role of organic modifiers and micelles - American Chemical Society

solvation ability of organic solvents In MLC (represented by the solvent strength parameter, S, of solutes) and the degree of solute Interactions with...
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Anal. Chem. 1092, 64, 1894-1900

Controlling Solvent Strength and Selectivity in Micellar Liquid Chromatography: Role of Organic Modifiers and Micelles Alireza S. Kordt and Morteza G. Khaledi' Department of Chemistry, North Carolina State University, P.O. Box 8204, Raleigh, North Carolina 27695

Slmultanrourenhancement In elutlon strength and seMlvlty whlch has been prevlourly observed In mlcellar llquld chromatography (MLC) for a varkty of compounds Is further Investlgatd. The reasons behind the occwcence ot tMs unlque phenomenon are studled, and the Influence of mlcelles and organk solvents on eluHonstrengthand 88IectMty Isdkcuswd. A model Is developed whlch explains the dependence of the rolvatlon ablllty of organlc solvents In MLC (represented by the solvent strength parameter, S,of solutes)and the degree of solute Interactlons wlth mlcelles. Whenever the dmerence In solvent strength parameter values of two solutes In micellar eluents, dS,Is porltlve, maxlmum sekctlvlty le observed at the weakest eluent strength. When dS C 0, there exkts an Inverse relatlonshlp between retentlon and solvent strength parameter so that selectlvlty monotonlcally Increases with volume fractlon of organlc solvent In mlcellar eluents. I t Is shown that usually there Is no dlrect relatlonshlp between the solvent strength parameter In MLC and retentlon. As a result, sekctlvlty enhancementdue to an IncreaseInthe concentratbn of organlc modlller (Le. solvent strength) occurs frequently In MLC. Interestingly,for cases where selectlvlty decreases wlth an Increase In organlc modllkr, rlmultaneow enhancement of selectlvlty and solvent strength can be observed by Increarlng mlcelle concentratlon. I n a sense, the concentrations of organlc modlller and mlcelles complement one another In lmprovlng selectlvlty at higher elutlon strengths. As a result of thls unlqm phenomenonbetter s e p a r a t h Inshorter analyrk tlmes can be observed. The mutual effects of mlcelles and organlc modlller on one another would also requlre a rlmultaneous optlmlzatlon of these two parameters.

INTRODUCTION Micellar liquid chromatography (MLC) has the capability of simultaneous separation of ionic and nonionic compounds. It also offers the advantages of the feasibility of optimizing parameters due to the linear retention behavior, performing gradient elution without a need for column reequilibration, the possibility of direct on-column injection of serum and plasma samples, enhancement of fluorescence and phosphorescence detection and gradient capability with electrochemical detectors, applications in quantitative structurebiological activity relationship, and low cost and toxicity.l-lO

* To whom correspondence should be addressed.

+ Present address: Mallinckrodt Medical, Inc., 675 McDonnell Blvd., P.O. Box 5840, St. Louis, MO 63134. (1)Armstrong, D. W. Sep. Purif. Methods 1985,14, 213. (2)Cline Love, L.J.; Habarta, J. G.: Dorsev, J. G. Anal. Chem. 1984, 56, 1132A. (3)Hinze, W. L. In Ordered Media in Chemical Separation; Hinze, W. L., Armstrong, D. W., Eds.; ACS Symposium Series 342;American Chemical Society: Washington, DC, 1987. (4)Dorsey, J. G.Adu. Chromatogr. 1987,27, 167. (5)Khaledi, M. C . Trends Anal. Chem. 1988,7,293. (6) Cline Love, L. J.; Arunyanart, M. J. Chromatogr. 1985,342, 293. 0003-2700/92/0364-1894$03.00/0

The effect of micelles on retention and selectivity in reversed-phase LC (RPLC)has been reported previously.llJ2 Generally, an increase in micelle concentration would result in a decrease in retention except for a few caseswhere retention might increase with micelle concentration.12 The rate of change in retention of different solutes varies with charge and hydrophobicity of solutes as well as the length of alkyl chain, charge, and concentration of micelles. The most noticeable drawback of MLC is slow mass transfer which leads to poor efficiency. Dorsey et al.I3 reported that the addition of a small amount of (35% ) propanol and raising the temperature to 40 "C would improve the column efficiency. Armstrong et al. noted that poor mass transfer from the surfactant-coated stationary phase is the predominant reason for the poor efficiency in MLC.14 Yarmchuk et al.15 reported that poor efficiency is due to slow mass transfer of solute from micelles as well as from stationary phase to the bulk solvent. They suggested that chromatographic efficiencycan be improved by increasing the operating temperature. They argued that the addition of organic modifier might reduce the role of micelles and bring the system closer to a hydroorganic system. Although the effect of organic modifiers on chromatographic efficiency in MLC has been studied by severalworkers, their role on selectivity has surprisingly been ignored until recently. Khaledi et al.'6J7 studied the effect of organic solvent on retention and methylene group selectivity in MLC. They showed that the retention behavior in micellar mobile phases was quite different from that of hydroorganic mobile phases. They further demonstrated that the retention behavior of a ternary mobile phase of organic solvent-water-micelles was similar to that of purely aqueous micellar mobile phases. It was shown that addition of propanol up to 20% to the micellar eluent does not create a hydroorganic system. Surprisingly, the methylene group selectivity remained constant upon the addition of propanol to micellar eluents, which caused a significant increase in solvent strength despite the fact that in RPLC solvent strength and selectivity are often reciprocally related. This was the first report on the "anomalous" relationship between the solvent strength and (7)Strasters, J. K.;Breyer, E. D.; Rodgers, A. H.; Khaledi, M. G. J. Chromatogr. 1990,5ZZ, 17-33. (8) Strasters, J. K.; Kim, S. T.; Khaledi, M. G. J. Chromatogr. 1991,

586,221-232. (9)Khaledi, M. G.;Breyer, E. D. Anal. Chem. 1989,61, 1040-1047. (10)Breyer, E. D.; Strasters, J. K.; Khaledi, M. G. Anal. Chem. 1991, 63, 828-833. (11)Yarmchuk, P.; Weinberger, R.; Hirsch, R. F.; Cline Love, L. J. Anal. CHem. 1982,54,2233. (12)Armstrong, D. W.; Stine, G. Y. Anal. Chem. 1983,55,2317. (13)Dorsey, J. G.;DeEchegaray,M. T.;Landy, J. %Anal. Chem. 1983, 55,924. (14)Armstrong, D. W.; Ward, T. J.;Berthod, A. Anal. Chem. 1986,58, 579. (15)Yarmchuk, P.; Weinberger, R.; Hirsch, R. F.; Cline Love, L. J. J. Chromatogr. 1984,283, 47. (16)Khaledi, M. G.;Pueler, E.; Ngeh-Ngwainbi, J. Anal. Chem. 1987, 59,2738. (17)Khaledi, M.G.Anal. Chem. 1988,60,876. 0 1992 American Chemlcal Society

ANALYTICAL CHEMISTRY, VOL. 64, NO, 17, SEPTEMBER 1, 1992

0.08 M SDS 3% PrOH

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14% PrOH

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EXPERIMENTAL SECTION Apparatus. The HPLC system consisted of a pump (Model 2350, ISCO, Lincoln, NE) and a variable-wavelengthabsorbance detector (V4, ISCO) set at 254 nm, controlled by Chemresearch chromatographic data management system controller software (ISCO) running on a PC-88 Turbo personal computer (IDS, Paramount, CA). The column (75 X 4.6 mm) was packed with Bakerbound CIS-bonded phase packing material (J. T. Baker, Inc., Phillipsburg, NJ) using an HPLC column slurry packer (Altech Associates Inc., Deerfield, IL). The column was thermostated at 40 "C by a water circulator bath (Lauda Model MT6, Brinkmann Instruments, Inc., Westbury, NY). A silica precolumn was used to saturate the mobile phase with silicates and to protect the analytical column. The column dead volume was determined from the retention times of peaks originated by multiple injections of pure water in the chromatographic system. Reagents. The stock solution of sodium dodecyl sulfate(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, Ann Arbor, MI). The sample solutions were prepared by diluting the stock solutions (10mg/mL in methanol or tetrahydrofuran) in the mobile phase. Amino acids, peptides, and SDS were purchased from Sigma (St Louis, MO). 2-Propanol (PrOH) and n-butanol (BuOH) were obtained from Fisher Scientific (Pittsburgh, PA).

~

RESULTS AND DISCUSSION

chromatographic selectivity in MLC with the hybrid mobile phases of micelles and organic solvents. Tomasella and Cline Love have examined the effects of organic modifier and temperature on retention in MLC based on thermodynamic properties.18 Recently, Hinze and Weber presented a quantitative treatment of retention behavior of homologous series on the basis of two independent equilibria in MLC.lg Khaledi et al.7,8920 also reported the effect of adding organic solvents to micellar eluents on chromatographic selectivity of polar and ionic solutes. Elution strength increasesin RPLC with an increase in organic solvent or micelle concentration. It was observed that, by addition of an organic solvent to micellar eluents, a simultaneous enhancement in solvent strength and selectivity for some ionic and nonionic compounds can be achieved. This selectivity enhancement was attributed to the competing partitioning equilibria in MLC system and/or to the unique characteristics of micelles to compartmentalize solutes and organic solvents. In this paper the results of a further study on the role of organic modifiers and micelles in controlling solvent strength and selectivity in MLC are reported. The reasons behind the simultaneous enhancement of solvent strength and selectivity as a result of the variations in the concentrations of an organic modifier and micelles in MLC are discussed. The term hybrid is used for the ternary eluents of water-organic solvent-micelles throughout the text.

With hybrid eluents of mice11es-water-2-propano1, a simultaneous enhancement in solvent strength and selectivity was observed for a large group of amino acids, small peptides, phenols, substituted benzenes, and benzoic acids. The strength of the mobile phase can be enhanced by increasing the concentrations of micelles and/or an organic cosolvent. Parts a-c of Figure 1show the reconstructed chromatograms for nine amino acids and peptides at a fixed micelle concentration (0.08 M SDS) a t different concentrations of 2propanol. It is shown that as the volume fraction of 2-propanol increases, analysis time decreases and selectivity significantly increases. Parts a and b of Figure 2 show the influence of 2-propanol concentration on selectivity for several pairs of amino acids and peptides and for substituted benzenes at a constant micelle Concentration. As is shown, selectivity variations occur systematically and monotonically for different peaks as a result of an increase in 2-propanol concentration. Parts a and b of Figure 3 illustrate the changes in selectivities of the same pairs of compounds due to the variations in micelle concentration at a constant 2-propanol composition. Interestingly, micelle concentration has an opposite effect on selectivity as compared to 2-propanol. For those pairs of peaks whose selectivities are reduced with increasing 2-propanol, an enhancement in selectivity is observed as a result of increasing micelle concentration and vice versa. These observations suggest that although solvent strength increases with concentrations of both micelle and organic solvent, the effect of these two parameters on selectivity could be quite different, even opposite. Micelles and 2-propanol compete to interact with solutes, and as a result they influence the role of one another in controlling retention and selectivity. Effects of Micelles on the Solvent Strength Parameter. In conventional RPLC, there is a linear relationship between In k' (solute's retention) and volume fraction of organic modifier (&,rg) over a limited range. The slope of this line is called the solvent strength parameter, S, which is generally proportional to retention and molecular weight of solutes in conventional hydroorganic RPLC.2*22 For two

(18)Tomaaella, F. P.; Cline Love, L. J. Anal. Chem. 1991, 63, 474. (19) Hinze, W. L.; Weber, S.G. Anal. Chem. 1991,63, 1808. (20) Khaledi, M. G.; Straaters, J. K.; Rodgers, A. H.; Beryer, E. D. Anal. Chem. 1990,62,130.

(21) Snyder,L. R.; Quarry, M. A.; Glajch,J. L. Chromatographia 1987, 24, 33. (22) Coenegracht,P. M. J.; Metting, H. J.; Smilde, A. K.; CoenegrachtLamers, P. J. M. Chromatographia 1989,27, 135.

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0 t (min) Flguro 1, Chromatograms of a mlxture of nine amino acids and pepMas. The chromatograms were reconstructed on the basis of the Individually measuredretention date collectedon a 7 . k m Cla column, 4.6mm i.d., 5-pm partkle she, thermosteted at 40 OC, assumlng 2500 plates and equal concentrations of all components, pH 2.5. Components: (1) Ala-Tyr (AY), (2) Tyr (Y), (3) Met (M), (4) Leu-Tyr (LY), (5) Asp-Phe (DF), (6) Glyleu-Tyr (GLY), (7) Trp 0, (8) Leu-Tyr (LW, (9) Phe-Phe (FF). Moblle phase: (a, top) 0.08 M SDS, 3% 2-propanol; (b, mlddle) 0.08 M SDS, 8% 2-propanol; (c, bottom) 0.08 M SDS,14% 2-propanol.

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992

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2-Propanol (%) Varlatlon of selectlvlty wlth the volume fraction of 2 9 0 panol at 0.02 M SDS for (a, top) amlno acids and peptides and (b, bottom) substkuted benzenes.

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solutes a and b, where k’b > k’a, s b is often larger than Sain hydroorganic eluents. As a result, the selectivity between these two solutes decreases with an increase in organic modifier concentration. Similar to conventional hydroorganic mobile phases, in hybrid systems the relation between retention and volume fraction of organic solvents can be expressed as eq 1, where S h y b is the solvent strength parameter in hybrid system and In k’o is the retention in a purely aqueous micellar eluent. In k’ =

+ In k’,

(1)

In MLC, where micelles form a pseudophase in the eluent, retention and selectivity are governed by three competing equilibria, namely partitioning (or binding) from bulk solvent to micelles and to the alkyl-bonded stationary phase or a direct transfer from micelles to the stationary phase. Retention in MLC can be described as eq 2,’ where K,, is the

solute-micelle binding constant, Ps, is the partition coefficient of compound between the mobile and stationary phases, Q is the phase ratio, and [MI is the micelle concentration. Equation 2 can be written in the logarithmic form as eq 3. The relationships between both In (Pa,@)and In k’ = In (Pa,@)- In (Km,[Ml

+ 1)

(3)

In (KmwIMl + 1)with r$org are linear, and one can write the following empirical linear relationships:

Varlatlon of selectlvlty with the mlcelle concentratton at 12% 2-propanol for (a, top) amlno acids and peptides (b, bottom)

Flguro 3.

substituted benzenes.

where K m d and P a d are respectively the solute-micelle binding constant and partition coefficient of solutes between mobile and stationary phases for purely aqueous micellar eluents (i.e. no organic modifier). The linear regression of the eqs 1, 4, and 5 resulted in high correlation coefficients (>0.99in most cases). Note that the slopes of eqs 4 and 5, Saand S,, represent the sensitivity of variations in the solutes partitioning from the bulk solvent into the stationary phase and into micelles with changes in qjOw Thus, the chromatographic S parameter, Shyb, is dependent upon S, and S, according to eq 6 which can be derived by combining eqs 1

and 3-5. The negative sign in eq 6 clearly reflects the competing nature of the two partitioning equilibria (into micelles and stationary phase). In the absence of micelles S, = 0 and S h y b = S, which represents the solvent strength parameter in conventional hydroorganic RPLC. This equation also shows that the S values in hybrid systems are generally smaller than those of hydroorganic eluents. The frequently observed phenomenon of simultaneous enhancement of selectivity with solvent strength can therefore be attributed to the existence of the competing equilibria in MLC which is reflected in the relationship shown in eq 6. Another important observation is the dependence of S, and consequently S h y b on micelle concentration (eqs 5 and 6).

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1902

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Flguro4. Variatlon of &value wlth micelle concentration for (a, top left)amino acids and peptides and (b, top right)substltuted benzenes. Variation of S, with micelle concentration for (c, bottom left) amino acids and peptides and (& bottom right) substltuted benzenes.

This shows that micelle concentration has an effect on how retention and selectivity in MLC are influenced by 4orrParts a and b of Figure 4 illustrate the variation of S h y b with micelle concentration for amino acids, peptides, and substituted benzenes, respectively. It is shown that the S values of all solutes decrease with micelle concentration. The degree of reduction in S h y b depends on the variation (an increase) of S, with micelle concentration (Figure 4c,d), which in turn is a function of solutemicelle interactions. As shown in Figure 4c,d, the degree of increase in S, for various solutes is different. As a result the difference between the S values (dS) for a given pair of solutes, which has a direct influence on the dependence of selectivity on (40rg), can vary with micelle concentration. In order to examine the validity of eq 6, we compared the experimentally measured S h y b and the S h y b calculated from eq 6 for all solutes. The results are listed in Table I. There is a good agreement between the measured and calculated S h y b values, considering errors in the measurement of Kmw’s and Pnw’s and also the regression errors. From eq 1,eq 7 can be derived for selectivity (CY= k’b/k’,, k’b > k’J between two solutes a and b in hybrid systems at volume fractions of organic modifiers, 1and 2. A combination of eqs 1, 6, and 7 gives eq 8, where dS, and dS, are the differences between the S, and S, values for two compounds

Table I compd

AY M

Y GLY W DF LW

FF LY

[Shybl’

SIUb

S,E

[Shyblcd

5.4 13.7 11.9 6.3 9.2 6.7 6.5 7.2 7.3

6.7 4.6 8.3 4.8 8.6 6.0 1.9 4.6 4.7

11.4 17.6 19.0 10.7 16.8 12.1

4.7 13.0 10.7 5.9 8.2 6.1 6.3 7.1 6.9

8.2

11.7 11.6

The average Svalues measuredas the slope of the linear regression of the retention vs the volume fraction of 2-propanol at 0.02, 0.04, 0.06,and 0.08M SDS. The slope of the linear regression of (&,[MI + 1) vs the volume fraction. The slope of the linear regression of Psw+vs the concentration of 2-propanol. Shyb calculated from S,S .,

a and b and a0 is the selectivity in a purely aqueous micellar eluent (Le. no organic modifier present). In ag- In a1 = - ( S h y b

- S h y b 1)(42- 41)

(7)

+ In a.

(8)

In a = (dS, - dS,)4,,,

From eq 8 one can conclude that In CY has a linear relation with dorg. In other words selectivity can monotonically

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Table 11. Values for as,, dS,, d s h y b , and

Shyb

at 0.02 M

SDS.

solutes

dS,

dS.

dShvb

Shvb

Aromatics 4.08

phenanthrene 0.14

-0.22

-0.45

0.43

0.70

0.46

0.42

0.80

0.30

-0.58

-2.88

2.59

0.26

-0.75

-0.82

-0.19

-0.001

0.06

-0.05

-0.17

-0.25

4.53

naphthalene

a = a,,/ff,,

4.07

toluene

3.77

benzene nitrobenzene

6.36 7.18

benzaldehyde

7.12

benzonitrile

7.37

benzyl alcohol Amino Acids and Peptides

FF

7.32 2.4

3.4

0.25

-4.9

-8.6

-4.73

LW

7.07

W

11.8 2.60

6.10

4.31

0.30

-0.90

-1.28

-0.80

-0.50

GLY

7.49

LY

8.77 0.00

DF

8.77 1.8

-5.6

-7.33

-2.4

1.4

0.50

-7.60

8.12

M

16.1

Y

15.6

0.10

AY

of dS, are larger than those of dS,. Despite the small values and overall range of S,, the existence of competing equilibria has an important impact on chromatographic selectivity in MLC which can be manipulated by concentrations of both organic solvent and micelle. on Competing Equilibria in MLC. From Effect of eq 2 one can derive eq 9 for selectivity in MLC as

7.48

a The compounds are ranked on the basis of the decreasing order of their retention factors a t 0% 2-propanol.

increase or decrease with the volume fraction of organic solvents. When dS, > dS, for two solutes 1 and 2, the selectivitybetween these compoundsincreases monotonically with the concentration of organic solvent. However in the case of dS, < dS,, the selectivity between solutes 1 and 2 decreases monotonicallywith qLrg. The dS,, dS,, dShyb,and S h y b Values of all solutes are listed in Table 11. In this table compounds are listed according to increasing order of their retention at 0% 2-propanol (no organic solvent present) (Pa&). As can be seen, for both uncharged and charged solutes, there is no direct relationship between retention and Shyb.

In fact it can be observed that generally for most benzenes, there is an inverse relationship between the retention and S h y b (Table 11). Longer retained compounds have an overall smaller Shyb. A similar behavior for amino acids and peptides was observed except for W and AY; for these two compounds pKa shift also plays an important role. It can also be seen that for most aromatic compounds, amino acids, and peptides S, and retention are inversely related while there is a direct relationship between S, and retention. The opposite behavior of S, and S, as a function of solute type is the reason behind the inverse relationship between S h y b and retention, since S h y b = S, - S,. The values and the range of S, variations wkh solute structure are much smaller than those of S,. For aromatics S, values are in the range 3-7.7 while the range of S, values is from 0.30 to 0.90. The S, values of amino acids and peptides are in the range 8.2-19, and the S, values are in the range of about 2-9. This shows that the variations in S h y b with solute structure are mostly due to changes in S,. As shown in Table 11,for most of the compoundslisted the values

(9)

where a, is the stationary-phasepartitioning selectivity (P, 2/ P,, 1) and a,, is a function of binding selectivity to micelles, {(Kmw I[MI + l)/(Kmw 2[Ml + 1)). The Paw and K,, values of all compounds decrease with an increase in the volume fraction of 2-propanol. The degree of decrease in these parameters, however, is usually not equal for different solutes. This can lead to changes in selectivity. The K,, and P,, values of the benzenes and peptides were measured at different 2-propanol concentrations. The procedure for these measurements, the difficulties, and the uncertainties have been reported elsewhere.23For those pairs of compounds that selectivity increased as a result of increasing 2-propanol concentration, the K,, and P,, of the less retained compound in the pair decreased to a higher degree as compared to those of more retained compounds. For other pairs of solutes for which selectivity decreased with an increase in percent 2-propanol, the reduction in Kmw and P,, of the compound with the higher retention in the pair was more than those of the compoundwith lower retention. Parts a and b of Figure 5 illustrate variations of K,, and P,, of three typical solutes (LW, W, AY) with increasing percent 2-propanol respectively. As shown, the rank of the slopes for these solutes is W > AY > LW. LW/W and LW/AY belong to the fiist group of solute pairs (for which selectivity increases as a result of an increase in organic solvent concentration), and W/AY belongs to the second group of solute pairs (for which selectivity decreasesas a result of an increase in organic solvent concentration). These observations suggest that a,, and a,, for LW/W and LW/AY should increase and those for W/AY should decrease systematically as a result of an increase in 2-propanol concentration. The variations of a,, and a,, of these three pairs of solutes with increasing of percent 2-propanol are shown in Figure 6a,b. Figure 6c illustrates the variation of chromatographic selectivity a = a8,/amW with the volume fraction of organic modifier. In this figure it is shown that there is a systematicchange in selectivity with the organic solvent concentration (an increase for LW/ W representing the first group and a decrease for W/AY from the second group). It is noteworthy to consider that both eq 9 and Figure 6c show that a,, and a,, have opposite effects on chromatographic selectivity. In other words, an increase or a decrease in selectivity with an increase in organic solvent concentration is controlled by the competition between these two parameters. Another important factor is the effect of micelle concentration on am, and therefore on chromatographic selectivity. For the first group of pairs of compounds like LW/W, both a,, and a,, increase with percent 2-propanol but the rate of increase of the former parameter is more than that of the latter (Figure 6a,b). When micelle concentration increases at fixed 2-propanol concentrations, the rate of increase in amwbecomes larger than the rate of the increase of a,, and selectivity decreases. For the second group of solutes like W/AY, both a,, and a,, decrease as a result of an increase in percent 2-propanol but the degree of decrease in as, is more than that of a,,. As a result of an increase in the mi(23) Kord, A. S.; Strasters, J. K.; Khaledi, M. G. Anal. Chim. Acta 1991, 246, 131.

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2-Propanol (%) flguro 5. (a, Top) linear regression of log ( K , [ M ] 4- 11vs the volume fractbn of 2-propanol for three solutes. (b, Bottom) linear regression of log (Psw'P) M the volume fraction of 2-propanol.

celle concentration at fixed percent 2-propanol, the degree of decrease in am, becomes larger than that of as, (which is independent of [MI), and as a result of this, selectivity increases. These observations explain the selectivity behavior of the two groups of test compounds with respect to variations in percent 2-propanol and [MI, which are shown in Figures 2a,b and 3a,b. Another parameter that should be considered is micellarinduced shift of ionization constants. Addition of 2-propanol would also cause a shift in the pK,'s of ionizable compounds. Because of this, a change in the concentration of 2-propanol can affect the degree of the solutes' pKa shifts. Obviously, any change in the ionization constant of the solutes can influence the charge and consequently their interactions with micelles (Kmw)and the surfactant-modified stationary phase (PBW).

It has been observed that pKa values of amino acids and peptides increase in the SDS micellar eluents.24 The magnitude of the micellar-mediated pKa shift is a function of the (24) Khaledi, M. G.; Rodgers, A. H. Anal. Chirn. Acta 1990,239,121.

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X PROH Flguro 6. (a, Top) Variation of aswvs the volume fraction of 2-propanol for LW/W and W/AY. (b, Middle)Variation of amW vs the volume fraction of 2-propanol for LWIW and W/AY. (c, Bottom) linear regression of asw/amw vs the volume fraction of 2-propanol for LW/W and WIAY.

Table 111. pKa of Amino Acids and Peptides Measured in (a) Micellar Solution (0.08 M SDS) and (b) Hybrid Solution (0.08 M SDS + 15% 2-Propanol) compd pK. micellar pKahybrid d(PKa) GLY LY LW W

4.67 4.75 4.97 5.57

4.17 4.19 4.52 4.8

-0.5 -0.56 -0.45 -0.77

micellar binding constants of acid-conjugate base pairs. The addition of 2-propanol to the aqueous solution changes the dielectric constant of both the bulk solvent and the microenvironment of micelles. It also reduces the K,, values of the acid-conjugate base pairs. Both of these factors would cause a further shift in pK,. The degree of a change in pKa shift due to the presence of 2-propanol in micellar eluents would not be the same for different solutes, depending on their structures. The pK. of four amino acids and peptides in micellar and hybrid eluents are listed in Table 111.

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992

CONCLUSIONS The addition of organic solvents to micellar eluents improves column efficiency, adjusts the elution strength of the generally weak micellar eluents, and often provides improved selectivity. The results presented above show that simultaneous enhancement of selectivity and solvent strength in MLC can be expected to occur often and to occur for almost any groups of compounds that interact with micelles. It is true that a similar phenomenon has been reported for other modes of RPLC such as conventional hydroorganic eluents and ion pair LC. However this is not the normal pattern for these techniques and occurs under certain conditions.20-22

The origin of this unique behavior in MLC stems from the fact that retention is influenced by the competing partitioning equilibria and the characteristics of micelles acting as organized media.

ACKNOWLEDGMENT We gratefully acknowledge the support of this work through a research grant from the National Institutes of Health (FIRST Award, GM38738). RECEIVEDfor review December 16, 1991. Accepted May 21, 1992.