Anal. Chem. 1991, 63, 1524-1529
1524
Parameter for Predicting Retention in Planar Chromatography David Nurok,* Lisa A. Julian, and Christopher E. Uhegbu Department of Chemistry, Indiana University-Purdue University at Indianapolis, 1125 East 38th Street, Indianapolis, Indiana 46205-2810
x,
A parameter, deslgnated for planar chromatography In either the normal- or reversed-phase mode applles to each of a serlos of weak solvents used as a blnary mlxture wlth a glven strong solvent or, atternatlvely, to a serles of strong solvents used wlth a glven weak solvent. A strong h e a r relatlonshlp between #her the average R, of solutes (sterdds or esters of dansyl amino aclds) or the R, of lndlvklual solutes and the value of for each of the serles of solvents Is found for many of the systems studled. Thls relationship can be used to predlct the retention of a solute when a mobile phase of known value Is used. The relatlonshlp between and establlshed solvent strength parameters Is also dlscussed.
x
x
x
Solvent strength is important in all forms of liquid chromatography, and parameters such as P' (1)and to (2) have been introduced to quantify it. Other parameters such as the Hildebrand parameter, 6 ( 3 ) , the solvatochromic polarity parameter, E ~ ( 3 0(4), ) and the solvatochromic dipolaritypolarizability parameter, A* (5),originally introduced for other purposes, have been applied to chromatography usually by a multiparameter approach (6-8)but also by a single-parameter approach (9). Both to (10) and ET(30) (9) have been used to accurately predict retention as a function of concentration in systems with defined components. Reference 10 includes a summary of reports on the use of toto predict retention, and ref 9 includes a summary of the use of solvatochromic parameters in chromatography. The cluster center is an empirical parameter for planar chromatography that has been briefly discussed previously with respect to the separation of either steroids or a mixture of azo dyes by normal-phase thin layer chromatography (TLC) (11, 12). In the initial report, the value of the cluster center was shown to decrease uniformly with increasing eluotropic strength for each of 14 solvent systems. This parameter differs from those noted above in that it applies only to binary mixtures of solvents in which one component is held constant, and it is defined with respect to the solutes of a given class. In spite of these constraints, it appears to be a useful parameter because, under defined conditions, it allows R, to be predicted in untried solvents. The current report discusses this use of the parameter and compares cluster center values of solvents to the corresponding values for several of the established parameters. EXPERIMENTAL SECTION The steroids and dansyl amino acids were purchased from the Sigma Chemical Company (St. Louis, MO), and the solvents were purchased from the Aldrich Chemical Company (Milwaukee, WI). The reagents for preparing the esters of dansyl amino acids were a gift from Pierce (Rockford, IL). The esters were prepared according to the procedures in the 1989 Pierce Handbook, which are based on the following references: methyl esters (13), p nitrobenzyl esters (14),phenacyl esters (15),and p-bromophenacyl esters (16). A single spot was obtained for all derivatives,except serine and threonine, which each yielded two spots. The R, of the higher spot was measured for these two compounds. TLC plates were a gift from Whatman Inc. (Clifton, NJ). These were K5 and K6 silica gel plates, Catalog Nos. 4850-820 and
486C-820, respectively,and KCle reversed-phase plates, Catalog No. 4801-800. The K5 and K6 plates were heated at 90 "C for 30 min and then maintained at a relative humidity of 60% until immediately before use. All normal-phase chromatography was performed on K5 plates, apart from one series of experiments that compared the behavior of the two types of plates. The KCl8plates were used without conditioning. The mobile phases for the latter plates were prepared with 0.5 M aqueous sodium chloride in order to protect the layer. Thin layer chromatography was performed in a twin-trough chamber (Camag Scientific Inc., Wilmington, NC) using plates cut in 10- x 10-cm sections, with a 15-min conditioning period after adding solvent. Overpressured layer chromatography was performed in a Chrompres 25 (Labor MIM, Budapest, Hungary) using 20- X 20-cm plates.
CHROMATOGRAPHIC RELATIONSHIPS Soczewinski has reported ( I 7), in a somewhat different format, the following linear relationship:
X,+ b (1) where k is the solute capacity factor, X,is the mole fraction log k = a log
of the strong solvent in a binary mixture of a strong and weak solvent, and a and b are experimentally determined constants for each solute. This relationship was originally reported for normal-phase chromatography on silica gel but has been found to be applicable also to reversed-phase chromatography (18). The relationship between R, and capacity factor is given by
The difference between two R, values, AR,, is
m, = R , l - R,2
(3)
and may be defined in terms of the corresponding capacity factors k, and k2:
k2 - k l
= (1
+ k,)(l + k,)
(4)
A plot of AR, for a pair of solutes versus solvent composition, expressed as the mole fraction of the strong solvent, may be constructed by using eqs 1 and 4 for those systems where the empirical constants, a and b, are known. Figure 1shows plots for each of the 10 solute pairs in a mixture of 5 dansyl amino acids, as the p-nitrobenzyl esters, separated on a silica gel plate using ethyl acetate/cyclohexane as the mobile phase. The maximum of each plot is referred to as (AR,), and the mole fraction a t which it occurs is designated (AR,)- values are distributed over a range of mole fractions as illustrated in Figure 2, which shows a plot of (AI$), versus mole fraction of strong solvent for the same system discussed above but with data for the p-nitrobenzyl ester of 15 dansyl amino acids. The plot includes a point for each of the 105 solute pairs in the system. There is substantial scatter about the central mole fraction, which is the cluster center, and it might be argued that this would preclude its use as a solvent strength parameter. Surprisingly this is not the case, and it
0003-2700/9 1 /0363-1524$02.50/0 C 199 1 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63,NO. 15, AUGUST 1, 1991
1525
0. 40
n
0. 32 0.24
E
a
0. 16
X 0. 08
0.00 0.0
0. 2
0.6
0. 4
0. e
1.0
xs Flgurr 1. Plot of AR,, for each solute pair, versus mole fraction of ethyl acetate, in the seperation of p-nitrobenzyl esters of ftve dansyi amino acids on a silica gel layer using ethyl acetate/cyciohexane as the mobile phase. 0.0
0.1
0.2
0.3
0.4
0.5
0.6
X 0.48
A
4
t
0.38
u 0.24
O.
I
0
X
.
0.8
0.7
*
0.60.0
0.0
0.2
0.6
0. 4
0.
e
1.0
xs Flgure 2. Plot of for each solute pair, versus mole fraction at which the letter occurs, in the separatlon of p-nitrobenql esters of 15 dansyi amino acids on a silica gel layer using ethyl acetate/cyciohexane as the moblie phase.
is shown below that this parameter allows an accurate estimation of Rf in certain systems. x is used to designate the cluster center in this report and is defined as
(5) where n is the number of solute pairs in a mixture. The x value is calculated over a mole fraction range of 0.01-1.0 for the strong solvent. Only the higher value of (At2f)- is used in the calculation for those solute pairs that undergo an inversion of retention with increasing concentration of the strong solvent. It should be noted that x is a parameter-or to be more precise, a statistic-in the statistical sense in the same way as the mean or variance are basic parameters of a population distribution (19). Thus, in contrast to solvent strength parameters such as P' and to, the value of x depends not only on the identity of the mobile phase but also on the identity of the mixture of solutes that is separated. The dependence of x on these identities allows prediction of Rf in different solvent systems as is discussed in this paper.
RESULTS AND DISCUSSION In the context of this paper, a series of weak solvents, each as a binary mixture with a given strong solvent, is referred to as a "constant strong solvent" series. A "constant weak solvent" series is defined in the converse manner. There is a relationship between average R, and x at a constant concentration of the strong solvent in either of these two series.
0.1
0.2
0.3 X
0.4
0.5
0.6
Flgwo 3. Plots of average Rt versus x for a mixture of 11 steroids separated on a silica gdi layer using tetrahydrofuran as the constant strong solvent with each of 13 weak solvents. The mole fractlon of tetrahydrofuran Is 0.1 (a), 0.3 (b), and 0.7 (c).
Table I. Steroids
set 1
set 2
A'-adrenosterone"
17a-aceto~yprogesterone~*~
5a-androstane-3,17-dioneo
cholesterol" corticosterone" cortisone" 1-dehydrotestosterone" diethylstilbestrol" epiandrosterone" ethisterone"
androsteroneo,* AlB-dehydropregnenoloneacetate oxime' Al8-dehydropregnenoloneoxime" ll-deoxycortisol".* 3-methyl-j3-estradiol"ether (3-methyl)ethynylestradiol0~* ether
17a-methyl-As-androstene-
j3-estradiolapb
3j3,17&diolU prednisone"
estrone"** hydrocortisone",* prednisolone" spironolactone",* testosterone"-*
5a-pregnane-3j3,20&diol"
pregnenolone" progesterone" stigmasterol"
5-androstene-3j3,17j3-diolb~
" Used with the acetonitrile, 2-butanone, butyl acetate, ethyl acetate, and methyl benzoate binary mixtures. *Used with the tetrahydrofuran binary mixtures. Not a member of either set 1 or set 2. Constant Strong Solvent Series. The relationship is very dependent on the concentration of the strong solvent as is illustrated in Figure 3, which shows a plot of the average Rf of a mixture of 11steroids listed in Table I versus the x values of 13 of the weak solvents listed in Table 11, each used as a binary mixture with tetrahydrofuran. The plot is curved at a low concentration of the strong solvent but is remarkably linear at intermediate concentrations, with the best correlation
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, AUGUST 1, 1991
Table 11. Weak Solvents carbon tetrachloride0* chlorobenzenea-' chl~robutane~*~*~J chloroform""@' ethylbenzeneOpb*d methylene toluenea-'
cyclohexane"*b*d-i decahydronaphthalenensbJ j decanea,b.eJh.i dodecane" hexanea,b,d-i isooctaneOFd-fh octanea*bJ*i Jvi
penMeo,dJ-i
"Used as a binary mixture with ethyl acetate for separating steroids. Used as a binary mixture with tetrahydrofuran for separating steroids. 'Used as a binary mixture with each of acetonitrile, 2-butanone, butyl acetate, and methyl benzoate for separating steroids. dUsed as a binary mixture with ethyl acetate for separating esters of dansyl amino acids. eValues of P' used are from ref 21. 'Values of to used are from ref 22. gValues of d used are from ref 23. hValues of A* used are from ref 5. 'Values of ET(30) are from ref 20.
(r = 0.995) occurring at a mole fraction of 0.3. The linearity is maintained at a high concentration of the strong solvent but is accompanied with considerably more scatter of the individual points. Such a high correlation-at an appropriate concentration-is found for several of the other series discussed in this paper. These correlations, and the corresponding mole fraction of the strong solvent, are listed in Table 111. The systems most studied were those with ethyl acetate as constant strong solvent and using as solutes, either the steroids in Table I, or esters of the dansyl amino acids in Table IV. The corresponding x values are listed in Table V, and it is seen, with reference to the steroids, that these values depend on the identity of the set of solutes being studied. Vide infra for a discussion of this topic. The plots of average Rf versus x for the systems in Table V exhibit a similar dependence on concentration to that noted above for the systems with tetrahydrofuran as the constant strong solvent. A point of interest is the very high correlation (Table 111)between average Rf and x,at an appropriate concentration, for each of the four series of esters of the dansyl amino acids. Four other constant strong solvent systems are listed in Table 111. The systems based on acetonitrile and 2-butanone exhibit a strong correlation between average Rf and x at an appropriate concentration of the strong solvent. These are stronger solvents, based on P'values, than either ethyl acetate
or tetrahydrofuran. The system based on butyl acetate exhibits a moderate correlation, while the system based on methyl benzoate exhibits virtually no correlation between Rf and x. These are the second-weakest and weakest, respectively, of the six "strong" solvents based on a comparison of Rf values (no literature P' values were available), of either steroids or p-nitrobenzyl esters of dansyl amino acids, using each solvent at a given concentration in a binary mixture with toluene. It thus appears important that the constant strong solvent be of sufficient strength, in order to obtain a good correlation between Rf and x. The data for the constant toluene series-i.e., a constant weak solvent series-were available from an independent study that was performed by overpressured layer chromatography (OPLC) for all the compounds apart from the methyl and phenacyl esters, which was performed by TLC. The solvents used in this series are listed in Table VI. There is a linear relationship between average Rf and the x value of the strong solvent, at an appropriate concentration, for both the steroids and the esters of the dansyl amino acids. The results for the constant toluene and the constant strong solvent series are not strictly comparable because of the different number of solvents studied. It is nevertheless noted that, where there is a difference, the r values for the above correlation are lower in the toluene series than in virtually all of the series with either acetonitrile, 2-butanone, ethyl acetate, or tetrahydrofuran as constant strong solvent. In the case of the steroids, the poorer correlation found between Rf and x in the constant toluene series compared to that found in the constant strong solvent series may be related to solvent demixing due to the use of OPLC and TLC, respectively, for the separations in the two series. Solvent demixing-due to the absence of preequilibration with solvent vapor-will have a more substantial effect on the former than on the latter technique. This interpretation should be treated with some caution because the methyl esters of the dansyl amino acids which were separated by TLC in the twin-trough chamber (i.e., with preequilibration) have a somewhat lower correlation between R, and x than the corresponding pbromophenacyl esters which were separated by OPLC. Aqueous Binaries. OPLC was used with bonded C18 plates and aqueous solutions of the organic modifiers listed in Table VI1 for the separation of the steroids and the p bromophenacyl and p-nitrobenzyl esters of the dansyl amino
Table 111. Correlation Between Average Rf a n d x const component in binary mixture
chromatographic mode
no. of binary mixtures
tetrahydrofuran ethyl acetate ethyl acetate ethyl acetate ethyl acetate ethyl acetate ethyl acetate ethyl acetate acetonitrile 2-butanone butyl acetate methyl benzoate toluene toluene toluene toluene toluene water water water
TLC TLC TLC TLC TLC TLC TLC TLC TLC TLC TLC TLC OPLC OPLC TLC OPLC TLC OPLC OPLC OPLC
13 12 15 12
" DAA:
dansvl amino acids.
10
10 10 10 5 5 5 5 9 6 6 6 6 6 5 5
no. 11 29 15 14 15 15 15 15 15 15 15 15 30 15 15 15 15 30 15 15
solutes class steroids steroids set 1 steroids set 2 steroids p-nitrobenzyl esters of DAA" methyl esters of DAA" p-bromophenacyl esters of DAA" phenacyl esters of DAA" set 1 steroids set 1 steroids set 1 steroids set 1 steroids steroids p-nitrobenzyl esters of DAA" methyl esters of DAA" p-bromophenacyl esters of DAA" phenacyl esters of DAA" steroids p-nitrobenzyl esters of DAA" p-bromophenacyl esters of DAA"
mole fraction strong solvent for best correlation correlation coeff 0.3 0.3 0.4 0.4 0.4 0.3 0.3 0.3 0.1 0.2 0.6
0.8 0.4 0.6 0.3 0.3 0.5 0.5 0.4
0.995 0.981 0.992 0.988 1.00 0.997 0.996 0.998 0.997 0.991 0.958
