Anal. Chem. 2003, 75, 371-378
Universal Two-Dimensional HPLC Technique for the Chemical Analysis of Complex Surfactant Mixtures Olivier P. Haefliger*
Firmenich SA, Corporate R&D Division, P.O. Box 239, CH-1211 Geneva 8, Switzerland
Two-dimensional HPLC was applied for the first time to the analysis of complex surfactant mixtures. In the first dimension, ion chromatographic-type separations were performed on a diol column eluted by an acetonitrilewater (0.1% trifluoroacetic acid) gradient. Using this new technique, cationic and amphoteric surfactants were not retained at all, nonionic surfactants exhibited a weak and essentially unspecific retention, and anionic surfactants were retained mainly according to their functional group. Rather than detecting the analytes immediately after this first separation, successive fractions were automatically and quantitatively transferred to parallel C2 (dimethyl) and C4 (butyl) reversed-phase columns using an innovative setup. The second dimension of the separation then took place, by which the analytes were separated according to their hydrophobicity. Surfactants from all four classes, cationic, amphoteric, nonionic, and anionic, were separated simultaneously in single 54-min two-dimensional HPLC runs. The suitability of the method for quantitative measurements was demonstrated. Surface-active compounds, more generally called surfactants, are one of the most widely used classes of chemicals. They are made up of an apolar lipophilic alkyl chain linked to a polar hydrophilic moiety and are usually classified in four families depending on the charge of the latter one: anionic, nonionic, cationic, or amphoteric. Surfactants are used in all home care products such as detergents or fabric softeners, and in all body care products such as shower gels, shampoos, or creams. Therefore, the development of efficient methods for the chemical analysis of complex surfactant mixtures throughout their life cycle is of high importance. For example, the surfactant composition of application products and their raw components needs to be accurately verified as small changes, which may be caused by batch-to-batch variability, can have a significant influence on their performance. Also, the fact that large amounts of surfactants are eventually released into the environment requires their concentrations to be monitored to characterize their persistence and degradation. Chemical analysis of surfactants, performed most often using high-performance liquid chromatography (HPLC), is well documented in the literature (for review see refs 1-3). Most of the * Tel: (+41) 22 780 3239. Fax: (+41) 22 780 3334. E-mail: olivier.haefliger@ firmenich.com. (1) Morelli, J. J.; Szajer, G. J. Surfactants Deterg. 2000, 3, 539-552. 10.1021/ac020534d CCC: $25.00 Published on Web 12/21/2002
© 2003 American Chemical Society
reported methods make use of reversed-phase (RP) systems, with or without an ion-pairing reagent. In these cases, the analytes are separated mainly according to the length of their alkyl chains, while the type of their hydrophilic moiety only has a weaker influence. Other stationary phases with different selectivities have been used as well, such as a cation exchanger,4 amino-derivatized silica under normal-phase conditions,5 polyphenol-derivatized silica,6 or ion chromatography.7 In these cases, the separations were achieved mainly based on the nature of the hydrophilic moiety and only to a lesser extent according to the alkyl chain length. Despite these numerous reports, HPLC analysis of complex surfactant mixtures, in particular when compounds that belong to different families are present simultaneously, is still not an easy task. One reason for this is the inherent complexity of the individual analytes, which most of the time are not a single welldefined molecule but rather a mixture of homologues. For example, the surfactant called cocamide DEA is in fact a mixture of diethanolamides derived from coconut oil fatty acids, which are themselves a mixture of eight main fatty acids produced in characteristic proportions by coconuts. Accordingly, HPLC separations performed on RP columns result in eight distinct peaks. This result is of interest when a detailed characterization of cocamide DEA is needed. However, it also means that there is a large probability for coelutions to occur when a sample is analyzed where other surfactants with similar or even greater complexities are also present. Therefore, a preseparation of complex surfactant mixtures to split them into fractions with fewer analytes is often needed. This task is frequently performed using solid-phase extraction (SPE) cartridges. In summary, to achieve the best possible separation, an HPLC method should associate the alkyl chain length selectivity provided by RP columns with an additional selectivity that would be dominated by the nature of the hydrophilic functional groups. To achieve this combination of two antagonistic properties, we report here the first two-dimensional HPLC (2D-HPLC, or LC/LC) technique applied to the analysis of surfactants. The versatility of (2) Morelli, J. J.; Szajer, G. J. Surfactants Deterg. 2001, 4, 75-83. (3) Schmitt, T. M. Analysis of surfactants, 2nd. ed.; M. Dekker: New York, 2001. (4) Matsuzaki, M.; Ishii, K.; Yoshimura, H.; Hashimoto, S. J. Soc. Cosmet. Chem. Jpn. 1993, 27, 494-497. (5) Shang, D. Y.; Ikonomou, M. G.; Macdonald, R. W. J. Chromatogr., A 1999, 849, 467-482. (6) Wilkes, A. J.; Jacobs, C.; Walraven, G.; Talbot, J. M. World Surfactant Congr., 4th, Vol. 1 1996, 187, 389-402. (7) Bevers, H. A. J. M.; Hulst, R. Chromatographia 2000, 52, 162-164.
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the method will be demonstrated by addressing all four families of surfactants simultaneously: anionics, nonionics, cationics, and amphoterics. This case is only rarely encountered in the literature, but is nevertheless of high importance in real applications where compounds from at least three families are often found simultaneously. For example, a typical shampoo or shower gel contains among others at least one anionic, one amphoteric, and several nonionic surfactants. Also, environmental analyses often require large numbers of surfactants and their metabolites to be analyzed in the same sample. The first dimension consists of an ion chromatographic-type separation performed on a diol column eluted by an acetonitrilewater (0.1% trifluoroacetic acid) normal-phase gradient, which is described here for the first time. Under these conditions, cationic and amphoteric surfactants are not retained at all. Nonionic surfactants exhibit a weak and essentially unspecific retention. Finally, anionic surfactants are retained according to their functional group, with sulfates eluting in general prior to sulfonates, and with the length of the alkyl chains exhibiting a clear yet weaker influence on the retention. The analytes are not detected immediately after this diol column. Rather, successive effluent fractions are transferred to C2 (dimethyl) and C4 (butyl) reversed-phase HPLC columns. A separation according to the length of the alkyl chain of the analytes then takes place that constitutes the second dimension of the analysis. The first LC/LC separation was reported in 1978 by Erni and Frei, who coupled gel permeation chromatography (GPC) with C18 reversed-phase HPLC to analyze senna glycosides.8 Their interface consisted of a two-position, eight-port valve equipped with two loops. The principle consisted of repeatedly filling one of the two loops with the effluent from the GPC column, eluted by a first pump, while the content of the other loop was loaded and then eluted on the RP column using a second pump. A prerequisite for this method is to use a solvent in the first dimension that is a weak eluent in the second dimension, to avoid dispersion effects when the loops are emptied into the second column. Another less important requirement is to keep the flow rate relatively low in the first dimension compared to the second dimension in order to avoid wasting too much time when the loop is being emptied. This type of interface, with some minor changes in the design of the valve, has remained in use by other researchers over time.9-11 A simplified version of the same interface consists of using a more common two-position, six-port valve equipped with a single loop.12-15 An improved interface was introduced in 1997 that no longer required storage loops. Instead, a two-position, four-port valve was used to load the first column effluent directly on one of two parallel RP columns utilized for the second dimension.16,17 The valve allowed the elution of one column to take place at the same time as the loading of the other one. A second two-position, four-port (8) Erni, F.; Frei, R. W. J. Chromatogr. 1978, 149, 561-569. (9) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 161-167. (10) Opiteck, G. J.; Lewis, K. C.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 1518-1524. (11) Murphy, R. E.; Schure, M. R.; Foley, J. P. Anal. Chem. 1998, 70, 15851594. (12) Holland, L. A.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3275-3283. (13) Moore, A. W.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3456-3463. (14) Venema, E.; deLeeuw, P.; Kraak, J. C.; Poppe, H.; Tijssen, R. J. Chromatogr., A 1997, 765, 135-144. (15) Holland, L. A.; Jorgenson, J. W. J. Microcolumn Sep. 2000, 12, 371-377.
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valve, synchronized with the first one, completed the setup to direct the effluent of the loaded column to the waste and the effluent of the eluted column to the detector. Further interface designs derived from this one have been proposed since then, and a convenient one consists of replacing the two 2-position, 4-port valves with a single 2-position, 10-port valve.18-20 Compared to these reports, the coupling we wanted to achieve presented specific difficulties that none of the setups described so far have addressed simultaneously. First, as explained in the Experimental Section, our eluent in the first dimension on the diol column always consists of a minimum of 80% acetonitrile that is a strong eluent in the second dimension as well. As a consequence, the eluted fractions cannot be loaded as such on a RP column. Several of the reports described above used size exclusion chromatography with an aqueous eluent in the first dimension and did not have to face this issue. Nevertheless, certain applications required this problem to be solved, and a simple way to do it consists of diluting the first column effluent with a large amount of water.12,13 Another one of our specific needs was that a solvent gradient is required in the first dimension, which is a case that has not really been addressed to date. Generating an accurate and reproducible gradient is a trivial procedure when working in one dimension, but is harder when the gradient pump needs to be matched with another pump for the water dilution. Finally, an essential requirement for the new setup was that it needed to be suited for routine work and provide reproducible and accurate quantitative results. EXPERIMENTAL SECTION Instrumentation for One-Dimensional Measurements. The 5-µL samples were injected using a 10-µL injection loop mounted on a thermostated autosampler/column oven (Thermo Separation Products, TSP, San Jose, CA, AS3000; samples at 20 °C, column at 30 °C). A quaternary pump (TSP P4000) equipped with a degasser (TSP SCM1000) was used for elution. Detection was performed using a photodiode array detector (DAD, TSP UV6000; discrete wavelengths only: 200, 230, and 280 nm) and an evaporative light scattering detector (ELSD, Sedex 75, Sedere, Alfortville, France; 40 °C, 3.5 bar, gain 9) mounted in series. All chromatograms displayed in this report have been recorded using the ELSD detector. UV detection is not suited to surfactant analysis as most of the molecules lack a chromophore but is convenient for peak assignment when the complex samples being worked with may contain other compounds. One-Dimensional Measurements. Analyses on a diol column were performed on a 25 cm × 4 mm Nucleosil 100-5 OH (diol) ChromCart column from Macherey-Nagel (Du¨ren, Germany) equipped with an 8 mm × 4 mm precolumn of the same material. The column was eluted at 1 mL/min with a normal-phase gradient of water-0.1% trifluoroacetic acid (H2O-0.1% TFA, solvent A) and acetonitrile-0.1% trifluoroacetic acid (ACN-0.1% TFA, solvent B): Hold for 5 min at 0% A, then go to 20% A in 10 (16) Opiteck, G. J.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 2283-2291. (17) Opiteck, G. J.; Jorgenson, J. W.; Moseley, M. A.; Anderegg, R. J. J. Microcolumn Sep. 1998, 10, 365-375. (18) Unger, K. K.; Racaityte, K.; Wagner, K.; Miliotis, T.; Edholm, L. E.; Bischoff, R.; Marko-Varga, G. J. High Resolut. Chromatogr. 2000, 23, 259-265. (19) Wagner, K.; Miliotis, T.; Marko-Varga, G.; Bischoff, R.; Unger, K. K. Anal. Chem. 2002, 74, 809-820. (20) Ko ¨hne, A. P.; Welsch, T. J. Chromatogr., A 1999, 845, 463-469.
Figure 1. Experimental setup for two-dimensional HPLC analyses. For explanations see text.
min, then hold for 25 min at 20% A, then go back to original conditions in 1 min, and recondition column for 5 min. Analyses on a C4 column were performed on a 25 cm × 4 mm Nucleosil 120-5 C4 (butyl) ChromCart column from MachereyNagel equipped with an 8 mm × 4 mm precolumn of the same material. The column was eluted at 1 mL/min with a gradient of H2O-0.1% TFA (solvent A) and ACN-0.1% TFA (solvent B): Hold for 2 min at 50% B, then go to 98% B in 24 min, then hold for 2 min at 98% B, then go back to original conditions in 1 min, and recondition column for 5 min. General Methodology for the LC/LC Measurements. The setup we used was made up of among others three HPLC pumps and a motorized 2-position, 10-port valve connected as illustrated in Figure 1. The first dimension consisted of the ion chromatographic-type separation performed on a diol column eluted by a water-acetonitrile gradient. The analytes were not detected right after their elution from the column. Rather, they were diluted with water supplied by an isocratic pump and subsequently directed by the valve toward a RP column placed in series for the second dimension of the separation. This dilution with water was an essential step that allowed successive chromatographic fractions of the analytes to be collected and refocused at the head of the RP column. Otherwise, the analytes would have eluted through the RP column immediately due to the large amount of acetonitrile present in the effluent of the diol column, and no efficient gradient elution would have been possible. A critical step when this 20fold dilution of the effluent was performed was to avoid exposing the RP column to too high a flow rate, which would have caused an unacceptable back pressure as well as column deterioration.
Therefore, the flow rate on the diol column had to be kept at 50 µL/min instead of the more usual 1 mL/min. To meet this condition, it was necessary to use a microbore (1-mm i.d.) diol column instead of a conventional 4-mm one. This type of HPLC could not be performed with a classical HPLC pump, and a dedicated micropump was required. Note that the frequently used microflow setups that consist of dividing a higher flow rate into two streams using a special flow splitter were not a satisfactory option. They are not accurate enough under the conditions of rapidly changing back pressures inherent to LC/LC and would cause unacceptable retention time fluctuations from one measurement to the next. The 2-position, 10-port valve was another key part of the LC/ LC system. After a certain time of analyte collection on the RP column, as described above, the valve was rotated. At this point, analyte collection started taking place on a second RP column placed in parallel to the first one. During this time, the analytes collected on the first RP column were eluted using a conventional HPLC gradient pump. This elution constituted the second dimension of the separation. Once it was completed, the valve was switched back to its previous position. A new fraction of analytes was collected on the first RP column, while the analytes collected on the second RP column were eluted. In our experiment, a total of four fractions were collected on the two RP columns. The first one contained the cationic and amphoteric surfactants, the second one the nonionic surfactants, the third one the sulfates, and finally the fourth one the sulfonates. Strategy for the Tuning of the LC/LC Method. The experimental parameters of the method were optimized in four successive steps. 1. Optimization of the gradient on the diol column. Particular attention was given to achieve a sharp separation of the cationics and amphoterics from the nonionics, while maintaining a highquality analytical separation of the anionics. 2. Determination of the times at which the valve had to be switched. The first switching time had to take place in the narrow time window between the end of the elution of the cationics and amphoterics and the beginning of the elution of the earliest eluting nonionics, which were the most apolar ones. There was more freedom for the setting of the second switching time. It could take place at any time during the several minutes between the end of the elution of the latest eluting nonionics, alkylolamide DEAs, and the elution of the first sulfate, octadecyl sulfate. A compromise had to be found for the determination of the third switching time knowing that large sulfonates may elute prior to small sulfates. We decided to collect all of the most widely used sulfonate surfactants (linear alkylbenzenesulfonates, paraffin sulfonates, and olefin sulfonates) in the fourth and last fraction, while keeping as many alkyl sulfates as possible and at least all of the components of laureth sulfate in the third fraction. Finally, the fourth and last switching time could take place as soon as octylsulfonate, the last sulfonate under investigation, had eluted. 3. Selection of the RP columns, knowing that the first fraction had to be eluted on the same column as the third one, and the second fraction on the same column as the fourth one. This selection included picking either a C2 (dimethyl) or a C4 (butyl) column. As will be explained more thoroughly in the Results and Discussion section, satisfactory analysis of nonionic and cationic surfactants with two alkyl chains requires a C2 column, but a C4 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003
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column gives a better resolution for the ones with only one alkyl chain. Another criterion was the choice of the length of the column, knowing that a 250-mm column offers a better resolution than a 125-mm column but at the cost of a longer dead time at the beginning of each gradient elution. We decided to elute the cationic/amphoteric and the sulfate fractions on a 125-mm C4 column and to elute the nonionic and the sulfonate fractions on a 250-mm C2 column. 4. Optimization of the gradients on the RP columns to achieve the best possible separation efficiency. LC/LC Measurements. Separations in the first dimension were performed on a 250 mm × 1 mm Nucleosil 100-5 OH (diol) microbore column (Macherey-Nagel). It was eluted at 50 µL/min using a dedicated micropump (Evolution 200, ProLab, Reinach, Switzerland) equipped with a degasser (ProLab) and connected to the autosampler/column oven described above, but equipped this time with a 5-µL loop on the injector. Stainless steel and PEEK tubing of the smallest available diameter (0.005 in. ) 127 µm) and the shortest possible length was used in the whole section of the instrument eluted at micro flow rates. An isocratic pump (TSP P100, 0.95 mL/min H2O-0.1% TFA) was used for the dilution, which took place in a plain stainless steel tee (U428, Upchurch, Oak Harbor, WA). The 2-position, 10-port valve used for the hyphenation of the two separations had bores of 0.25 mm (C2-1000 EP, Valco, Houston, TX). It was electronically controlled by a microactuator triggered by external digital outputs of the micropump. The RP columns used in the second dimension were a 125 mm × 4 mm Nucleosil 120-5 C4 (butyl) ChromCart column (Macherey-Nagel) for the analysis of the cationics and amphoterics (fraction 1) and of the sulfates (fraction 3) and a 250 mm × 4 mm Nucleosil 100-7 C2 (Macherey-Nagel) ChromCart column for the analysis of the nonionics and the sulfonates (fractions 2 and 4, respectively). They were eluted at 1 mL/min using the instrumentation described above in the case of one-dimensional measurements. This particular combination of columns was selected due to its optimized combination of speed, versatility, and resolution for most applications. The same solvents, H2O-0.1% TFA (solvent A) and ACN0.1% TFA (solvent B), were used in both dimensions. The gradients used and their analytical significance are given in Figure 2. During collection, the eluent through the RP columns always consisted of a minimum of 95% H2O-0.1% TFA, which ensured that no elution of the surfactants took place during the extended time for sample delivery to column 2. Elutions in the second dimension always commenced with a rapid change within 30 s from the low-strength transfer eluent to the starting composition of the gradient eluent. This means that the RP columns were not equilibrated, a fact that must be taken into consideration when the second-dimension gradients are optimized. The return to the original conditions of 95% H2O-0.1% TFA after the end of each gradient always took place in 1 min, followed by another 1 min of reequilibration prior to column switching. Although this time was too short to completely reequilibrate the column, it was sufficient to ensure complete collection of the analytes of the next fraction. The 2-position, 10-port valve was switched after 3.6, 13.0, 32.0, and 43.0 min. 374 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003
Figure 2. Description of the events occurring in both dimensions during the two-dimensional HPLC analyses. Top: in the first dimension, on the diol column. Bottom: in the second dimension, on the C2 and C4 reversed-phase columns. The vertical bars indicate switching of the 2-position, 10-port valve. For explanations, see text.
Chemicals. The HPLC solvents were Millipore water (Millipore, Bedford, MA) and HPLC gradient grade acetonitrile (SDS, Peypin, France). TFA was purchased from Acros (Geel, Belgium) and had a purity of 99%. The measured surfactant standards were obtained by three different means. Whenever possible, well-defined compounds were purchased at a high purity, which was always at least 97% and in general more than 99%. Chemicals were purchased from the following companies. Fluka (Buchs, Switzerland): octyl-, decyl-, dodecyl-, hexadecyl-, and octadecyltrimethylammonium bromides; didodecyldimethylammonium bromide; dioctadecydimethylammonium chloride; heptadecanoic, octadecanoic, nonadecanoic, and eicosanoic acids; sodium decyl and dodecyl sulfates; sodium decane, undecane, and tetradecane sulfonates. From Aldrich (St. Louis, MO): tetradecyltrimethylammonium bromide, octadecanol, and eicosanol. From Acros: octanoic, nonanoic, decanoic, dodecanoic, tetradecanoic, and pentadecanoic acids; sodium octane, dodecane, and hexadecane sulfonates. From Sigma (St. Louis, MO): undecanoic, tridecanoic, and hexadecanoic acid; sodium nonane sulfonate. From Avocado (Heysham, U.K.): sodium octyl sulfate. From Lancaster (Morecambe, U.K.): sodium tridecyl, tetradecyl, hexadecyl, and octadecyl sulfates. In other cases, pure standards were synthesized. Sixteen alkylolamides, corresponding to all possible combinations of dodecanoic, tetradecanoic, hexadecanoic, and octadecanoic acid with ethanolamine (MEA), diethanolamine (DEA), 2-propanolamine (MIPA), and di-2-propanolamine (DIPA) were synthesized from the fatty acid methyl esters and the corresponding alkano-
lamines in the presence of catalytic sodium methylate.21,22 Also, four alkyl betaines (CH3(CH2)nN+(CH3)2COO-) were synthesized from the same four fatty acids reacted with sodium chloroacetate.23 Finally, other surfactants were purchased in the form of technical products from the following companies. From Impag (Zurich, Switzerland): coco betaine (Dehyton AB30, Cognis). From Scheller (Zurich, Switzerland): cetearyl octanoate (mixture of hexadecyl and octadecyl octanoate, Tegosoft Liquid, Goldschmidt), decyl oleate (Tegosoft DO, Goldschmidt), and isocetyl palmitate (Tegosoft HP, Goldschmidt). From Brenntag Eurochem (Mu¨hlheim an der Ruhr, Germany): sodium laureth sulfate (CH3(CH2)n(OCH2CH2)mSO4-Na+, Marlinat 242/70), cocamide DEA (CH3(CH2)nCON(CH2CH2OH)2, Marlamid DF 1218), and C13-17 paraffin sulfonates (mixed secondary alkanesulfonates, Marlon PS65). From CFS (Schweizerhalle, Switzerland): octyl hydroxystearate (Wickenol 171, Alzo). From Aldrich: polyoxyethylene10 octadecyl ether (Brij 76). RESULTS AND DISCUSSION One-Dimensional Measurements on a Diol Column. Figure 3 displays the chromatograms recorded on the diol column for six surfactant standards selected for discussion. All of the components of a mixture of monoalkyltrimethyl- and dialkyldimethylammonium ions (A) were observed to elute exactly at the elution time that corresponds to the dead volume. These positively charged species are therefore not retained at all by a diol phase conditioned at an acidic pH. This finding was general for all cationic species and included among others cationic surfactants of the esterquat family (data not shown). Coco betaine (B) yielded an interesting pattern of peaks. The largest peak was observed at the retention time that corresponds to the dead volume, as was the case for the cationic surfactants, but an additional broad shoulder was observed immediately thereafter. Injection of standards demonstrated that the largest peak is the one of the alkyl betaines, which are cationic under the measurement conditions, while the shoulder was caused by NaCl, which is poorly soluble in the 100% acetonitrile eluent. NaCl was present in the sample because it is a byproduct of the industrial synthesis that consists of reacting an alkyldimethylamine with sodium chloroacetate. Panels C and D of Figure 3 display the chromatograms of two very different fatty acid-based nonionic surfactants, a very apolar one (cetearyl octanoate) and the one with the strongest retention observed (cocamide DEA). In both cases, homologues with different alkyl chains were not resolved, which clearly demonstrates that the retention is dominated by the hydrophilic group. The earliest eluting nonionic surfactant, represented here by cetearyl octanoate, eluted later than the last eluting cationic and amphoteric surfactants, that is, after 2.1 min. This sets the basis for the class separation of cationic and amphoteric surfactants from nonionic surfactants. Also, the chromatogram of cocamide DEA shows that all of the fatty acid-based surfactants studied had exited the column after 6 min. Panels E and F of Figure 3 display two examples of anionic surfactants, which were all strongly retained. The order of sulfates eluting prior to sulfonate, with carboxylates (data not shown) (21) Meade, E. M. British Patent 631637, 1949. (22) Meade, E. M. U.S. Patent 2464094, 1949. (23) Cipiciani, A.; Primieri, S. J. Chem. Soc., Perkin Trans. 2 1990, 1365-1367.
Figure 3. Chromatograms of selected surfactant samples recorded using a conventional one-dimensional HPLC method based on a diol column. For detailed conditions, see text. The numbers indicate the number of carbon atoms in homologue chains. (A) Mixture of octyl-, decyl-, dodecyl-, tetradecyl-, hexadecyl-, and octadecyltrimethylammonium, and didodecyl- and dioctadecyldimethylammonium standards. (B) Coco betaine, technical product. (C) Cetearyl octanoate, technical product, a mixture of hexadecyl octanoate and octadecyl octanoate. (D) Cocamide DEA, technical product, the nonionic surfactant that exhibited the strongest retention. (E) Sodium laureth sulfate, technical product, with components of the general formula CnEOmSO4Na, with n ) 12, 14, 16 and m ) 0 f 8. (F) C13-17 paraffin sulfonates, technical product.
behaving like nonionics, can be understood by considering the pKa values. For those compounds with the same functional group, the analogues with the longest apolar chain eluted first. Analogues that differ only by one carbon unit in their chains were almost baseline separated (F). In the chromatogram of sodium laureth sulfate (E), the largest peak can be assigned with confidence to ethoxylated dodecyl sulfates that coelute with tetradecyl sulfate, while the one on the right is characteristic of dodecyl sulfate. This means that the presence of ethoxylate subunits in the molecule leads to shorter retention times. The exact number of ethoxylates, which varies in this case from one to more than eight (RP-HPLC, Figure 4 F), however, has little influence on the retention time. A striking consequence is that sodium laureth sulfate, despite its complexity, yields a compact group of only four peaks. While this prevents a detailed characterization of the raw product, it is a favorable property when quantitation of this analyte in a complex mixture is required as the whole block of peaks can be integrated together and is therefore less likely to overlap with other analytes. During method development, the weakest retention of anionic surfactants was observed with a mixture of approximately 40% H2O-0.1% TFA and 60% ACN-0.1% TFA, while these compounds Analytical Chemistry, Vol. 75, No. 3, February 1, 2003
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Figure 4. Chromatograms of selected surfactant samples recorded using a conventional one-dimensional HPLC method based on a C4 reversed-phase column. For detailed conditions, see text. The numbers indicate the number of carbon atoms in homologue chains. (A) Mixture of octyl-, decyl-, dodecyl-, tetradecyl-, hexadecyl-, and octadecyltrimethylammonium standards. (B) Coco betaine, technical product. (C) Mixture of 16 alkylolamides standards derived from dodecanoic, tetradecanoic, hexadecanoic, and octadecanoic acid, and MEA, DEA, MIPA, and DIPA. (D) Mixture of all linear chain fatty acid standards with 8-20 carbon atoms. (E) Polyoxyethylene(10) octadecyl ether (C18EO10), a mixture of octadecyl ethers with an average of 10 ethoxylate units. Homologues with the largest number of ethoxylates elute first. (F) Sodium laureth sulfate, technical product. Homologues with the lowest number of ethoxylate units elute first.
were strongly retained with both eluents taken separately. Therefore, gradient elution is essentially possible starting both from a high water percentage and from a high acetonitrile percentage. When starting from a high water percentage, most of the retention must be caused by reversed-phase properties since the analogues with the shortest chain eluted first, in contrast with the behavior described above. Also, nonionic and amphoteric surfactants exhibited strong retention, which was not the case with the gradient described above. Overall, gradients starting with high water contents were found to lead to poorer separations than the optimized gradient and did not exhibit the desired specificity for anionic surfactants. Good chromatographic separation of anionic surfactants, with the same specificity as when the optimized gradient was used, was also observed when simply eluting isocratically with a mixture of 20% H2O-0.1% TFA and 80% ACN-0.1% TFA (data not shown). Such a procedure would be perfectly suitable for those cases where only the analysis of anionic surfactants is required. The advantage of the gradient procedure is that it also allows the class 376
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separation of cationic and amphoteric surfactants from nonionic surfactants, which was a prerequisite for LC/LC analyses. One-Dimensional Measurements on a C4 ReversedPhase Column. Figure 4 displays the chromatograms recorded on the C4 (butyl) column for six surfactant standards selected for discussion. Alkyltrimethylammonium ions (A), which were selected as an example of cationic surfactants, were well resolved although significant tailing was observed. The same effect was observed in the case of coco betaine (B), the selected example of amphoteric surfactants. In this case, a strong peak was observed at the dead time, which was assigned to NaCl as explained previously. The distribution of the peak intensities of the homologues was typical for surfactants derived from hydrogenated and fractionated coconut oil fatty acids. All 16 alkylolamide standards under investigation were baseline resolved, a result that demonstrates the efficiency of the separation method (C). This was also true for fatty acids differing by only one methylene unit (D). In this case, the relative intensities of the peaks were especially interesting. The injected sample actually contained all fatty acids from C8 to C20, but only the ones with at least 14 carbon atoms were detected by the evaporative light scattering detector, and maximal detection efficiency was only reached with at least 16 carbon atoms. The chromatogram of polyoxyethylene10 octadecyl ether (E) showed that ethoxymers differing by only one ethoxylate unit could still be separated. Comparison of this chromatogram with other ones obtained for samples with different average numbers of ethoxylate units revealed that the largest ethoxymers elute first. Finally, efficient separation was achieved for anionic surfactants as well. Paraffin sulfonate homologues differing by one methylene unit were baseline resolved, and certain regioisomers were separated as well (data not shown). Sodium laureth sulfate (F) produced a broad range of peaks, due to the complexity of this type of surfactant made up of homologues that can have three different (C12, C14, and C16) alkyl chains and any number between zero and more than eight ethoxylate units. Interestingly, the opposite effect was observed as compared to the case of alkylethoxylates (see above), where the homologues with the lowest number of ethoxylate units eluted first this time. Close consideration of the six chromatograms displayed in Figure 4 clearly shows that this method is not really suited to the analysis of surfactants with more than 20 carbons in their alkyl chains, such as the ones that contain two alkyl chains in the same molecule. Such analyses should rather be performed on a C2 (dimethyl) column with a weaker retention. For this reason, we decided to use a C2 column as one of the two parallel RP columns during the LC/LC measurements. LC/LC Measurements. Figure 5 displays chromatograms of six surfactants selected as being representative of the different classes. Each chromatogram consists of four sections, which correspond to the four successive gradient elutions performed on the two parallel RP columns, as explained in the discussion of Figure 2. As a consequence of the criteria taken into account during the development of the method, the cationic (C8-C18 alkyltrimethylammoniums, A) and amphoteric (coco betaine, B) surfactants, which are not retained at all on the diol column, were observed in the first section. The second section corresponded to the elution of the nonionic surfactants (cocamide DEA, C, and
Figure 5. Two-dimensional chromatograms of selected surfactant samples. For detailed conditions, see text. The numbers indicate the number of carbon atoms in homologue chains. Peaks labeled with a * are caused by contamination. (A) Mixture of octyl-, decyl-, dodecyl-, tetradecyl-, hexadecyl-, and octadecyltrimethylammonium standards. (B) Coco betaine, technical product. (C) Cocamide DEA, technical product. (D) Cetearyl octanoate, technical product, a mixture of hexadecyl octanoate and octadecyl octanoate. (E) Mixture of sodium octyl, decyl, dodecyl, tridecyl, tetradecyl, hexadecyl, and octadecyl sulfate standards. (F) Mixture of sodium octane, nonane, decane, undecane, dodecane, tetradecane, and hexadecane sulfonate standards.
cetearyl octanoate, D). The third subsection was the one of the sulfates (C8-C18 alkyl sulfates, E), with the exception of octyl sulfate, which is not actually used as a surfactant and was included in the mixture only for method characterization purposes. Finally, the last section contained the sulfonate surfactants (C8-C16 alkylsulfonates, F). Compared to the one-dimensional RP chromatograms displayed in Figure 4, the resolution was slightly lower. This was not caused by the LC/LC technique itself but rather by deliberate instrumental choices. A short C4 column was used expressly for the cationic/amphoteric and sulfate fractions in order to keep the method fast. Also, a C2 column with an inherent lower resolution was applied to the separation of the nonionic and sulfonate fractions for the sake of versatility with respect to the most apolar nonionics. Cationization by acidification of the sample solutions containing amphoterics was found to be critical to ensure that these analytes
Figure 6. Chromatogram of a complex mixture of surfactants. For detailed conditions (same as for Figure 5), see text. The mixture contained 20 mg each of dodecyl betaine (peak 1), tetradecyl betaine (2), hexadecyl betaine (3), octadecyl betaine (4), dodecyltrimethylammonium bromide (5), tetradecyltrimethylammonium bromide (6), hexadecyltrimethylammonium bromide (7), octadecyltrimethylammonium bromide (8), 12 alkylolamides derived from dodecanoic, tetradecanoic, hexadecanoic and octadecanoic acid, and MEA, DEA and MIPA (C12MEA 9, C14MEA 10, C16MEA 11, C18MEA 12, C12DEA 13, C14DEA 14, C16DEA 15, C18DEA 16, C12MIPA 17, C14MIPA 18, C16MIPA 19, C18MIPA 20), octadecanol (C18OH), eicosanol (C20OH), octadecanoic acid (C18COOH), isocetyl palmitate (iC16C16ate), octyl hydroxystearate (C8C18OHate), decyl oleate (C10C18(c9)ate), sodium octyl sulfate (C18SO4), sodium decanesulfonate (C10SO3), sodium dodecanesulfonate (C12SO3), sodium tetradecanesulfonate(C14SO3),sodiumhexadecanesulfonate(C16SO3), and 200 mg of 70% sodium laureth sulfate (CnEOmSO4), dissolved in 100 mL of methanol with 1% TFA. (A) Whole chromatograms. (B), (C), and (D) zooms on different regions of interest.
were exclusively present in the first fraction. No such pH dependence was observed for any other analyte beside the amphoteric surfactants. Note that even under these conditions traces of alkylamidopropyl betaines (data not shown) could still be detected in the second fraction. Cetearyl octanoate (D), a very apolar surfactant, was observed to elute late in the gradient of the nonionics fraction. It is a typical example of a compound that could not be properly eluted from a C4 column. Also, the tendency of the peaks to become broader at the end of the gradient was general. The chromatographic behavior was not subject to matrix effects, and no consequences of hypothetical overloading of the microbore diol column were observed. This is proven by Figure 6, the chromatogram of a very complex mixture of all types of surfactants, in which the same behavior was observed for Analytical Chemistry, Vol. 75, No. 3, February 1, 2003
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Table 1. Figures of Merit for the Quantitative Analysis of Surfactants Using the LC/LC Method and the Evaporative Light Scattering Detector (40 °C, 3.5 Bar, Gain 9)a
analyte
LOD (ng injected)
detector satn threshold (ng injected)
hexadecyltrimethylammonium bromide octadecyltrimethylammonium bromide dodecyl betaine tetradecyl betaine dodecyl diethanolamide tetradecyl mono-2-propanolamide dodecyl sulfate octadecyl sulfate hexadecyl sulfonate polyoxyethylene(10) dodecyl ether sodium laureth sulfate 70% linear alkylbenzenesulfonates 75%
15 25 15 15 10 15 40 65 25 10 500 150
3000 4500 2000 2000 2100 2300 3500 6500 3200 4300 26000 12000
a
Limits of detection (LOD) are defined by S/N ) 3.
surfactants analyzed together as for surfactants analyzed separately. The purpose of the mixture was to put as many compounds together that could still be somehow separated. No real application product would simultaneously contain MEA, DEA, and MIPA alkylolamides. Being able to separate them with 0.3-min broad peaks is a very satisfactory result. The fact that all of the components of sodium laureth sulfate were found in the same section is of particular interest for the same reasons already explained above. The suitability of the method for quantitative measurements was then assessed by successively injecting sequentially diluted surfactant mixture solutions. Table 1 displays figures of merit for a few surfactants belonging to all four classes. The lower sensitivity achieved for sodium laureth sulfate and linear alkylbenzenesulfonates than for the other compounds is due to the broad distributions of peaks generated by these analytes. The dynamic ranges of the evaporative light scattering detector, which has a nonlinear response, were always ∼2 orders of magnitude. However, the truly useful ranges for quantitative measurements were actually smaller since good linearity of the data plotted on loglog scales was typically only observed for concentrations greater than at least three times the LODs. Note that the gain of the
378 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003
detector was set at a value of 9 on a scale of 12, which we found to be a convenient setting for all-purpose measurements as it prevents detector saturation from occurring at too low concentrations. It was, however, not the most sensitive one. The results should therefore only be interpreted as a clear demonstration that quantitation using the new LC/LC technique is possible and as a comparison of the relative responses of different analytes, but not as an attempt to achieve maximal sensitivity. CONCLUSIONS AND PERSPECTIVES We have developed a new and versatile method for the chemical analysis of complex surfactant mixtures. A run is performed in only 54 min, all equilibration times included, which is very satisfactory when considering the number of analytes that have to be separated. A drawback is that three HPLC pumps are required. This statement, however, needs to be qualified in parallel with the fact that tedious steps such as collection of fractions at the exit of a classical HPLC system, or extractions using SPE cartridges, are no longer needed. Another weakness is that during the 54 min, only four elutions could be performed in the second dimension due to the relatively long time required by the gradients. This is, however, not something typical for LC/LC, and other authors have reported separations in the second dimension that only required a few minutes.9-12,15-17,20 To achieve this, they used fast HPLC techniques that require dedicated short columns, which are currently not readily available with the C4 and C2 modifications that we needed. Using such columns and our same run time of 54 min, it would have been possible to perform more than 10 elutions in the second dimension. ACKNOWLEDGMENT We thank Jacques Sulzer for his valuable collaboration. We are also grateful to Sina Escher and Eric Fre´rot for helpful discussions. We thank Scheller (Zurich, Switzerland) for the kind donation of cetearyl octanoate, decyl oleate, and isocetyl palmitate samples. We thank CFS (Schweizerhalle, Switzerland) for the kind donation of an octyl hydroxystearate sample.
Received for review August 16, 2002. Accepted November 3, 2002. AC020534D
