Temperature dependence of retention in reversed-phase liquid

plicitly the role of the stationary phase in the retention process. In 1983 Martire and Boehm published the first retention model to consider the effe...
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Anal. Chem. 1992, 64, 1317-1323

1917

AC RESEARCH

Temperature Dependence of Retention in Reversed-Phase Liquid Chromatography. 1. Stationary-Phase Considerations Lynn A. Cole+and John G. Dorsey' Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172

The retentlon mechanism In reversed-phase llquld chromatography (RPLC) has been investigatedby examlningthe temperature dependence of retentlon, wlth emphasls on the role of the statlonary phase In the retention process. Both chromatographktemperature studles and differentialscanning calorimetry were used to examlne the role of alkyl chaln bondlng denslty on the retentlonmechanism In RPLC. Phase tranrltloneof reversebphasestatlonary phaseswere observed at bondlng denslties greater than 2.84 MmoI/m*. Thermodynamic constants for the transfer of a solute from the moblle phaseto the stationary phase (AH0 and ASO) were calculated for low bonding denslty columns, and comparison of these values to prevlowly reported values for the partltloning of a nonpolar solute from the bulk organic llquidto water lndlcated that the chromatographicretentlon processIsnot welhodeied by bulk-phase oll-water partltlonlng processes. I n addltlon, this data showed that the entropic contrlbutlon to retention becomes more slgnHlcant with respect to the enthalpic contrlbutlon as the stationary-phase bondlng denslty Is Increased, provlding addltionaisupport that partltioning, rather than adsorption, Is the relevant model of retention.

INTRODUCTION Reversed-phase liquid chromatography (RPLC) has been the most widely used liquid chromatographic technique in recent years,l yet the retention mechanism in RPLC remains unclear. Many studies have been conducted that examine the retention mechanism in RPLC, and two main theories have evolved. Solvophobic theory, first applied to RPLC by Horvath and co-workers in 1976, proposes that retention is primarily related to hydrophobic interactions between the mobile phase and solutes.2-4 The role of the stationary phase is minimized by solvophobic theory, and retention is thought to occur through an adsorption rather than partitioning process. This theory describes a two-step mechanism for retention which involves creation of a solute-sized cavity in + Present address: The' Procter and Gamble Co., Sharon Woods Technical Center, Cincinnati, OH 45241. (1)Majors, R. E. LC-GC 1988, 6, 298-302. (2)Horvath, Cs.; Melander, W.; Molnar, I. J. Chronatogr. 1976,125, 129-156. (3)Melander, W. R.;Horvath, Cs. InHigh-Performance Liquid Chromatography, Aduances and Perspectiues; Horvath, Cs.,Ed.; Academic Press: New York, 1980;Vol. 2,pp ,113-319. (4)Horvath, Cs.; Melander, W. R. Am. Lab. 1978, 17-36.

0003-2700/92/0364-1317$03.00/0

the mobile phase and transfer of the solute to or from this cavity. The driving force for retention as described by solvophobic theory is the free energy change associated with the two-step solute-transfer mechanism.2 The partitioning model of retention considers more explicitly the role of the stationary phase in the retention process. In 1983 Martire and Boehm published the first retention model to consider the effects of stationary-phase chain organi~ation.~ This statistical mechanical model described the stationary phase as a "breathing" surface which could expand or collapse depending on the mobile-phase composition. More recently Dill has proposed a partitioning model of retention based upon mean-fieldstatistical thermodynamic theory, which describes a three-step molecular process by which the solute transfers from the mobile phase to the stationary phase.6-8 This three-step process involvescreation of a solute-sized cavity in the stationary phase, transfer of the solute from the mobile phase to the stationary phase, and closing of the solute-sized cavity in the mobile phase. In the partitioning model, the solute is approximated to be fully embedded in the stationary-phase chains rather than adsorbed on the surface. Since configurational constraints are imposed upon the alkyl chains through their attachment to the silica surface, Dill describes the stationary phase as an interphase,which differs from a bulk system in that the surface to volume ratio of an interphase is high and its properties vary with depth from the surface. The organization of the interphase is influenced by geometrical constraints of the interface, including the length and bonding density of the alkyl chains attached to the silica surface, as well as the solvent which contacts the chains. In addition, the chains will adopt as much disorder as possible with the geometrical and solvent constraints in keeping with the second law of thermodynamics.6 Partitioning here implies that the solute "embeds" in the constrained stationary-phase chains and differs from bulk-phase partitioning only in the nature of the second liquid, here the constrained alkyl chains. Dill's partitioning model predicts that the retention process will be primarily driven by two forces. One is the difference in the contact free energy of the solute in the mobile phase and the stationary phase. This prediction has been examined (5) Martire, D. E.; Boehm, R. E. J. Phys. Chem. 1983,87, 1045-1062. (6) Dill, K.A. J. Phys. Chem. 1987,91, 1980-1988.

(7) Dill, K.A.; Naghizadeh, J.;Marqusee,J. A. Annu.Reu.Phys. Chem.

1988, 39, 425-461.

(8) Dorsey, J. G.; Dill, K. A. Chem. Reu. 1989,89, 331-346.

0 1992 American Chemical Society

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992

for a large data base of almost 350 sets of experiments? and in agreement with theory it was found that the dependence of retention on the mobile-phasecompositioncan be correlated to binary interaction constants of solutes with solvents. The second driving force for retention predicted by Dill concerns the partial ordering of the grafted alkyl chains, leading to entropic expulsion of the solute from the stationary phase at sufficiently high bonding density. At low alkyl chain bonding densities, the partitioning of solutes should increase linearly up to a critical bonding density of about 2.7 pmol/m2. However, above the critical alkyl chain bonding density configurational constraints become increasingly important and partitioning becomes "entropically expensive", leading to a decrease in the partitioning of solutes. The effect of alkyl chain bonding density on the partitioning of small nonpolar solutes has been tested,lO and a maximum in partition coefficient was observed a t a bonding density of about 3.1 bmol/m2, supporting Dill's theory. In a separate study, the volume necessaryto reequilibrate a reversed-phase stationary phase followinggradient elution was determined as a function of CIS bonding density,11 and a critical bonding density of about 2.9 pmol/m2 was observed. This indicates that partitioning of the organic modifier occurs similarly to the partitioning of small solutes and is also a function of stationary-phase bonding density. Valuable information about the retention mechanism in RPLC may be gained by examining the temperature dependence of retention. The temperature dependence of retention is given by

where k' is the capacity factor of the solute [k' = (t,- t,)/t,], AHo is the enthalpy of transfer of the solute from the mobile phase to the stationary phase, AS" is the entropy of transfer of the solute from the mobile phase to the stationary phase, R is the gas constant, T is temperature, and 0 is the phase ratio of the chromatographic column (the volume of the stationary phase divided by the volume of the mobile phase). This expression shows that a plot of In k' versus 1 / T (called a van't Hoff plot) has a slope of -AH"/R and an intercept of AS/R In 0 if AHo is invariant with temperature (Le., a linear van't Hoff plot is obtained). This provides a convenient way of calculating the thermodynamic constants AH" and ASo for a chromatographic system if the phase ratio is known or can be calculated. These constants are useful in assessing the thermodynamic driving force for retention. The temperature dependence of retention has been previously explored.12-17 Linear van't Hoff plots have been observed for various monomeric and polymeric commercially available (218 columns over temperature ranges of about 30 deg using hydroorganic mobile phases.12-15J7 In these studies AHo values were calculated using the slope of the van't Hoff plots, but ASo values were generally not provided due to ambiguity in the calculation of the phase ratio for commercial c~lumns.~s In one study ASo values were provided, although detailed conclusionsabout the retention mechanism in RPLC

+

(9) Ying, P. T.; Dorsey, J. G.; Dill, K. A. Anal. Chem. 1989,61,25402546. (10)Sentell, K.B.; Dorsey, J. G. Anal. Chem. 1989,61,930-934. (11)Cole, L. A.;Dorsey, J. G. Anal. Chem. 1990,62,16-21. (12)Grushka, E.;Colin, H.; Guiochon, G. J. Chromatogr. 1982,248, 325-339. (13)Tchapla, A.;Heron, S.; Colin, H.; Guiochon, G. Anal. Chem. 1988, 60,1443-1448. (14)Yamamoto, F. M.; Rokushika, S.; Hatano, H. J. Chromatogr. Sci. 1989,27,704-709. (15)Issaq, H. J.;Jaroniec, M. J. Liq. Chromatogr. 1989,12,2067-2082. (16)Issaq, H. J.; Fox, S. D.; Lindsey, K.; McConnell, J. H.; Weiss, D. E.J . Liq. Chromatogr. 1987,10,49-70. (17)Sander, L. C.; Field, L. R. Anal. Chem. 1980,52,2009-2013.

were not drawn since "the structural details of the bonded alkyl phase and the silica gel used as base materials of the commercially available packing materials are usually unkn0~1P.14 In another study, the phase ratio of a commercial column was calculated by using models based on information provided by the manufacturer, and both ASo and AH" were determined.17 Nonlinear van't Hoff plots have also been observed for temperature studies of reversed-phase stationary Nonlinear van't Hoff behavior may be indicative of a change in the mechanism of retention. Phase transitions of stationary phases may cause nonlinear van't Hoff behavior, but morphological changes in the bonded layer do not necessarily change the intrinsic mechanism. Typically, temperature ranges of 45 deg or more have been evaluated in the studies showingnonlinear van't Hoff plots. Deviationsfrom linearity for monomeric C18 stationary phases have been observed at about 22 "C in the absence of solvent using a gas chromatographic techniq~e.~9*~0 These phase transitions have been observed to be much more pronounced on high bonding density monomeric stationary phases (bonding densities greater than 4.0 pmol/m2) than on those with low bonding densities.m The transitions observed by Morel and Serpinetlg were reversible, showing the same deviation from linearity when the column was heated or cooled, but irreversible "phase transitions" have been observed for polymeric columns in the presence of s01vent.2~~~~ However, a separate study of monomeric phases suggested that these irreversible phase transitions were the result of a solvent release process26 and were different from the reversible, melting-like phase transitions previously observed.l*2l Differential scanning calorimetry (DSC) has been used to investigate phase transitions of reversed-phase stationary phases.27*%Melting-type transitions, evidenced by a distinct endothermic peak in a DSC scan, have been suggested for monomericreversed-phase stationary phases.1*21.26-% In one study, Cla stationary phases with bonding densities less than 2.5 pmol/m2failed to show distinct phase transitions, which was attributed to their low bonding densities.27 However, the CISstationary phases did show distinct phase transitions a t about 20 "C after being treated with free C18 alkane. Since the transition observed for the alkane-treated CISstationary phase was quite different from that observed for the pure Cl8 alkane (transition temperature 31 OC), the authors concluded that the transition observed at 20 "Cwas aresult of the mixed surface monolayer, although this could also be explained by the difference between a bulk-phase alkane and an interphase with one end of the chain tethered to the silica surface. In another study, DSC was used to study phase transitions of monomericC22 stationary phases ranging in bonding density from 1.78 to 4.3 pmol/m2.28Phase transitions were found to become more distinct and shifted to higher temperatures as the bonding density was increased, Phase transitions were not observed for stationary phases with bonding densities less than 2.6 pmol/m2. Glass transitions have also been ~~

~

~

(18)Hammers, W. E.;Verschoor, P. B. A. J. Chromatogr. 1983,282, 41-58. (19)Morel, D.; Serpinet, J. J. Chromatogr. 1980,200,95-104. (20)Morel, D.; Serpinet, J. J. Chromatogr. 1981,214,202-208. (21)Morel, D.; Serpinet, 3. J. Chromatogr. 1982,248,231-240. (22)Van Miltenburg, J. C.;Hammers, W. E. J.Chromatogr. 1983,268, 147-1 - - . - 55 - -. (23)Kessaissia, Z.;Papirer, E.; Donnet, J.-B. J.Colloid Interface Sci. 1981,79,257-263. (24)Gilpin, R. K.;Squires, J. A. J. Chromatogr. Sci. 1981,19,195-199. (25)Yang, S. S.;Gilpin, R. K. J. Chromatogr. 1988,449,115-118. (26)Morel, D.; Serpinet, J.;Untz, G. Chromatographia 1984,18,611614. (27)Hansen, S.J.; Callis, J. B. J . Chromatogr. Sci. 1983,21,560-563. (28)Claudy, P.;Letoffe, J. M.; Gaget, C.; Morel, D.; Serpinet, J. J. Chromatogr. 1985,329,331-349.

ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992

proposed for monomeric reversed-phase stationary phases studied by using a subambient gas chromatographic technique.23 Although much valuable information has been gained from these temperaturestudies of reversed-phase stationary phases, there are several issues which remain unresolved, and the driving force for retention remains unclear. A discrepancy apparent in these studies is the observation of linear or nonlinear van't Hoff plot behavior, which has led to debate about the presence of phase transitions in reversed-phase stationary phases. Since many of the studies showing linear van't Hoff behavior were conducted over a narrow temperature range (typically 20-50 "C), it is possible that the deviation from linearity usually observed a t about 20-25 "C might have been missed. Also, many studies showing both linear and nonlinear van't Hoff behavior were conducted on commercial columns. Since information about the bonding chemistry, starting silica, and surface coverage for commercial stationary phases is usually not available, it is typically not possible to calculate the phase ratio, and thus AS" values, for commercial columns.13J4 In addition, since the bonding density of commercial columns is usually not accurately known, it is virtually impossible to assess the role of surface coverage, and more broadly the stationary phase, on the retention mechanism in RPLC. Morel and Serpinet,20Hansen and calli^,^^ and others noted that the observation of phase transitions of reversed-phase stationary phases depends greatly on bonding density. In this study the retention mechanism in RPLC has been examined by conducting a temperature study over a wide temperature range on well-characterized, monomeric C18 columns of known high and low bonding density. By examining the behavior of well-characterized stationary phases it is possible to eliminate many of the ambiguities present in previous studies. The role of the mobile phase on the retention mechanism in RPLC has also been investigated, and will be discussed in part 2 of this work.29

EXPERIMENTAL SECTION Apparatus. All retention measurements were made with a Spectra-Physics SP8800 ternary pump (Spectra-Physics, San Jose, CA) and UV detection at 254 nm with an AB1 Analytical Spectroflow 757 variable-wavelength detector (AB1 Analytical Kratos Division,Ramsey, NJ). Sample injection was performed using a Rheodyne Model 7125 manual injector (Rheodyne, Inc., Cotati, CA) fitted with a 20-pL sample loop, and detector output was recorded using a Hewlett-Packard HP 3394A integrator (Hewlett-Packard, Avondale, PA). Temperature control was achieved with a Fisher ScientificIsotemp Mode19000refrigerated circulator (Fisher Scientific, Fair Lawn, NJ) using a circulating mixture of 50/50 (v/v) ethylene glycol/water. In addition to the thermostated analytical column, a mobile-phase static mixer placed just prior to the injector was temperature-controlled so that the mobile phase was preequilibrated to the appropriate temperature before reaching the analytical column to avoid thermal gradients in the column.30J1 In addition, the interconnecting tubing between the static mixer, injector, and analytical column was minimized, and the injector was wrapped in foam and duct tape to help maintain temperature control. Mobilephase flow rate for most experiments was 1.0 mL/min. For experiments performed at -5.0 "C the mobile-phase flow rate was increased to 2.0,4.0,or 6.0 mL/min as appropriate to decrease the elution time of the solutes. Deuterium oxide was used as a to marker. DSC studies were performed using a Mettler TA3000 system. Each sample was run over the temperature range -10 to +80 "C, cooled, and then rerun over the same temperature range. (29) Cole,L.A,; Dorsey, J. G.; Dil1,K. A. Anal. Chem. 1992,64,following paper in this issue. (30) Poppe, H.; Kraak, J. C.; Huber, J. F. K.; van den Berg, J. H. M. Chromatographia 1981, 14, 515-523. (31) Perchalski, R. J.; Wilder, B. J. Anal. Chem. 1979, 51, 774-776.

1910

Solvents and Columns. HPLC-grade acetonitrile (Fisher Scientific, Fair Lawn, NJ) was used without further purification. Water was obtained from a Barnstead Nanopure I1 water purification system (Barnstead Co., Boston, MA) fitted with a 0.45-pm filter. The mobile phase used for these studies was 601 40 (v/v) acetonitrile/water. Mobile-phase components were sparged with helium and mixed in the appropriate proportions by the SP8800 pump. Benzene (Mallinckrodt, Inc., Paris, KY), toluene (Eastman Organic Chemical, Rochester, NY), ethylbenzene (Fisher Scientific), and propylbenzene (AlfaProducts, Danvers, MA) samples were prepared using HPLC-grade methanol (Fisher Scientific) to a final concentration of 1000 ppm each. 1-Nitropropane (Matheson, Coleman, and Bell, Norwood, OH), 1-nitrobutane (Aldrich Chemical Co., Milwaukee, WI), and 1nitrohexane (Aldrich) samples were prepared in HPLC-grade methanol with a final concentration of 2000 ppm each. Naphthalene (Eastman) was prepared to a final concentration of 40 ppm, and anthracene (Matheson) had a final concentration of 1 ppm, both in HPLC-grade methanol. Each solute was injected in triplicate, and the retention times were measured by the HP 3394A integrator. Monomeric CIS stationary phases prepared, packed, and previously used in our laboratory1°were used for all studies. The Cl8 bonding densities of these columns were calculated as previously described32and ranged from 1.60 to 4.07 pmol/mZ. All stationary phases except the stationary phase with bonding density 4.07 pmol/mz were prepared on the same starting silica [20-30-pm irregular Davisil (Grace, Baltimore, MD), pore size 147 A] to eliminate differences in chromatographic behavior associated with different silica ~ubstrates.3~ The column with bonding density 4.07 pmol/m2was prepared using 10-pmDavisil silica with identical chemical and physical properties to the 2030-pm Davisil silica. Detailed synthesis procedures for the preparation of these stationary phases may be found elsewhere.a The columns were 15 cm x 4.6 mm i.d. Temperature Studies. Capacity factors were determined over the temperature range -5.0 to +80.0 "C. The chromatographic system was allowed to equilibrate at each temperature for at least 1 h prior to every experiment. This equilibration time period was validated by examining the retention time of benzene as a function of column equilibration time at 0.0 "C using a 60/40 acetonitrile/water mobile phase and the 3.56 pmol/ mz bonding density column. The retention time of benzene was measured every hour for 7 h and then again after 22,23, and 24 h. Over this 24-h time period, the retention time of benzene ranged from 4.96 min to 5.06 min, with an average retention time of 5.01 min i 0.735% . Since the retention time of benzene after only 1 h of equilibration was 5.02 min (near the 24-h average), the chromatographic system was sufficiently thermally equilibrated for use after 1 h of equilibration time. Calculation of Phase Ratio. The volume-phase ratio of a chromatographiccolumn (0)is the volume of the stationary phase ( V,) divided by the volume of the mobilephase ( Vm). The volume of the stationary phase was determined using the following equation35

where % C is the carbon loading as determined from elemental analysis, M is the molecular weight of the bonded alkyl ligand (g/mol), W , is the weight in grams of the bonded packing contained in the chromatographic column, n, is the number of carbons in the alkyl ligand, and p is the density (g/cmS) of the bonded alkyl ligand. This equation determines the actual volume of the alkyl chains, which is the relevant volume for the chromatographic process. % C values were determined from duplicate, in-house elemental analysis of the bonded phases at the University of Florida Chemistry Department. The weight (32) Berendsen,G. E.; de Galen, L. J.Liq. Chromatogr. 1978,1,561586. (33)Sander, L. C.; Wise, S. A. J. Chromatogr. 1984, 316, 163-181. (34) Sentell, K. B.; Barnes,K. W.; Dorsey, J. G. J.Chromatogr. 1988, 455,95-104. (35) Sentell,K. B.; Dorsey, J. G. J. Liq. Chromatogr. 1988,11,18751885.

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992

Table I. Am, AS", and TAP Values for All Solutes on the 1.60 pmol/m2 Column with Mobile Phase 60/40 Acetonitrile/Water

1

0

L

E

-1

ASo = -6.36 cal mol" K-' -2 i ' i . , . , . 0.0028 0.0030 0.0032 0.0034 0.0036 0.0038 1IT I 84.1

I

60.3

I

I

39.5

21.1

I

1

4.8

-9.8

solute benzene naphthalene anthracene 1-nitropropane 1-nitrobutane 1-nitrohexane toluene ethylbenzene propylbenzene a

AHo9

ASo,

TASo,b

r2

kcal mol-'

cal mol-' K-l

0.9971 0.9971 0.9868

-3.07

-6.36

kcal mol-' -1.93

0.9982 0.9984

0.9952 0.9983

0.9963 0.9898

-4.68 -3.37

-2.85 -2.80 -3.92 -3.48

-3.05 -2.87 -2.68 -2.56

-1.42

-1.02

-9.89 -8.10

-3.00

-5.74

-1.74

-5.21 -4.13 -3.27

-1.25 -0.99

-2.45 -1.58

*

r2 value for the linear fit of the van't Hoff plot. For T = 303

K.

T ("C)

van't Hoff plot for the solute benzene on the column with bondlngdensity 1.60 pmol/m2andthe mobile phase 60140 acetonitrile/ water.

Figure 1.

of the packing contained in the 15-cmX 4.6-mm i.d. columnswas determined previously%and was found to be 1.1705 f 0.0003 g (average of five determinations). Densities for the trimethylsilyl and octadecylsilyl groups were 0.8638 and 0.8607 g/cm3, respectively, as reported by Cheng.37 For the end-capped stationary phase with a Cle bonding density of 1.60 pmol/m2,the total V , was taken to be the sum of the volumes of the trimethylsilyl and octadecylsilyl alkyl groups. V , was determined by using the gravimetric method with methylene chloride and methanol as the two pure liquids. For the columns prepared using 20-30-pm irregular Davisilsilicawith column dimensions 15 cm X 4.6 mm i.d., V , was previously determined to be 1.805 f 0.002 mL.38 It was assumed that V , for these columns would be essentially constant since they were all prepared using the same starting ~ilica.3~ Individual column variations in V , are small and well within the experimental error, and these minimal variations donot influencethe trends observed for this data. V , for the column with bonding density 4.07 pmol/ m2 was previously determined by Ying.3s

RESULTS AND DISCUSSION van't Hoff plots were prepared for all solute and stationaryphase combinations investigated. Linear van't Hoff behavior was obtained for all solutes on the column with a bonding density of 1.60 pmol/m2 using the mobile phase 60/40 acetonitrile/water. Figure 1 is a van't Hoff plot for the solute benzene on this column. The correlation of determination (r2) for the linear fit of this plot was 0.9971. Since this data was linear, it was possible to calculate AHo and AS" for this system. AH"was determined to be -3.07 kcal mol-', and AS" was calculated to be -6.36 cal mol-' K-l. The value of -3.07 kcal mol-' for AH" correlates very well with previous values obtained for the enthalpy of transfer of small, nonpolar solutes from hydroorganic mobile phases to CIS reversed-phase stationary phases.12 The ASo value of -6.36 cal mol-' K-' also fell within the range of values determined by others.14J7 Table I contains a complete list of the AH"and ASo values obtained for all the solutes on the 1.60 pmol/m2column with the mobile phase 60/40 acetonitrile/ water. The r2values listed for each solute demonstrate good linearity of this data over the temperature range -5 to +80 "C. All of the AH" and ASo values compare well to previous values for similar solutes reported in the literature. Also listed in Table I are TAS" values a t 30 "C (T = 303 K). It is interesting to note that for (36) Sentell, K. B. Doctoral Thesis, University of Florida, 1987. (37) Cheng, W. Anal. Chem. 1985,57,2409-2412. (38) Ying, P. T. Doctoral Thesis, University of Florida, 1989.

0.8-

r2

=

0.9952

-

0.6

L

-c

-

0.4

-

0.2

AHo =

O.(

-2.19 kcal mol-'

A S o = -2.61 cal mol-' K*

I

1

-0.2 0.0028 0.0030 0.0032 0.0034 0.0036 0.0038 1 IT I

84.1

I

60 3

I

I

39.5

21.1

I -9.8

1

4.8

T ("C)

van7 Hoff plot for the solute benzene on the column with bondlngdensity 2.84 pmol/m2andthe mobile phase 60140 acetonitrile/ water. Figure 2.

every solute evaluated when AH" is compared to TAS" over the temperaturerange studied,the magnitude of AH" is always greater than that of TAS". This indicates that enthalpy plays a greater role in the transfer of a solute from the mobile phase to the stationary phase, and therefore in the retention process, than does entropy. Linear van't Hoff plots were also obtained for every solute on the 2.84 pmol/m2 column with the mobile phase 60/40 acetonitrile/water. Figure 2 is a van't Hoff plot for the test solute benzene on this column. The r2value for this plot was 0.9952. AH" was determined to be -2.19 kcal mol-', and AS" was found to be -2.61 cal mol-' K-l. These values for AHo and AS" again agree with previously reported literature v a l ~ e s . ~ ~Values J ~ J ~for AHo, AS", and TASO (at T = 303 K) for all solutes tested on the 2.84 pmol/m2column appear in Table 11. Good linear correlation was obtained for most van't Hoff plots as evidenced by the r2 values. The AH"and AS" values calculated in this chromatographic study may be compared to the thermodynamic values for the dissolution of small, nonpolar solutes in water. Gill and Wads039 reported a AHo value of 2.08 kJ mol-' (0.497 kcal mol-') and a AS" value of -57.8 J mol-' K-' (-13.8 cal mol-' K-l) for the dissolution of liquid benzene in water at 298 K. Since the dissolution of benzene in water corresponds to the transfer of the solute from the stationary phase (nonpolar environment) to the mobile phase (polar environment) in a chromatographic system, the values reported by Gill and ~

~

~~~

(39) Gill, S. J.; Wadso, I. Proc. Natl. Acad. Sci. U.S.A. 1976,73,29552958.

ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992

Table 11. A P , A P , and TAP Values for All Solutes on the 2.84 pmol/m2 Column w i t h Mobile Phase 60/40 Acetonitrile/Water

AH",

As",

solute

r2a

kcal mol-1

cal mol-' K-l

benzene naphthalene anthracene 1-nitropropane 1-nitrobutane 1-nitrohexane toluene ethylbenzene propylbenzene

0.9952 0.9825 0.9802c 0.9898 0.9971 0.9844 0.9828 0.9547 0.9223

-2.19 -2.17 -2.74d -2.91 -2.61 -2.33 -2.04 -1.92 -1.94

-2.60 -1.20 -1.47d -6.44 -4.73 -2.32 -1.34 -0.270 -0.482

TAP,b kcal mol-'

'

-0.79 -0.36 -0.45 -1.95 -1.43 -0.70 -0.41 -0.082

-0.15

r2 value for the linear fit of the van't Hoff plot. For T = 303 K. Van't Hoff plot is slightly curved, so linear fit may not be acceptable. d Values may be somewhat erroneous due to slightly curved van't Hoff plot.

Wadso are expected to be of the opposite sign from those determined in this study. Taking this into account, the AH" value reported by Gill and Wadso is similar to those found in this study for the solute benzene on either the 1.60 or 2.84 pmol/m2 column, although the values found in this chromatographic study are of slightly greater magnitude. The AS"values found in this study, however, are of the same sign as the AS"values reported by Gill and Wadso. In the process evaluated by Gill and Wadso, the nonpolar solute is inserted into the hydrogen-bonded, polar environment resulting in a decrease in entropy (increase in order). This decrease in entropy is known as the hydrophobic effect. In this chromatographic study the solute is transfered from a polar to a relatively nonpolar environment, resulting in a decrease in entropy, and representing an increase in the order of the system. The decrease in entropy is evidence of the interphase nature of the nonpolar stationary-phase chains and demonstrates the difference between a partially ordered interphase and a bulk system. Although some previous studies have compared the chromatographicretention process to oilwater partitioning, these ASo values indicate that oil-water partitioning is not a complete descriptor of the retention process in RPLC. Gill and Wadso also reported AH" and AS"values for the dissolution of toluene, ethylbenzene, and propylbenzene in water. Similar trends are observed for these solutes when compared to the AHo and ASo values determined in this chromatographic study. Although small differences in AH" and AS" values were observed for various solutes on the same column with the same mobile phase, these differences were found to be essentially insignificant when compared to a change in stationary-phase bonding density using enthalpy-entropy compensation. Enthalpy-entropy compensation is a term used to describe a compensation temperature which is systemindependent for a class of similar experimental systems.40 Enthalpy-entropy compensation has been applied to chromatographic systems to evaluate the retention mechanism.13J4J7,41,42 Equation 3 relates the compensation temperature (8) to the capacity factor a t some temperature T

(k'd:

If a plot of In k'T versus -AHo for a set of solutes is linear, (40)Boots, H.M. J.; de Bokx, P. K. J . Phys. Chem. 1989,93,82408243. (41)Kuchar, M.; Kraus, E.; Rejholec, V.; Miller, V. J. Chromatogr. 1988,449,391-401. (42)Melander, W.R.;Campbell, D. E.; Horvath, Cs. J.Chromatogr. 1978,158,215-225.

1321

then enthalpy-entropy compensation exists for the system, which means that the mechanism of retention is similar for all cases evaluated. A plot of In k'T (for T = 313 K) versus -AHo was prepared for all solutes on the 1.60pmoVm2 column using the 60/40 acetonitrile/water mobile phase. The r2 value obtained when all solutes were plotted was 0.737, which is only slightly lower than previouslyreported values," and may be considered adequate to verify enthalpy-entropy compensation for chromatographic systems. The linear fit is better if smaller groups of solutes are considered. For example, the r2 value is 0.984 when In k'313 versus -AHo is plotted for the group of solutes benzene, toluene, ethylbenzene, and propylbenzene. This high degree of correlation indicates that the retention mechanism is the same for these solutes using this chromatographic system. A plot of In k'313 versus -AHo for all solutes except anthracene on the column with bonding density 2.84 pmol/m2 had an rz value of 0.839, again demonstrating enthalpy-entropy compensation for this group of solutes. An alternative method for evaluating enthalpy-entropy compensation is to determine the compensation temperature as13940

p = - AH

(4) AS This method was used to evaluate the effect of stationaryphase bonding density on the retention mechanism in RPLC. The compensation temperature was determined for each solute on the 1.60 and 2.84 pmol/m2 columns. For the 1.60 pmol/m2 column, the @ values ranged from roughly 400 to 800 "C, and on the 2.84 pmol/m2column the @ values ranged from about 500 to 1800 "C. Although these ranges are somewhat broad, the difference between the @ value for a particular solute on the 1.60 pmol/m2column compared to the value on the 2.84 pmol/m2column is greater than the differences in p values between different solutes on the same column. For example, the @valuedetermined for toluene on the 1.60pmol/ m2 column was 549 "C, and on the 2.84 pmol/m2 column it was 1517 "C. The difference between these two values is 968 "C, which is larger than the range of values found on the 1.60 pmol/m2 column for all solutes, and approaches the range of the values found on the 2.84 pmol/m2 column. This comparison indicates that differences in stationary-phase bonding density contribute more significantly to the retention mechanism in RPLC than do differences between solutes for the small, nonpolar solutes considered in this study. This is in agreement with previous studies in which the mechanism of retention was found to be the same for a series of similar solutes using enthalpy-entropy although these studies considered only one stationary phase and the role of the stationary phase in the retention process was not evaluated. On columns with bonding densities above 2.84 pmollm2, linear van't Hoff plots were no longer obtained over the temperature range evaluated. Figure 3 is a van't Hoff plot for the test solute benzene on the column with bonding density 3.06 pmo1/m2,using the mobilephase 60140 acetonitrile/water. A deviation from linearity is apparent a t about 20 "C. Figures 4 and 5 are van't Hoff plots of benzene on the columns with bonding densities 3.56 and 4.07 pmol/mZ, respectively, using the 60140 acetonitrile/water mobile phase, and also show the deviation from linearity at roughly 20 OC. These deviations from linearity may suggest phase transitions of the reversedphase stationary phases and resemble the reversible phase transitions observed by Morel and Serpinet19.20a t about 22 OC on densely packed CIS stationary phases. This work supports the observation that phase transitions are more pronounced on high bonding density columns than on low bonding density columnsz0since deviations from linearity in

1322

ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992

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the van't Hoff plots were only observed on columns with bonding densities greater than 2.84 pmol/m2 when using 60/ 40 acetonitrile/water as the mobile phase. It is interesting to note that in all the van't Hoff plots for any column bonding density, the data is linear over the narrow temperature range

20-50 "C. Many previous temperature studies of reversedphase stationary phases were conducted over this narrow temperature range and linear van't Hoff plots were obtained, but this work shows that in data obtained over a narrow temperature range deviations from linearity may be missed. DSC was performed on all the stationary phases used in this study, as well as the native silica from which they were made, to verify the presence of phase transitions in the reversed-phase stationary phases. Upon the first heating of each sample over the temperature range -10 to +80 "C, a broad, exothermic peak was observed between 20 and 40 OC. This peak disappeared after the sample was cooled and immediately rerun, indicating that the peak was the result of loss of adsorbed water or some other solvent from the sample. Figure 6 shows the second heating DSC scans for the native silica and the stationary phases with bonding densities 2.84and 4.07 pmol/m2. The native silica scan shows no peaks or changes in baseline over the temperature range examined. (The dip in baseline observed for all the samples a t -10 "C is the result of initial temperature equilibration of the sample and reference pans.) The DSC scan of the stationary phase with bonding density 2.84 pmol/m2 shows a gradual exothermic change in baseline ending at 26.7 "C. This gradual change in baseline of the DSC scan is indicative of a glass transition-type phase transition. The stationary phase with bonding density 4.07 pmol/m2also shows this glass transition-like change in baseline on the DSC scan, this time ending at 35.7 "C. The other two stationary phases examined, with bonding densities 3.06 and 3.56 pmollm2, showed this change in baseline at 27.8 and 29.7 OC, respectively. Two trends are apparent in this data. First, the transition temperature increases with an increase in stationary-phase bonding density, which correlates with previous studiesa28 The increase in transition temperature for stationary phases with higher bonding densities may be attributed to the greater energy necessary to effect the glass transition, or free rotation of the C18 chains, for high bonding densities in which more intermolecular chain interactions must be overcome. The second trend evident in these DSC data is that the change in baseline becomes more distinct and has greater magnitude as the bonding density is increased. This supports previous studies27328 in which phase transitions of reversed-phase

ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992

stationary phases were barely discernible on DSC scans of low bonding density stationary phases, but became more distinct as the bonding density was increased. Chromatographically, phase transitions of low bonding density materials are not observed, as is the case in this study, since phase transitions of these materials are not of great enough magnitude to cause a distinct and obvious change. In this study, both chromatographic temperature studies and DSC have been used to obtain information which better elucidates the role of stationary-phase bonding density on the retention mechanism in RPLC and also addresses some of the discrepancies previously found in the literature concerning the role of the stationary phase on the retention mechanism in RPLC. Phase transitions of the reversed-phase stationary phases have been observed chromatographically for stationary phases with bonding densities greater than 2.84 pmol/m2. The glass transition-like phase transitions were verified using DSC, which showed that the phase transition became more distinct and shifted to slightly higher temperatures as the stationary-phase bonding density was increased. Phase transitions of these stationary phases are not obvious chromatographically over the narrow temperature range 2050 "C frequently studied by others but become apparent if the temperature range examined is broadened. Also, phase transitions are not obvious chromatographically for low bonding density stationary phases, and linear van't Hoff behavior was obtained in these cases. The AH" and ASo values calculated for the retention of small, nonpolar solutes on these low bonding density columns were compared to AH" and AS" values obtained by Gill and Wads039 for the transfer of the nonpolar solute from the bulk organic liquid to water. Comparisonof these values revealedthat the chromatographic process is not directly modeled by bulk oil-water partitioning processes, since the nonpolar stationary phase is an inter-

1329

phase (anchored at one end) rather than a bulk medium. For the same solute using the same mobile phase, both AH" and ASo become more positive as the stationary-phase bonding density is increased, which indicates that the entropic contribution to retention becomes more significant with respect to the enthalpic contribution as the stationary-phase bonding density increases and provides evidence for the second driving force for partitioning (retention)predicted by Di11.6 This general trend is evidenced over the entire range of stationary-phasebonding densities examined. Enthalpyentropy compensation revealed that differencesin stationaryphase bonding density contribute more significantly to changes in the retention mechanism than do differences in the test solutes examined. The results of this work support the partitioning model of retention proposed by Dillm and show that the nature of the stationary phase, particularly stationary-phase bonding density, must be considered in order to most accurately describe the retention process.

ACKNOWLEDGMENT We are grateful to Jeff Grothaus for providing the DSC scans and to Frank Meeks and Ken Dill for helpful discussions. We are grateful for support of this work by grants from the Air Force Office of Scientific Research, 91-0254, and the National Institutes of Health, GM-33382. RECEIVED for review October 24, 1991. Accepted March 16, 1992. Registry No. Acetonitrile, 75-05-8;benzene, 71-43-2; naphthalene, 91-20-3; anthracene, 120-12-7;1-nitropropane,108-032; 1-nitrobutane,627-05-4;1-nitrohexane,646-14-0;toluene, 10888-3; ethylbenzene, 100-41-4;propylbenzene, 103-65-1.