Interactive Gel Networks Chromatographic and Analytical Properties with a Pyridine Type Functional Group David H. Freeman, Rosalie
4. Angeles, Delmo P. Enagonio, and Willie May
Analytical Chemistry Division. National Bureau of Standards, Washington, D. C. 20234
The gel is a copolymer of 2-methyl-5-vinyl pyridine with DVB cross-linking. The composition, infrared absorption, swelling, and gel permeation properties were measured. The pyridine moiety is basic, as indicated by its ability to complex ROH and RCOOH solutes. Chromatographic affinity is directly related to the strength of the solute:gel complex. Solvents mask the pyridine gel in the order EtOH > CHC13 > CCI4. The resulting solute affinity varies oppositely with the strength of the solvent:gel complex. ROH affinity correlates systematically with the proton donor strength. Gel capacity is high and several applications are described.
Interactive gel chromatography offers a new and systematic basis for approaching the separation of chemical compounds ( I ) . The required networks exhibit at least two fractionative mechanisms. The first arises because the network is composed of cross-linked and randomly oriented polymer chains in an irregular beehive configuration. The limiting apertures exclude large molecules while allowing smaller ones to permeate on a “space available” basis. Second, functional groups of a specific kind are permanently attached to the network. These groups interact with conjugate molecules of solute (or solvent). Such interactions have been classified by Mulliken (2). The same principle of electronic complementarity may underlie information transfer mechanisms in biological systems (3). Although specific interactive effects have been observed (4, 5), an unscrambling of the mixed steric and interactive contributions was only recently done ( I ) . A more detailed theoretical basis for interweaving the effects of molecular size with the competitive gel, solvent, and solute interactions has been described (6); this will be referred to as part I. The liquid chromatographic application is of particular interest because the liquid itself exerts a powerful role as solvent as well as its ability to regulate the intensity of solute-gel affinity. The present study (Part 11) concerns the properties of poly-2-methyl-5-vinylpyridine/divinylbenzene which is readily swollen by various solvents to become “PVP gel.” We have already reported our observation of the orderly correspondence between solute affinity and the hydrogen donor strength of the solute compounds ( I ) . The proton donor strength of a compound regulates the intensity of its possible role as solute or solvent, and both will be considered here. The gel in turn becomes an effective materi-
D. H. Freeman and E. P. Enagonio, Nature, Phys. Sci., 230, 135 (1971). R. S. Mulliken, J. Phys. Chem., 56, 801 (1952). Y . Kyogoku, R. C. Lord. and A. Rich, Proc. Nat. h a d . Sci., U S . ,
57 (2), 250 (1967). J. T. Ayers and C. K. Mann, Anal. Chem., 36,861 (1966). L. Sweetman and W. L. Nyhan, J. Chromatogr., 56, 349 (1971). D . H. Freeman,Ana/.Chem., 44, 1 1 7 (1972).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
a1 for measuring this strength, and the orderly behavior of such measurements will be described. Our interest also springs from the need for practical improvements in the separation and characterization of marginally stable organic compounds, including those intended for use as clinical Standard Fkference Materials. The PVP gel has been found to be useful in several ways. It is a moderately firm gel, somewhat softer than conventional ion exchange resins, but readily adapted for efficient chromatography at moderate pressures. As expected from its three-dimensional structure in the gel state, PVP gel exhibits good performance with large sample loads. This is especially important to purity measurements, and the use of PVP gel for isolating impurity fractions has been developed. We have studied the fundamental analytical chemical properties of the present PVP network and gel. The composition and swelling behavior have been measured and related to cross-linking. The theory in part I requires an assessment of the purely steric effects, so hydrocarbon solute compounds have been used to achieve a gel permeative characterization of the network porosity. Estimates of ROH-PVP interaction constants have been obtained and used to describe compound behavior as solute and as solvent.
EXPERIMENTAL PVP-Network Preparation. Suspension copolymerization of 2-methyl-5-vinylpyridine with divinylbenezene (ca. 55% w/w DVB purity) was carried out under the direction of A. H. Greer (Ionac Chemical Company, Birmingham, N.J.) and, independently, under Roy Wood (Bio-Rad Laboratories, Richmond, Calif.). The reaction can be expressed as follows:
f I
CH,
P DVB
VP
x ( e )
@-
Network
(1)
EVB The DVB cross-links give the network its three-dimensionality and its insolubility. The available technical grade DVB contains EVB (E = ethyl) which also becomes polymerized. The basic 2,5 disubstituted pyridine moiety comes from the corresponding vinyl pyridine (VP) monomer. A KBr pellet was prepared of the dry network by the usual technique of low temperature powdering (7), and the infrared ab( 7 ) N. B. Colthup, L. H. Daly, and S. E. Siberley, “Introductionto Infrared and Raman Spectroscopy,” Academic Press, New York, N . Y . , 1964, p 66.
Figure 1. Infrared absorption spectrum of PVP matrix used in this study. Preparation: low temperature powdering, KBr pellet
sorption spectrum was obtained, as shown in Figure 1. The bands a t 1186, 1488, 1566, and 1597 c m - l are characteristic of the aromatic pyridine ring. The band a t 1027 cm-1 is that used by Laskorin (8) as an internal standard for measuring the presence of DVB. The rneta-DVB moiety is apparent a t 712 and 798 cm-1. The aliphatic hydrocarbon backbone of the network is indicated by CH stretch a t 2850-2950 cm--l, methylene deformation at 1444 cm-1, and methine deformation a t 1348 cm-1. Small particle "fines" were removed in ethanol by repeated 45-minute sedimentation and decantation in 20-cm high beakers. The particle size was measured with a Coulter counter and found to be over 90% by size frequency in the range 16-36 pm. Microscopic examination indicated the presence of over 99% optically transparent and spherical particles. With darkfield illumination, the transparency was maintained. Examination with cross polarizers showed the absence of strain birefringence. Cracked or fragmented beads were absent. No multiparticle clusters were evident with the beads immersed in ethanol. Solid probe mass spectrometry showed that the PVP network does not undergo appreciable decomposition below 200 "C. Similar stability is known for polystyrene/DVB (9). Evacuated oven drying of PVP was done a t 135 "C. Elemental analysis and calculated compositions were obtained, respectively, as 82.2 (82.3) %C, 7.5 (7.7) %H, and 10.1 (10.0) O/cN for an original mixture of 794/ 134 by weight ratio for the vinyl-substituted pyridine/divinylbenezene (55% pure, rneta- and para-DVB) monomers. This corresponds to 7.3 mol 90DVB, or 7.9 weight 7'0 DVB, in the network. The nitrogen content of the network should vary as a function of the incorporated monomer ratio where the weight fraction of nitrogen f v is calculated using
where x is the mole fraction DVB in the combined vinylbenzene isomers, and [ ] refers to molecular weight. The average value [DVB] is taken as the sum z [DVB] + (1 z ) [EVB], where z is the mole fraction of DVB in the mixture of DVB and EVB. Using the measured value of f~ we can solve Equation 2 to obtain the apparent cross-linking. The elemental composition measurements were thus found to provide important confirmations, but they only give a rough measure of the apparent compositional crosslinking as 6 f 2 mol ?& DVB. The swelling properties of a polymer network are a measure of the structural extendability (10). The weight swelling ratio was determined using the centrifugal filtration method. The retention of excess liquid was corrected using the expression:
-
r =
r (apparent) l + e
where r(apparent) is the measured apparent relative solvent content of the gel. On a weight basis, it is given by the ratio [W(gel) - W(matrix)]/W(matrix). The value of W(gel) is affected by a small amount of surface liquid which adheres to the beads. Then, r is the corrected weight of gel per unit weight of matrix and e describes the retention, as previously defined (11). The evaluation of the weight retention factor, e, has been done following Professor Scatchard (22) that the corresponding volumetric factor e, is the same for gel as for non-porous glass beads. Then, e = e , p s / p g where p is density. The subscripts s and g refer to solvent and gel. Values of e , from 0.043 to 0.062 were reported for various spherical beads by Fricke and coworkers (13). Our retention corrections were based on the average meaiured values of e , = 0.051 for water and 0.050 for ethanol using 5-30 pm glass beads. Standard Reference Material 1003, and with centrifuging a t lo00 times the force of gravity. We assume that the swelling of network by liquid involves only small departures from volume additivity, so the gel density can be calculated:
where the subscript rn refers to unswollen matrix. An algorithm for calculating r and pg from Equations 3 and 4 was used as follows. A starting value of e = 0.05 is assumed. (*) Then, r is calculated from Equation 3. Next, p g is obtained from Equation 4. Then, an improved value of e is calculated from e , and the available density ratio. Return to (*). We found the convergence of this calculation to be rapid. Other important quantities are related to swelling and are derived from the values of r and p g . The volume swelling ratio of the gel is 9 = u(swollen)/u(unswollen) = r pm/pg and the solvent volume fraction in the gel is 4 = ( 9 - l ) / 9 . The stoichiometric gel phase molarities were defined in part I. The functional group molarity is given by
Mi
1000 p s
(5)
=
For the present PVP network, &,v is the network equivalent weight with respect to aromatic nitrogen, g network/mole of the vinylpyridine moiety. This is calculated from the network equivalent weight per mole of aromatic rings (14) given by
(3)
(8) 6 .N. Laskorin. N. P. Stupin. L. A. Fedorova, G. N . Nikul'skaya, and A. I . Yuqhin. lnd. Lab., 34, 966 (1966). (9) S. Straus and S. L. Madorsky. J. Res. Nat. Bur. Stand., Sect. A , 65, 243 (1961). (10) J. R . Parrish. J. Appl. Chern., 15, 280 (1965).
(1 1 ) D. H. Freeman, J. Phys. Chem., 64, 1048 (1960). (12) G. Scatchard and N. J. Anderson, J. Phys. Chem., 6 5 , 1536 (1961) . (13) G. H. Fricke, D. Rosenthai. and G . A . Welford, Anal. Chem., 43, 648 (1971). (14) D. H. Freeman, L. A. Currie, E. C. Kuehner. H. D. Dixon. and R. A. Paulson. Anal. Chern., 42, 203 (1970).
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Table I. Weight Regain, Gel Density, VolumeSwelllng Ratio, Functional Group Molarity, and Solvent Molarity in PVP Gel Solvent r PB 9 M'G Mas
cc14 CHC13 EtOH THF C6H6
1.43 2.93 1.94 1.82 1.26
1.32 2.54 2.41 2.10 1.35
1.28 1.37 0.95 1.03 1.11
0.242 0.606 0.585 0.524 0.259
6.23 3.23 3.40 3.91 6.07
2.5 7.5 10.0 6.5 2.9
where x is mole fraction cross-linking agent, and z is mole fraction cross-linking agent in the DVB mixture. The equivalent weight is then obtained: EN = & / ( l x / z ) . For our x = 0.08 matrix, t was 0.55. The average [DVB] is 131, (VP) is 119.2, and this gives & N = 141.5 g/mole of aromatic nitrogen, Imbibition of the pure solvent liquid into the gel can also be expressed in terms of the solvent molarity in the gel where [SI refers to solvent molecular weight.
-
The measured swelling properties for the gel are reported in Table
I. The chromatographic apparatus has been described elsewhere in detail (15) and was assembled in the order: solvent reservoir, pump, depulsator, stream splitter for column and reference stream, 0.25-ml sample injection value, precision bore glass column 0.25-inch internal diameter, refractive index detector, and recorder. The volume of each apparatus component was determined where possible by the difference between its empty weight and that measured after filling with water. The detector cell volume was calculated from the geometry of its components. The total extra-column dead volume of the system (16) was approximately 0.4 ml with an uncertainty of ca. 0.05 ml. This dead volume is subtracted from the chromatographic measurement of total mobile phase volume V m to give the value which applies inside the column. On this basis, we noted that the void volume fraction in the column was within the range 0.35 to 0.37 throughout the investigation. This agrees closely with the findings of Professor Rieman and coworkers (17). The overall column length for the analytical columns was 60 cm and the end fittings left a column bed length of 58 cm. The bed was packed under a fluid pressure which exceeded that for the normal rate of elution at 0.5 ml/min. On occasion, bed compression occurred, and the presence of clear liquid void in the top of the column would appear. This was accompanied by increased peak dispersion. To correct this, the gel would be removed from the column, reconditioned, and repacked. In c c l 4 , it was difficult to obtain sharp peaks with this length column. We believe this results from inadequate swelling in this solvent. HBnce, the hydrocarbons measurements were obtained in CCla with a bed depth of 20 cm, and the remaining chromatography in CC14 was carried out with a 7-cm bed depth. The elution volume measurements were obtained as follows. Flow rate was monitored using buret and timer. Elution time was measured on a chart recorder. The elution measurements were obtained a t least in triplicate and the results were averaged. The volume measurements are considered accurate to within about 3% of relative error. Although higher precision was usually demonstrated for a given system, we measured the stationary phase volume only indirectly and the estimate of reliability considers the reproducibility with different and independently assembled chromatographic systems. T o within experimental error, we believe t h a t the chromatographic affinities are independent of packing, cleanup, repacking, and extended use of a single column material during several months of observation. Nat. Bur. Stand. ( U . S . ) Tech. Note 589, S. D. Catalog No. c 13.46:589, U S . Government Printing Office Washington, D.C. 20402. D. B. Bly, K. A. Boni, M . J . R. Cantow. J. Cams. D. J. Harmon, J. N . Little, and E. D. Weir, Polym. Lett., B, 401 (1971). G . D. Manalo, R. Turse, and W. Rieman, Anal. Chim. Acta, 21, 383 (1959). ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
Solvent preparation included selection to meet specific low residue requirements for the evaporation of solvent from collected fractions. The CCl4, and CHC13 (with preservative ethanol), and anhydrous ethanol were obtained as reagent grade. The presence of UV-absorbing impurities had to be negligible since these otherwise interfered with subsequent measurements in the laboratory. The methanol was distilled prior to use. The solvent reservoir for the chromatography was kept a t 50-60 "C to promote degassing, and to discourage bubble formation in the detector loop The measurement of elution volume was usually carried out with 1- to 3-mg samples. On occasion, slightly large samples were used to improve the sensitivity. Peak dispersion measurements were made in ethanol using a 0.63- X 27-cm column at flow rates in the range 0.2 t o 1.4 cm3/ min, or a calculated linear rate of 0.03 to 0.23 cm/sec. These rates are typical (18) for gels used in high resolution ion exchange, or steric exclusion chromatography. At a flow rate of 0.5 cm3/min, the effective plate heights H (cm) were for hexane 0.08, dihydroxyacetone 0.08, acetic acid 0.10, and benzoic acid 0.13. With the hexane and acetic acid at 1.0 or 1.4 cm3/min, no significant increase in H was observed, although a decrease by 10 or 20% was found a t 0.2 cm3/min. These efficiencies are not as good as those obtained by Hamilton (19). This suggests the potential two- or three-fold reduction in plate height if careful sizing procedures were carried out. Similar considerations of polydivinylbenzene type networks have been reported (20). With the higher sample loads, the dispersion increased, as will be discussed, and it was then advantageous to reduce the flow rate to 0.05 to 0.1 cm3/min.
RESULTS AND DISCUSSION Physical Properties of the PVP Gel. T h e e l e m e n t a l network composition c a n be referred to the "average" s t r u c t u r e . S i n c e vinyl m o n o m e r s a r e used, t h e network must form w i t h a hydrocarbon b a c k b o n e c o n t a i n i n g e q u a l numbers of m e t h y l e n e and m e t h i n e groups. The pendant a r o m a t i c rings f r o m V P , DVB, and EVB m o n o m e r s a r e a t t a c h e d t o the m e t h i n e groups. T h e e x t e n d e d hydrocarbon chains a r e linked t o g e t h e r for x = 0.08 b y DVB vertices which attach to one s i x t h of the m e t h i n e groups. A n equal fraction ( a p p r o x i m a t e l y ) connects to the reacted EVB. An average c h a i n between cross-links c o n t a i n s five m e t h y l p y r i d i n e and o n e e t h y l benzene groups. T h e PVP beads appear to be perfectly transparent w h e n e x a m i n e d e i t h e r unswollen or in the presence of a swelling solvent. Dark field microscopic illumination of the latter gave no evidence for milkiness d u e t o light scattering. This i n d i c a t e s t h e presence of an optically homogeneous gel state where t h e n e t w o r k s t r u c t u r e is uniformly dispersed in the swelling liquid. This is very different f r o m the evidence for heterogeneity that is readily d e m o n s t r a t e d in dark-field illumination of the macroscopically porous s t r u c t u r e s o b t a i n e d from solvent modified polymerization (21). The swelling m e a s u r e m e n t s a r e r e p o r t e d in T a b l e I. There appear to be good solvents ( 4 values f r o m 0.5 t o 0.6) and bad solvents (6 < 0.3). The swelling is higher than would be e x p e c t e d on the basis of the a m o u n t of cross-linking a g e n t . T h i s can b e s h o w n b y reference to the toluene swelling properties of polystyrene/DVB networks. Our previous m e a s u r e m e n t s (22) of the latter were correlated using the f u n c t i o n q In (1 l / q ) which is suggested i n Flory's work (23). On that scale, 8 mole per c e n t D V B
-
P. 8. Hamilton, D. C. Eogue, and R. A. Anderson, Anal. Chem., 32, 1782 (1960). P. B. Hamilton, "Handbook of Biochemistry," H. A. Sober, Ed., Chemical Rubber Company, Cleveland, Ohio, 1968; p 5-47, see Figure 5 . R. N . Kelley and F. W. Bilimeyer. Jr.. "Gel Permeation Chromatography," K. H. Altgelt and L. Segal, Ed., M. Dekker, New Y o r k , N.Y., 1971;p47etseq. J. C. Moore, J. Poiyrn. Sci., Part A , 2, 835 ( 1964), D. H. Freeman, V . C. Patel, and M. E. Smith, J. Polyrn. Sci., Part A, 3, 2893 (1965). P. J , Flory, "Principles of Polymer Chemistry," Cornell University Press, Ithaca, N . Y . , 1953; p 5 7 6 etseq.
in ethanol-swollen PVP gel corresponds to 4.4 mole per cent DVB in a toluene-swollen polystyrene/divinylbenzene gel. This suggests that the present PVP gels are twice as swollen, and twice as “soft,” as the corresponding polystyrene/DVB gels. P V P Gel Permeability. An effective characterization of gel network porosity is provided by classical gel permeation chromatography. This requires, the use of non-interactive solutes whose sizes are accurately known. The normal alkanes have been adopted for this purpose and our experimental approach is similar to that of Hendrickson and Moore (24). This calibration procedure gives an excellent first approximation, but it does neglect the effect of solute conformation (25). Solute size estimates have been referred to the calculated number ( N e )of carbon atoms in the respective normal hydrocarbon molecules (24). The elution value measurements in the present work have been referred to the corrected elution volume per unit of total column volume:
This is easily adapted to any size column since, with reference to Equation 26 of part I, the mobile phase volume in the column is estimated by V , = iVcol and the stationary phase volume Vs is the total volume of swollen gel in the column and is estimated by V , = (1 - i)Vcol.For spherical beads, i has an average value of 0.36 and is independent of bead size (17). Note that E = (1 - i)D; again referring to Equation 26 of part I. An elution scale for interactive gel chromatography can be set forth in the following way. The column void volume can be referred to the elution of a polymer molecule that is large enough to give E = 0. If a solute permeates the gel, then this occurs with E > 0. Inert solutes permeate gels to an extent which varies oppositely with their size. Interactive solutes permeate in the same way, but their affinity for the gel is amplified by the chemistry of the interaction. Explicit concepts should be used to distinguish between steric and interactive affinity. Consider two “isosteric” molecules, meaning they are equal in their ability to occupy space. If one of these is inert, chemical interaction is defined to be zero and the elution value will be denoted by the subscripted Eo. Assume the other is able to exhibit chemical interaction. The amount of the interaction is explicitly described by the partitioning ratio
which was defined in part I. Since the R value is corrected for the steric contribution, purely interactive affinity is implied by R > 0. The origin of selectivity for a mixture of compounds can now be traced in forms of these definitions. Purely steric selectivity means separation based upon a difference in the Eo values. Purely interactive selectivity is based upon the difference in the R values. The elution value measurements for hydrocarbons and ROH compounds are listed in Table 11. The solute size N e is given for each compound on the “carbon atom” scale followed in reference (24). For convenience, the Eo value is presented for each interactive compound. This was obtained by interpolation of the hydrocarbon measurements ( 2 4 ) J. G. Hendrickson and J. C. Moore. J. Polym. Sci., 4, 167 (1966). (25) W . W. Schultz. J. Chromatogr., 55,73 (1971).
to obtain the reference elution value for an inert compound of the same size as that of the interactive solute. The k values refer to Equation 11 and are calculated using the expression k = ( E - Eo)/EoMc” which is applicable to carbon tetrachloride where solvent masking effects are negligible. The measurement of low molecular weight hydrocarbon permeation of the PVP gel is given in Table 11. The results are shown graphically in Figure 2. The well known general trend is obtained; the hydrocarbon solute permeation varies oppositely with solute molecular size. This is the typical pattern of non-interactive gel permeation chromatography. Compare these results to those obtained in Larsen’s (26) study of polydivinylbenzene networks. Another distinguishing feature of the hydrocarbons is their obviously small chromatographic affinity. The obvious effect of chemical interaction ( E > Eo) is now seen in Table I1 as a major basis for extending the elution scale in gel chromatography . Prediction of Solute-PVP Gel Chromatography. The prediction of interactive gel chromatography can be achieved if the experimental conditions are adequately represented by Equation 19 of part I with the “b” term set equal to zero. In that way so1ute:gel association is the only important complexation process involving the solute. It may occur alone or be accompanied by so1vent:gel masking. In either case, the interactive solute partitioning ratio is simply:
R
=
ah
( b = 0)
(10)
The value of “a” is the effective functional group concentration, M G I Mc;’, where the maximum is obtained with no masking, and less than that when masking does occur. First, we shall illustrate the calculation without masking. Consider next the monofunctional alcohols ROH whose interaction with the PVP-gel is described by the following equation: -CH-CH2-
CH,
Although the chromatography will be governed by the above, we can take advantage of the apparent correspondence ( I ) between gel phase interactions and those which have been to date more thoroughly studied in homogeneous liquid media. To illustrate, the ability of ROH compounds to form complexes with various amine bases has been investigated by Gramsted (27) and by other authors. This work has been summarized by Davis (28). Acceptor-donor complexation often occurs on a 1:1 basis which we will assume here exclusively. (Since some ROH self-association ( k 1) tends to occur (29), some inaccuracy can be expected due to this.) From the available measurement (27) of ROH :pyridine association we have the following equilibrium:
-
(26) F. N. Larsen, Appl. Polym. Symp. 8, 111 (1969). (27) T. Gramsted, Acta Chem. Scand., 16,807(1962). (28) M. M. Davis, “Acid-Base Behavior in Aprotic Organic Solvents.” Nat. Bur. Stand. (U.S.) Monogr., 105, 126 (1968). (29) G. C. Pimentai and A. L. McClellan, “The Hydrogen Bond,” W. H. Freeman and Company, (San Francisco, Calif., 1960: Appendix B.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
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Table 11. Elution Measurements CCI,
Solvent Solute
N
nCi6” nCioHzn nCaHI8 nC6H14 nCdH10 C3H8 CZH6 CH4 CHClj PhNH2 t-BuOH i-PrOH nPrOH EtOH MeOH PhCHnOH PhOH
16 10 8 6 4 3 2 1 3.3 3.8 4.7 3.7 3.7 2.7 1.7 4.5 3.5
Eo
E
k
0.167 0.157 0.145 0.161 0.161 0.177 0.194 0.147
Mr;
PhCHzOH Pyridine 5.2” 6.23‘ 32.4 4.5 0.147 4.9 15
R=kMT: Solute size ( N C ) € 0 (interpolated) E (predicted)e E (obsd), Table I I a See ref. (28). bSee ref. ( 2 7 ) . c No masking. See Table I. d Includes masking. See text. e E = ( R I ) En from Equation 9.
E
0.228 0.268 0.277 0.300
0.115 0.139 0.172 0.217 0.228 0.271 0.293
0.080 0.100 0.127 0.170 0.191 0.204 1.o 2.4 2.5 4.0 4.2 6.9 12 15
PhOH CHCi3 2.4-Lutidine 10Ah 0.654d 68 3.5 0.22 15 28
0.322 0.337
. . . .
0.8 2.3 2.6 3.8 4.0 6.1 10 16
0.215 0.195 0.218 0.21 8 0.246 0.276 0.198 0.222
0.4
cc14
Eo
E
EO
0.042
Table 111. Model Compound Estimates of ROH Affinity for PVP Gel Solute Solvent Model Cpd Used k (1. mol- l )
EtOH
CHC13
0.373 0.470 0.655 0.818 1 .05 7.92 1.69 27.8
0.306 0.316
0.332 0.346
. . . . 0.339 0.309 0.31 9
0.359 0.704 1.37
I
I
I
I
I
I
I
2
4
6
8 Nc
IO
12
14
0.3
E
0.2
+
With pyridine as a model compound, Table 111 shows an illustrative prediction of benzyl alcohol chromatography based upon the reported (28) equilibrium constant for Equation 12. In the carbon tetrachloride, there is no significant solvent masking so the functional group has its stoichiometric concentration, MG’, taken from Table I. The size is calculated for benzyl alcohol following Hendrickson and Moore (24) with the sum 2.8(phenyl) 1.0 (methylene) + 0.7 (hydroxyl) giving 4.5 on the “carbon atom” scale. By interpolating the hydrocarbon permeation measurements given in Table JJ we find Eo. This leads to the prediction of E and comparison to the measured value. A similar calculation for phenol in chloroform requires an estimate of the masked functional group concentration, which will be described shortly. As described previously but without elaboration ( l ) , this type of estimation is able to predict the chromatographic affinity with some success. The calculation depends mainly upon the non-chromatographic model for the interaction. It also requires the steric calibration which should be applicable to many compounds. The difference between prediction and observation is not accounted for by the simplifying assumptions which we have used. If we had taken these into account, the predicted value of E would be still lower. Hence, we can infer that the model compound, pyridine, is insufficiently basic to provide an exact model for the PVP gel. Regardless, the k determinations now available from the chromatography
+
772
0
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
0.1
0 0
16
Figure 2. Permeation measurements for PVP gel using straight chain hydrocarbons in various solvents
provide a calibration that should be characteristic of a variety of PVP gel matrices. R0H:PVP Affinities in CC14, CHCI3, and EtOH. Measurements of various alcohol affinities for PVP gel were obtained in the individual solvents cc14, and CHC13, and EtOH. In any proton donor solvent, the R0H:PVP affinity is affected by the competitive strength of the solvent:PVP masking. This is clearly indicated by the trend in the experimental results given in Table 11. The R0H:PVP affinity is strongest in CC14 which is most nearly inert, and it is about five-fold weaker in CHC13. In ethanol, the solute affinities seem to be nearly indistinguishable when the proton donor strength is less than or comparable to that of the solvent. For benzyl alcohol and phenol. however, some interaction with the gel is evident.
2n
i
0
-I
I
?-
I
R
[ CSHS 1 I
50 rng/COMPONENT ?OH
14.7 1
1
10.7
/
I /
.' /
/ /
/
/'
'
J
25 40 V (CHCI, ,ml)
CORTISOL: 21 bc
0
/'
I
I
18.4
SAMPLE: 104mgOF CORTISOL IN toed Of EtOH
1
CHCl3
L: 58cm
t BuCH
I
I
5
IO
I L
L-58crn F.0.092 crn3/min EACH FRACTION N0.-2.3crn3
15 FRACTION NO. 6 MERCAPTOPURlNE
uv
/'EtOH
h
/
1
0
11.0
3540
21
48
V ( E t O H , rnl)
Figure 4. Illustrations of poly(2-methyl-5-vinylpyridine/DVB) gel chromatography (0.6 X L-cm columns)
We can now summarize on the ability of the simple theory to predict chromatographic affinity. Starting from solution data, the predictions are roughly accurate. It is certain that they are no better than the preciseness of the correspondence between model compound and gel functionality. The calculated affinity ratios for different solvents seem to be much more reliable. This depends only upon the internal consistency of the theory. The foregoing analysis is subject to testing from still another point of view. In Figure 3, we have plotted the loga. rithmic values of R for the alcohols measured in various solvents and listed in Table 11. The Taft CT* value (31) for the substituent group is used as the correlating abscissa variable. The resulting straight lines parallel the predictions of Figure 1 of part I. The absence of curvature indicates that solute self-association or solvent-solute association are not significant. In other words, the b term in Equation 19 of part I is close to zero. It would be of interest to determine the accuracy with which this correlation can be extrapolated to predict the chromatographic behavior of compounds which we have not measured in the present study. The solid lines in Figure 3 represent the empirical relation In R
=
r
+
SO*
(13)
We assume that the r-term depends upon the solvent and s upon the network. The data in Table I1 were treated by least squares analysis to Equation 13. The slopes were 3.87 for CC14 and 4.95 for CHC13, giving the average value 4.41 which we assigned to s. Then, the average r-values were determined as 3.96 for CCll and 1.65 for CHC13. The results suggest a close correlation between k and g*. Since In M G , we have the same relationIn R is given by In k ship for the different solvents.
+
In k
=
2.1 + 4.41 u*
(31) R . W. Taft, Jr.. "Steric Effects in Organic Chemistry," M. S. Newman. Ed.. John Wiley & Sons. New York, N.Y., 1956; Chap. 13.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, A P R I L 1973
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I
I
0.6
w
0.5
ga
i 5
0.4
0
50
IO0
Weight of Sample (ma)
Figure 5. Effect of sample size upon elution value (€) and upon plate height H ( m m ) . Test compound: hydroxyacetone. Solvent: ethanol
This would be interesting for future study. It is possible, if not likely, that well-defined interactive gels are able to provide a basis for rapidly determining the acid-base strength of chemical compounds. Analytical Applications for PVP-Gel Chromatography. Our principal application of this work has been to assist in the determination of chemical purity of marginally stable compounds. The study of a particular compound involved measurement of elution values for the likely contaminants. These measurements were also referred to small sample size. The analytical column was kept the same for larger sample sizes. This was not due to preference, but to the lirhited availability of stationary phase throughout the main part of the study. Early evidence was obtained that the PVP-gel could be used gainfully with substantial sample size. The first observation was that elution values did not seem to vary appreciably while component mass was varied in the range 0.5 to 5 mg. An effort to extend this was made using the five compounds shown in the top chromatogram in Figure 4. Excellent resolution is achieved with 50 mg per sample component for a total of 250 mg entered. The chromatography of dihydroxyacetone in ethanol was studied with varied sample input. As the latter is increased the peaks are continually broadened, and the elution volume steadily decreases. These effects are also shown in Figure 5 . The variation of elution volume should be attributed to the effect of masking by the most significant component capable of causing the masking, that being of course the solute. Brief descriptions of applications to specific problems of purity analysis are describd next. Cholesterol was chromatographed in chloroform with inputs of 100-mg samples. A single impurity peak
774 e ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
emerged before the cholesterol peak. The impurity peak was later examined by thin layer chromatography and six or more individual impurities were then resolved. This cooperation of the two methods was gratifying since the TLC did not show the presence of the impurities without the chromatographic preconcentration. Hydrocortisone chromatography was carried out in ethanol as the moving phase. This keto-steroid derives its protogenic strength ( E = 1.38) from the OH groups in the 17 and 19 positions where they are adjacent to the ketone carbonyl group. The structure and acidity of this steroid should be similar to that of 1,3-dihydroxyacetone where E = 1.1 is the measured value. Other compounds (and E values) were measured in ethanol: water (0.52), hydroxyacetone (0.55), 2-chloroethanol (0.69), mannitol (0.9), sorbitol (0.9), cortisol (1.08), acetic acid (1.18), hydrocortisone-21 acetate (1.291, phenol (1.37), benzoic acid (2.5), and trichlorophenol (2.9). Several of these elution values were checked in methanol where they were found to be approximately 30% less. The limited solubility in ethanol of hydrocortisone (15 mg/ml) presented a problem with regard to achieving the needed increased sample size. Despite this, useful results were obtained with 100 mg entered from a IO-ml sample loop. Fraction collection, solvent evaporation, and measurement of the residue weights were carried out. The result is the chromatogram in Figure 4. The “detector” in this case is the analytical balance. This gives a chromatographic “mass balance” which is useful in the metrology of chemical purity. The chromatography of 6-mercaptopurine was also found to be feasible in ethanol. The major component was easily resolved from the precursor impurities as shown in Figure 4. An NMR spectrum of the purified component was run in DMSO-de solvent with T M S added as a marker. The valuable purine compound was later easily recovered from these additives. Since DMSO is frequently used in NMR work and it is often difficult to separate from a fragile polar compound, this simple chromatographic recovery method should be a useful aid to the spectroscopic laboratory.
SUMMARY The PVP-gel is a polymer reagent whose protophilic properties are closely parallel t o established acid-base behavior in homogeneous media. Its affinity for diffusible solutes originates from a combination of permeative and complexative processes. The finding of an orderly relationship between measured affinity and solute proton donor strength suggests a new framework for studying hydrogen bonding, and for measuring the proton donor strength of chemical compounds. The high capacity of the gel makes it equally useful for performing chemical separations.
Received for review September 25, 1972. Accepted December ll, 1972.
