Anal. Chem. 1994,66,450-457
Liquid Chromatographic Study of Solute Hydrogen Bond Basicity Lay Choo Tan and Peter W. Carr’ Department of Chemistry, Smith and Kolthoff Hall, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455 Jean M. J. Frbchet and Vladimir Smigol Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York 14853
The purpose of the present work was to investigate a liquid chromatographic method for the measurement of relative hydrogen bond basicities of dilute species. This type of determinationcannot be done with conventionalreversed-phase liquid chromatography due to the silanophilic interactions of basic solutes with the silica packing material. The studies were done on a polymeric stationary phase with pendant phenol groups that act as powerful hydrogen bond donors. Solute retention was evaluatedin terms of two hydrogen bond basicity scales, BzH and @zC, and a steric hindrance parameter, 4.BzH and BzC are basicity scales based on the free energy of forming 1:l hydrogen bond complexes and the retention on a strong hydrogen bond donor gas chromatographicphase, respectively. The 4 parameter characterizes the steric effect experienced by the solute acceptorsite. It is shown that retention correlates very strongly with BzH and less strongly with BzC. The log k’ values need only two descriptive parameters, Le., BzH and 4, to give a good fit. As a whole, retention on the phenolic polymeric phase provides an efficient method for the measurement of relative hydrogen bond basicities. Hydrogen-bonding basicity has been implicated as one of the parameters needed to explain many physicochemical and biochemical properties such as water solubilities,14 octanolwater partition coefficient^,^-^ adsorption from water onto carbon,1° gas-liquid partition coefficients,ll retention in reversed-phase liquid chromatography,12-15 partitioning between blood and various body tissues,1”18 and the toxicity of (1) Taft,R. W.;Abraham,M.H.;Doherty,R.M.;Kamlet, M. J.Nature(London) 1985, 13, 384. (2) Kamlet, M. J.; Doherty, R. M.; Abboud, J.-L.; Abraham, M. H.; Taft, R. W. J . Pharm. Sci. 1986, 75, 338. (3) Kamlet, M. J.; Doherty, R. M.;Abboud, J.-L.; Abraham, M. H.; Taft, R. W. CHEMTECH 1986, 566. (4) Kamlet, M. J.; Doherty, R. M.;Abraham, M. H.; Carr, P. W.; Doherty, R. F.; Taft, R. W. J. Phys. Chem. 1987, 91, 1996. ( 5 ) Kamlet, M. J.; Abraham, M. H.; Doherty, R. M.; Taft, R. W. J . Am. Chem. SOC.1984, 106, 464. (6) Taft, R. W.; Abraham, M. H.; Famini, G. R.; Doherty, R. M.; Abboud, J.-L. J . Pharm. Sci. 1985, 74, 807. (7) Kamlet, M. J.; Doherty, R. M.; Carr, P. W.; Mackay, D.; Abraham, M. H.; Taft, R. W. Enuiron. Sci. Techno/. 1988, 22, 503. (8) Kamlct, M. J.; Abraham, M.H.; Carr, P. W.; Doherty, R. M.; Taft, R. W. J . Chem. Soc. Perkin Trans. 2 1988, 2087. (9) Kamlet, M. J.; Doherty, R. M.; Abraham, M. H.; Marcus, Y.;Taft, R. W. J . Phys. Chem. 1988, 92, 5244. (10) Kamlct, M. J.; Doherty, R. M.; Abraham, M. H.; Taft, R. W. Carbon 1985, 23. . 549. . (11) Kamlet, M. J.; Taft, R. W.; Carr, P. W.; Abraham, M. H. J. Chem. SOC. Faraday Trans. I 1982, 78, 1689. (12) Sadek,P.C.;Carr,P.W.;Doherty,R.M.;Kamlet,M.J.;Taft,R. W.;Abraham, M. H. Anal. Chem. 1985, 57, 2978.
-..
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many compounds to various organi~ms.l~-~l The hydrogen bonding basicities of bases, which make up the fundamental blocks of a drug, greatly affect the drug’s biological activities. In view of this, there have been many efforts by physical organic and inorganic chemists to establish scales of relative solute basi~ity.~~-~O Kamlet and Taft defined a j3 scale of solvent hydrogen bond (HB) acceptor strength based on the spectroscopic behavior of a set of judiciously chosen indicators in bulk solvents.22 They found that the j3 values for the bulk solvent (except for amphiprotic substances that self-associate through hydrogen bonding interactions) could be used, to good approximation, as an estimate of the corresponding property of an infinitely dilute and were therefore taken as a measure of a solute hydrogen bond basicity, denoted P2. (B was no subscript denotes bulklsolvent HB basicity, while P with the subscript 2 denotes solute HB basicity at infinite dilution.) Subsequently, Kamlet et al. back-calculated many additional j32 values from regressions of octanol-water partition coefficients (KO,,,) and reversed-phase liquid chromatographic capacity factors or estimated them based on a set of parameter (13) Carr, P. W.; Doherty, R. M.; Kamlet, M. J.; Taft, R. W.; Melander, W.; Horvath, C. Anal. Chem. 1986,58, 2674. (14) Park, J. H.; Carr, P. W.; Abraham, M. H.; Taft, R. W.; Doherty, R. M.; Kamlet, M. J. Chromatographia 1988, 25, 373. (15)Leahy, D. E.; Carr, P. W.; Pearlman, R. S.; Taft, R. W.; Kamlet, M. J. Chromatographia 1986, 21, 473. (16) Kamlet, M. J.; Abraham, D. J.; Doherty, R. M.; Taft, R. W.; Abraham, M. H. J. Pharm. Sci. 1986, 75, 350. (17) Kamlet, M. J.;Doherty,R. M.;Fiserova-Bergerova,V.;Carr,P. W.;Abraham, M. H.; Taft, R. W. J. Pharm. Sci. 1987. 76, 14. (18) Abraham, M. H.; Kamlet, M. J.; Taft, R. W.; Doherty, R. M.; Weathersby, P. K. J . Med. Chem. 1985, 28, 865. (19) Kamlet, M. J.; Doherty, R. M.; Veith, G. D.; Taft, R. W.; Abraham, M. H. Enuiron. Sci. Techno/. 1986, 20, 690. (20) Kamlet, M. J.; Doherty, R. M.; Abraham, M. H.; Taft, R. W. Quanr. Struct.Act. Relat. 1987, 7, 71. (21) Kamlet, M. J.; Doherty, R. M.; Taft, R. W.; Abraham, M. H.; Veith, G. D.; Abraham, D. J. Enuiron. Sci. Techno/. 1987, 21, 149. (22) Kamlet, M. J.; Taft, R. W. J . Am. Chem. SOC.1976, 98, 377. (23) Taft, R. W.; Kamlet, M. J. J . Am. Chem. Soc. 1976, 98, 2886. (24) Terent’ev, V. A. Rum. J . Phys. Chem. 1972, 46, 1103. (25) Sherry, A. D.; Purcell, K. F. J . Phys. Chem. 1970, 74, 3535. (26) Sherry, A. D.; Purcell, K. F. J. Am. Chem. SOC.1972, 94, 1853. (27) Abboud, J. M.; Frange, B.; Benamou, C.; Bellon, L. J . Org. Chem. 1982,47, 4553. (28) Zeegers-Huyskens, T. J . Mol. Liq. 1986, 22, 191. (29) Drago, R. S.; Wayland, B. B. J. Am. Chem. SOC.1965, 87, 3571. (30) Abboud, J. M.; Sraidi, K.; Guiheneuf, G.; Negro, A.; Kamlet, M. J.; Taft, R. W. J . Org. Chem. 1985, 50, 2870. (31) Taft, R. W.; Abboud, J. M.; Kamlet, M. J.; Abraham, M. H. J . Solurion Chem. 1985, 14, 153. (32) Abraham, M. H.; Whiting, G. S.; Fuchs, R.; Chambers, E. J. J . Chem. Soc. Perkin Tram. 2 1990, 291. (33) Frange, B.; Abboud, J. M.; Benamou, C.; Bellon, L. J . Org. Chem. 1982.47, 4553. 0003-2700/94/03650450$04.50/0
0 1994 Amerlcan Chemical Soclety
estimation Although the 02 values determined therein are in accord with chemical intuition, they must be regarded as highly empirical estimates and not direct experimental measurements. Maria and Gal et a1.36were able to rationalize a wide variety of different solute basicity scales via a principal components analysis of different basicity dependent properties (BDPs). A BDP can be identified by two abstract factors, F1 and F2 through a double regression (eq 1): BDP = BDP,
+ SIFl+ S2F2
(1) F Irepresents a combination of electron delocalization (covalent) and electrostatic effects, while F2 represents only electrostatic effect. The ratio of constants S1 and S2 reflects the sensitivity of a BDP to the ratio of covalent vs electrostatic effect and, thus, provides information about family dependencies between classes of basic compounds, e.g., nitrogen vs oxygen acceptors. Based on this work, Abraham et al.37 showed that hydrogen bond formation generally corresponds to a specific combination of F1 and F2 and that hydrogen bonding is primarily, but not exclusively, an electrostatic interaction. Subsequently, Abraham et al. developed a solute HB basicity scale, 8zH,based on the measurement of the 1:l hydrogen bond complex formation constants 6, between a series of HB acceptors (i) and a number of HB donors in a relatively inert solvent such as carbon tetrachloride:38J9
+
-
A-H B A-H...B (2a) where A-H is a hydrogen bond donor (acid) and B is a hydrogen bond acceptor (base). They found that for a large number of bases, plots of log 6 for a set of acids and a given base vs log Ki for the same set of acids and an arbitrary reference base are straight lines that intersect in a relatively narrow intervalat logb=-1.1 (kO.1). Basedontheexistence of these linear relationships, they then derived a set of log K! values that define relative hydrogen b a s i ~ i t i e s , 3where ~+~~ L ~ a n D~characterize d thedonor (seeeq2b). Asolute basicity scale ,82H was then constructed from the K! values using eq 2c: log K‘ = LA lOgKi + D A
02H= [logK:
+ 1.1]/4.636
(2b) (2c)
Later, Abraham et al.40 used ”inverse” multiple linear regression analysis to develop a set of effective solute basicity parameters denoted using the extensive gas chromatographic data of McReynolds and others. &9zH differs from @zH in which the former considers the situation in which a basic solute molecule is surrounded by an excess of HB donors. Thus, a solute with several acceptor sites can form HB (34) Kamlet, M. J.; Doherty, R. M.; Carr, P. W.; Mackay, D.; Abraham, M. H.; Taft, R. W. Enuiron. Sci. Technol. 1988, 22, 503. (35) Kamlet, M. J.; Abboud, J. M.; Abraham, M. H.; Taft, R. W. J . Org. Chem.
1983,48,2m. (36) Maria, P.-C.; Gal, J.-F.; Franceschi, J.; Fargin, E. J. J . Am. Chem. Soc. 1987, 109, 483. (37) Abraham, M. H.; Grellier, P. L.;Prior, D. V.; Morris, J. J.; Taylor, P. J.; Maria, P.-C.; Gal, J.-F. J. Phys. Org. Chem. 1989, 2, 243. (38) Abraham, M. H.; Grellier, P. L.; Prior, D. V.; Morris, J. J.; Taylor, P. J.; Laurence, C.; Berthelo, T. M. Tetrahedron Lett. 1989, 30, 2571. (39) Abraham, M. H.;Grellier, P. L.; Prior, D. V.; Morris, J. J.; Taylor, P. J. J . Chem. Soc. Perkin Trans. 2 1990, 521. (40)Abraham, M. H.; Whiting,G.S.;Doherty, R. M.;Shuely, W. J.J. Chromarogr. 1991, 587, 213.
complexes with stoichiometries higher than 1:l. While 02H only encompasses the HB basicity due to 1:1 complexes, results from the total basicity of all HB acceptor sites in a solute. Recently, Li et al.41 developed an empirical free-energyrelated scale of relative HB acceptor basicity, @, for a wide variety of solutes based on their retention in gas chromatography. This chromatographic basicity scale was deduced from the differential solute retention on two stationary phases that differ only in their hydrogen bond donating ability. 4-Dodecyla,a-bis(trifluoromethy1)benzylalcohol, an extremely powerful HB acid, was used as the active hydrogen bond donor stationary phase while a related ether, 4-dodecyl-a,a-bis(trifluoromethy1)benzyl methyl ether, was taken as a chemically similar but hydrogen bond inert reference stationary phase. In that work, test solutes were present at infinite dilution in contact with pure bulk donor (stationary phase), thus HB complexes higher than 1:l stoichiometry could be formed. In the present study, fl2H and /3zC scales are used to evaluate the experimental data. These two basicity scales are different due to the nature of their derivation. In Abraham’s approach, there was little or no excess of donor relative to the acceptor, thus only 1:1 complexes could be formed at the most favorable site on the solute. In contrast, in Li’s the test solutes were present at infinite dilution in the pure bulk donor, thus the great excess of the donor concentration over that of the solute allowed multicomplex formation with multiacceptor site solutes. Thus, for virtually all compounds with two or more nonbonded pairs of electron, 0zCvalues are larger than OzH values. As a result, both scales can be represented in terms of F1 and F2,41but do not mutually correlate well in general. In addition, due to the bulky trifluoro groups near the O H donor, Li’s gas chromatographic approach may be more sensitive to front-strain effects than is the complex formation in Abraham’s 1:1 complex approach. In this paper, we explore the potential of liquid chromatography as a method for developing a relative HB solute basicity scale. In addition to its comparable precision and simplicity, liquid chromatography is not limited to volatile compounds, as is gas chromatography. This advantage is very important because basic compounds that are of biological, medicinal, and environmental interest are frequently nonvolatile. For the purpose of studying solute HB basicity, we hoped to design a liquid chromatographic system such that the retentions are mainly, if not exclusively, due to the basicity nature of the solutes. Ideally, such a liquid chromatography system has to fulfill three requirements. First, the stationary phase should provide strong HB donor sites while the mobile phase is incapable of donating hydrogen bonds. Then, retention will be a strong function of solute HB basicity. Second, the ability of both the stationary and mobile phases to interact with solutes via processes other than HB donating interaction must be similar. This condition will force other effects to cancel out and leave the solute HB basicity as the only factor influencing the retention. Third, the capacity factors should vary significantly. In this study, we designed both the stationary and mobile phases such that the above requirements will be fulfilled. The stationary phase used here is a macroporous vinylphenolethylstyrene-divinylbenzene polymeric resin (see 1)where the (41)Li,J.;Zhang,Y.;Ouyang,H.;Carr,P.W.J.Am.Chem.Soc.1992,114,9813.
Analytical Chemistty, Vol. 66,No. 4, February 15, 1994
451
~~
Q
‘“6-
components stationary phase
OH
CHjCHp
~
Table 1. Solvatochromlc Parameters of Components of Stationary and Moblle Phases.
CHz-CH
0.45
I
mobile phase
ff
benzeneb phenolc hexaneb ethylacetateb
0.00 0.69 0.00 0.00
B
**
0.10
0.59 O.Wd 0.00 0.55
0.23e
0.00 0.45
*
a All values are from ref 42 unless otherwiseindicated. Acting aa solvent. Acting as solute. Reference 43. e Reference 41.
pendant phenol group is a powerful HB donor. The numbers 0.12, 0.43, and 0.45 in I indicate the weight percentage of ethylstyrene, divinylbenzene, and vinylphenol, respectively. The mobile phases were mixtures of hexane and ethyl acetate. The solvatochromic parameters of model species were considered to estimate the chemical properties of both the stationary and mobile phases. The HB acidity (a),HB basicity (p),and dipolarity/polarizability (a*)of the main components of the stationary and mobile phase are given in Table 1. Again notice that solvatochromic parameters with no subscript denote bulk properties, while those with subscript 2 denote solute properties at infinite dilution. The pendent phenolic functionalities on the stationary phase contain acidic hydrogen atoms (a= 0.69),42 while both hexane and ethyl acetate are incapable of donating hydrogen bond (a= 0.00). This large difference in HB acidity between the stationary and mobile phases is highly desirable and ought to cause retention to be strongly dependent on solute basicity. On the other hand, the hydrogen bond basicities of the mobile-phase components (@hexane = 0.00, @ethyl acetate = 0.45) and stationary-phase components (Pbnzene= 0.10, @phenol = 0.23) do not differ significantly. Thus, the dependence of solute retention on the complimentary property, Le., the solute HB acidity (a2) should be insignificant. Finally, benzene and phenol have higher a* values than hexane and ethyl acetate. This makes the stationary phase slightly more dipolar/polarizable than the mobile phase, and we expect a small positive dependence of solute retention on solute dipolarity/polarizability (7r*2). Overall, we anticipate solute HB basicity to be the major retention-governing factor. In the present study, we analyze the experimental data based on the linear solvation energy relationships (LSER) developed by Kamlet, Taft, and their c o - w o r k e r ~ .This ~~~~~ approach has been successfully used to correlate, rationalize, and explain more than 600 different chemical systems.44 Among them, reversed-phase liquid chromatography has been studied extensively by the LSER m e t h ~ d . ~ J ~Based - l ~ on this approach, a free-energy term can be correlated with various fundamental molecular properties. In the present study, the logarithmic capacity factors on the phenolic column, which are free-energy terms, can be deconvolved into several molecular interaction terms: log k’ = SPo + m(V,/ 100)
+ sa*2+ a a 2 + bP2
(3)
where V, is the intrinsic molar volume of solute calculated using McGowan’s m e t h ~ d ;while ~ ~ ,r*2, ~ ~ ( ~ 2 and , 8 2 are the solute dipolarity/polarizability, HB acidity, and HB basicity, respectively. SPo is the intercept of the LSER equation. In ~~
~
(42) Marcus, Y. J . Solution Chem. 1991, 20, 929. (43) Li, J.; Zhang, Y.; Dallas, A.; Carr, P. W. J . Chromatogr. 1991, 550, 101. (44) Kamlet, M. J.; Taft, R. W. Acta Chem. Scand. B 1985, 39, 611. (45) McGowan, J. C. J . Chem. Technol. Biotechnol. 1984, 34A, 38. (46) Abraham, M. H.; McGowan, J. C. Chromatographia 1987, 23, 243.
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this paper, we used the a*2, and a2 parameters obtained from gas chromatographic measurement^?^ which were designated a*zCand azC,respectively. For the 02 parameter, we used @2c 41 and p2H,39which werediscussed above. The superscripts C and H denote the origins of the parameters. In addition to these conventional solvatochromic parameters, a scale of the steric hindrance effect, E, is included in this work. E,, or steric substituent constant, was developed by Taft4’ as a semiquantitative measure of the total steric effect associated with a given substituent relative to a standard. E , of a substituent, R in RCOOR’, is expressed as E, log (k/ko) where k and ko are the rate constants for the acidic hydrolysis of a substituted ester (RCOOR’) and a reference ester (CH3COOR’), respectively,under the same experimental conditions. In the present study, we adopt and extend the E, scale such that it applies for compounds of different classes such as pyridines, amines, anilines, and amides. However, only a few modifications are needed. Previously, the E, parameters have been used to successfully correlate retention of 2-alkylpyridines on silica and alumina columns.48 Note that E , is quite different from the length/breadth ratio49which accounts for the shape and bulkiness of an entire molecule.
EXPERIMENTAL SECTION The packing materials used here are monodisperse 10-gm spherical, macroporous poly (vinylphenol-co-ethylstyrene-codivinylbenzene) beads. The composition of the monomer units in the copolymer is 45.0 wt % vinylphenol (3.75 mmol/g), 42.7 wt % divinylbenzene (3.28 mmol/g), and 12.3 wt % ethylstyrene (0.93 mmol/g). The specific surface area and average pore size determined using the BET method are 151 m2/g and 11 1 A, respectively. The design and synthesis of the monomers and polymers were discussed in detail elsewhere.5c52 The polymer beads were packed into HPLC columns using a pressurized upward-slurry technique. About 0.7 g of polymer was suspended in 15 mL of a “balanced-density solvent mixture” with the aid of sonication. The balanced-density solvent mixtures3 used here was 1.2:1.O (v/v) 2-propanolcarbon tetrachloride. Columns ( 5 cm X 4.6 mm i.d.) were packed for 1 h at a pressure of 3000 psi with 2-propanol as the packing solvent. The column was then brought to the analytical mobile-phase composition via a gradient. (47) Taft, R. W., Jr. Steric Effects in Organic Chemistry; Newman, M. S.,Ed.; John Wiley & Sons, Inc.: New York, 1956; pp 597603. (48) Chumakov, Yu. I.; Alyabyeva, M. S.;Kaboulov,B. D. Chromatographia 1975, 8, 242. (49) Wise, S.A.; Bonnett, W. J.; Guenther, F. R.; May, W. E. J . Chromatogr. Sci. 1981, 19, 457. ( 5 0 ) Rolls, W. A.; Frkhet, J. M. J . Am. Chem.SOC.Polym. Mater. Sei. Eng. 1987, 56, 745. (51) Rolls, W. A.; Svec, F.; FrQhet, J . M. J . Polymer 1990, 32, 165. (52) FrQhet, J. M. J.; Eichler, E.; Ito, H.; Wilson, C. G. Polymer 1983, 24, 995. ( 5 3 ) Rolls, W . A.; Frkchet, J . M . J . J . Chromatogr. 1990, 5 0 4 , 97.
All liquid chromatographic measurements were made at 40.0 (k 0.05) “ C in three different mobile-phase compositions: 10/90,20/80, and 90/10 (v/v) ethyl acetate-hexane. The reported capacity factors were averages of at least triplicate determinations. The void volume of the system was taken as the peak produced by a hexane-enriched mobile phase. All measurements were made with a Hewlett-Packard 1090 liquid chromatograph with an external refractive index detector (HP 1047A). Retention times were taken as the peakmaxima reported by a Hewlett-Packard 9 153data system. The organic solvents hexane and ethyl acetate used were HPLC-grade and were obtained from EM Science (Cherry Hill, NJ) and Mallinckrodt, Inc. (Paris, KY), respectively. All solutes were obtained commercially. All samples were prepared in the mobile phase under study. For the analysis of proton-transfer effect, UV spectra of equimolar mixtures of phenol-pyridine and phenol-triethylamine in 90/ 10 (v/v) ethyl acetate-hexane were obtained and compared to that of the sodium phenoxide solution. Samples were scanned using a Hewlett Packard 8452A diodearray spectrophotometer, and a 90/10 (v/v) ethyl acetatehexane mixture was used as a blank solution.
RESULTS AND DISCUSSION Preliminary studies of retention on the phenolic column described above were carried out with mobile phases comprised of 10/90 and 20/80 (v/v) ethyl acetatehexane. Least-squares regression, based on the LSER model, such as given in eq 3, showed that the log k’values at 10/90 (v/v) ethyl acetatehexane depend mainly on solute HB basicity (@2c) and polarity/polarizability (.lr*zC) and much less significantly on solute HB acidity (wC)and intrinsic molar volume ( Vx).Upon increasing the volume fraction of ethyl acetate in the mobile phase to 20%, the dependence of the log k’values on ( ~ and 2 ~ 7r*zC weakened slightly due to the increased HB basicity and dipolarity/polarizability of the stronger mobile phase (see Table 1). Based on the above analysis, we tried to eliminate the dependence of log k‘on solute HB acidity and dipolarity/ polarizability by increasing the amount of ethyl acetate in the mobile phase. Upon further investigation, we found that the log k’ values in 90/ 10 ethyl acetate-hexane do not depend significantly on any solute parameters other than solute HB basicity (see below). We have now collected retention data for 53 judiciously chosen compounds whose log k’values and corresponding solute solvatochromic parameters are given in Table 2. Most of these compounds are very strong HB acceptors (high values of @zC and @zH) and span a wide range in other solute properties, such as V,, azC,and r*zc. In addition, they are all retained with reasonable k‘values (0.220) with a 90/10 ethyl acetate-hexane mobile phase. More importantly, these compounds span a variety of families of bases, including pyridines, anilines, amines, amides, alcohols, diazines, and cyclic oxygenic compounds; thus we will be able to test for the existence of class dependencies of the log k’ values. For simplicity, we first study the retention of pyridine derivatives that have only a single HB acceptor site (1-13 in Table 2). Because @2H is a measure of HB basicity where only one acceptor site is i n v o l ~ e d , ~it~was J ~ chosen as the explanatory parameter in the LSER regression of the log k’
values (see eq 4). In eq 4, and elsewhere, n is the number of test solutes, while sd and r indicate the average residual and correlation coefficient of the fit: log k‘ = (-2.20 f 0.15) + (3.98 f 0.26) n = 13,
sd = 0.082,
(4)
r = 0.9777
The above regression equation indicates that the dependence of log k’ on /3zH is extremely strong. It confirms prior qualitative statements as to the importance of solute HB basicity as a retention-controlling factor on a phenolic stationary phase.53 This is hardly surprising because Abraham’s @2H scale is consistent with and partly based on the formation of hydrogen bonded complexes with p h e n o ! ~ . ~ ~ . ~ ~ This strong dependence is highly desirable if the method is to be used for estimating unknown @2H values. However, the regression fit is not as good as has been observed in other studies of @zH and @2c.38,39,41 The modest correlation coefficient (0.9777) suggests that additional explanatory parameters may be needed to describe the log k’values. As mentioned above, neither V,, L Y ~ nor ~ , a*lC are significant variables. However, a detailed inspection of log k’values of compounds with identical @zH values leads us to hypothesize that steric hindrance due to substituent group(s) adjacent to the acceptor site suppress retention. First, 2,6-dimethylpyridine (log k’ = 0.255, see Table 2) was distinctly less retained than the analogous 2,4-isomer (log k’ = 0.460) despite their identical @zHvalues. We rationalize this as steric hindrance introduced by the presence of both the 2- and 6-substituted methyl groups in contrast to the 2,4-isomer whose acceptor site is only hindered by the 2-substituted methyl group (see below). The hypothesis of steric hindrance is further confirmed by the fact that 2,6-di-tert-butylpyridineeluted at the column dead volume (k’ = 0). The @2H value for 2,6-di-tert-butylpyridineis not available in the literature. However, it ought to be a better base than either of the two dimethylpyridines because a tertbutyl substituent is a better electron-donating group than is a methyl substituent. On theother hand, the tert-butyl groups are extremely bulky and totally hinder the access of the phenolic group to the acceptor site on the pyridine derivative, and thus this compound elutes at the dead volume. As explained above, Abraham’s @zH value^^^^^^ were derived from solution-phase experiments in which both the HB donor and the HB acceptor are dissolved and are totally free to move about and to interact via hydrogen bonding. Thus, the effect of steric hindrance exerted by the substituent groups adjacent to the acceptor site is smaller than encountered here with a tethered donor and does not so seriously influence @zH. This situation is vastly different from the HB interaction between the pendant phenolic groups of the stationary phase and the acceptor solutes in the present study. The pendant phenolic groups are bonded to a semirigid and nonliquid polymeric support. Their freedom of movement is highly limited. To interact via hydrogen bonding, an acceptor solute has to come to a definite orientation such that its acceptor site aligns with the donor site of the phenolic groups. An acceptor solute that has bulky substituent group(s) adjacent to its acceptor site has less chance of aligning its acceptor site with the donor site. As a result, hydrogen-bonding interaction between solute and stationary phase is suppressed and retention is reduced. AnaWicaIChemistry, Vol. 66,No. 4, February 15, 1994
453
Table 2. Solute Retention Data and Descrlptlve Parametem
no.
compound
log k’ 5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
pyridine 2-methylpyridine 3-methylpyridine 4-methylpyridine 2,4-dimethylpyridine 2-fluoropyridine 2-chloropyridine 3-chloropyridine 2-bromopyridine 3-bromopyridine 2-ethylpyridine 2,6-dimethylpyridine 2,4,6-trimethylpyridine 3-cyanopyridine aniline p-ethylaniline m-toluidine p-toluidine benzonitrile quinoline pyrimidine (1,3-diazine) pyrazine (1,l-diazine) pyridazine (1,a-diazine) tetrahydrofuran 1-butanol benzylalcohol dioxane 1-hexanol tetrahydropyran diethylamine triethylamine dibutylamine diisopropylamine tripropylamine tributylamine dimethylformamide dimethylacetamide NJV-diethylformamide N-methylacetamide NJV-diethylacetamide NJV-diphenylacetamide N-methylpyrrolidin-2-one N-methyl-2-pyridone N-methylimidazole dimethylsulfoxide 3-methoxyaniline 2-aminopyrimidine 1,1,6,6-tetramethyluea 4-ethylpyridine 3,5-dimethylpyridine 2,6-tert-butylpyridine triazine 4-cyanopyridine
0.327 0.328 0.376 0.453 0.460 -0.562 -0.426 -0.218 -0.413 -0.168 0.140 0.255 0.393 -0.336 -0.600 -0.641 -0.612 -0.551 -0.663 0.258 0.067 -0.044 0.567 -0.270 -0.386 -0.465 -0.369 -0.511 -0.405 0.684 0.186 0.326 0.363 -0.442 -0,381 0.558 0.783 0.412 0.682 0.612 -0.030 0.949 1.009 1.237 1.143 -0.544 0.318 0.633 0.392 0.445 -0.405 -0,299
vx
67.53 81.62 81.62 81.62 95.71 69.30 79.77 79.77 85.03 85.03 95.71 95.71 109.80 83.00 81.62 103.24 95.71 95.71 87.11 104.43 63.42 63.42 63.42 62.23 73.09 91.60 68.10 101.27 76.32 77.20 105.38 133.56 105.38 147.65 189.92 64.68 78.77 92.86 64.68 106.95 250.87 82.00 87.49 67.72 61.26 101.58 73.40 102.84 95.71 95.71 180.25 59.31 83.00
BZH
B2C
a# b
u*2c b
E6
0.62 0.62 0.62 0.66 0.64 0.43 0.45 0.49 0.44 0.51 0.60 0.64 0.69 0.44 0.38 0.42 0.40 0.42 0.42 0.63 0.53 0.48 0.64 0.51 0.45 0.42 0.47 0.45 0.48 0.70 0.67 0.71 0.67 0.58 0.60 0.66 0.74 0.67 0.72 0.73 0.64 0.76 0.76 0.81 0.78 0.40 0.61 0.74
0.90 0.98 0.98 1.07 1.07 0.68 0.72 0.76 0.70 0.74 0.98 0.75 0.75 0.79 0.42 0.47 0.45 0.45 0.40 0.90 1.13 1.05 0.95 0.61 0.52 0.51 0.79 0.51 0.61 0.93 0.64 0.87 0.90 0.61 0.58 0.97 1.06 0.97 1.06
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.20 0.17 0.19 0.19 0.00 0.00 0.00 0.00 0.00 0.00 0.31 0.43 0.00 0.34 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.45 0.00 0.00 0.22 0.25 0.00
0.60 0.60 0.60 0.60 0.61 0.67 0.73 0.73 0.76 0.76 0.61 0.62 0.64 1.16 0.76 0.77 0.76 0.76 0.85 0.65 0.90 0.90 0.90 0.27 0.30 0.71 0.45 0.33 0.27 0.10 0.02 0.13 0.12 0.03 0.05 0.81 0.80 0.85 0.80 0.84 1.35 0.45 1.00 0.88 1.00 0.99 1.37 1.20
0
1-06 0.85 1.10 1.00 1.20 1.42 0.44 1.20 1.20
-0.07 0 0 -0.07 -0.21 -0.24 0 -0.27 0 -0.36 -0.49 -0.49 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 -0.72 -1.08 -1.88 -1.86 -1.95 -2.82 -0.47 -0.715 -1.98 -0.48 -2.225 -3 -0,245 -0.245 -0.245 -0.49 -0.245 0 -0.94
*
a Log k’ values were obtained in a 1 0 (v/v) ethzl acetatehesane. Vxvalues were calculated wing McGowan’s method614 &H waa from ref 39. % !f, waa from ref 41 unless estimated. a2 and **aC were from ref 43 unless estimated. E, waa from ref 47.
The above explanation is very consistent with the well-known sensitivity of liquid-solid chromatography to geometric isomeri~ation.~~ If the above hypothesis concerning the effect of steric hindrance on retention is true, then the addition of a steric hindrance factor as an explanatory parameter to eq 4 should substantially improve the fit. We adopted Taft’s steric substituent constant,E,ara$fex$snddit to t h p m e n t purpose. The Eo values used are sbws is Table 2. Notice that E, values are negative becauscsterkhindrance suppresses the interactions of interest. In the present study, the hydrogen bonding interaction between the pendant phenolic groups on (54) Snyder, L. R. Principles of Adsorption Chromatography: The Separarion of Nonionic Organic Compounds; Marcel Dekker, Inc.: New York, 1968; pp 307-3 15.
454
Analytical Chemistry. Vol. 66,No. 4, February 15, 1994
the stationary phase with the acceptor site on the solutes, and thus retention, ought to diminish as the steric factor increases. The LSER equation for log k’vs /3zH and E8 is shown below: log k‘ = (-2.18
f 0.09)
+ (4.05 f 0.16)bZH+ (0.34 f 0.O8)Es (5)
n = 13,
sd = 0.050,
r = 0.9927
The regression fit was greatly improved from sd = 0.082 (in eq 4) to sd = 0.051 (in eq 5 ) upon addition of Es as an explanatory parameter. The e coefficient (that is the dependence on E,) is significant but much smaller than the bH coefficient (dependence on pzH). This implies that the steric hindrance effect is real but minor. The fact that the e
~
Tabh 3. LSER Equatlonr of Different Ch.mloal Subgroups
eq
chemical groups
SP,.
bHb
5 6 7 8 9 10
one-site pyridines diazines oxygenic acceptor site compds amines amides amides
-2.18 f 0.09 -2.01 f 0.11 -1.41 f 0.37 -3.85 f 1.27 -2.22 f 0.81 -1.94f 0.41
4.05 f 0.16 4.03 f 0.48 2.18 f 0.80 6.48 f 1.71 4.24 f 1.13 -e
bcb
eb
sdc
IC
nd
0.34 f 0.08
0.050 0.054 0.055 0.160 0.095 0.057
0.9927 0.9931 0.8046 0.9604 0.9667 0.9882
13 3 6 6 6 6
10
2 -O
-e
2.59 f 0.39
10
0.16 f 0.12 0.14 f 0.04 0.09 f 0.03
a SP,is the intercept of the LSER equation. bH, bc, and e denote the dependence on BsH, &c, and E,, respectively. sd and r denote the average residual and correlation coefficient of the fit, respectively. d n is the number of compounds. e Deliberately exclude these terms in regressions.
coefficient is positive in sign confirms the idea that steric hindrance adjacent to an acceptor site suppresses its ability to interact with the stationary-phase phenolic groups and thereby reduces retention. Note that the regression intercept and the bH coefficient (eq 5) remain unchanged, within their standard deviations, relative to those given in eq 4. Equation 5 is an amazingly good result in that both the explanatory parameters ,9zH and E , were derived from experiments that involve solution-phase measurements and are totally independent of chromatography. Up to this point, we show that retention on the phenolic column is predominantly induced by the solute HB basicity and was suppressed by the steric hindrance adjacent to the acceptor site. Table 3 lists the LSER equations obtained in the study of various chemical classes, including the pyridine derivatives (eq 5 ) . The LSER equations for d i a ~ i n e s , ~ loxygenic -~~ acceptor site ~ o m p o u n d s amines,3G35 ,~~~~ and amides3"' subsets are given in eqs 6-9, respectively. Coefficients bH,bc, and e denote the dependence of log k'values on j3zH, and E,, respectively. The acceptor sites of the diazinesand oxygenic acceptor site compounds are free from steric hindrance and thus eqs 6 and 7 have no eE, term. For amines, the alkyl groups adjacent to the :NE site are responsible for steric effects that limit access to the acceptor site. For amides, both C=O and N are hydrogen bond acceptors. Because the C=O site is reported to be a stronger acceptor than the N site,56g57 the E, values for amides were assigned according to the steric hindrance effects experienced by the C=O site. Overall, the goodness of fit for those four equations is satisfactory in view of the small data size. However, most of the correlations are much poorer than that obtained in eq 5 . The diazine$'-23 have two nitrogen atoms, each of which bears a lone pair that functions as an acceptor site (see 11).
telrahvdroluran 1,3-diazine
II
CH3 N,N-dimethyl acetamide
lv
trialkyl amine
V
The bH coefficient for the diazines subset is the same as that for the pyridine subset. Since the bH coefficient is a measure of the strength and intensity of hydrogen-bonding interactions (55) Laurence, C.; Nicolct, P.: Helbert, M. J . Chem. Soc. Perkin Tram. 2 1986, 1081. (Sa) Pauling, L. Nature ofthe Chemfcal Bond; Cornel1 University Press: Ithaca, NY, 1948; pp 284-334. ( 5 7 ) Schciner, S.; Wang, L. 1. A m . Chem. Soc. 1993, 215, 1958.
between the solutes and the stationary phases, this leads us to consider two concepts. First, the bH coefficients for compounds with similar acceptor sites (aromatic N atom for both pyridines and diazines) are similar. In other words, the hydrogen bond interactions involved may show a family dependence. Second, if both of the acceptor sites on the diazines were to hydrogen bond with the stationary-phase phenolic groups, then the logarithmic k' would have to be approximately twice of that of a simple pyridine. However, this was not observed. Thus, the results suggest that the hydrogen bond interactions between the solutes and the phenol groups in the present study are mainly limited to a 1:l stoichiometry. For the oxygenic acceptor site compounds, each oxygen atom carries two lone pairs (see 111). The bH coefficients for both the oxygenic compounds and amines are different from that of the pyridine subset. The oxygenic compounds have two basic sites but a much smaller bH coefficient, whileamines with a single basic site have a much larger bH coefficient. This again suggests that the hydrogen bond interaction of this phenolic column is rather specific and shows a significant family dependence. Such family dependencies for hydrogen bond interactions have been observed e l s e ~ h e r e . ~ ~ ~ ~ ~ . ~ ~ Amides have both a nitrogen and an oxygen site that together provide three potential acceptor sites (see IV). However, the >C=O site is reported to be a stronger acceptor than the NR2 site.56J7 The bH coefficient for theamide subset is in the same range as the pyridine subset. Because bH shows family dependency, we cannot conclude whether the pendant phenol group hydrogen bonds to only the strongest acceptor site of an amide or to more than one acceptor site. To investigate this further, we regressed log k' values for the amides against ,9zC and E , (see eq 10 in Table 3). Recall that /3zC can account for both 1:1 and higher HB complexes/ and in the process of determining pf, an IR study showed that 1 mol of dimethylacetamide interacts with 2 mol of a hydrogen bond donor. Equation 10 gave a better fit than did eq 9. This result suggests that some degree of multiple hydrogen bond interaction took place. The e coefficient for the amine subset is virtually insignificant. This may be due to the molecular configuration (see V) such that the substituted alkyl groups are positioned far from the :N= site and thus exhibit minimal steric hindrance. The e coefficient for the amides subset is significant but much smaller than that of the pyridine subset. Again, the need for a steric hindrance term indicates that the stationary-phase polymer is rigid and the pendant phenol groups, relative to solution-phase phenol moieties, are limited in their motion. Analytical Chemistry, Vol. 00, No. 4, Februaty 15, 1994
455
Table 4. LSER Equatlons of Subsets Accordlng to Number of Acceptor Sltes and Sterlc Hlndrance
eq
subsets
SP,'
bH"
11 12 13 14 15 16 17 18 19 20
single site unhindered single site unhindered single site single site single site single site multi-site unhindered multi-site unhindered multi-site multi-site
-2.33 f 0.11 -1.40f 0.08 -2.11 f 0.19 -1.43 f 0.17 -2.37 f 0.11 -1.47 f 0.17 -2.51 f 0.28 -1.02 f 0.22 -2.40f 0.10 -1.44f 0.24
4.23 f 0.22
bc" 1.78f 0.11
3.68f 0.34
5
1.86f 0.22
4.33 f 0.20
9
1.86f 0.22
*
4
*
9
4.71 0.56
9
0.21 f 0.03 -0.06 f 0.06 4
1.08 f 0.27
4.50 0.18 9
5
9
9
9
9
4
5
5
ea
1.82 f 0.26
5
0.15 f 0.03 -0.03 f 0.09
sd'
0.075 0.088 0.182 0.225 0.099 0.224 0.125 0.223 0.104 0.324
P
na
0.9878 0.9832 0.9159 0.8685 0.9772 0.8756 0.9418 0.7994 0.9855 0.8501
11 11
25 25 25 25 11 11
23 23
Footnotes are as described in Table 3. Table 5. LSER Equatlonr of All Compound8 wlth Mnerenl Explanatory Parameters
eq 21 22 23 24 a
chemical groups all compounds all compounds all compounds all compounds
SP,.
b?f'
-2.22 f 0.12 -1.42 f 0.12 -2.44 f 0.08 -1.44 f 0.13
3.98 f 0.20 5
4.52 f 0.15 9
5
1.82 f 0.15 4
1.82f 0.16
ea 4 9
0.19 f 0.02 -0.05 f 0.05
sd'
P
na
0.177 0.260 0.113 0.267
0.9455 0.8784 0.9780 0.8709
48 48 48 48
Footnotes are as described in Table 3.
Table 4 gives the LSER equations of subsets subdivided according to the number of acceptor sites and steric hindrance experienced by the solutes. We first examined the regression of the log k 'values of all single-site (regardless of functionality), sterically unhindered compounds against PzH and @, separately (see eqs 11 and 12). These compounds include benzonitrile, quinoline, various pyridines, and aniline derivatives. The regressions fit for both the equations are satisfactory and equally good. For single-site compounds, both /32H and /32c measure the HB basicity for 1:l HB complexation. We next investigated all single-site acceptors, including both sterically hindered and unhindered compounds against &H and /32c (see eqs 13 and 14). This subset is primarily comprised of pyridines, anilines, and amines. The fits are much poorer because steric hindrance was not taken into account. Upon addition of E, as an explanatory parameter, the fit using P2H improved greatly (compare eqs 13 and 15). However, the fit for eq 15 is not as good as that shown in eq 5 . Nonetheless, the intercept, the bH coefficient, of eq 15 is similar to that observed with the pyridine subset. On the other hand, the use of E, does not improve the goodness of fit for the equation based on @2c (see eq 16). The eE, term for eq 16 has the wrong sign and is not significant at all. This suggests that Abraham's PzH is superior to Li's /3zC in this system comprised of single-site acceptors. We next examined multisite, sterically unhindered compounds against PzH and /3zC, separately (see eqs 17 and 18). These compounds include diazines, alcohols, and other oxygenic compounds. The regression fit for both equations are poor, and the one based on /32c is extremely bad. The bH coefficient for eq 18 is only slightly larger than that for the pyridine subset, although these are multiacceptor species. This again indicates that most of the hydrogen bond complexes formed with the phenol phase are limited to a stoichiometry of 1:l. We then investigated all multisite, sterically hindered and unhindered compounds (see e q s 19 and 20). Compounds like pyrrolidinone, pyridone, and imidazole, urea, and amide 456
bc'
Analyticai Chemistry, Vol. 66,No. 4, February 15, 1994
derivatives were added to the regression. The fits based on
PzH and E, are satisfactory but poorer than those of eq
5.
Nonetheless, both the bH and e coefficients of eq 19 are different from those seen in the pyridine subset. The full data set (1-48 in Table 2) was then studied. We excluded solutes with small k'values (less than 0.2) and those for which P2H values are not available. The LSER equations against P2H, @2c, and E, are given in Table 5 . Notice the range of log k'is much wider with the full data set as compared to the limited sets described in Table 3. Comparison of eq 21 and eq 22 shows that the dependence of log k' values on P2H is much stronger than on P2c. In addition, the regression fit is considerably better. To take steric hindrance into account, E, was added as a descriptive parameter to both the equations (see eqs 23 and 24). The goodness of fit for the equation against PzH was significantly improved, but the bH and e coefficients are now somewhat different from those ineq 5 . The addition of E, to the regression against P2c had no effect on the regression fit nor on the intercept or the bC coefficient. This confirms the idea that PzC is not a suitable explanatory parameter in this case and indicates that higher complexes were not important with this phenolic stationary-phase system. In previous we suggested that 8zCallows for the formations of both 1:1 and higher complexes where HB donors are in excess. In the present case, the basic solute is at infinite dilution relative to the phenolic groups which are present in vast excess. However, formation of higher complexes seems to be limited because the phenol groups are attached to a more rigid solid. On the whole, eq 23 gave the best fit in describing the retention of basic compounds on the phenolic stationary phase. Notice that the b coefficient is much higher than the corresponding coefficients obtained in any solubility, octanolwater partitioning, or RPLC study. 1-91,2-15 This indicates a very strong dependence on solute basicity as desired. However, the goodness of fits in eq 23 was not nearly as good as those obtained in prior work,38,39v41 and this suggests the need for additional explanatory factors. Other solute explanatory variables denoting hydrogen bond acidity (cyzc), dipolarityl
polarizability ( ~ * 2 ~ and ) , size (V,), were added separately to eq 23. The regression results are shown below. log k' = (-2.47 f 0.09) + (4.55 f 0.15)&H+ (0.18 f 0.02)Es (0.12 f 0.15)~~:
+
n = 48,
sd = 0,114,
r = 0.9783
(25)
log k' = (-2.54 f 0.08) + (4.48 f 0.13)BzH+ (0.17 f 0.02)Es + (0.17 f 0.05)~*:
n = 48,
sd = 0.100,
r = 0.9834
(26)
log k' = (-2.17 f 0.12) + (4.35 f 0.15)j3,H+ (0.10 f 0.04)E, + (-0.23 f 0.08)Vx/100
n = 48,
sd = 0.105,
r = 0.9814
(27)
For eqs 25-27, the improvements in the goodness of fit compared to eq 23 are not statistically significant at the 95% confidence interval. The insignificant dependence of retention on cqC suggests that the hydrogen bond basicity of both phases are similar and cancel, as predicted above. A rather weak dependence on a*2Cis expected because the phenyl rings of the stationary phase and the polar phenolic groups are susceptible to dipolarity/polarizability interactions with the solutes. In eq 27, the dependence on V, appears to be significant because of its covariance with E,. Notice the e coefficient became smaller upon adding V, to the variable set (compare with eq 23). Overall, neither CY^, ~ * 2 nor ~ , V, significantly improves the fit. Thus, we conclude that retention on the phenolic column depends primarily on solute HB basicity and less so on the steric effect experienced by the acceptor site@). To discern whether any proton transfer took place between the phenols and the basic solutes, a UV spectroscopic study was carried out. Phenoxide cations absorb at 294 nm. However, there was no absorbance observed for the phenolpyridine and phenol-triethylamine mixtures at that wavelength. Thus, we conclude that there was no proton transfer between phenol and pyridine or triethylamine. Because triethylamine is one of the strongest Br~nstedbases among (58) Wehrli, A,; Hildcbrand, J. C.; Keller, H. P.; Stampfli, R.; Frei, R. W. J . Chromatop. 1978, 149, 199.
all the compounds used in this study, we conclude that the proton-transfer phenomenon does not take place between the solutes and phenolic functions on the stationary phase. Overall, use of retention on a phenolic phase is superior to use of retention in RPLC9g34J5for the estimation of solute HB basicity. In view of the very large value of the slope vs @zH compared to the other coefficients, back-calculated j32 values will be less sensitive to errors in estimation of V, and r*zCin comparison to results in RPLC. Perhaps more importantly, retention of pyridines and amines on this phenolic phase were not complicated by the very strong silanophilic interactions between the column supports and the solutes, as would have happened in the case of silica-based RPLC columns.58
CONCLUSIONS Retention of single-site, nonsterically hindered compounds on the phenolic polymer column depends only on the solute hydrogen bond basicity, &H. The regression fit for data limited to one chemical group was excellent and deteriorates upon incorporation of many chemical classes in a single equation. For a wide variety of chemical compounds, retention depends on solute hydrogen bond basicity (@zH) and the steric hindrance adjacent to the acceptor site (Es). Other solute parameters such as solute size, dipolarity/polarizability, and hydrogen bond acidity are not significant in governing retention. There is evidence for family dependence for nitrogenic and oxygenic acceptor site compounds. There is also evidence for a minor amount of multiple hydrogen bond complexation. However, the bH coefficients for the single acceptor and multiacceptor site subsets do not differ significantly. As a whole, retention on the phenolic polymeric packing provides an efficient method to the rapid estimation of the relative hydrogen bond basicity of strongly basic compounds. We project that this method will provide a means for measuring hydrogen bond basicities for many nonvolatile compounds that are of pharmaceutical and biological interest. ACKNOWLEDGMENT This work was supported by Grant CHE-8616597 from the National Science Foundation to the University of Minnesota and by Grant GM44885-01 from the National Institutes of Health to Cornel1 University. Received for review May 24, 1993. Accepted November 17, 1993." ~~
* Abstract
published in Aduonce ACS Abstrocts, January 1 , 1994.
Analytical Chemistry, Vol. 66, No. 4, February 15, 1994
457
