Anal. Chem. 1002, 64, 1901-1907
1001
Chromatographic Characteristics of Surfactant-Mediated Separations: Micellar Liquid Chromatography vs Ion Pair Chromatography Alireza S. Kordt and Morteza G. Khaledi’
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204
The effects of concentrations of organlc solvent and surfactant on eiutlon strength and selectlvlty In MLC and IPC are studled. I t Is observed that rekctlvlty between most palrs of solutes used in thls study Increases In MLC and elther docreases or passes through a mlnlmum In IPC, wlth the volume fractlon of organlc modlfkr. I n both MLC and IPC, selectlvlty varles wlth surfactant concentration;however, the overall variatlon in rekctlvlty and eiutlon order are more pronounced In MLC. The solvent strength decreases In IPC and Increases In MLC as a result of an Increase in surfactant concentration. An Iterativeregredon d d g n k usedto predlct the optlmum mobile-phase comporltlons In terms of solvent strengthand sdecthrlty. The corrdatlonbetweenthe predlcted and measured chromatograms is excellent In MLC and poor In IPC. Thk k due to a more regular and reprodudbk retentkn behavlor In MLC whlch greatly facllltates the development of robust methodologks. For a mlxture of amino acids and pep tldes, a large retentlon gap between the first and the last elutlng solutes Is observed In IPC, whlch makes the use of organlc solvent gradient Inevltabie. However, a better reparation for the same mlxture of solutes can be achleved In MLC kocratlcally. Apparently, the general elution problem can be alleviated In MLC by uslng an optlmum eluent compodtlon. I t is observed that the efflclencies of MLC and IPC are comparable. The above observatlons Indlcate that MLC can be a powerful alternatlve to IPC In order to achieve optimlzed separatlons In shorter analyds time.
INTRODUCTION In recent years, the use of secondary chemical equilibria in reversed-phase liquid chromatography (RPLC) has been greatly increased.lP2 The existence of these equilibria in the mobile phase provides an enhancement of selectivity, two important examples being ion pair chromatography (IPC) and micellar liquid chromatography (MLC) in which surfactants are used in the mobile phase. IPC has provided a powerful alternative to ion-exchange chromatography and has extended the capability of RPLC by allowing simultaneous separation of ionic and nonionic compounds. In ion pair chromatography a small concentration of an ion pairing reagent, which has an opposite charge to the ionic solutes, is added to the aqueous mobile phase and its concentration is intentionally kept low in order to avoid formations of micelles. Addition of hydrophobic ion pairing reagents to mobile phases in RPLC generallyimproves selectivitywhile it usually extends the analysis time.
* To whom correspondence should be addressed.
+ Present address: Mallinckrodt Medical, Inc., 675 McDonnell Blvd., P.O. Box 5840, St. Louis, MO 63134. (1) Karger, B. L.; LePage, J. N.; Tanaka, N. In High Performance Liquid Chromatography: Advances and Perspectives; Horvath, Cs., Ed.; Academic Press: New York, 1980; Vol. 1, p 113. (2) Foley, J. P.; May, W. E. Anal. Chem. 1987, 59, 102.
0003-2700/92/0364-1901$03.00/0
Surfactants have also been utilized at micelle-forming concentrations in a variety of separation techniques for a number of years. The popularity of micellar-mediated LC separations has increased since 1980, when Armstrong and Henry pioneered in using surfactant solutions at concentrations above a critical micelle concentration (cmc) as mobile phases for RPLC.3 In the past 11 years, the usefulness of micellar mobile phase for RPLC has been widely explored.‘-10 Most of the significant MLC advantages, however, are unrelated to the separation process, for example, direct injection of physiological fluids, detection enhancement, no need for column regeneration after gradient elution, safety, cost, etc. From the early publications, the role of micelles in the mobile phases of RPLC has been compared to that of organic modifiers mainly because of the capability of micelles to enhance aqueous solubility of uncharged solutes. Micelles were thought to replace organic modifiers in RPLC and in fact many attempts were made to demonstrate the unique advantages of MLC over hydroorganic RPLC. Despite the capabilities of MLC, however, this technique has not received enough attention in solving the “real world” problems in analytical laboratories for two apparent reasons. Firstly, RPLC with hydroorganic eluents is a powerful and popular technique that would be very difficult for any other HPLC method to replace; in fact one might reasonably argue the need for an alternative technique. Secondly, the two techniques have been often compared in an area in which MLC has clear disadvantages as a separation technique, that is for the separation of uncharged and hydrophobic compounds. Ironically, MLC is a poor choice in this respect from the three important aspects of efficiency, solvent strength, and selectivity. Column efficiency in MLC is poor, inferior to hydroorganic systems, and deteriorates as the hydrophobicity of solutes increases. Micellar eluents are generally weak; thus long analysis times for hydrophobic solutes are observed. Separation selectivity for hydrophobic compounds is small (ascompared to hydroorganic mobile phases at similar solvent strengths) due to the similar environments of the mobile and stationary phases in MLC.11J2 Interesting selectivities have been reported in the early publications of MLC mostly in the (3) Armstrong, D. W.; Henry, S. J. J. Liq. Chrornatogr. 1980, No. 3, 657. (4) Armstrong, D. W. Sep. Purif. Methods 1986, 14, 213. (5) Hinze, W. L. In Organized Media in Chemical Separation; Hinze, W. L., Armstrong, D. W., Eds.;ACS Symposium Series 342; American Chemical Society: Washington, DC, 1987. (6) Dorsey, J. G. Ado. Chrornatogr. 1987, 27, 167. (7) Khaledi, M. G. BioChromatography 1988,3, 20. (8) Hinze, W. L.; Singh, H. N.; Baba, Y.; Harvey, N. G. Trends Anal. Chem. 1984, No. 3,193. (9) Yarmchuk,P.;Weinberger,R.; Hirsch, R. F.; Cline Love, L. J. Anal. Chem. 1982,54, 2233. (10) Breyer, E. D.; Starsters, J. K.; Khaledi, M. G. Anal. Chem. 1991, 63, 828. (11) Khaledi, M. G.; Pueler, E.; Ngeh-Ngwainbi, J. Anal. Chem. 1987, 59, 2738. (12) Khaledi, M. G. Anal. Chem. 1988,60, 876. 0 1992 American Chemical Society
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form of elution reversal@-15 however, this does not necessarily translate into enhanced separations. The problem with the efficiency,solvent strength, and selectivity can be improved by the addition of a small concentration of an organic modifier to the micellar mobile phases; however, even this system is not as suitable as hydroorganic systems for the separation of hydrophobic compounds. The main strength of micellar LC lies in its capability of separating mixtures of ionic and uncharged solutes due to the amphiphilic nature of surfactants. In this respect, micellar mobile phases offer a clear advantage in terms of chromatographic selectivity over the conventional hydroorganic eluents. It is therefore more relevant to compare the capabilitiesof MLC to those of ion pair chromatography (IPC), the only HPLC technique that has similar characteristics. In both MLC and IPC, surfactants provide electrostatic sites of interaction, thus influencing the retention behavior of the charged solutes. Organic modifiers control solvent strength and in some cases affect separation selectivity. There are, however, important differences between the two systems. In MLC, the mobile phase is composed of surfactant molecules in the form of monomers and aggregates as compared to the existence of only monomer surfactants in the IPC mobile phases. In addition, the amount of adsorbed surfactants onto the stationary phase in MLC is nearly constant and independent of the changes of surfactant concentration in the micellar mobile phase, while the concentration of surfactants on the stationary-phase surfaces in IPC increases with that in the mobile phase. Both of these factors have a dramatic influence on the chromatographiccharacteristics of MLC and IPC. As a result, the retention behavior of charged and uncharged solutes in the two systems can be drastically different despite the apparent similarities between the two systems. In this paper, the roles of organic modifier concentration and surfactant concentration on solvent strength and selectivity in MLC and IPC are discussed. It is shown that MLC provides several advantages over IPC such as simultaneous enhancement of selectivitywith elution strength which results in better separations in shorter analysis times, a more robust and predictable retention behavior which facilitates the optimization of a separation, and alleviation of the general elution problem.
EXPERIMENTAL SECTION Apparatus. The HPLC system consisted of a pump (Model 400, Applied Biosystems, Foster City, CA) and a variablewavelength absorbance detector (Model 783 A, Applied Biosystems) set at 210 nm, controlledby Chemresearchchromatographic data management system controller software (ISCO, Lincoln, NE) running on a PC-88 Turbo personal computer (IDS, Paramount, CA). The retention behavior of individualsolutes was studied using a 5-pmparticle size Ultremex Clecolumn (Phenomenex,Torrance, CA) 100 X 4.6 mm. The column dead volume (0.6 mL) was measured by making multiple injections of water samples. The test mixture was separated using a longer (250- X 4.6-mm) U1tremex column (dead volume, 2.1 mL) in order to generate a larger number of theoretical plates. The columns were thermostated at 40 “C by a water circulator bath (Lauda Model MT6, Brinkmann Instruments, Inc., Westburry, NY). A silica precolumn was used to saturate the mobile phase with silicates and to protect the analytical column. The software to evaluate the separation at different mobilephase compositions in both MLC and IPC was based on an extended version of the iterative regression optimization strat(13)Armstrong, D.W.; Stine, G. Y. Anal. Chem. 1983,55,2317. (14)Arunyanart, M.;Cline Love, L. J. J. Chromatogr. 1985,342,293. (15)Berry, J. P.;Weber, W. G. J. Chromatogr. Sci. 1987,25,307.
egy.16J7The simulated chromatograms are based on a Gaussian peak shape, using the plate-count and dead volume observed in chromatographicexperiments. The boundaries of the parameter space are determined by practical limitations of the chromatographic system. In MLC the lower surfactantconcentration must be well above the cmc (8mM at ambient temperature and without organic modifier) and must be strong enough to cause elution of all solutes. The upper surfactant concentration is determined by a combination of solubility of the surfactant, the viscosity of the resulting mobile phase, and degradation of efficiency at higher concentrations. The volume fraction of 2-propanol (PrOH) was limited to a maximum of 153’6 to protect the integrityof micelles. In IPC the lower surfactant concentration was adjusted at a level which ensures adequate retention of all components (i.e. k’ > 1). The upper surfactant concentration was well below the cmc to avoid any formation of micelles. The maximum concentration of PrOH was limited to 25% because of the lack of sufficient retention for early eluting components, and the lower volume fraction of PrOH was limited to 7 % due to long and impractical retention for late eluting solutes in IPC. Reagents. The stock solution of sodiumdodecylsulfate (SDS) was prepared by dissolving the required amount of surfactant in doubly distilled deionized water and filteringthrough a 0.45-pm Nylon membrane filter (Gelman Sciences Inc., Ann Arbor, MI). The test solutes were tyrosine (Y), methionine (M), alanyltyrosine (AY),tryptophan (W),aspartylphenylalanine (DF),leucyltyrosine (LY), glycylleucyltyrosine (GLY),leucyltryptophan (LW), phenylalanylphenylalanine (FF). The sample solutions were prepared by dilutingthe stock solutions(10mg/mL in water or tetrahydrofuran) in the mobile phase. The ionic strength was adjusted by adding phosphate buffer such that the total buffer concentrationof the finalsolution was 0.02 M. After the required amount of PrOH was added, the pH was adjusted to 2.5.
RESULTS AND DISCUSSIONS In both MLC and IPC a variety of parameters (such as the type/concentration of surfactant and organic cosolvent, pH, ionic strength, temperature, etc.) can influence the elution strength and selectivity. In this study we focused on two of the most important parameters, i.e. surfactant concentration and organic modifier concentration. The rest of the parameters mentioned above were kept the same for both MLC and IPC. Organic Modifier Concentration. In conventional RPLC with hydroorganic mobile phases, IPC, and MLC an increase in organic solvent concentration increasesthe solvent strength and decreases the solutes’retentions. The degree of reduction of a solute’s retention can be demonstrated by a solvent strength parameter, S, which is the slope of the linear plot of In k’ vs q50rg as is expressed by eq 1,where k’ is retention In k’ = -&bo-
+ In k’,
factor, q50rg is the volume fraction of organic solvent, and In k’0 is the retention factor of a solute in a purely aqueous mobile phase. This is illustrated in Figure la,b for IPC and MLC, respectively. In conventional RPLC with hydroorganic mobile phases the S values of many compounds depend on their molecular weights and contact area with the stationary phase. Thus, the S parameter is directly related to the solute retention as shown by eq 2,18where p and q are constant empirical values. S =p
+ q In k’,
(2)
When the volume fraction of organic solvent increases from (16)Drouen, A. C. J. H.; Billiet, H.A. H.;Schoenmakers, P. J.; de Galan, L. Chromatographia 1982,16, 48. (17)de Galan, L.;Billiet, H.A. H.Adu. Chromatogr. 1986,25,63. (18)Schoenmakers,P.J.OptimizationofChromatographicSelectiuity; Journal of Chromatography Library, Vol. 35;Elsevier: Amsterdam, 1986; Chapter 3.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992 5,
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2-Propanol (“A) Flgwe 1. Influence of organlc modifier on retention in (a, top) IPC (0.5 mM SDS) and (b, bottom) MLC (20 mM SDS). 41 to 42, the change in selectivity between compounds a and b (a = k’dk’,) can be obtained from eq 1 as
In a2- In a1= -(Sb - Sa)(& - $1) (3) By definition a > 1or k’b > k’a, as a result s b > S a and a2 < al. In other words, for a large group of compounds for which S is proportional to In k’,selectivity decreases as a result of an increase in organic modifier concentration (solvent strength). In general, eqs 1and 3 can also be applied for both MLC and IPC systems. In the case of IPC a good correlation between S and In k’0 was observed for all solutes studied in this work with one exception. Therefore eq 2 can be applied, as shown in Figure 2a. As a result, in IPC an increase in volume fraction of organicmodifier usually leads to a decrease in selectivity (Figure 3a). This is a behavior similar to that in conventional RPLC. In MLC, micelles control the solvation ability of organic modifiers and eq 2 is not generally valid which is in contrast with the behavior observed in IPC.19920 In fact the S parameter in MLC is often inversely related to retention (Figure2b).19920 This implies a selectivity enhancement as a result of an increase in volume fraction of organic solvent. This is illustrated in Figure 3b. As the concentration of PrOH increases from 3 5% (v/v) to 15 5% (v/v), selectivity for most pairs of solutes under study increases. The simultaneous enhancement of separation selectivity and solvent strength in MLC using hybrid eluents of micellewater-organic solvents was previously observed for different (19) Khaledi, M. G.; Strastars, J. K.; Rodgers, A. H.; Breyer, E. D. Anal. Chem. 1990,62,130. (20) Kord, A. S.; Khaledi, M. G. Anal. Chem., preceding article in this issue.
1
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groups of ionic and nonionic compounds with a variety of functional groups and both for anionic and cationic micellar e1~ents.l~The reasons for the occurrence of this unique phenomenon in MLC was attributed to the existence of the competing equilibria in MLC and the influence of micelles on the role of organic modifiers19920 This selectivity enhancement occurs systematically; i.e., peak separation increases monotonically with volume fraction of organic solvent added to micellar eluents. According to several reports, the variation of either the concentration or the type of organic modifier in IPC did not result in any significant selectivity changes for mixtures of ionic solutes.21-23 However, other workers have demonstrated that certain ionic solute mixtures can be separated by varying the concentration24-26and/or the typeZ7-31of organic modifier in the presence of the pairing ion in IPC. To the best of our knowledge, there has not been any report of a monotonic enhancement of selectivity with volume fraction of organic (21) Snyder,L.R.;Glajeh,J.L.;Kirkland, J. J.A.acticalHPLCMethod Development; Wiley: New York, 1988; Chapters 4 and 5. (22) Bieganowska,M.; Soczewinski,E.;Janowska, M. Chromutographia 1984, 18, 99. (23) Dihuidi,K.;Kucharski, M. J.; Roeta, E.;Hoogmartens,J.;Vanderhaeghe, H. J. Chromatogr. 1985, 325,413. (24) Jandera, P.; Engelhardt, H. Chromatographia 1980,13, 18. (25) Lindberg, W.; Johansson, E.: Johansson, K. J . Chromtogr. 1981, 211, 201. (26) Coenegracht, P. M. J.; Tuyen, N. V.; Metting, H. J.; CoenegrachtLamers, P. M. J. J. Chromutogr. 1987, 389, 351. (27) Riley, C. M.; Tomlinson, E.; Jefferies, T. M. J. Chromtogr. 1979, 185, 197. (28) Goldberg, A. P.; Nowakowska, E.; Antle, P. E.; Snyder, L. R. J . Chromatogr. 1984,316, 241. (29) Skulie, S.;Haddad, P. R.; Lamberton, C. J. J . Chromatogr. 1986, 363, 125. (30) Dong, M. W.; Lepore, J.; Tarumoto, T. J.Chromatogr. 1988,442, 81. (31) Bartha, A.; Vigh, Gy.; Stahlberg, J. J. Chromatogr. 1989, 485, 403-419.
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solvent in IPC with the same generality that has been observed for MLC.19~20 Surfactant Concentration. In IPC the addition of surfactant usually causes an increase in the retention of oppositely charged solutes due to the electrostatic attraction. In this study the experiments were performed at pH 2.5; therefore the amino acids and peptides carried a net partial positive charge. When the concentration of the anionic ion pairing agent (SDS) was increased from 0.5 to 3.5 mM the retention of all solutes with the exception of FF and LW, increased. The reason behind the drastic decrease in the retention of these two most hydrophobic peptides is not known. Similar anomalous behavior has been reported for the small peptides with SDS.32 The elution order of the compounds did not change with an increase in SDS concentration in IPC. In MLC, unlike IPC, an increase in surfactant concentration leads to a decrease in retention and an increase in elution strength. The elution order of four of the nine compounds changed as a result of a change in micelle concentration. In IPC an increase in surfactant concentration in the mobile phase would lead to an increase the amount of adsorbed surfactant monomers on the stationary phase, which usually causes an increase in retention gnd a change in selectivity (Figure4a). In MLC the amount of ionic surfactant monomers which modify the stationary phase is approximately constant and an increase in the total surfactant concentration leads to (32) Hancock, W. S.; Bishop, C. A.; Meyer, L. J.; Harding, D. R. K.; Hearn, M. T. W. J. Chromatogr. 1978, 161, 291.
Figure 4. Influence of surfactant concentration on seiectlvlty in (a, top) IPC in the presence of 7 % PrOH and (b, bottom) MLC In the presence of 3 % PrOH. The connecting llnes show the relatlve posltlon of the peak selecthrlty for different pairs.
an increase in the concentration of micelles in mobile phase. This results in a decrease in retentions of solutes as well as a change in selectivity (Figure 4b). The degree of decrease in retention for different compounds varies depending on their partition coefficients into micelles (P,,) and into the stationary phase (Psw). The overall variation in selectivity and elution order are more pronounced in MLC. More importantly, the change in selectivity with concentration of surfactant in MLC is concurrent with an increase in solvent strength. However, an increase in the concentration of ion pairing reagent results in a change in selectivity as well as a decrease in elution strength. In other words, better selectivity in IPC stems from the use of weaker eluents. Thus, the use of surfactant concentration to manipulate selectivity is less advantageous in IPC as compared to that in MLC.
OPTIMIZATION OF PARAMETERS As mentioned above, concentrations of surfactant and organic modifier in the mobile phase have significant effects on elution strength and selectivity in both MLC and IPC. Obviously, a change in elution strength would have a direct influence on selectivity and vice versa. As a result, the effects of concentrations of surfactant and organic solvent on solvent strength and selectivity should be evaluated simultane0usly.~3J~In cases such as this where the parameters are interactive, optimizing one mobile-phase variable at a time (33) Strasters, J. K.; Breyer, E. D.; Rodgers, A. H.; Khaledi, M. G . J. Chromatogr. 1990, 511, 17. (34)Strasters, J. K.; Kim, S. T.; Khaledi, M. G. J. Chromatogr. 1991, 586, 221.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992
parameter space
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b: measured
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Flgure 5. Parametw space for iterativeregression optimlzation deslgn for IPC and MLC. The five Initial measurements were performed using the following moblie phases. For 1% (A) 0.5 mM SDS + 7 % PrOH (B) 3.5 mM SDS 7 % ROH; (C) 3.5 mM SDS + 25% PrOH; (D) 0.5 mM SDS + 25% ROH (E) 2 mM SDS 16% PROH. For MLC: (A) 0.02 M SDS + 3% ROH; (B) 0.2 M SDS + 3% ROH (C) 0.2 M SDS + 15% ROH; (D) 0.02 M SDS + 15% PrOH; (E) 0.11 M SDS + 9%
+
+
1
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28 time (min) Figure 6. Predicted (a)and measured (b) chromatograms at optimum mobilephase compositlon (0.17 M SDS 12.6% PrOH) In MLC. The solutes identities are as follows: (1) y; (2) M; (3) AY; (4) W; (5) DF; (6) LY; (7) GLY.
+
PrOH.
can be an ineffective and inefficient procedure and the optimum conditions can be easily missed. For this purpose, we used iterative regression optimization design which has been successfully applied for the multiparameter optimization in MLC.33934 This procedure was originally developed for optimization of mobile-phase parameters in RPLC16J' and in IPC.35v3S The optimization procedure is based on the assumption that retention (In k') is a linear function of the parameters within a selected portion of the parameter space. The experimental design for the optimization of the two parameters of surfactant and organic modifier concentrations was based on five initial experiments, four at the corners of a square and one at the center, as shown in Figure 5 for IPC and MLC. The retention of all amino acids and peptides in the mixture were measured at these five mobile-phase compositions. Using the initial data, the linear models of In k' vs the parameters for all solutes are built. In the case of these two parameters the linear model is a plane. The retention of all solutes at other mobile-phase compositions would then be predicted in each of the four triangle subspaces through linear interpolation. Using the predicted In k' at each parameter value (i.e. at each [SDSI and &&, the corresponding chromatogram is simulated by the computer and the predicted quality of separation (e.g. minimum resolution or analysis time) is calculated. Consequently, the mobile-phase compositions for the optimal chromatogram can be predicted. The accuracy of the predicted optimum depends upon the correctness of the assumption of linear behavior of retention as a function of the two parameters as well as the reproducibility of retention data. Deviations from linearity and/or irreproducible retention behavior would cause erroneous predictions,thus requiring more experiments to be performed in order to locate the optimum. Note, however, that one important coneiderationin method development is to optimize a separation with a minimum number of experiments. A more detailed discussion of the application of the iterative regression strategy for conventional RPLC and IPC can be found in refs 16, 17, and 35-31 and for MLC in refs 33 and 34. The iterative regression analysis predicted that the optimum resolution in MLC would be obtained at a mobile phase (35)Billiet, H.A. H.; Drouen, A. C. J. H.; de Galan,L. J. Chrornatogr. 1984,316, 231. (36)Billiet, H.A. H.; Vuik, J.; Strastere, J. K.; de Galan,L. J. Chromatogr. 1987, 384, 153. (37)Billiet, H.A. H.; de Galan,L. J. Chrornatogr. 1989,485, 27.
/I
IPC
a: predicted
b: measured 4
0
135
time (min)
Flgure 7. Predicted (a) and measured (b) chromatograms at optimum mobile-phase compositlon in IPC (2 mM SDS 16% PrOH). The solutes identities are as those in Figure 6.
+
composed of 0.17 M SDS and 12.6% PrOH. The optimum condition for the IPC system was predicted to be at a mobilephase composition of 2 mM SDS and 16% PrOH. The optimum which was predicted on the basis of only five initial experimentsand the measured chromatograms in MLC are illustrated in Figure 6. It is shown that there is an excellent agreement between the predicted and measured chromatograms. Apparently, the assumed linear model of In k' vs the parameters is reasonable. This is even more impressive considering the fact that the initial data (for the simulated chromatogram, Figure 6a) were collected on a short, 10-cm column, while the optimum chromatogram (Figure 6b) was measured on a different 25-cm column (from the same manufacturer). The retention times on the latter column were longer than on the former column, but the retention factors correlated well. Column to column reproducibility is important in HPLC method development. Perhaps, the adsorption of a constant amount of surfactant onto the alkylbonded stationary phase would improve retention reproducibility in MLC. The predicted optimum and measured chromatograms for IPC are shown in Figure 7. It is shown that there is not a good correlation between the predicted and measured chromatograms. These observations suggest that the assumed linear dependence of In k' on &g and surfactant concentration are not as reasonable as those in MLC. In IPC a wider range of organic modifier concentration is
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992
needed as compared to that in MLC. As a result the plot of Ink’ vs r # is~ more ~ ~ likely ~ to deviate from a linear to a quadratic relation. It has been shown that there is a linear relation between In k‘ and In [surfactant] in IPC for a limited range of surfactant concentration (below cmc). However, the concentration of adsorbed surfactant on the stationary phase changeswith the variations in organic modifier concentrations. The presence of organic solvent would influence solute-ion pairing reagent interactions in the mobile phase and on stationary phases. As a result of a simultaneous variation of concentrations of both surfactant and organic modifier, the relation between In k‘ and surfactant concentrations may deviate from linearity. Another reason behind the poor correlation in Figure 7 might be due to the poor retention reproducibility in IPC. These results indicate that due to a regular (Le., linear or slightly curved) retention behavior and perhaps because of a more reproducible retention behavior in MLC as compared to that in IPC, the optimization of parameters that influence solvent strength and selectivity is much more feasible in the former technique. As a result, better separation can be achieved in MLC with a minimum experimental effort. General Elution Problem. The retention gap between the first and the last components in IPC is considerably larger than that in MLC, which can also be seen from Figures 6 and 7. The ratio of the retention factors of the last eluted compound over that of the first eluted solute was 3.21 for MLC (k‘lut = 5.04, k’rmt = 1.57) (Figure 6) and 9.40 for IPC (k’lut = 51.35, k’first = 5.46) (Figure 7), respectively. It is expected that, for a mixture of solutes which contains more hydrophobic and/or oppositely charged ionic components, the gap between the first and the last peaks in IPC becomes even larger. In order to further investigate this phenomenon, we added two more hydrophobicdipeptides (FF and LW) to the previous mixture of seven components. On the basis of five initial measurements, the optimum chromatograms for this ninecomponent mixture in both MLC and IPC were predicted by the iterative regression procedure. The optimum mobile phases were 0.17 M SDS + 12.6% PrOH in MLC, and 2 mM SDS + 16%PrOH in IPC, which are similar to those obtained for the mixture of seven components. The measured chromatograms at these optimum conditions were determined. Again, an excellent agreement between the predicted and measured chromatograms was observed for MLC, while for IPC there was not a good correlation between the predicted and measured chromatograms. The ratios of the retention of the last peak over the first peak are 7.31 for MLC (k’bt = 11.46, k’fint = 1.57) and 42 for IPC (k’ht = 229.54, k’fmt = 5.46). Obviously, in the case of IPC the large gap between retentions of the first and the last solutes as well as the overlapping of the early eluting peaks shows the existence of a general elution problem. This makes the IPC separation of the nine-component mixture by the isocratic method to be impractical. In order to increase the elution strength and to reduce the retention of the late eluting components, the volume fraction of PrOH was increased from 16% to 28% which also adjusted the solvent strength in IPC at a level similar to that in MLC. The resulting chromatograms are shown in Figure 8a for MLC and Figure 8b for IPC. In IPC, the retention gap between the first and the last solutes was reduced to 9.76 (k’lut = 10.73, k’fint = 1.1); however, as expected, the separation of the early eluting peaks was deteriorated. It can be seen that peaks 1 and 2 and also peaks 4-6 overlapped. These observations illustrate the necessity for the use of a gradient elution in IPC. The organic solvent gradient changes the amount of surfactant monomers which are adsorbed onto the alkyl-bonded stationary phase
MLC
3
28
time (min)
IPC 6 51
0.0
I
time (min)
28
Flguro 8. Separation of the nine-component mlxture by (a, top) MLC (0.17 M SDS 12.6% PrOH) and (b, bottom) IPC (2 mM SDS 28% !+OH). Solutes: (1) Y; (2) M; (3) AY; (4) W; (5) DF; (6) LY; (7) QLily; (8) LW; (9) FF.
+
+
and therefore would disturb the column equilibration. The column reequilibration times in IPC can be prohibitively long. In the isocratic MLC separation shown in Figure 8a, the time gaps between peaks 4 and 8 as well as 8 and 9 can be improved by performing either a flow gradient and/or mobilephase composition gradient (i.e. increasing micelle concentration and/or organic modifier volume fraction). It is important to note that in MLC even after performinga mobifephase composition gradient (micelles or organic solvent), the reequilibration time is either very short or not needed.38139 Bartha et al.3I also reported the existence of a large gap between the retention of the first and the last peaks in a mixture of solutes with the same charge and different hydrophobicity in IPC. They suggested that, as a general rule, when the charge type of the first and last components are the same and the retention gap is excessive, it is advantageous to try a different organic modifier. When the retention gap remains unacceptably large, one must consider either gradient elution or a change in the phase system.31 However, a comparison of chromatograms in Figure 8a,b suggests that, by replacement of the IPC system with MLC, a better separation in shorter analysis time can be achieved under optimum isocratic conditions. In the preceding paper the interactive role of micelles and organic modifiers in controlling soIvent strength and selectivity was discussed.m Organic solvents compete with solutes to interact with the active site of micelles, and micellescontrol the solvation ability of organic modifiers. As a result, the solvent strength parameter, S (slope of log k’ vs values of all solutes in MLC are smaller than those in IPC. Consequently, an increase in volume fraction of organic solvent reduces the retention of early eluting solutes in MLC to a smaller extent than that in IPC. This would alleviate the general elution problem. (38)Dorsey, J. G.; Khaledi, M. G.; Landy, J. S.; Lin, J. L. J. Chromatogr. 1984, 316, 183. (39)Madamba, L.; Khaledi, M. G. Unpublished results.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992
Efficiency. Perhaps the major shortcoming of MLC is ita poor chromatographic efficiency.u*41The column efficiency in MLC has been traditionally compared to that in conventional RPLC with hydroorganic eluents. However, the chromatographic capabilities of MLC and IPC for the separation of ionic solutes is remarkably better than that of conventional RPLC. Thus, the type of separation problem which can be solved by these two methods is different from that of conventional RPLC. Consequently, a comparison between the efficiencies in MLC and IPC is more relevant. Using a hydroorganic eluent (755% ACN/25% water) a new 25-cm CU column had about 14 000 theoretical plates. Using the same column, the efficiency was decreased to about 5500 for both ion pairing and micellar eluents with similar elution strengths. The major cause of the poor efficiencyin MLC as compared to conventional RPLC is thought to be the slow mass transfer of solutes from the surfactant-modified stationary phase.@+ However, in IPC, similar to MLC, the hydrophobic surfactant molecules are adsorbed onto the alkyl-bonded stationary phase, and the kinetics of the solutes’ mass transfer should be more or less similar to that in MLC. One might observe a better efficiency in IPC by using different types of surfactant (e.g. shorter chain length than SDS) and organic modifier (e.g. methanol). This would not, however, change the other chromatographic characteristics described above. (40) Dorsey, J. G.; DeEchegaray, M.T.;Landy, J . S. Anal. Chem. 1983, 55, 924. (41) Yarmchuk, P.; Weinberger, R.; Hirach, R. F.; Cline Love, L. J. J. Chromatogr. 1984,47, 283. (42) Armstrong, D. W.; Ward, T. J.;Berthod, A. Anal. Chem. 1986,58, 579. (43) Borgerding, M. F.; Hinze, W. L.; Stafford, L. D.; Fulp, G. W.; Hamlin, W. C. Anal. Chem. 1989,61, 1353. (44) Berthod, A.; Borgerding,M. F.; Hinze, W. L.J.Chromatogr.1991, 556, 263.
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The use of SDS and 2-propanol for IPC (and in MLC) in this study was merely because of comparative reasons.
CONCLUSIONS The surfactant-mediated chromatographic techniques (i.e. MLC and (IPC) are the only HPLC methods which are suitable for separation of mixtures of charged and uncharged solutes. Similar parameters influence solvent strength and selectivity in these two systems. Despite these apparent similarities, the chromatographic characteristics of these HPLC techniques are significantly different. This is mainly due to the existence of micelles in the MLC eluents. In addition, due to the presence of micelles in the mobile phase, certain parameters such as concentration of organic modifier have a more pronounced effect on the chromatographic selectivity in MLC. Due to the better reproducibility and regularity of the retention behavior, it is much easier to optimize a separation in MLC than in IPC. Therefore one can achieve a better separation in MLC with a minimum amount of experimental effort. The use of organic modifier concentration gradient, for the separation of a mixture of hydrophilic and hydrophobic solutes with the same charge, is essential in IPC. In such cases IPC can be replaced by MLC in order to achieve a better separation isocratically. ACKNOWLEDGMENT We thank J. K. Strasters for his assistance in extending and using the IR program for MLC. This work was supported by a grant from the National Institutes of Health (FIRST Award, GM 38738). RECEIVED for review December 16, 1991. Accepted May 21, 1992.
