Anal. Chem. 1989, 61, 1749-1754
From this study it may be concluded that both neutral and charged amino acids in the microenvironment surrounding a histidine center can secondarily influence chromatographic retention of a protein by moderating histidine interaction with an IMAC column. The exact mechanism by which other amino acids moderate the interaction of histidine with an IMAC column remains to be determined. It may be concluded that IMAC can be a powerful technique for recognizing changes in protein structure within 15 A of histidine centers in proteins providing the histidine residue is recognized by the IMAC column. Moreover, it has been demonstrated that IMAC is an effective analytical technique for the separation of geneticay engineered proteins differing by as little as a single methylene group in a protein of 27 500 M,.
ACKNOWLEDGMENT The gracious gift of the subtilisin samples from Genencor, Inc., was appreciated. We are indebted to Tom Porter for his technical assistance. LITERATURE CITED Purdue University, 1988, personal communication. “Therapeutic Peptide and Proteins: Assessing the New Technlques“ Banbury Report #29, D. R. Marshad, Ed.. 1988. (2) -thing. MJ.; McCammon, K.; Sambrook. J. CeU 1988.48.939-950. (3) Garnick, R. L.; Solli. N. J.; Papa, P. A. Anal Chem. 1988, 60, 2546-2557. (4) Regnier, F. E. Chromtcgraphfb 1987, 24, 241-251.
1749
(5) Porath. J.; Carlson, J.; Olsson, I.; Belfrage, 0.Nature 1975, 258, 598-599. (6) Hemden, E. S.; Porath, J. J . Chromatogr. 1985, 323, 255-264. (7) Porath, J.; Olln, B.; Granstrand, B. Arch. Blochem. Blophys. 1983, 225, 543-547. (8) Sulkowski, E. TI‘&, Blotechnol. 1985. 3 , 1-7. (9) Hemden, E. S.; Porath, J. J . Chrmtogr. 1985, 323, 285-272. (10) Kawasaki, 1.; Takahashi, S.; Ikeda, K. EW. J . Bbchem. 1985, 152, 361-371. (11) Bernardi, G. Methods €mymo/. 1973. 27. 471-479. (12) oorbmff, M. J. Anal. & & e m . 1984, 736 425-432. (13) GOrbUnOff, M. J. Anal. Bkchem. 1984, 736,433-439. (14) Gorbunoff, M. J.; Timasheff, S. N. Anal. Blochem. 1984. 736, 440-445. (15) Chicr. R. M.; Regnler. F. E. J . Chromatogr. In press. (18) Belew. M.; Yip, 1. T.; Anderson, L.; Ehmstrijm, R. Anal. Biochem. 1987. 764, 457-465. (17) Nabgawa, Y.; Yip, 1.-1.; Belew, M.; Porath, J. Anal. Biochem. 1988, 768, 75-81. (18) Yamakawa, Y.; Chlba, J. J . L/q. Chfomtcgr. 1988, 1 7 , 665-681. (19) Wright, C. S.; Alden. R. A.; Kraut, J. Nature 1969, 227, 235-242. (20) Russell, A. J.; Fersht, A. R. Nature 1987, 328, 496-500. (21) Chlcr, R. M.; Regnier. F. E.. submitted for publication in Anal. Chem. (22) Cossu. G.; Righetti, P. G. J . Chromtogr. 1987, 398. 211-216. (23) Codte. R.; Kuntz. I. D. Annu. Rev. Blophys. Bioeng. 1974, 3 , 95-128. (24) Monjon, B.; Solms, J. Anal. Blochem. 1987. 160, 88-97. (25) Alden, R. A.; Birktoft, J. J.; Kraut, J.; Robertus. J. D.; Wright, C. S. Bbchem. Blophys. Res. Commn. 1971, 45, 337-344. (26) Sawyer, L.; James, M. N. G. Nature 1982, 295, 79-80.
(1) Dixon, J.,
RECEIVED for review February 21,1989. Accepted May 17, 1989. Thii work was supported by NIH Grant Number 25431. This is paper No. 11929of the Purdue University Agricultural Experiment Station.
Subambient Temperature Modification of Selectivity in Reversed-Phase Liquid Chromatography Lane C. Sander* and Stephen A. Wise
Center for Analytical Chemistry, National Institute of Standards and Technology (formerly National Bureau of Standards), Gaithersburg, Maryland 20899
The effect of column temperature on selectlvlty was studied for the separatlon of polycycllc aromatlc hydrocarbon (PAH) mlxtures. Three commerclal columns prepared by uslng monomerlc and polymerlc surface modlflcatlon chemistry were employed. Selectlvlty was evaluated through the use of a three-component PAH mixture previously developed for phase type evaluation. Column selectivity was found to vary CO~~~LIOUSIY with temperature, regardless of the type of phase used. The shape recognltlon ablllty of each phase, Le., the ablllty of the phase to separate closely related Isomers, was observed to be highest at subambient temperatures. A model for the temperature Induced selectivity changes Is proposed based on the morphology of the bonded phase.
INTRODUCTION Unlike in gas chromatography, temperature often serves only a minor role in the development of separation methods in liquid chromatography (LC) (1). Instead, solute retention is usually controlled by easily adjusted parameters such as mobile phase composition and flow rate. In contrast to gas chromatography,the allowable range for column temperatures in LC is restricted by the properties of the mobile phase. Increased viscosity and freezing are limiting factors at low
temperatures; at high temperatures boiling is a limiting consideration (2). Separations are commonly carried out under ambient conditions, although greater reproducibility in retention and quantitation result when isothermal conditions are established (3). The use of elevated column temperature has been reported as a way of increasing separation efficiency ( 4 ) , particularly for large molecular weight solutes (5). Elevated column temperatures have also been used to advantage with solutes with limited solubility in the mobile phase, and in ion-exchange chromatography (5). Thermodynamic aspects of solute retention have been studied in some detail (6-11). In general, solute retention is inversely related to temperature, i.e., retention decreases with increasing temperature. Morel and Serpinet (22-26) and others (17-19) have presented evidence for the existence of phase transitions within the bonded phase, occurring over a narrow temperature interval. The presence of a “break” or offset in a plot of log k’vs 1/T (Van’t Hoff plot) indicates a change in the way in which the solute interacts with the stationary phase, i.e., a phase transition. Although the effect is of considerable theoretical interest, in practice the shift in retention times caused by the transition is small and does not apply if isothermal conditions are maintained. Other temperature-dependent changes have been reported. Stalcup et al. calculated relative enthalpies and entropies of transfer for
This article not subject to U.S. Copyright. Published 1989 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 81, NO. 15, AUGUST 1, 1989
various polycyclic aromatic hydrocarbons (PAHs) to describe retention mechanisms in methanol and acetonitrile environments (20). Chmielowiec and Sawatzky (11) calculated thermodynamic parameters for PAHs and observed changes in elution order as a function of temperature. These changes were attributed to “entropy dominated retention effects”. Changes in phase morphology that occur with temperature have been investigated by a variety of techniques including calorimetry (12,13,21,22),inelastic neutron scattering (23), nuclear magnetic resonance (NMR) spectroscopy (24-28), and infrared (IR) spectroscopy (29). Using an NMR technique, Claudy and co-workers (13) concluded that bonded phase chain mobility is increased at elevated temperatures. As might also be expected, chain mobility increases with the distance along the chain from the silica surface (27). Sander et al. (29) compared the IR spectra of alkyl-bonded silicas a t various temperatures to the spectra of the corresponding alkyl silanes and alkanes. Spectral changes resulting from crystal lattice ordering were observed in the neat liquid/solid phase spectra but not in bonded phase spectra. However, overall bonded phase ordering resulting from a reduction in gauche-gauche (tggt, bend), gauche-trans-gauche’ (gtg’, kink), and end gauche conformations was observed at low temperatures. In this paper, the effect of column temperature on selectivity will be examined for the separation of PAH mixtures. Observed changes in selectivity will be compared with selectivity differences previously reported for C18phases prepared by monomeric and polymeric surface modification chemistries. A model for temperature induced selectivity changes is proposed based on the morphology of the bonded phase. This representation, which can be considered an extension of the “slot model” (30,31),suggests that changes in selectivity are a result of changes in phase rigidity that occur at different temperatures.
EXPERIMENTAL SECTION Reagents and Columns. HPLC grade solvents were used in (PhPh) was all separations. Phenanthr0[3,4-~]phenanthrene obtained from Aldrich Chemical Co. (Milwaukee, WI), 1,23,45,67,8tetrabemnaphthalene (TBN, dibenzo[g,p]chrpene) was obtained from Rutgers (Castrop-Rauxel, Federal Republic of Germany),and benzo[a]pyrene( B e ) was obtained from BCR (Community Bureau of Reference, The Netherlands). Limited quantities of a solution containing these three solutes are available from the authors upon request. A mixture containing 16 priority pollutant PAHs, SRM 1647a, was obtained from NIST (Gaithersburg, MD). Three commercial C18columns were employed in this work: Zorbax C18 (a monomeric C18 column; MAC-MOD Analytical, Wilmington,DE), Bakerbond wide pore C18(a heavily loaded polymeric C18 column; J. T. Baker Chemical Co., Phillipsburg, NJ), and Vydac 201TP C18(a polymeric CLB column; The Separations Group, Hesperia, CAI. All columns were of the same physical configuration,i.e., 25 cm X 4.6 mm, with 5 pm diameter spherical particles. The Vydac 201TP C18column was packed in our laboratory with commercial C18silica (Vydac 201TP C18 phase). Apparatus. Column temperature was regulated through the use of an insulated column jacket. Ethanol was circulated through the jacket by an external refrigerated fluid bath. Column temperatures within the range of -30 to 55 “C were possible with this apparatus; for temperatures greater than 55 OC, a column block heater was used. Column temperature was measured by using a thermometer immersed in the column jacket and temperatures were maintained to within h0.2 “C ( f l OC for the block heater). Separations were carried out with a chromatograph consisting of an autosampler with loop injector, reciprocating piston pump, and UV detector (254 nm). RESULTS AND DISCUSSION In previous work we have shown that significant differences in selectivity exist among commercial and experimental C18 columns for the separation of polycyclic aromatic hydro-
carbons (30-36). These differences result from variations in a number of parameters in the preparation of the phase, including alkyl chain length (31),phase loading (34,37,38), and, most importantly, the type of surface modification chemistry employed (32, 39). Phases prepared under anhydrous conditions, or using monochlorosilane reagents (i.e., “monomeric phases”), have properties distinctly different than phases synthesized using polymeric surface modification procedures (i.e., “polymeric phases”). Typical polymeric synthesis schemes employ trichlorosilane reagents in conjunction with measured quantities of water (32). This type of phase is distinct from and should not be confused with cross-linked or immobilized phases of the type used in capillary GC columns. An empirical classification scheme has been developed for grouping phases with similar selectivity toward PAHs (35). The elution order of a mixture of three PAH solutes (PhPh, TBN, BaP; see Experimental Section) varies with bonded phase type and gives an accurate assessment of phase properties for the separation of a wide variety of PAH samples. By calculating the selectivity factor a m N / B a p , a numerical measure of selectivity can be made, and columns can be grouped into similar classes. We have classified phases as follows: (~mN/Bap 2 1.7, “monomeric-like”selectivity; C Y ~ N I W < 1, “polymeric-like” selectivity; and 1 IC Y T B N / B ~ ~ 1.7, “intermediate” selectivity. In the present work, a mixture of PhPh, TBN, and BaP was chromatographed on polymeric (Vydac 201TP C18)and monomeric (Zorbax C18) columns at various temperatures. Dramatic shifts in solute elution were observed over the temperature interval investigated. Characteristic separations for the polymeric phase are illustrated in Figure 1. Separations were carried out at elevated temperatures using 7525 acetonitrilewater mobile phase; subambient separations were made with 85:15 acetonitrile-water. Under ambient conditions, the mixture was fully resolved with three nearly equally spaced peaks. At elevated temperatures, the elution of BaP decreased relative to TBN; thus the selectivity factor (YTBN/W increased. By use of the classificationscheme described above, phase selectivity was more ”monomeric-like” at elevated temperatures. At subambient column temperatures, the opposite trend was observed. The retention of BaP increased relative to TBN, decreasing C X ~ NPolymeric-like / ~ column selectivity increased with decreasing temperature. Similar trends were observed for the separation of the phase selectivity test mixture at various temperatures on the Zorbax C18 column. Overall column selectivity, as designated by C U ~ ~became / W , more “polymeric-like” at low temperatures. At elevated temperatures phase selectivity changed only slightly, becoming nearly constant above 45 OC. Selectivity is plotted as a function of temperature for polymeric and monomeric columns in Figure 2. Monomeric data points reflect measurements carried out at a mobile phase composition of 85:15 acetonitrilewater; polymeric data points are for multiple mobile phase compositions. Selectivity was observed to be nearly independent of mobile phase composition for the polymeric column; however, the slight scatter in the polymeric data in Figure 2 is the result of a minor compositional dependence. Several trends are apparent in Figure 2. The selectivity of both monomeric and polymeric columns becomes more “polymeric-like” at reduced temperatures. At elevated temperatures, the two columns have similar “monomeric-like” phase selectivity. The selectivity plots suggest that for a finite range of selectivities, column retention behavior of one column type (e.g., polymeric) can be achieved by using a column of the other type (e.g., monomeric) a t a different temperature. For example, at ambient temperature (-26 “C) the selectivity
ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
1751
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perature for monomeric and polymeric CI8 columns: mobile phase, Zorbax C,,, 85:15 ACN-H,O; Vydac 201TP C,,, various ACN:H,O compositions. coefficient a T B N / B e for the polymeric phase is 0.71. About the same value for C Y T B N / B is ~ ~ obtained for the monomeric phase at -4.6 OC, and it is expected that PAH separations at this temperature would be comparable to separations carried out at ambient conditions on the polymeric column. To test this idea, a mixture of 16 PAHs was separated on the Zorbax C18column at ambient and subambient conditions (Figure 3). Gradient elution conditions were adjusted in each case to give similar overall retention times. At 25 OC, partial resolution of the components was achieved, characteristic of that observed for most monomeric phases (32,X). Fourteen of the 16 components were resolved at -8 OC, with two early components coeluting (fluorene and acenaphthene). The elution order and overall spacing of the components in the chromatogram are very similar to separations achieved on polymeric CI8 phases (32, 36).
Another possibility is suggested by the selectivity plots in Figure 2. At low temperatures, C Y T B N / Bvalues ~ are reduced and phase selectivity is more "polymeric-like" for both of the phase types. In general, a high degree of shape recognition ability ("shape selectivity") exists for phases with low C Y ~ N / W values (30). Separations of isomers and other similarly structured compounds can sometimes be achieved with such columns that cannot be achieved for columns with "monomeric-like" selectivity. The use of polymeric phases at low temperatures should result in phase selectivity and shape recognition ability not obtainable in any other way. Although, in theory, comparable phase selectivity might also be obtained with monomeric phases at low temperatures, in practice the temperature necessary to achieve such a change in selectivity is below the working limits for most mobile phase solvents. To examine this possibility, a mixture of the six possible methylchrysene isomers was used to probe phase shape recognition ability at ambient and subambient temperature conditions. Because these isomers differ only in the ring position of the methyl group, it is difficult to separate all six methylchrysene isomers by liquid and gas chromatography. The quantification of the individual isomers is of some epidemiological interest, since 5-methylchrysene is significantly more carcinogenicthan the other isomers. The first attempt to separate methylchrysene isomers by LC was made by Wise and co-workers (40). Four of the six isomers were resolved with a Vydac 201TP C18 column; however 5- and 6-methylchrysene were not resolved. Separation of all six isomers was first achieved by Markides et al. using capillary GC and experimental smectic ( 4 1 ) and nematic (42) liquid crystalline polysiloxane stationary phases. Such liquid crystalline GC phases are highly shape selective and in fact have retention properties closely corresponding to polymeric C18phases in LC (43). Resolution of these isomers is not possible on more conventional GC phases, e.g., with SE-52 or SE-54 phases.
ANALYTICAL CHEMISTRY, VOL. 61, NO.
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15, AUGUST 1, 1989 4
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mLlmin.
Separations of the methylchrysene isomers at ambient and subambient temperatures are illustrated in Figure 4. An isothermal separation of a mixture of the six methylchrysene isomers was carried out at 27 OC, with gradient elution, using a heavily loaded polymeric C18phase (Bakerbond wide pore C18). Four of the six isomers were resolved (5- and 6methylchrysenecoeluted), similar to the separation previously reported (40). Under the same conditions, only three isomers were resolved by using the monomeric column (not shown). In Figure 4, all six isomers are resolved with the Bakerbond C18 column under isocratic conditions using a subambient temperature program. Separation of 5- and 6-methylchrysene could only be achieved at low temperature. However, under isothermalconditions (-20 O C ) the 1-,2-, and 3-methylchrysene isomers were strongly retained, even using a pure acetonitrile mobile phase. By use of a temperature program, it was possible to separate the critical isomers, while permitting the more easily separated later eluting isomers to be eluted in a reasonable period of time. This separation demonstrates not only an exceptional degree of shape selectivity but the feasibility of temperature programming as an alternative to
Figure 4. Separation of methylchrysene isomers on a Bakerbond wide pore C,, column: (a) temperature 27 OC, gradient elution, 60:40 ACN-H20 to 8515 ACN-H20 over 40 min, 1 mL/min; (b) isocratic
elution (100% ACN), temperature program, -25 to 20 OC over 40 min. gradient elution in liquid chromatography.
RETENTION THEORY In previous studies we have advanced a picture of solute retention referred to as the "slot model" (30,31). This model suggests that ordering within the bonded phase layer may be represented schematically as slots in a layer. Stated simply, solute penetration into these slots results in retention. Viewed in this way, predictions can be made on the effects that solute shape and bonded phase morphology will have on retention. A few trends can be summarized. Long narrow solutes are retained longer than square solutes of the same molecular weight. Similarly, planar solutes are retained longer than nonplanar solutes with similar overall structure. To view these trends in terms of the model, imagine a surface with a mixture of narrow and wide slots. Long narrow solutes, i.e., solutes with large length-to-breadth ratios (L/B) (44), will penetrate a larger number of the slots than square solutes. Because of this enhanced interaction with the bonded phase, retention is increased for long narrow solutes. A similar argument can be made for planar and nonplanar solutes with similar L / B ratios. Nonplanar solutes are more restricted from the slots than corresponding planar solutes. Interaction with the bonded phase is reduced, and retention is decreased for nonplanar molecules. Phase morphology is an important aspect of this model. It is clear that changes in phase structure in some way affect the chromatographicproperties of LC columns. For example, monomeric and polymeric C18phases are both prepared from silanes with the same (C18) alkyl substituents, yet the two
ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
phases differ markedly in selectivity toward PAH and other classes of solutes. In previous work we suggested that these differences originate primarily from phase order (30,43). In a comparison of phase selectivity between selected LC and GC columns, a close correspondencein retention behavior was observed between polymeric C18columns in LC and a smectic liquid crystalline phase in GC (43). A high degree of shape recognition ability was observed for each column. Various PAH isomer mixtures were separated, and the elution orders for the two columns were nearly identical. In contrast, little shape recognition ability was found for monomeric C18 LC columns or methylsilicone GC columns and little separation of isomers was possible. Liquid crystalline phases have a higher degree of order resulting from alignment of the rodlike substituent molecules. By analogy, this suggests that, as with liquid crystal GC phases, phase ordering within polymeric CIS LC phases may be responsible for the selectivity exhibited by these phases. This order may result from loss of alkyl chain mobility within a rigid polymer layer. Bonded phase length is another aspect affecting phase selectivity. In previous work we have shown that the highest degree of shape recognition was achieved with long chain length alkyl phases (31). This trend was observed for both monomeric and polymeric phases but was most dramatic with polymeric phases. As with liquid crystalline GC phases, the more extended alkyl phases exhibited the greatest shape selectivity for PAH isomers. This observation is consistent with the changes in selectivity that result from changes in phase morphology occurring at low temperatures. The temperature-dependent change in retention behavior for monomeric and polymeric C18phases observed in this study can thus be explained in terms of chain mobility and order. For pure long chain alkanes (e.g., octadecane), chain mobility decreases with temperature, ultimately forming an ordered crystalline solid. In the crystalline state, alkyl chains exist in the all-trans conformation, i.e., fully extended. Such crystalline ordering is not possible for bonded phases since alkyl chain spacing is constrained by the covalent point of attachment to the surface. However, reduction in bonded ligand mobility with temprature has been clearly demonstrated by NMR (13) and inelastic neutron scattering (23). Furthermore, bonded alkyl chains have been shown to straighten at reduced temperatures (29). Bends and kinks in alkyl chains give rise to specific transitions in IR spectra. These transitions decrease as a function of temperature. It can be concluded that alkyl bonded phases become more rigid and more rodlike at low temperatures and thus take on some of the characteristics of liquid crystals. This low-temperature ordering is most pronounced for polymeric bonded phases, for which chain motion is somewhat constrained even at ambient temperature. It is informative to examine Van't Hoff plots for the three probes used in this work (Figure 5 ) . Evidence for a phase transition is present in the plots for monomeric (Zorbax C18) retention data. The transition appears to occur over an interval from about 40 to 50 "C. This transition temperature is similar to that reported by Morel and Serpinet (14) for densely bonded monomeric C18 phases. The presence of a transition is not observed for the polymeric phase. The lack of a transition may reveal much about polymeric phase morphology. Over the temperature interval investigated, alkyl chain association does not change abruptly with temperature. This suggests that constraints to chain mobility exist for polymeric phases that do not exist with monomeric phases. In contrast, it appears that sufficient freedom exists in monomeric phases to permit a transition in which alkyl chains are more associated with each other below the transition temperature. Selectivity does not change abruptly as a con-
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sequence of the transition. This is contrary to the proposed retention theory of Jinno et al. (45). They predicted that a critical change in selectivity toward PAHs would occur near the phase transition, due to a change from "solidlike" to "liquidlike" behavior. We have observed instead gradual changes in shape selectivity over the range -20 to 100 "C, for both monomeric and polymeric CIS phases (Figure 2). It should be noted, however, that the probes employed by Jinno et al. are substantially larger than those used in this work, and the results may not be directly comparable. Because changes in selectivity occur continuously as a function of temperature, it seems likely that the presence or absence of a phase transition is of secondary importance to the ordering of individual chains. The possibility of solvent-induced conformational changes for large nonplanar PAH has been advanced by FeWr (46).He suggests that changes in retention behavior may be partly due to such conformational changes for the solute. However, because the conformation of planar PAH would be unaffected by changes in temperature, changes in selectivity for planar solutes that occur as a function of temperature would still be attributed to conformational changes of the bonded phase. A clear example is illustrated in Figure 3 for the separation of chrysene and benz[a]anthracene. The elution order observed a t -8 "C (benz[a]anthracene, chrysene) is characteristic of polymeric C18 phases; at 25 O C the solutes coelute (characteristic of monomeric C18 phases). Further insight can be obtained through thermodynamic evaluation of the data. In k' has been shown to be related to temperature through the following relation: In k' = -AH,,JRT AS,,,/R In 4
+
+
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
where AH,,,, and ASrnq are respectively enthalpies and entropies of transfer (mobile to stationary phase), R is the ideal gas constant, T is the absolute temperature K, and 6 is the phase ratio (volume stationary phase/volume mobile phase). The slopes of the curves in Figure 5 are related to the enthalpy of the retention process, whereas the intercepts are related to the sum of the entropy change in solute retention plus the phase ratio. Numerical evaluation of the phase ratio is difficult at best, since the volume of the stationary phase available to the solute during retention must be estimated. Consequently, the calculation of absolute entropies of transfer is probably not justified. Enthalpies of transfer may be calculated without difficulty since knowledge of the phase ratio is unnecessary. Such calculations have been carried out for retention data at elevated temperatures (20). Below about 25 "C, slopes of the Van't Hoff plots for PhPh and TBN are similar, indicating that the enthalpies of transfer for these solutes at subambient temperatures are similar for the monomeric and polymeric C18 columns. For BaP, this condition is achieved at temperatures lower than 12 OC. Above these temperatures the curves diverge, indicating different enthalpies of transfer, perhaps due to dissociation of bonded phase chains with the monomeric phase at higher temperatures. The slopes of the BaP curves are significantly greater than for the other solutes. Thus, the enthalpy of transfer for BaP is greater than the other solutes. This suggests that BaP is able to interact more strongly with the bonded phase than with the other solutes, perhaps as a result of the planar nature of BaP compared to the nonplanar nature of PhPh and TBN. The asymptotic nature of the Van't Hoff plots for BaP may be of some consequence, yet the significance is difficult to prove rigorously, since absolute entropies of transfer cannot be calculated. It is interesting, however, to assume that the phase ratios for the monomeric and polymeric columns are about the same. For PhPh and TBN, this means that these solutes are more ordered (Le., the entropies of transfer are smaller) in the polymeric stationary phase than in the monomeric phase, at any given temperature. For BaP, the degree of ordering would be about the same for both phases, below about 12 "C. This ordering is consistent with the observations presented earlier for the polymeric phase compared to the monomeric phase. It can be stated without assumption that the sum AS,,,,/R + In 4 is about the same for BaP on the two columns (
