Anal. Chem. 198g36 1 , 780-781
780
Hydrophobicity Parameter for Aqueous Size Exclusion Chromatography Gels Sir: Retention in size exclusion chromatography (SEC) is described by the chromatographic partition coefficient
K = (V, - Vo) (V, - VO)-'
(1)
where V , is the measured peak elution volume, V , the total column volume, and Vothe exclusion or void volume. In the absence of adsorption or partitioning, K may not exceed 1. However, for amphiphilic solutes, such as long-chain alcohols (1-3),phenols ( 4 ) , detergents ( 5 ) ,and dyes ( 6 ) ,values of K in excess of unity are commonly observed on Sephadex gels, revealing the partitioning of such solutes onto this stationary phase. Other aqueous SEC packings, such as Spheron ( 7 )or PW Gel (8),also display preferential binding of hydrophobic solutes. The molecular basis of such interactions was discussed thoroughly by Marsden (9) and more recently reviewed in detail by Janado (10). The hydrophobic properties of aqueous gel stationary phases may be employed advantageously for separations of biological macromolecules. Derivatization of relatively hydrophilic SEC gels with apolar reagents (11,12) leads to amphiphilic packings such as Phenyl-Superose (Pharmacia) or Butyl Toyopearl (Toyo Soda). Such columns offer a way to separate proteins under mild, nondenaturing conditions and exemplify the technique of "hydrophobic interaction chromatography" (13). Siliceous bonded phases may show similar effects. Phenylethanol, used as a hydrophobic probe by Pfannkoch et al. (14), eluted with values of K ranging from 1.4 to 10 on all such packings studied: SynChropak, TSK SW, LiChrosorb Diol, pBondage1, and Shodex OH pak. Indeed, the chromatographic partition coefficient on Cs- or CI6-stationary phases has been used to ascertain polarity indices for numerous solutes (15). These partition coefficients of course may also reflect hydrogen-bonding interactions with underivatized silanol groups or coulombic interactions with their dissociated (SiO-) forms. In this report we compare the hydrophobicity of several aqueous SEC gels based on cross-linked hydrophilic polymers: Sephadex (an epichlorohydrin cross-linked dextran); Superose (a cross-linked agarose) (16); Biogel (a cross-linked polyacrylamide); and PW gel (prepared by the multifunctional polymerization of a hydroxylated ether monomer) (17). The dependence of K on the solubility of a series on normal alcohols suggests a quantitative parameter for characterizing the hydrophobicities of SEC gel packings.
EXPERIMENTAL SECTION The chromatographic system was comprised of a Milton Roy Minipump, a Rheodyne Model 7010 injector equipped with a 100-pLloop, and a Waters R401 differential refractometer. PW Gel (Toyo Soda Ltd.) and Superose 6 (Pharmacia) columns were commercial items, with dimensions 30 X 0.78 cm i.d. and 30 X 1.0 cm i.d., respectively. Biogel A-50 (BioRad) was packed into a 40 cm X 1.00 cm i.d. column (Pharmacia C 10/40) with an efficiency of 600 plates m-l. Sephadex G-75 was packed in a similar column with an efficiency of 800 plates m-l. Normal alcohols (butyl to octyl) were all reagent grade, obtained from Aldrich. The total column volume Vt was determined by injection of DZO; the exclusion volume V, was determined from the elution of blue dextran. All chromatographywas carried out with distilled water as eluant. The flow rate was maintained at 0.5, 0.2, and 0.15 mL min-' for Superose, Biogel A-50, and Sephadex G-75, respectively. Flow rates, determined by weighing timed collections of eluant, were reproducible to better than 0.2%.
RESULTS AND DISCUSSION The dependence of K on the solute's molar solubility in pure water, S, is shown in the double logarithmic plot of Figure 1. Along with the current data for Superose, Biogel, and Sephadex G-75 are included results for PW Gel (8),Sephadex G-100 ( 3 ) ,and Sephadex G-10 (2, 3). Within experimental error, all the data conform to the linear relationship In K = A - B In S
(2)
as observed previously for PW Gel (8). The empirical relationship of eq 1may be explained on the basis of the known linear dependence on carbon number ( n ) of the standard free energy of aqueous solution of alcohols (18). In so doing, we may obtain some insight into the physical significance of the constants A and B. Let us assume, with Dawkins (19,20), that the measured partition coefficient is comprised of steric and partitioning terms, i.e. In K = In Ki
+ In Kp
(3)
where Ki is the "ideal" partition coefficient. For small solute molecules, we assume provisionally that Ki z 1, and the measured K may be considered essentially equal to Kp (this approximation may fail for the highest molecular weight alcohols in the smallest pore-size gel). The observed linear dependence of alcohol solubility on n may be semiempirically expressed as In S = -AGo,/RT =
-AG
Os(
OH) /R
T - nAGo ,( CH2) /R T
(4)
where the standard free energy of solution, AGO,, is divided into contributions from the alcohol head group and the methylene units (18). Recognizing that Kp corresponds to the transfer of the solute from the bulk mobile phase to the gel environment, we may write a similar expression In K p = -AGOsEC,,/RT = -AG"sEc(OH)/RT-
nAGOsEc(CHz)/RT (5)
where AGOsEC,,is the standard free energy of transfer of solute from mobile phase to packing. We now assume that A G o s ~ ~ ( C Hand 2 ) -AGos(CH2) are closely related: the first describes the contribution of a methylene group to the transfer of solute from water to gel, and the second the transfer from water to neat alcohol; the magnitude of both presumably reflects the hydrophobicity of gel and neat liquids, respectively. The two parameters may be related by AG"SEc(CH,) = -rAG",(CH,)
(6)
where y is a gel-specific constant that describes the hydrophobicity of the packing relative to that of the neat alcohols. Equation 6 allows us to combine eq 4 and 5 to yield In K , = -7 In S - [rAG",(OH) - AGosEc(OH)]/RT ( 7 ) Comparison of eq 2 and 7 reveals that B = y, i.e. the slopes of Figure 1,are equivalent to the dimensionless parameter y, which we define as the gel hydrophobicity index. Also, we see that A = (RT)-'[AGosEc(OH) - rAGo,(OH)]; thus, the observation that the lines are vertically displaced at In S = 0 occurs in part because a change in y leads to a change in the magnitude of the second term of eq 7. Specific interactions
0003-2700/89/0361-0780$01.50/0 'C 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 7, APRIL 1, 1989
In
Ki - 2 p n / d ,
781
(11)
Proceding as before, except with K = KiK,,we obtain In K = A - B In S - 2 p n / d ,
(12)
Equation 12 takes into account geometric size exclusion and shows that the observed dependence of In K on In S contains an additional contribution of opposite direction to the partitioning effect. This result is seen most clearly when d, is small, i.e. for the data with Sephadex G-10, which display a small but discernibly negative slope.
CONCLUSION
A-4
Ill
11
I
I
i
7 5
Figure 1. Double logarithmic plots of the chromatographic partition coefficient vs the molar solubility of the alcohol for (m) PW Gel (from ref 8); (A)Superose 6 (this work); (A)Sephadex G l O O (ref 3);(0) Sephadex 0 7 5 (this work); (0)Sephadex G10 (ref 2 and 3); and (0) Bio-gel A50 (this work). Data for heptanol and octanol on PW gel conform to line shown.
Table I. Values of the Dimensionless Gel Hydrophobicity Parameter for Stationary Phases of This Study stationary phase
PW G e l B i o G e l A-50M Superose 6 Sephadex G-10 Sephadex G-75 Sephadex G-100
chemical t y p e
Y
h y d r o x y ether polyacrylamide agarose dextran dextran dextran
1.13
LITERATURE CITED
0.015
0.084 0.23 0.080
