3 Highly Sensitive and Selective Method for the Determination of Anionic Surfactants Using 30 Suppressed Conductivity Detection

The Acclaim PolarAdvantage II (PA2) column provides excellent 4 hydrolytic stability in both acidic and alkaline conditions, and is ideal for highly selective and sensitive analysis for anionic surfactants using µS 5 RPLC and suppressed conductivity detection. Conductivity detection in 1 2 suppressed mode provides excellent selectivity and sensitivity for ionic species, making it suitable for trace-level analysis and sample detection 3 in complex matrices. Figure 4 demonstrates the separation of a series 0 of alkyl sulfates on the Acclaim PA2 column. The developed method has good linear responses in a wide dynamic range (0.1 to 1000 ppm), under both isocratic and gradient conditions. With the injection 0 3 6 9 12 15 volume of 25 μL, the LOD is estimated to be approximately 20 ppb Minutes (S/N = 3) and the LOQ is estimated to be approximately 50 ppb (S/N = 10). Column: Acclaim PA2, 5 µm Dimensions: 4.6 × 150 mm Mobile Phase: (A) Acetonitrile (B) Borate buffer (6.2 g boric acid in DI water, adjust to pH 8.3 with 50% NaOH aqueous solution) (C) DI water Gradient: Time (min) A B C -15 25 30 45 0 25 30 45 10 70 30 0 15 70 30 0 Temperature: 30 °C Flow Rate: 1 mL/min

Inj. Volume: 25 µL Instrument: ICS-3000 chromatographic system Detection: Suppressed conductivity (AMMS III 4 mm suppressor, chemical mode at 1.5 mL/min with 20 mN sulfuric acid)

Peaks: (10 to 100 µg/mL each) 1. Decyl sulfate 2. Dodecyl sulfate 3. Tetradecyl sulfate 4. Hexadecyl sulfate 5. Octadecyl sulfate

24240

Figure 4. Separation of alkyl sulfates with the Acclaim PA2 column and conductivity detection.

4 The Optimization Strategy ofof Surfactantthe Separation Analysis Power by ofHPLC 1-D Nano LC Analysis of Proteomics Samples

Table 1. Summary of Methods for Surfactant Analysis

Application Separation Column Detection Mobile Phase* Instrument

Acetonitrile/ammonium acetate Acclaim Surfactant UV, ELSD, CAD, MS HPLC Anionic Surfactants (or formate) buffer Acclaim PolarAdvantage II Suppressed conductivity Acetonitrile/borate buffer IC Acetonitrile/ammonium acetate UV, ELSD, CAD, MS HPLC Cationic Surfactants Acclaim Surfactant (or formate) buffer Suppressed conductivity Acetonitrile/acetic (formic) acid IC Acetonitrile (or methanol)/water or Acclaim Mixed-Mode HILIC-1 UV, ELSD, CAD, MS HPLC ammonium acetate (or formate) buffer Nonionic Surfactants Acetonitrile (or methanol)/water or Acclaim Surfactant UV, ELSD, CAD, MS HPLC ammonium acetate (or formate) buffer Acetonitrile (or methanol)/water or Acclaim PolarAdvantage II UV, ELSD, CAD, MS HPLC ammonium acetate (or formate) buffer Amphoteric Surfactants Acetonitrile (or methanol)/ammonium acetate Acclaim Surfactant UV, ELSD, CAD, MS HPLC (or formate) buffer Acetonitrile/ammonium acetate Acclaim Surfactant UV, ELSD, CAD, MS HPLC Hydrotropes (or formate) buffer Acclaim PolarAdvantage II Suppressed conductivity Acetonitrile/borate buffer IC Mixture of Surfactants (anionic, Acetonitrile (or methanol)/ammonium acetate Acclaim Surfactant UV, ELSD, CAD, MS HPLC cationic, nonionic, amphoteric, etc.) (or formate) buffer

*When UV is used to detect surfactants that have a chromophore, phosphate buffer can also be used.

5 CONCLUSION REFERENCE The proven chromatographic methods, including separation column, 1. Schmitt, T.M. Analysis of Surfactants; Marcel Dekker: New York, 2001. detection method, mobile phase selection, and instrumentation platform (summarized in Table 1), provide a general guideline for surfactant analysis 2. Liu, X.; Pohl, C.; Weiss, J. New Polar-Embedded Stationary Phase for using HPLC. Surfactant Analysis. J. Chromatogr., A 2006, 1118, 29–34.

3. Liu, X.; Pohl, C. A Versatile Column for Surfactant Analysis by HPLC. American Laboratory 2005, 37 (20), 27–30.

4. Liu, X.; Pohl, C. New Hydrophilic Interaction/Reversed-Phase Mixed-Mode Stationary Phase and Its Application for Analysis of Nonionic Ethoxylated Surfactants. J. Chromatogr., A 2008,1191, 83–89.

5. Liu, X.; Bordunov, A.; Pohl, C. Preparation and Evaluation of a Hydrolytically Stable Amide-Embedded Stationary Phase. J. Chromatogr., A 2006, 1119, 128–134.

Acclaim, AMMS, CAD, Chromeleon, and CSRS are registered trademarks of Dionex Corporation. Milli-Q is a registered trademark of Millipore Corporation. Brij is a registered trademark of ICI Surfactants. Triton X-100 is a registered trademark of The Dow Chemical Company.

Passion. Power. Productivity.

Dionex Corporation North America Europe Asia Pacific 1228 Titan Way U.S./Canada (847) 295-7500 Austria (43) 1 616 51 25 Benelux (31) 20 683 9768; (32) 3 353 4294 Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735 P.O. Box 3603 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190 6 Sunnyvale,The Optimization CA Strategy ofof Surfactantthe Separation Analysis PowerIreland (353)by of1HPLC 644 1-D 0064 ItalyNano (39) 02 LC 51 62 Analysis 1267 Sweden (46) of 8 473Proteomics 3380 TaiwanSamples (886) 2 8751 6655 South America LPN 2525-01 7/10 94088-3603 Switzerland (41) 62 205 9966 United Kingdom (44) 1276 691722 Brazil (55) 11 3731 5140 ©2010 Dionex Corporation (408) 737-0700 www.dionex.com