878
Anal. Chem. 1988, 60,876-887
Hydrophobic Selectivity in Micellar and Hydro-Organic Reversed-Phase Liquid Chromatography Morteza G . Khaledi
Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148
Selectlvlty for homologous serles In reversed-phase llquld chromatography (RPLC) uslng mlcelleJs and organk solvents as the modifiers for the aqueous mobUe phase was studied. Slgnlflcant differences were observed between mlcellar and hydro-organlc RPLC systems. Wlth hydro-organlc moblle phases, methylene group(8) selectlvlty depends only on the eluent compoenlon for a glven statlonary phase chain length. For mkellar eluents, however, the selectlvtty for homologous serles Is a functlon of the sokae type. ThIs can be attrlbuted to the varlous solutes’ locatlons Won mlcelles wlth different mlcroenvlronment polarities In a glven micellar eluent composltlon. For a hydro-organlc mobile phase, methylene selectlvlty Is slightly larger for a C-18 than for a C 8 statlonary phase, while It Is the same on both columns for a mlcellar mobHe phase. I n micellar LC, the overall nonspeclflc selectlvlty values are small and the range of thelr varlatlon Is Ibnlted for a rather large range of solvent strength. ThIs shows the existence of a dmllar envkomnent for a methylene group In micelles as compared to that In alkyl-bonded statlonary phase. Surprlsingly, In the presence of rnldks, the addnlon of up to 20% of 2-propanol had vlrtuaUy no effect on methylene group(s) selectivity, desplte a large Increase In eluent strength. Thls le attrlbuted to a slmllar solvation of both mlcelles and statlonary phase wlth propanol. I n llght of these results, It Is concluded that the elutroplc solvent strength scale, eo, is lnapproprlate to quantltate mlcellar eluent strengths.
In a chromatographic method, separation occurs because of various molecular interactions between a solute with mobile and stationary phases. Establishing a rational foundation for predicting retention demands an in-depth knowledge of the nature, as well as the mechanism, of these interactions. Reversed-phase liquid chromatography (RPLC) with different mobile phase “modifiers” such as organic solvents, micelles, or a hybrid of the two have several similar characteristics. For example, an increase in the modifier concentration (i.e. volume fraction of organic solvent or micelle concentration) would result in a decrease in retention of nonionic compounds. Micellar and hydro-organic systems share the basic components of a reversed-phase LC system, that is a nonpolar stationary phase and a polar aqueous mobile phase. Thus, hydrophobic interactions can play an important role in governing retention of nonionic compounds in these systems. In addition, retention and selectivity are most easily controlled by adjusting solute-mobile phase interactions in all RPLC systems. Despite these similarities, however, there exist distinct differences between the RPLC methods with various mobile phase modifiers (I). Firstly, hydro-organic solvents are homogeneous, while micellar media are microscopically nonhomogeneous. Micelles provide several sites of interactions with different microenvironment “polarities”. The interactions of nonionic solutes with micelles occur through different mechanisms such as surface adsorption, pseudophase ex-
traction (partitioning), and solute-surfactant coassembly (comicellization) (2). The type of interaction mechanism which depends upon the solute and micelle properties, determines the location(s) of a compound in/on micelle. This creates a unique situation in which solutes occupying various locations in/on micelle may experience different microenvironment “polarities” in a given micellar mobile phase composition. The free energy of transfer from bulk solvent to micelle reflects the “solvation” experienced by a solute and is highly sensitive to variations in compound location in the micelle (3). As a result, characterization of microenvironment “polarity”of a solute in micelle and its relationship to retention and selectivity in RPLC with micellar mobile phases are of prime importance in a retention mechanism study. The characteristics of the alkyl-bonded stationary phases in RPLC with micellar and hydro-organic mobile phases are another noteworthy difference. The composition and structure (conformation) of the hydrocarbonaceous stationary phase is a function of the hydro-organic mobile phase composition. The extraction of an organic solvent by the grafted alkyl phase has a profound effect on retention and selectivity (4,5).With micellar eluents, however, the alkyl-bonded phase is modified with an approximately constant concentration of free (monomers) ionic surfactant which is equal to the critical micelle concentration (cmc) (6, 7). As a result, the composition and structure of a stationary phase are independent of mobilephase composition. In other words, solutes would experience a stationary phase with unchanged characteristics at different mobile phase compositions. A knowledge of the exact role and significance of these parameters in causing different behavior in micellar and hydro-organic RPLC is of prime importance in a retention mechanism study. The exact retention mechanism in hydro-organic RPLC is still not clear; however, the enormous amount of information that exists about this system makes it a valuable “reference” point for studies of retention behavior in micellar LC. A study of the chromatographic behavior of homologous series provides information of great use for a better characterization of the mobile and stationary phases effects on retention and selectivity in RPLC with micellar’ and hydroorganic mobile phases. The existence of a linear relationship between retention factor and a structural parameter (i.e. number of carbons) makes the retention study of homologous series particularly attractive for comparative purposes. The chromatographic behavior of these compounds with hydroorganic RPLC is well established (8-13).The regular increase of retention due to addition of a methylene group allows one to differentiate between contribution of nonspecific interactions to retention from that of specific interactions of the polar functional group of homologous series with mobile and stationary phases. In a previous paper, the retention behaviors of two alkyl homologous series were compared in RPLC using micellar, hydroorganic, and hybrid (micelles in hydroorganic) mobile phases (I). The presence of micelles in the aqueous mobile phase of RPLC has a profound effect on the chromatographic
0003-2700/88/0360-0876$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
characteristics of homologous series. With conventional hydro-organic mobile phases a linear relationship exists between log k’ and the number of carbons, N,, in the normal chain. For micellar and hybrid mobile phases (with one exception), i t is the retention factor, k’, and not log k’, which provides a better linear relationship with the carbon number (I). The retention characteristics of the homologous series in hybrid mobile phases (SDS and ClGTABmicelles in propanol/water solvents) is similar to that with a purely aqueous micellar mobile phase, despite the fact that the stationary phase in a hybrid system resembles more a hydro-organic than a micellar system. A study of retention behavior of homologous series as a function of organic modifier concentration in the presence and absence of micelles provides interesting results. In conventional binary mobile phases, the linear relationship between logarithm of retention factor and volume fraction of O log k,) is usually valid only organic modifier (Le. log k’ = S for a small range of concentrations and a quadratic equation is observed for a larger range (14,15).For water-rich eluents (below 10% organic solvent), even a quadratic fit is inadequate. With hybrid mobile phases, in the presence of a constant micelle concentration, however, excellent linearity exists between log k ’and 0 even for a range of small concentrations of 3% to 20% of 2-propanol (Le. water-rich eluents). Secondly, for hydro-organic mobile phases, the slope of log k’ vs 0, also known as Snyder’s solvent strength, changes drastically with solute size. The variations in solvent strength S values with an increase in solute size is minimized in the presence of micelles. Thirdly, the overall S values for 2propanol in hybrid mobile phases are much smaller than those measured in hydroorganic solvents (1). Since the stationary phases in the binary propanol-water systems and in the hybrid systems are very similar, these observations reflect the impact of the presence of micelles. The constancy of the solvent strength, S, with the variations in solute size and the small values of S in the hybrid mobile phases are probably due to localization of solutes and propanol in micelles which reduces the size factor as far as the solvation of a solute by propanol is concerned (1). In this paper, we examine the methylene group(s) selectivity, also known as hydrophobic selectivity, in different micellar, hydro-organic, and hybrid systems. The study of chromatographic selectivity is advantageous in retention mechanism research since it provides useful information about the mobile and stationary phase effects and is not affected by the column phase ratio. Also, the methylene selectivity is not influenced by the presence of the silanol groups on bonded phase surfaces.
+
EXPERIMENTAL SECTION Apparatus. A Rainin (Woburn, MA) gradient liquid chromatograph incorporating three Rainin Rabbit HP pumps, an Apple Mcintosh computer as the controller, a Model 7125 Rheodyne injection valve, and a Rainin Dynamax mixer were used. The detector was a model 4VISCO (Lincoln,NE)W-vis variable wavelength with a built-in chart recorder. The analytical columns used were Rainin Microsorb 5pm octyl(4.6 X 150 mm) and a 3-pm ODS (4.6 X 50 mm) for hydroorganic and SDS experiments and an Altex Octyl Ultrasphere (4.6 X 150 mm) from Beckman was used for CTAB experiments. A silica precolumn and a dry-packed C-18 guard column were used to saturate the mobile phase with silicates and to protect the analytical column. The silica precolumn, the guard column, and the analytical column were water jacketed and thermostated at 31 “C with a Hauk (Karhruhe,GFR) circulating bath. Reagents. Stock solutions of sodium dodecyl sulfate, SDS (Polysciences), and cetyl trimethylammonium bromide, CTAB (Fluka), were prepared in deionized, doubly distilled water and were filtered through a 0.45-pm Nylon-66 membrane fiiter (Rainin Instruments). Alkylbenzenes and alkylphenones were obtained from different sources.
877
1.77 IO
m
T 1.23
1.28
’
,
L6.7B 8.88 Nc 5.88 Figure 1. log k’vs number of carbons, alkylbenzenes: (*) 0.072 M C,,TAB micellar mobile phase; (0)60% methanol In water. ,
The void volume of the system with Microsorb columns was measured by injecting different solutions such as CH,CN-H,O (l:l),urea, two uracil solutions dissolved in methanol and SDS, and a mixture of aqueous SDS-methanol at several SDS and hydroorganic mobile-phase compositions. The time equivalent of the void volume was measured from the time of injection to the first deviation from the base line. The average value of 1.01 mL was obtained for the 15-cm Microsorb with a relative standard deviation of 5 % from 16 measurements. The void volumes with the hydro-organic mobile phases (with the same column) were *5-10% different from the above value, however, due to inherent uncertainties in void volume measurements, the value of 1.01 mL was used for all k’measurements. The void volumes for the 5-cm C-18 Microsorb column (used in SDS experiments) and for 15-cm C-8 Ultrasphere (CTAB column) were 0.35 and 1.08 mL, respectively. For CTAB column, a mixture of water-methanol (8515) waa used for void volume measurements. The regression calculations were done with the program Curve Fitter (InteractiveMicroware, PA) on an Apple IIe microcomputer. R E S U L T S A N D DISCUSSION Measurement of Hydrophobic Selectivity. Hydrophobic or methylene selectivity is the contribution of a methylene group to retention and is defined as the ratio of retention factors of two solutes differing from one another by a methylene group, i.e. a(CHJ = k;+,/kk. For hydro-organic mobile phases, it is usually calculated from the slope of the linear plots of log k’ vs N , for a given homologous series, since log k’ = log a(CHJN,
+ log 0
(1)
For micellar and hybrid systems, however, the curvature in the log k’vs N, plots implies a variation of the slope with the number of carbons (Figure 1). A quadratic equation provides the best correlation, that is log k’ = (log r)N:
+ (log a)N, + log 0
(2)
On the basis of the interaction index model, Jandera has suggested that the relationship between log k’and N , (for hydro-organic LC) should be explained through a quadratic equation (16). For alkylbenzenes and alkylphenones with hydro-organic mobile phases, the coefficient of the second degree is small enough so that the quadratic term may be neglected, thus a linear correlation is observed. Apparently, this is not the case for the micellar and hybrid mobile phases studied. Thus, the other two alternatives to the slope method for a(CHZ)measurement are, firstly, the use of average values of methylene selectivities of successive members in the series and, second, the use of coefficient to the first degree term in the quadratic equation (eq 2). The a(CH,) values measured by using the three methods are compared in Table I for selected mobile phases. As expected, the a(CH,) values for hydroorganics obtained by averaging are very similar to those
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
Table I. Comparison of Methods for a(CH2)Measurements (Homologous Compounds, n -Alkylbenzenes; Stationary Phase, C-8) mobile phase
av (fSD)O
slope ( r 2 ) b
first degree coefF
40% propanol 50% propanol 65% methanol 90% methanol 50% acetonitrile 80% acetonitrile
A. Hydro-Organic 1.455 (f0.03) 1.295 (f 0.01) 1.590 (f0.08) 1.150 (f0.03) 1.530 (f0.04) 1.218 (f0.03)
1.466 (0.999) 1.298 (0.999) 1.578 (0.999) 1.144 (0.995) 1.517 (0.999) 1.210 (0.996)
1.505 1.296 1.478 1.110 1.458 1.161
0.15 M SDS 0.30 M SDS 0.50 M SDS 0.07 M C16TAB 0.10 M Cl6TAB 0.15 M C16TAB
B. Micellar 1.285 (f0.07) 1.175 (f0.04) 1.125 (f0.05) 1.174 (f0.06) 1.166 (f0.05) 1.150 (f0.04)
1.329 (0.979) 1.196 (0.981) 1.144 (0.969) 1.211 (0.948) 1.186 (0.971) 1.168 (0.975)
1.586 1.301 1.243 1.414 1.312 1.271
0.10 M CIBTAB+ 3% PrOH 0.10 M C16TAB+ 10% PrOH 0.10 M C16TAB+ 20% PrOH 0.10 M SDS + 3% PrOH 0.10 M SDS + 10% PrOH 0.10 M SDS + 20% PrOH
C. Hybrid 1.171 (f0.07) 1.162 (f0.06) 1.157 (f0.06) 1.270 (f0.07) 1.258 (f0.07) 1.235 (10.06)
1.183 (0.971) 1.177 (0.962) 1.177 (0.964) 1.308 (0.979) 1.287 (0.978) 1.267 (0.978)
1.294 1.309 1.309 1.496 1.457 1.426
Average a(CH2)of successive members of alkylbenzenes; SD, standard deviation. degree coefficient of quadratic eq 2. measured from the slope of log k’vs N, plots and are relatively close to the first degree coefficients. Interestingly, for micellar and hybrid systems, the a(CH,) values from the slope and the averaging methods are reasonably close, despite a clear curvature in the log k’vs N , plots for these mobile phases (Figure 1). The values of the first degree coefficients for these systems are, however, not as close as those obtained from the other two methods. The methylene selectivity values obtained by averaging were used in the other parts of this work. Effect of Solute Type. The nonhomogeneous environment of micelles creates a unique situation in which different solutes might experience various microenvironment “polarities” in a given mobile phase composition. The type of solute-micelle interaction mechanism such as surface adsorption, partitioning, etc. depends upon the characteristics of both solute and surfactant (2). The observed curvature in the log k’ vs N , plots for alkylbenzenes with micellar and hybrid mobile phases is an example of the effect of different locations of the members of a homologous series in micelles (Figure 1). Mukerjee has measured the effective ”polarities“ of different benzenes in micelles. He has shown that the dielectric constants of the solutes microenvironment in SDS micelles decrease systematically on the progressive alkyl substitution. For example, the dielectric constants for benzene, ethylbenzene, n-butylbenzene, and hexamethylbenzene in SDS solutions were reported as 49, 41, 36, and 23, respectively (17, 18). As mentioned earlier, the free energy of transfer reflects the solvation experienced by a solute (or a functional group) upon transferring to a different medium. The hydrophobic selectivity value represents the free energy of transfer of a methylene group from mobile phase to the nonpolar stationary phase since
AAGo(CH2) = -RT In (a(CH2))
(3)
The hydrophobic selectivity decreases as the difference between mobile and stationary phase “polarities” is reduced. Likewise, for a given micellar mobile phase composition, a compound located in a more “polar” media experiences a larger change in its microenvironment upon transferring from a micellar mobile phase to the bonded alkyl stationary phase. This is shown in Table I1 where a(CHz) decreases as the
Slope of log k’ vs N , plots; r2 for the plots.
First
number of carbons is increased with an exception of alkylphenones with SDS micelles (1). Thus, the methylene selectivity between n-pentyl- and n-butylbenzenes is smaller than that between ethylbenzene and toluene since the former pair is located in a more nonpolar environment than the latter. These changes are quite small and in certain cases their statistical significance might even be questionable. However, the decrease in a(CHz)values with an increase in the molecular size is observed for almost all cases. I t is in fact the small magnitude of the variations that results in correlation coefficients of as good as 0.99 for log k’ vs N , plots, though a nonlogarithmic fit (i.e. k’vs N,) consistently provides a better correlation for these systems (1). For hydro-organic mobile phases, the variations in a(CH,) measured from different successive pairs is also observed, but these are rather random changes without a regular trend. For hydro-organic eluents, the curvature is observed outside a range between two “critical carbon numbers” (8-12). For long chain homologous compounds (n > 16) the phenomenon of folding and configurational changes could cause a curvature in log k’vs N , plots. Also, it has been suggested that the effect of the addition of a methylene group becomes constant only when it is sufficiently removed from the residual group; therefore below a “critical carbon number” a curvature might be observed in log k’vs N, plots (10). This is apparently not the exact reason for the curvature with micellar eluents since we have not observed any curvature in these plots for hydro-organic mobile phases despite the use of short chain length homologous series (Figure 1). However, the effect of polar functional group of the series on the a(CHz) values with micellar eluents should be examined from the perspective of its contribution to the overall “polarity” of the nonionic molecules. Since a(CH,) is the ratio of retention factors of two compounds differing only in a -CHz- group, it should be independent of the series type for a given mobile and stationary phase system, such is the case for the hydroorganic mobile phases with small Aa(CH,) values (Table 111). The carbonyl group of the alkylphenones makes them more “polar” than alkylbenzenes. Therefore it is logical to assume that the alkylphenones are located in/on a more “polar” medium of micelles. As a result, the methylene groups of alkylphenones would see a different mobile phase environment than those of alkylbenzenes; a
ANALYTICAL CHEMISTRY, VOL. 60, NO.
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Table 11. Methylene Selectivity between Successive Members of Homologous Series, C-8 Column, with SDS and CTAB Eluents ~ ( C H Z=) k'n+l/k'n SDS Micellar Mobile Phases [SDS] = 0.06 M
compound
[SDS] = 0.08M
[SDS] = 0.15 M
[SDS] = 0.10 M
[SDS] = 0.20M
[SDS] = 0.30 M
[SDS] = 0.40 M
[SDS] = 0.50 M
Alkylbenzenes n-hexylbenzene NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1.20
1.18
1.16
1.12
1.09
1.07
NA
NA
1.27
1.22
1.20
1.17
1.14
1.12
1.39
1.34
1.31
1.27
1.24
1.19
1.17
1.13
1.47
1.40
1.36
1.30
1.26
1.22
1.19
1.18
1.76
1.63
1.55
1.44
1.38
1.30
1.26
1.23
1.43
1.37
1.29
1.24
1.22
1.18
1.15
1.13
n-pentylbenzene n-butylbenzene n-propylbenzene ethylbenzene toluenea benzene a(CHz), avb
Alkylphenones n-hexaphenone 1.40
1.40
1.37
NA
1.36
1.34
1.33
1.32
1.46
1.44
1.41
NA
1.39
1.37
1.35
1.34
1.56
1.52
1.49
NA
1.43
1.41
1.39
1.38
1.59
1.54
1.50
NA
1.41
1.37
1.35
1.34
1.50
1.48 0.11
1.44 0.15
1.40 0.18
1.37 0.19
1.36 0.21
1.35 0.22
n-valerophenone n-butyrophenone n-propiophenone acetophenone a(CHZ),avb Aa(CH2)'
0.07
a(CHZ) = k'n+l/k'n CTAB Micellar Mobile Phases compound
[CTAB] = 0.072 M
[CTAB] = 0.10 M
[CTAB] = 0.153 M
[CTAB] = 0.180M
Alkylbenzenes n-hexylbenzene 1.14
1.12
1.11
1.11
1.13
1.13
1.11
1.12
1.13
1.16
1.16
1.13
1.21
1.19
1.18
1.18
1.26
1.24
1.19
1.20
1.53
1.33
1.29
1.23
1.17
1.17
1.15
1.15
n-pentylbenzene n-butylbenzene n-propylbenzene ethylbenzene toluenea benzene a(CHZ)avb
Alkylphenones hexaphenone 1.27
1.25
1.23
1.23
1.31
1.30
1.27
1.26
1.41
1.37
1.32
1.31
1.62
1.54
1.45
1.42
1.40 0.23
1.37 0.20
1.32 0.17
1.31 0.16
valerophenone butyrophenone propiophenone acetophenone (r(CHzavb Aa(CH2)'
Methyl selectivity, a(CH8)= k',l,,,/k'bmne. Average value for a(CHZ)between successive homologues excluding toluene/benzene, i.e., 4CHd. ' A ~ C H Z=) ~ ( C H -Z ( ~) ( C ~ H~z )~b a~~ ~~~ ~~ . ~ situation that does not exist in hydro-organic solvents. As shown in Table 11, for micelles the a(CH2) values are dependent on the type of series.used as the selectivity for alkylphenones are consistently larger than those observed for alkylbenzenes. Again, based on eq 3, a methylene group of
alkylphenones undergoes a larger change in its microenvironment "polarity" as it is transferred from micellar eluents to stationary phase. Several workers have shown that the incremental free energy of transfer per methylene group (AAG"(CH,)), from water to SDS micelles, depends upon the
880
ANALYTICAL CHEMISTRY, VOL. 60,
NO. 9,
MAY 1, 1988
Table 111. Methylene Selectivity between Successive Members of Homologous Series, C-8 Column, Hydro-Organic Mobile Phases 4CHJ = k’,,+i/k’,, 50%
CHSCN
70%
CH3CN
60%
CH30H
90%
CHSOH
35%
PrOH
50%
PrOH
Alkylbenzenes n-pentylbenzene 1.56
1.34
1.77
1.16
1.55
1.30
1.53
1.31
1.75
1.19
1.58
1.29
1.55
1.30
1.72
1.13
1.66
1.31
1.48
1.27
1.61
1.12
1.61
1.28
1.47
1.25
1.62
1.13
1.67
1.31
1.52
1.29
1.69
1.15
1.61
1.30
n-butylbenzene n-propylbenzene
ethylbenzene toluenea benzene a(CH2)*av
Alkylphenones
hexaphenone 1.54
1.31
1.73
1.13
1.68
1.33
1.51
1.27
1.69
1.13
1.67
1.31
1.50
1.26
1.59
1.08
1.65
1.32
1.56
1.22
1.56
1.12
1.68
1.30
1.53 0.01
1.27 -0.02
1.64 -0.05
1.12 -0.03
1.67 +0.06
1.32 +0.02
valerophenone
but yrophenone propiophenone
acetophenone a(CH2)av Aa(CH2)av
Methyl selectivity, a(CHJ = k &,,,,/k Lmne.*Averagevalue for a(CH2)between successive homologues excluding toluene/benzene, i.e., a(CH3). Aa(CH2) = a(CH2)phsnonea- a(CWknzenea* class of homologous compounds (e.g. alkanes, alkylbenzenes, etc.) (19-22). Also, the AAG”(CH2)for the same group of compounds (e.g. phenols) changes with the surfactant type (i.e. anionic vs cationic) (22). These differences have all been attributed to different locations (i.e. different microenvironment polarities) of solubilization in/on micelles. Interestingly, the Aa(CH2) values are very similar for different micellar and hybrid mobile phases (Tables I1 and VII). This is important especially since the SDS micelles with a C-12 chain length surfactant provide a less hydrophobic environment than CI6TAB micelles. Apparently, the effect of surfactant type (chain length, charge) on the methylene selectivity is the same for both homologous series. The situation remains the same with hybrid mobile phases, since the addition of 2-propanol to micellar solutions has a negligible effect on hydrophobic selectivity (vide infra). In order to better characterizethe effecta of surfactant type, homologous series, micelle concentration, etc. on hydrophobic selectivity, the selectivities of multiple-methylene groups, a(CHz),, were measured for different cases. Toluene and acetophenone were used as the parent compounds for alkylbenzenes and alkylphenones, respectively. The relative retention of other homologous compounds in the series were defined as a(CHp)n,BZ = ~ ’ ( C & ~ ( C H Z ) ~ C /k’(C&CH3) H~) (4) a(CHJn,pH = k’(C&6CO(CH2)nCHJ /k’(C&5COCH3) (5) The a(CHZ),, values for different micellar, hybrid, and hydro-organic mobile phases are listed in Table IV. Note that the changes in a(CH,), with carbon number for benzenes are far smaller than those for phenones in micellar eluents. Therefore, the difference in a(CH,), between phenones and benzenes increases with the number of carbons, i.e. compare a(CH2I1and C X ( C H values ~ ) ~ for the two homologous series. This is clearly demonstrated in Table V where the a(CH2), ratios of phenones/benzenes for different mobile phases are
listed. The selectivity ratio of phenones/benzenes can be expressed as
The first term of the right-hand side of this equation is the selectivity of a carbonyl group and the second term is the inverse of carbonyl selectivity. For hydro-organic solvents, the carbonyl selectivities measured from the first and the second terms of the right-hand side of the above equation are almost the same; thus the selectivity ratio remains nearly constant at different carbon numbers and is equal to 1.00 (Table V). This is not the case, however, for micelles since the carbonyl group selectivity depends on the microenvironment “polarities” of the compounds. That is-the carbonyl selectivity between toluene and acetophenone (the second term) is different from that of the first term with more hydrophobic compounds since the latter are located in a more “nonpolar” environment than the former compounds. A noteworthy observation from Table V is that, with SDS mobile phases the selectivity ratios of phenones over benzenes increases with SDS concentration which is in contrast to the expected behavior such as that with CTAB micelles. The “peculiar” selectivities in micellar mobile phases have been represented by the frequent elution reversal as a result of a change in micelle concentration (23, 24). The role of micellar mobile phases in controlling functional group selectivity will be published elsewhere (25). Effect of Mobile Phase Modifier. In a RPLC system, cu(CH,) decreases with an increase in modifier concentrations in the aqueous mobile phase. There exist, however, distinct differences between micelles and organic solvents in influencing the hydrophobic selectivity. With hydro-organic solvents, the RP stationary phase is enriched with organic solvent so that the concentration of an
ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
881
Table IV. Multiple Methylene Groups Selectivity, a(CH2),
ffl
ffz
0.20 M SDS 0.30 M SDS 0.50 M SDS
1.26
1.56 1.45 1.34
0.07 M CTAB 0.15 M CTAB 0.18 M CTAB
1.26 1.19 1.20
0.10 M SDS + 3% PrOH 0.10 M SDS + 6% PrOH 0.10 M SDS + 8% PrOH 0.10 M SDS + 10% PrOH 0.10 M SDS + 15% PrOH 0.10 M SDS + 20% PrOH
1.22 1.18
"3
"4
Micellar 1.87 1.69 1.50
ff1
"2
"3
ff4
2.16 1.89 1.60
1.41 1.37 1.34
2.02
1.93 1.85
2.80 2.65 2.49
3.80 3.55 3.28
1.96 1.81 1.81
1.62 1.45 1.42
2.28 1.92 1.86
2.99 2.44 2.35
3.78 3.00 2.88
2.21
2.32 2.35 2.38 2.41 2.39
3.11 3.25 3.28 3.34 3.35 3.38
4.22 4.35 4.34 4.40 4.37 4.41 3.49 3.49 3.52 3.62 3.65 7.80 3.90 2.98
1.53 1.41 1.42
1.73 1.64 1.60
1.35 1.34 1.33 1.34 1.33 1.31
1.75 1.74 1.73 1.70 1.68 1.65
Hybrid 2.18 2.13 2.13 2.04 2.02 1.97
2.59 2.54 2.54 2.47 2.38 2.33
1.50 1.56 1.57 1.59 1.60 1.61
0.10 M CTAB + 3% PrOH 0.10 M CTAB + 6% PrOH 0.10 M CTAB + 10% PrOH 0.10 M CTAB + 15% PrOH 0.10 M CTAB + 20% PrOH
1.24 1.23 1.24 1.24 1.24
1.45 1.46 1.48 1.47 1.47
1.73 1.66 1.70 1.68 1.67
1.83 1.85 1.90 1.87 1.87
1.56 1.57 1.58 1.61 1.59
2.17 2.17 2.26 2.28
2.81 2.82 2.86 2.95 2.99
35% PrOH in H20 45% PrOH in Hi0 50% PrOH in H20
1.61 1.36 1.28
2.67 1.89 1.67
7.30 3.48 2.82
1.68 1.42 1.30
2.77 1.98 1.71
4.63 2.75 2.25
.
Hydro-Organic 4.22 2.56 2.16
Table V. Selectivity Ratio a(CH2), (phenones)/cr(CH,), (benzenes) mobile phase
dCHz)i ratio
a(CHA ratio
ff(CH213 "(CHZ)~ ratio ratio
0.50 M SDS
Micellar 1.30 1.14 1.38
1.50 1.66
1.76 2.05
0.07 M CTAB 0.18 M CTAB
1.28 1.18
1.49 1.31
1.73 1.47
1.93 1.59
1.11
Hybrid 1.26
1.43
1.63
1.23
1.45
1.72
1.89
1.28
1.49
1.70
1.89
1.28
1.55
1.79
1.95
1.10
0.20 M SDS
0.10 M SDS + 3% PrOH 0.10 M SDS + 20% PrOH 0.10 M CTAB + 6% PrOH 0.10 M CTAB + 20% PrOH 35% PrOH in H20 50% PrOH in H20 70% CH30H in H2O 90% CH30H in H2O 50% CH&N in H2O 80% CH&N in HzO
1.12
Hydro-Organic 1.04 1.04 1.02 1.02 1.05 0.96
1.04 0.92
1.07 1.06 0.89
1.00
0.94
0.91
0.90
1.05
1.02
1.01
1.00
1.12
1.03
1.01
0.99
organic modifier in the alkyl-bonded phase is higher than that in the aqueous mobile phase (4,5). Due to this solvation effect, the discrimination ability of the stationary phase for a change in one methylene group is reduced. In other words, a methylene group experiences a similar environment in an organic-rich RP system as it is transferred to a stationary phase. As a result, methylene selectivity decreases with an increase in organic solvent concentration until i t usually reaches a plateau at high organic compositions (26). Solvation of a
2.21
bonded phase by eluents with different solubility parameter create a stationary phase with different solvent strengths. Thus, the methylene selectivity will be greatest for the largest difference in solvent strength between stationary phase and mobile phase. For a purely aqueous mobile phase, a(CH2) is about 4 and the corresponding value for a 100% organic solvent, e.g. methanol, is around 1.1-1.2 (8-12, 26). In a water/methanol mobile phase, a(CH,) changes roughly 0.05 unit for each 10% of water (IO). With micellar eluents, the overall a(CH,) values are much smaller than the corresponding values for hydro-organic mobile phases with a similar solvent strength. Note that selectivity and eluent strength are reciprocally related. Also, the variation of the methylene selectivity with micelle concentration is rather small and the overall selectivity range is very limited. For micelles, the typical selectivities for alkylphenones and -benzenes range between 1.1and 1.6 for SDS concentrations between 0.5 and 0.06 M, which is similar to the values observed for organic-rich eluents in hydrc-organic systems. The overall smaller a(CH,) value is again an indication of how closely the environment of a methylene group in the mobile phase resembles that of in the alkyl-bonded stationary phase. In micellar LC, retention and selectivity are a result of two competing equilibria, namely, solute partitioning (or binding) in the stationary phase and that into micelles in the mobile phase (27, 28). A mathematical expression describing the relationship between group selectivity and the two equilibria provides a better picture of the factors controlling selectivity in micellar LC. The relationship between retention factor and micelle (or surfactant) concentration has been expressed as D
(7) Where P,, is partition coefficient of the compound in a stationary phase and Kmwis a micelle-water binding constant per surfactant monomer, [MI is the micelle or total surfactant concentration in the mobile phase, and 4 is the chromatographic phase ratio (27, 28).
882
ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
The relationship between selectivity and micelle concentration can then be derived from eq 7 as
For methylene selectivity, the subscripts 1 and 2 refer to two homologous compounds with n and n + 1 carbons in the normal chain, respectively. Divide the numerator and the denominator of this equation by (Kmw,lpsw,l)
a.95
(84
8.28
iSDSi -
8.58
8.35
2
FIoufe 2. Effect of micelle concentration on methylene selectivity: (e) alkylbenzenes; (m) alkylphenones.
[MI + Define the methylene selectivity of binding to micelle as ~(CHZ),, = Kmw,z/Kmw,l and the corresponding value for partitioning into stationary phase as a(CHz)sw= Psw,z/P,,,l, thus
Table VI. Effect of Micelle Concentration on a(CH,),, Equation 11 plot
(slope f CI”) x lo2
l/[SDS] l/[SDS] l/[SDS] l/[SDS]
l/[SDS] a2 vs l/[SDS] a3 vs l/[SDS] ( ~ vs g l/[SDS]
vs a2 vs ( ~ vs 3 CY^ vs ( ~ 1
(9) For a pair of compounds with large K,, values and/or at large micelle concentrations, the second term of eq 9 can be neglected and thus the chromatographic selectivity is simply a difference between the logarithms of micelle and stationary phase selectivities, i.e. log a(CH2) = log (u(CH~),,- log 4CHJrnw (10) In such cases the group selectivity becomes independent of micelle concentration. On the other hand, at larger concentrations, solute-micelle binding constant, Kmw,might change due to variations of certain micelle properties such as cmc and aggregation number. This would lead to small changes in selectivity with concentration even under the conditions that eq 10 is applicable. Equation 10 indicates that, the net free energy of transfer of a methylene group from mobile phase to the stationary phase is a difference in the free energy of transfer from bulk solvent (e.g. water) to stationary and from bulk solvent to micelle. Micelles and grafted alkyl-bonded phases have a similar molecular organization and have been termed “interphases”that is, interfacial phases of chain molecules (29). Such systems have a large surface/volume ratio and their property varies with depth from the interface (29). In addition, a constant amount of free surfactant which is adsorbed on the alkyl-bonded stationary phase makes the environments in the two systems even more similar. The “micelle-like” appearance of the stationary phase has also been discussed by other workers (30). Therefore, it is not surprising that a methylene group does not find much difference between its environment in surfactant aggregates and that in the alkylbonded phase. This situation is similar to that in an organic-rich, hydro-organic RPLC where the enrichment of an alkyl-bonded phase by organic solvent makes the stationary phase environment more like that of the mobile phase. There is, however, a large difference between the solvent strengths of these two systems, that is, micellar eluents are generally much weaker (vide infra). Figure 2 shows the plots of a(CH,) vs SDS concentration for both alkylbenzenes and alkylphenones. The changes in selectivity is larger a t lower micelle concentrations and it approaches a plateau.
CY^ vs
ag
vs l/[CTAB]
al vs l/[CTAB]
l/[CTAB] 0 3 vs l/[CTAB] a4 vs l/[CTAB] ( ~ vs 2
intercept f CI
r2
n
A. Alkylbenzenes, SDS 1.89 (k0.49) 1.16 (f0.02) 4.51 (10.42) 1.31 (f0.05) 9.42 (f1.5) 1.36 (f0.08) 13.9 (f2.5) 1.40 (f0.14)
0.987 0.990 0.985 0.980
9 10 6 6
B. Alkylphenones, SDS 1.69 (f0.16) 1.32 (f0.02) 4.18 (f0.26) 1.79 (f0.03) 7.54 (f0.44) 2.39 (f0.05) 12.1 (f0.91) 3.12 ( f O . l )
0.990 0.995 0.996 0.995
C. Alkylbenzenes, CTAB 2.92 (f0.75) 1.83 (A0.07)
0.980
D. Alkylphenones, CTAB 2.34 (f0.4) 1.30 (f0.04) 5.05 (f0.7) 1.59 (f0.07) 7.72 (fl.1) 1.94 ( A O . l l ) 10.86 (11.32) 2.29 (f0.13)
0.993 0.994 0.994 0.995
4
’CI, 95% confidence interval. A linear correlation exists between hydrophobic selectivity and the inverse of micelle concentration if KmW,,[M]>> 1,then eq 8 can be written for a(CHz), as
or
Therefore a plot of ( Y ( C Hvs ~ )1/[M] ~ should be linear (Figure 3). Table VI lists the slopes, intercepts, the 95% confidence intervals, and the correlation coefficients of the linear fit of the plots a(CHZInvs 1/[M] for the homologous compounds. The excellent correlations show that eq 11is applicable. As can be seen from the slopes listed in Table VI, the variations of a(CH2) with micelle concentration are small; however, the changes are more pronounced for multiple-methylene selectivity, a(CH2),,, as the slope increases with carbon number in compound 2. On the other hand, the variations of a(CH2),, of benzenes with CTAB mobile phases are almost nonexistent which shows eq 10 is applicable for these cases, while these changes for phenones are more noticeable. The changes of a(CHdnwith CTAB concentration are smaller than those with SDS mobile phases due to the more hydrophobic environment
ANALYTICAL CHEMISTRY, VOL. 80, NO. 9, MAY 1, 1988
883
Table VII. Methylene Selectivity for SDS and CTAB Hybrid Mobile Phases 4 C H J = k ',t+i/k ',, compound
3%
0%
0.10 M SDS in aqueous 2-pronpanol; % PrOH 6% 8% 10%
15%
20%
Alkylbenzenes n-pentylbenzene 1.20
1.19
1.19
1.19
1.21
1.18
1.18
1.27
1.25
1.23
1.23
1.20
1.20
1.19
1.31
1.29
1.29
1.30
1.28
1.26
1.26
1.36
1.35
1.34
1.33
1.34
1.33
1.31
1.55
1.51
1.50
1.48
1.47
1.44
1.43
1.29
1.27
1.26
1.26
1.26
1.24
1.24
n-butylbenzene n-propylbenzene ethylbenzene toluenea benzene a(CH2)avb
Alkylphenones hexaphenone 1.37
1.32
1.34
1.32
1.32
1.30
1.30
1.41
1.41
1.39
1.40
1.40
1.39
1.41
1.49
1.48
1.49
1.49
1.49
1.51
1.48
1.50
1.50
1.56
1.57
1.59
1.60
1.61
1.44 0.15
1.44 0.17
1.45 0.19
1.45 0.19
1.45 0.21
1.45 0.21
1.45 0.21
valerophenone butyrophenone propiophenone acetophenone a(CHZ)avb Aa(CH2) a?
dCHA 0%
k'n+i/k'n
0.10 M CIBTABin aqueous propanol, % PrOH 3% 6% 10% 15%
20%
Alkylbenzenes n-hexylbenzene 1.12
1.10
1.09
1.11
1.11
1.11
1.13
1.12
1.12
1.12
1.11
1.12
1.16
1.13
1.13
1.14
1.14
1.14
1.19
1.17
1.19
1.19
1.19
1.18
1.24
1.24
1.23
1.24
1.24
1.24
1.33
1.32
1.32
1.32
1.33
1.32
1.17
1.17
1.15
1.16
1.16
1.16
n-pentylbenzene n-butylbenzene n-propylbenzene ethylbenzene to1uene benzene a(CHZ)av
Alkylphenones hexaphenone
1.25
1.25
1.24
1.23
1.23
1.22
1.30
1.30
1.30
1.29
1.31
1.31
1.37
1.39
1.38
1.40
1.40
1.43
1.54
1.56
1.57
1.58
1.61
1.59
1.37 0.20
1.37 0.20
1.37 0.22
1.38 0.22
1.39 0.23
1.39 0.23
valerophenone butyrophenone propiophenone acetophenone a(CHZ)av Aa(CH2)av
a Methyl selectivity; a(CHJ = k $,,~wne/k Lwnv'Average value for a(CHZ)between successive homologues excluding toluene/benzene, i.e., (Y(CHB).e AdCH2) = a(CH2)phenonw- (Y(CH2)bwnew
of the former. In general, the overall effect of surfactant type (i.e. head group charge and chain length) on methylene selectivity is small (Table 11). Hybrid vs Hydro-Organic. The addition of small percentages of propanol to micellar mobile phases was recommended by Dorsey et al. to enhance chromatographic efficiency (31). There has been some concern, however, that adding propanol to micelles might reduce the role of micelles
in a separation process, that is, sacrificing the chromatographic selectivity (32). In a previous paper, we have indicated that the retention behavior of the homologous series with hybrid mobile phases (i.e. micelles in hydro-organic solvents) is the same as that with micelles in a purely aqueous solvent (1). In fact i t was demonstrated that the presence of micelles in a water-propanol solvent can have a profound effect on retention behavior of the series as a function of propanol con-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
884
Table VIII. Stationary Phase ,Effect, Methylene Selectivity Ratio, a(CH&.,8/a(CHz)c.s
mobile phase 8
alkylbenzenes
alkylphenones
ac-18lffc.8
aC-18/@c.8
Mice11ar
l o : 0 54
i
0.50 M SDS 0.40 M SDS 0.30 M SDS 0.20 M SDS 0.10 M SDS
1.003 1.001 0.995 0.994 1.006
0.995 0.995 0.991 0.996 0.938
av f SD
0.999 f 0.005
0.983 f 0.025
Hydro-Organic b
e: .---
.
o-., 6
,
.
.
,
.
. 8
--
, 13
17
1
/[so$/
Flgure 3. Multiple methylene group selectivlty vs l/[SDS]: (a) alkylbenzenes; (b) alkylphenones. a(CH2), *; a(CH2),, 0;a(CH,J3, A; a(CH2)4, X .
centration in mobile phase. The methylene selectivity values for hybrid mobile phase of SDS and CTAB micelles in water-propanol solvents are listed in Table VII. As shown, despite the addition of up to 20% of 2-propanol to the micellar mobile phases a (CH,) remains almost unchanged (Table VII). Note that for the water-propanol mobile phases, the methylene selectivity changes by almost 0.30 unit for an increase in propanol concentration from 35% to 50% (Table 111). A similar situation exists for multiple-methylene groups selectivity. As shown in Table IV, an increase of 15% in propanol for hydroorganic eluents would cause a large change (by several units) in, e.g., a(CH2)&The addition of up to 20% propanol would reduce a(CH2)4for benzenes in SDS micelles by only a few tenths of a unit and has virtually no effect for CTAB micellar mobile phases. Interestingly, the a(CH2), for alkylphenones is increased slightly upon the addition of propanol to micelles (Table IV). With the hybrid eluents, solute binding constants to micelles and their partitioning into stationary phase both decrease as a result of 2-propanol addition and therefore the eluting power of the mobile phase increases (1). Apparently, both the alkyl-bonded phase and the micelles are solvated with 2-propanol to a similar (or the same) extent so that methylene groups still find the same difference in their microenvironment “polarities” upon transferring to stationary phase. This is quite remarkable that in a reversed-phase LC system a dramatic change in the eluents strength has virtually no effect on the hydrophobic selectivity. One of the noteworthy disadvantages of micellar eluents is their weak solvent strengths (6). These results indicate, however, that the strength of micellar mobile phases may be enhanced by adding propanol without sacrificing the nonspecific chromatographic selectivity. Effect of Stationary Phase. There has been a disagreement about the effect of stationary phase chain length on methylene selectivity (12, 13). The results in this work indicate that for hydro-organic mobile phases, the a(CH2) values on a (2-18 stationary phase are consistently larger than those on a C-8 column. Other workers have also reported a
35% 2-propanol 40% 2-propanol 45% 2-propanol 50% 2-propanol 55% 2-propanol
1.077 1.069 1.056 1.069 1.056
1.099 1.086 1.094 1.080 1.101
av f SD
1.056 f 0.009
1.092 f 0.009
60% methanol
1.105 1.097 1.091 1.101
1.114 1.100 1.068 1.086
1.099 f 0.006
1.092 f 0.002
1.078 1.046 1.096 1.094
1.105 1.098 1.089 1.053
1.079 f 0.0230
1.086 f
methanol methanol methanol av f SD 65% 70% 75%
50%
CHBCN
60% CH&N 70%
CH&N
80% CHSCN av f SD
0.023
greater methylene selectivity for ODS columns with hydroorganic solvents ( 4 , 5 , 1 2 ) . Melander and Horvath, however, have concluded that the difference in a(CH2)on the C-18 and C-8 columns is statistically insignificant and that the a(CH2) values were sufficiently similar to consider identical retention energetics for homologous compounds on the two alkyl chain lengths (13). The ratios of methylene selectivities on a C-18 over that on a C-8 column are listed in Table VIII. As shown in this table, for SDS micellar mobile phases this ratio is about 1.00, which indicates identical a(CH,) values for the two stationary phases. The ratio is very similar for the three organic solvent systems in light of the fact that the alkylbonded phases enriched by different organic solvents have different “solvent strength” characteristics ( 4 , 5). For alkylbenzenes at different SDS micellar concentrations the log k’ (C-18) is linearly related to log k’(C-8) as
log k’(C-18) = 1.02 log k’(C-8) + 0.058
r2 = 0.995, n = 30 For hydro-organic mobile phases at five different concentrations using methanol, acetonitrile, and 2-propanol as the organic modifiers, the corresponding relationship is log k’(C-18) = 1.11log k’(C-8)
+ 0.141
r2 = 0.980, n = 28 The constancy of methylene selectivity on the C-8 and C-18 columns with micelles might be the result of the adsorption of a constant amount of free surfactant on the two stationary phases. This would further reduce any difference in characteristics, if any, that might affect discrimination capability of the two stationary phases for a methylene group. Elutropic Solvent S t r e n g t h Scale. Perhaps an overall weaker solvent strength for micellar mobile phases as compared to hydro-organic solvents in RPLC is one of the very first and notable observations made by many workers. This is based on comparing the overall retention data between the
ANALYTICAL CHEMISTRY, VOL. 60, NO.
9, MAY 1, 1988 885
Table IX. Relative Eluent Strengths Measured from Equations 12 and 13; Effect of Different Reference SolutesaVb co =
based on strongest eluent
weakest eluent
-
-
benzene
weakest
hexaphenone
1.90% MeOH 2. 80% ACN 3. 70% ACN 4.75% MeOH 5.55% PrOH 6.70% MeOH 7. 60% ACN 8.50% PrOH 9.65% MeOH 10. 45% PrOH 11. 60% MeOH 12. 50% ACN 13. 40% PrOH 14. 0.5 M SDS 15. 0.4 M SDS 16. 35% PrOH 17. 0.10 M CTAB + 20% PrOH 18. 0.10 M SDS + 20% PrOH 19. 0.10 M CTAB + 15% PrOH 20. 0.30 M SDS 21. 0.18 M CTAB 22.0.10 M SDS + 15% PrOH 23.0.10 M CTAB + 10% PrOH 24. 0.15 M CTAB 25. 0.10 M CTAB + 6% PrOH 26. 0.10 M SDS + 10% PrOH 27. 0.20 M SDS 28.0.10 M SDS + 8% PrOH 29. 0.10 M CTAB + 3% PrOH 30.0.10 M SDS + 6% PrOH 31. 0.10 M CTAB 32. 0.15 M SDS 33. 0.10 M SDS + 3% PrOH 34. 0.07 M CTAB 35. 0.10 M SDS
based on strongest
(log k’ref - log k’J/V (eq 12Ia
-
1.90% MeOH 2. 80% ACN
3. 55% PrOH 4. 70% ACN 5.75% MeOH 6. 50% PrOH 7. 60% ACN 8.45% PrOH 9.70% MeOH 10. 65% MeOH 11.40% PrOH 12. 50% ACN 13. 0.10 M CTAB + 20% PrOH 14. 0.50 M SDS 15. 0.10 M SDS + 20% PrOH 16. 0.10 M CTAB + 15% PrOH 17. 60% MeOH 18. 0.40 M SDS 19. 35% PrOH 20. 0.18 M CTAB 21. 0.10 M SDS + 15% PrOH 22. 0.10 M CTAB + 10% PrOH 23. 0.15 M CTAB 24. 0.30 M SDS 25.0.10 M CTAB + 6% PrOH 26. 0.10 M SDS + 10% PrOH 27. 0.10 M SDS + 8% PrOH 28. 0.10 M CTAB + 3% PrOH 29.0.10 M SDS + 6% PrOH 30. 0.10 M CTAB 31. 0.20 M SDS 32.0.10 M SDS + 3% PrOH 33.0.072 M CTAB 34. 0.10 M SDS 35. 0.08 M SDS
a(CH2) benzenes
a(CH2) phenones
1. 0.50 M SDS 2. 0.40 M SDS
1.90% MeOH 2. 80% ACN 3.55% PrOH 4.50% PrOH 5. 0.18 M CTAB 6. 70% ACN 7. 0.15 M CTAB 8. 0.50 M CTAB 9. 0.40 M SDS 10. 0.10 M CTAB 11. 45% PrOH 12. 0.30 M SDS 13. 75% MeOH 14. 0.20 M SDS 15. 0.07 M CTAB 16. 60% ACN 17. 40% PrOH 18. 70% MeOH 19. 50% ACN 20. 65% MeOH 21. 35% PrOH 22. 60% MeOH
3. 0.18 M CTAB 4. 0.16 M CTAB 5.90% MeOH 6. 0.10 M CTAB 7. 0.07 M CTAB 8. 0.30 M SDS 9. 0.20 M SDS 10.80% ACN 11. 55.76 PrOH 12. 50% PrOH 13. 70% ACN 14.45% PrOH 15. 75% MeOH 16.60% ACN 17.40% PrOH 18.70% MeOH 19. 50% ACN 20. 65% MeOH 21.35% PrOH 22. 60% MeOH
-
“Reference mobile phase 90% MeOH; cod = 0.0. bReference mobile phase: 100% water; a(CH2) = 4.0 for eq 13: MeOH, methanol; ACN, acetonitrile; PrOH, 2-propanol. two systems. There is a need to develop a quantitative scale for a better evaluation of the solvent strengths of different RPLC mobile phases. There exist at least three solvent strength scales for hydroorganic mobile phases (IO,33,341. Althoueh these scales are semiauantitative. they Drovide a generalpicture of the strengths of different organic solvents for RPLC. The question is whether these scales can do the I
__
same for micellar mobile phases. The elutropic strength scale, eo scale, has been used for hydro-organic solvents and is defined as log
k’(S1)
- log k ’ ( S 2 ) =
V(€O(Sz)
- +I))
(12)
where k‘(si)is the retention factor of a solute in the solvent si and V is its mdlecular volume. This is a similar definition
886
ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
to that of Snyder’s treatment of adsorption on polar surfaces. With a solvent (e.g. Si) chosen as the reference and eo = 0, eq 12 can be used for comparing solvent strengths. It has been reported that for hydrmrganic RP systems, eq 12 is not only solute dependent (Le. is valid only for nonpolar solutes) but it is also influenced by stationary phase (IO). The problem of solute dependency is even more pronounced for micellar eluents. As mentioned earlier, different solutes which even belong to the same homologous series find different microenvironment “polarities” in a given micellar mobile phase. It becomes even more problematic once this equation is applied to develop a single elutropic scale for various surfactant systems with different chain lengths and head groups as well as for hydro-organic solvents. This discrepancy is shown in Table IX where different eluents are ranked based on their strengths by using eq 12, using benzene and hexaphenone as the reference solutes. In addition, the frequently observed elution order reversal upon a change in micelle concentration demonstrates the inadequacy of developing an elutropic scale based on the retention of one solute in an arbitrarily selected reference system (23,24). Colin et al. have later reported a similar equation based on methylene selectivity (IO). Thus eq 12 for a methylene group, with water as the reference solvent, can be expressed as log ~ ( C H Z )-Hlog ~ ~a(CHz)s, = v o t 2 O
(13)
Note that for hydro-organic solvents, methylene selectivity is independent of the solute type and varies slightly with the carbon chain length of the alkyl-bonded phase. Therefore, it has been recognized that a(CH2) is a “very convenient” means for measuring the solvent strength which depends only on the mobile phase composition. However as mentioned earlier, eo scale is at best a semiquantitative scale. It has been acknowledged for example, that most often the use of isoelutropic (i.e. same solvent strength) binary solvents does not yield the same retention factors due to the solvation effects of a specific organic solvent (IO). In light of the results presented above several fundamental problems can be recognized in applying the eq 13 to quantitate micellar solvent strength. Firstly, the methylene selectivity with micellar mobile phaaes is a function of the homologous series type. This would then lead to dependency of elutropic scales on the class of homologous series. The analyses of to scales based on a(CHz) values of benzenes and phenones indicate the seriousness of discrepancies. As shown in Table IX, an eo scale based on methylene selectivity of alkylbenzenes predicts that 0.50 M SDS, 0.40M SDS, and 0.18 M CTAB are the three strongest solvents among all hydro-organic and micellar solvents and a binary solvent of 90% methanol in water is ranked fifth after these micellar eluents. On the other hand, a similar scale based on a(CHz) of alkylphenones shows that the three mentioned micellar mobile phases are ranked eighth, nineth, and fifth, respectively, after hydro-organics and the 90% methanol in water is the strongest solvent according to this scale. This scale also fails even if it is limited only to comparing micellar eluents. For to scale based on selectivity of benzenes, 0.5 M SDS and 0.40 M SDS are stronger than 0.18 M CTAB, while using eo based on phenones results in a scale that predicts 0.50 M SDS and 0.40 M SDS are even weaker than 0.153 M CTAB. Secondly, the overall selectivity values are small and the range of their variation with micellar concentration is limited. For example, the methylene selectivity range for 0.05-0.50 M SDS almost corresponds to that of 6540% methanol in water eluents. The overall retention factors of benzenes and phenones with the SDS eluents are much greater than those of the methanol/water. In addition, the retention factors of “isoelutropic” (i.e. equal to or a(CH,)) eluent systems such
as 0.05 M SDS and 65% CHSOHin H 2 0 or 0.20 M SDS and 80% CH&N/H20, differ by as much as a factor of 10. These discrepancies arise from the fact that the methylene selectivities with micellar mobile phases do not quantitatively (or even semiquantitatively;like hydrmrganics) reflect the actual solvent strength of mobile phase, although the general notion of the reciprocal relationship between selectivity and eluent strength is observed. Lastly, and perhaps more importantly, the unchanging selectivity values for the hybrid mobile phases upon the addition of up to 20% propanol-which dramatically increases the eluents strength-totally discredits the validity of such a scale for these systems. CONCLUSION The resultapreaented in this work and in a previous paper c o n f i i the general belief that in RPLC, solute-mobile phase interactions play an important role in controlling retention and selectivity. Indeed, the uncommon retention behavior and separational selectivity in micellar eluents as compared to that in hydro-organic mobile phases are due to different nature of solute-mobile phase interactions. The significance of different interaction mechanisms in causing various behavior is even more remarkable in light of the fact that the driving force for retention and methylene group selectivity is very similar for these RP systems. Although the role of stationary phase cannot be underestimated, the similarities between hybrid and micellar eluents in the one hand and the differences between hydrmrganic and hybrid mobile phases on the other hand point out that the stationary phase is not the main source of different behavior. The general assumption that micelles play the same modifier role as organic solvent is not accurate and does not reflect the differences between the two R P systems. ACKNOWLEDGMENT The author is grateful for the computer curve fittings and calculations by Ms. Shahrzad Afshinpour and for the typing skills of Ms. Francis Caldwell. Registry No. SDS,151-21-3;CISTAB,57-09-0; n-hexylbenzene, 1077-16-3;n-pentylbenzene, 538-68-1; n-butylbenzene, 104-51-8; n-propylbenzene, 103-65-1; ethylbenzene, 100-41-4; toluene, 108-88-3;benzene, 71-43-2;n-hexaphenone, 1671-75-6;n-valerophenone, 1009-149;n-butyrophenone, 495-40-9; n-propiophenone, 93-55-0; acetophenone, 98-86-2; propanol, 71-23-8; methanol, 67-56-1; acetonitrile, 75-05-8. LITERATURE CITED (1) Khaledl, M. 0.; Peuler. E.; NgehNgwalnbi, J. Anal. Chem. 1987, 59, 2738. (2) McIntlre, 0. L.; Chappardi, D. M.; Casselberr, R. L.; Blount, H. N. J . phvs. Chem. 1982, 86, 2632. (3) Blrdl, K. S.;Ben-Naim, A. J . Chem. Soc., Faraday Trans. 7 1981, 77, 741. (4) Yonker, C. R.; Zwier, T. A.; Burke, M. F. J. Chromafogr. 1982, 247, 257. (5) Yonker, C. R.; Zwier, T. A.; Burke, M. F. J . Chromatogr. 1982, 247. 269. (6) Dorsey, J. G.; Khaledi, M. G.; Landy, S.L.; Lin, J.-L. J . Chromafogr. 1084, 376, 183. (7) Berthod, A.; Girard, 1.; Gonnet, C. Anal. Chem. 1988, 58, 1356. ( 8 ) Colin, H.; Guiochon. G. J. Chromatogr. Scl. 1880, 78, 54. (9) Grushka, E.; Colin. H.; Guiochon. 0. J . Chromafogr. 1982, 248, 325. (10) Colin, H.; Gulochon, 0.; Yun, 2.; Dlez-Masa, J. C.; Jandera, P. J . Chromatogr. Scl. 1983, 27, 179. (11) Colin, H.; Krstulovic, A. M.; Gonnord, M. F.; Guiochon, 0.; Yun, 2.; Jandera, P. Chrometographla 1983, 17, 9. (12) Krstulovic, A. M.; Colin, H.; Tchapla, A.; Guiochon, G. Chromatographk 1983, 77, 228. (13) Melander, W. R.; Horvath, Cs. Chromafographla 1982, 15, 86. (14) Jandera, P.; CoUn. H.; Qulochon, 0. Anal. Chem. 1982, 54, 435. (15) Shoenmakers, P. J.; Bllllet, H. A. H.; DeGalan, L. J . Chromatogr. 1989, 282, 107. (16) Jandera, P. J. Chromafogr. 1984, 374, 13. (17) Mukerjee, P.; Cardinal, J. R. J. phvs. Chem. 1978, 82, 1620. (18) Mukerjee, P. I n Sdunbn Chemktty of Surfactants; Mittai, K. L., Ed.; Plenum: New York, 1978; Vol. 1, pp 153-174. (19) Wlshnla, A. J. Phys. Chem. 1983. 6 7 , 2079. (20) Pramauro, E.; Pellzzettl, E. Anal. Chlm. Acta 1981, 726, 253.
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(21) Birdl, K. S. In Mlcelllzatlon, Solublllzatlon and Mlcroemulslons; M b i , K. L., Ed.; Plenum: New York, 1977; Voi. 1 p 151. (22) Bunton, C. A.; Sepuiveda, L. J . Phys. Chem. 1979, 83, 680. (23) Yarmchuck, P.; Welnberger, R.; Hlrsch, R. F.; Cline Love, L. J. Anal. Chem. 1982, 5+, 2233. (24) Armstrong, D. W.; Stifle, G. Y. AnalChem. 1983, 55, 2317. (25) Khaiedi, M. 0.;Breyer, E. D., unpublished work. (26) Karger, B. L.; a n t , J. R.; Hartkoff, A,; Welner, P. J . Chromatogr. 1976, 728, 65. (27) Armstrong. D. W. Sep. PurM. Methods 1985, 14, 213. (28) Arunyanart, M.; Cline Love, L. J. Anal. Chem. 1984, 56, 1557. (29) Dill, K. A. J . Phys. Chem. 1987, 9 1 , 1980. (30) Armstrong, D. W.; Ward, T. J.; Berthod, A. Anal. Chem. 1986, 58, 579. (31) Dorsey. J. G.; De Echegarary, M. T.; Landy, J. S. Anal Chem. 1083, 55, 924. (32) Yarmchuk, P.; Weinberger, R.; Hlrsch, R. F.; Cline Love, L. J. J . Cbromafogr. 1984, 283, 47.
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RECEIVED for review August 18, 1987. Accepted December 29, 1987. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. The author also gratefully acknowledges the financial support by the LSU System Biotechnology Institute and Cancer Association of Greater New Orleans. This work was presented at the 194th National Meeting of the American Chemical Society, New Orleans, LA, Aug 30-Sept 4,1987.
Determination of Polycyclic Aromatic Hydrocarbons in a Coal Tar Standard Reference Material Stephen A. Wise,* Bruce A. Benner, Gary D. Byrd, Stephen N. Cheder, Richard E. Rebbert, and Michele M. Schantz Organic Analytical Research Division, Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, Maryland 20899
A new Standard Reference Material (SRM) has been certified for use In the determination of polycycllc aromatic hydrocarbons (PAH). SRM 1597 Is a natural, complex, combustlon-related mMure of PAH Isolated from coal tar. Concentratlons of lndlvldual PAH were determined by using capillary column gas chromatography and reversed-phase llquld chromatography with fluorescence detectlon. Certified concentrations, based on the agreement of results from both gas chromatography (GC) and llquld chromatography (LC), are reported for 12 PAH. Noncertifled concentrations are reported for an addltlonal 18 compounds based on measurements by only one analytkal technique. Dmerences between the GC and LC measurements ranged from a low of less than 1% for phenanthrene, fluoranthene, and pyrene to 4-7 % for naphthalene, benz[a ]anthracene, benrqa Ipyrene, perylene, and Indeno[1,2,tcd]pyrene. Approximately 65 compounds were Identifled In SRM 1597 based on molecular welght and/or GC retention information. Thls SRM Is Intended for use In the evaluation and validation of analytical methods for the determlnatlon of PAH In complex mixtures and In the evaluation of elflclency and selectMty of Chromatographic systems.
Since 1933 (1) when several polycyclic aromatic hydrocarbons (PAH) were isolated from coal tar and found to be highly carcinogenic, PAH have been one of the most widely measured groups of environmental pollutants. A number of analytical techniques, including gas chromatography and liquid chromatography, have been used for the separation, identification, and quantification of PAH in a variety of complex environmental mixtures (2-4). To assist in validating the accuracy of PAH measurements, several Standard Reference Materials (SRM’s) have been issued by the National Bureau of Standards since 1980 for use in the determination of these compounds (5-12). These SRMs range in analytical difficulty from a simple calibration solution of the 16 priority pollutant PAH (SRM 1647) (5) to several complex, natural matrix materials such as shale oil (SRM 1580) (6, IO), crude
oil (SRM 1582) (7,111, air particulate material (SRM 1649) (8,12,13),and diesel particulate matter (SRM 1650) (9). A new SRM, which is intermediate in complexity when compared to existing SRMs, has been prepared and certified for use in the determination of PAH. SRM 1597, “Complex Mixture of Polycyclic Aromatic Hydrocarbons from Coal Tar”, consists of a natural mixture of PAH isolated from a crude coke oven tar and dissolved in toluene. This complex combustion-related PAH mixture is suitable for direct analysis (i.e., without sample cleanup or concentration) using such techniques as liquid chromatography (LC), gas chromatography (GC), or gas chromatography/mass spectrometry (GC/MS). Coal tar samples have long been analyzed for the determination of PAH (14-18). In 1979 Borwitzky and Schomburg (15) reported an extensive qualitative characterization of coal tar by GC/MS and suggested the use of coal tar as an inexpensive test mixture for the chromatographic determination of PAH in a nonartificial matrix. In recent years Lee and co-workers (19-25) and others (26)have used the PAH fraction from a coal tar sample as a test mixture for the evaluation of gas chromatographic separations of PAH and particularly for the evaluation of new stationary phases and columns. Vassilaros et al. (27)used coal tar PAH for the evaluation of a GC retention index system for PAH identification. The majority of the previous reports on the analysis of coal tar have been primarily qualitative or semiquantitative in nature (14-18). SRM 1597 has been characterized both qualitatively and quantitatively for the determination of PAH. Concentrations of individual PAH were determined by using capillary GC (on both nonpolar and liquid crystal stationary phases) and reversed-phase LC with fluorescence detection. Identifications were made based on comparison of GC retention data with authentic standards and molecular weight information obtained from GC/MS analysis. Certified concentrations are reported for 12 PAH based on agreement of both the GC and LC measurements. Concentrations for an additional 18 compounds are reported as informational values. In this paper the detailed analyses of SRM 1597 are described, which were performed by GC, LC, and GC/MS as
This artlcle not subJectto U.S. Copyright. Published 1988 by the American Chemical Society
