Anal. Chem. 1003, 65, 1819-1826
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Deuterium Nuclear Magnetic Resonance Spectroscopy as a Probe for Reversed-Phase Liquid Chromatographic Bonded Phase Solvation. 2. Aqueous Solvation In Methanol and Acetonitrile Binary Mobile Phases David M. Bliesnert and Karen B. Sentell' Department of Chemistry, University of Vermont, Burlington, Vermont 05405-0125
Using the inversion/recovery method, deuterium (aH) longitudinal relaxation times, (TI),of deuterium oxide were measured for binary solutions of methanol and D20 and acetonitrile and DaO and for these mixtures in contact with two different reversed-phaseliquid chromatographicstationary phases. Changes in the D2O TIfor the solutions in contact with the stationary phase compared to those observed for the solutions alone give a qualitative understanding of the degree of association of water with the stationary phase. The results for two stationary phases with greatly different Cle-bonding densities are reported and discussed. The results are also compared and contrasted to our previous work, which examined changes in aH2'1 for deuterated methanol and acetonitrile in the same binary hydroorganic solutions as well as for these mixtures in contact with the same two stationary phases. INTRODUCT10N Separations in reversed-phase liquid chromatography (RPLC) are complex thermodynamic processes involvingthe transfer of solutes between the mobile and stationary phases. Although these transfer processeswere initiallymodeled after simple bulk-phase partitioning processes, such as found in octanoVwater systems, it is now clear that partitioning in RPLC is a more complicated process.13 The alkyl ligands of reversed-phase stationary phases exhibit ordering due to their covalent attachment to the rigid silica substrate; this ordering increases with alkyl chain surface coverage. The structure at the stationary-phase surface is not a distinct interface but an interphase which exhibits an order gradient that varies along the bonded alkyl chains as a function of distance from the silica surface.1-9 Formation of a solvation layer a t the stationary-phase surface results from preferential sorption of mobile-phase components by the covalently attached alkyl chains as well as by the silica support. Due to varying degrees of bonded ligand interactions along the chain length, mobilephase components will penetrate into the stationary phase in a nonuniform manner.' For any type of stationary phase, the overall structure of the solvation layer is a function of factors such as bulk mobile-phase composition; the degree of alkyl derivatization of the silica surface or bonding density, which is often expressed in units of micromoles of bonded t Presentaddrew ZenecaPharmaceuticaleGroup,ZenecaInc.,NLW2, W i l m i i n , DE 19897. (1) Marquee, J. A.; Dill, K. A. J. Chem. Phys. 1986,85,434. (2) Dill, K. A. J. Phys. Chem. 1987,91,1980. (3) Dorsey, J. G.;Dill, K. A. Chem. Reo. 1989,89,331.
0003-2700/93/0365-1819$04.00/0
alkyl ligand per square meter of bonded-phase surface (rmoll m2); and the numbers and types of residual silanolsremaining on the silica surface following derivatization." Although the nature of stationary-phase/mobile-phaseinteractions and the resultant stationary-phase solvation structure continues to be debated, a better understanding of these phenomena is slowly being gained. This knowledge is crucial to a better molecular level understanding of solute-transfer interactions that occur during the course of a RPLC separation. As an analytical technique for studying stationary-phase/ mobile-phaseinteractions in RPLC systems, nuclear magnetic resonance spectroscopy (NMR)offers several advantages. Via studies of various nuclei, the problem can be approached from the perspective of the stationary phase or the mobile phase. NMR techniques allow studies of RPLC systemsto be carried out under chromatographic solvation and temperature conditions with minimal perturbation to the system from the measurement. The present paper is a discussion of some of our solution-state NMR studies of stationary-phase/mobilephase interactions. Other researchers have undertaken such experiments in the past and have obtained interesting results,'% however, they did so using uncommon RPLC s o l v e n t s ~ ~ or ~ ~they ~ ~encompassed ~ ~ ~ ~ 2 3 only ~ ~ a~ small range of mobile-phase compositions.11,1"1~."-22~2~ Our goal was to (4) Martire, D.E.; Boebm, R. E. J. Phys. Chem. 1983,87,1046. (5) McCormick, R. M.; Karger, B. L. Anal. Chem. 1980,62,2249. (6) Yonker, C. R.; Zwier, T.A.; Burke, M. F. J. Chromatogr. 1982,241, 257. (7) Yonker, C. R.; Zwier, T.A.; Burke, M. F. J. Chromatogr. 1982,241, 269. (8) Scott, R. P. W.; Simpson, C. F. Faraday Symp. Chem. SOC.1980, 15,69. (9) Gilpin, R. K., Gangoda, M. E. J. Chromatogr. Sci. 1983,21,362. (10)Gilpin, R. K.; Gangoda, M. E. Anal. Chem. 1984,56,1470. (11) Gilpin, R. K.; Gangoda, M. E. J. Magn. Reson. 198S, 64, 408. (12) Gangoda, M.; Gilpin, R. K. J. Magn. Reson. 1987, 74, 134. (13) Gangoda, M. E.; Gilpin, R. K. Langmuir 1990,6,941. (14) Marshall, D. B.; McKenna, W. P. Anal. Chem. 1984, 56,2090. (16) Ellison, E. H.; Marshall, D. B. Anal. Chem. 1991,96,808. (16) Maciel, G.E.; Zeigler, R. C.; Taft,R. K. In Silanes, Surfaces and Interfaces, Vol. 1; Chemically Modified Surfaces; Leyden, D. E., Ed.; Gordon and Breach Scientific Publishers: New York, 1986; p 413. (17) Zeigler, R. C.; Maciel, G. E. In Chemically Modified Surfaces in Science and Industry, Vol. 2; Chemically Modified Surfaces; Leyden, D. E., Ed.; Gordon and Breach Scientific Publishers: New York, 1988; p 319. (18) Zeigler, R. C.; Maciel, G.E. J. Am. Chem. SOC.1991,113,6349. (19) Albert, K.; Evers, B.; Bayer, E. J. Magn. Res. 1988,62,428. (20) Bayer, E.; Paulus, A.; Peters, B.; Laupp, G.;Reiiere, J.; Klaus, A. J. Chromatogr. 1986, 364,26. (21) Albert, K.; Pfleiderer, B.; Bayer, E. In Chemically Modified Surfacesin Science and Industry, Vol. 2; Chemically Modified Surfaces; Leyden,D. E., Ed.;Gordon and Breach ScientificPublishers: New York, 1988; p 287. (22) McNally, M. E.; Rogers, L. B. J. Chromatogr. 1986,331,23. (23) Shah, P.; Rogers, L. B.; Fetzer, J. C. J. Chromatogr. 1987,388, 411. (24) Claeseens, H. A.; van de Ven, L.; de Haan, J.; Cramere, C. A.; Vonk, N. J. High Resolut. Chrornatogr. Chromatogr. Comrnun. 1989,6, 433. (26) Kelusky, E. C.; Fyfe, C. A. J. Am. Chem. SOC.1986,108, 1746. 0 1993 American Chemical Sockty
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
use NMR relaxation techniques to obtain a more comprehensive understanding of stationary-phase solvation by binary solvent mixtures commonly used in RPLC. This has been accomplished by measuring solution-state deuterium PH) longitudinal relaxation times, TI, of deuterated mobile-phase components both in binary solutions and when these solutions were placed in contact with the stationary phases of interest. Because deuterium is a quadrupolar nucleus, its solutionstate longitudinal relaxation time, T1, is described by the following equation:26
Since both the spin quantum number, I , and the quadrupolar coupling constant, x,are constants for a particular nuclewZ6 the inverse of the relaxation time (or the rate of relaxation) is directly proportional to T ~ The . molecular correlation time, f C ,is a measure of how long it takes a molecule to rotate through one radian and therefore is a measure of molecular motion. From eq 1it is evident that as the correlation time increases (e.g., molecular motion decreases) the longitudinal relaxation time, TI, decreases. Therefore, a greater degree of molecular association or restriction of molecular motion causes a decrease in TI. Our current study is based upon work first conducted by Marshall and McKenna,14in which they measured 2Hsolutionstate longitudinal relaxation times in DzO/acetonitrile mixtures as a function of their volume-to-volume (v/v) ratios over a composition range of 0-50% DzO. Marshall and McKenna plotted T1versus percent DzO in these mobile-phase mixtures for the binary solutions alone and when placed in contact with reversed-phase stationary phases and thereby qualitatively gauged the degree of water association with the stationary phases.14 In the samples studied, they found that the relative amount of water associated with the stationary phase in acetonitrileiwater mobile phases was a function of the amount of water in the solution. In addition to D2O relaxation times, they also measured T1 for acetonitrile-ds under a few of their experimental conditions. However, they were unable to perform measurements for mobile phases with acetonitrile contents less than 50 % by volume, since uniform wetting of the stationary phase by predominantly aqueous solvent mixtures was not possible when the stationary phase and mobile-phase mixtures were combined at atmospheric pressure.14 Although Ellison and Marshall have more recently measured 2H and 14Nlongitudinal relaxation times of mobilephase components to determine the surface fluidity in RPLC systems,l5 to our knowledge no further experiments of either type have since been performed. Marshall and McKenna's initial TI experiments14 over a limited range of acetonitrileiwater solution compositions demonstrated the potential of using NMR to study mobileand stationary-phase interactions, and therefore, we decided to utilize this approach in more extensive studies. In our investigations, the solution-state longitudinal relaxation times of deuterated mobile-phase components in binary aqueous mixtures with acetonitrile and with methanol are measured over the entire volume-to-volumecomposition range. These mixtures are then placed in contact with Cls stationary phases. Decreases in 2H T1 for the samples in contact with stationary phase compared to those measured in the bulk solution imply association of the deuterated mobile-phase component with the stationary phase.l4925 Small decreases in T1 imply more limited association, whereas large changes imply a significant degree of association of that component with the stationary phase. Our studies are also unique because they overcome the experimental problem of wetting the stationary-phase (26) Harris, R. K. Nuclear Magnetic Resonance Spectroscopy: A Physicochemical View; Pitman Books: London; 1983.
samples with highly aqueous mobile phases, allowing us to encompass the entire mobile-phase binary composition range in both solvent systems. Since our sample preparation method equilibrates the stationary and mobile phases at pressures comparable to those attained under chromatographic operating conditions, it also much more nearly approximates wetting conditions and volume phase ratios in real chromatographic systems. In order to also investigate the effects of stationary-phase surface modification on solvation layer formation, we have measured 2H spin-lattice relaxation times for mobile-phase components in contact with two well-characterized Cle modified stationary phases with significantly different alkyl ligand surface densities. The complex association behavior between the acetonitrile and methanol mobile-phase components and these stationary phases, as well as their correlation with bulk solution structure in the binary hydroorganic mixtures, is discussed in a previous ~ a p e r . 2In~order to better understand the contribution of the aqueous component of the mobile phase to stationary-phase solvation, this study has been further extended to examining the degree of water association with the stationary phase for both methanoliwater and acetonitrile/water mobile phases over the same range of compositions previously studied. In the present paper, the aqueous results are discussed as well as compared and contrasted to our previous work.27 In addition, the influence of mobile-phase interactions on RPLC stationary-phase structure is discussed.
EXPERIMENTAL SECTION NMR Measurements. TIvalues for deuterium oxide in methanol/DzOand acetonitrile/DZO mobile-phasemixtures, both in bulk solution and in contact with stationary phase, were measured with a Bruker WM-250 NMR spectrometer operating at a field strength of 5.875 T and a frequency of 38.4 MHz for 2Hwith a 10-mm broad-band probe (observation on inner coil and decoupling on outer coil). The standard inversion/recovery sequence [(180'-~-90'-acquire-delay),l was used to acquire the T I values, where T ranged from 0.005 to 2 s. The sequence employed no less than 10 T values, and each acquisition necessitated from 2 t o 32 scans. A relaxation delay (3-4 s) in excess of 5 TI was used to allow the system to return to equilibrium. Total acquisition times ranged from 10 to 50 min. Care was taken to minimize reflected power for each sample in order to minimize errors in the 180' and 90' pulses. For our probe, optimum 180' and 90' pulse lengths were slightly sample dependent, with approximate values of 30 and 15 ps, respectively. All spectra were acquired using automatic field frequency lock and temperature was held constant at 30 "C (303 K). T1 values were calculated by manual measurement of peak height for each T value followed by a three-parameterleast squares exponential fit of the peak height versus T plot. Relative error for replicate measurements was in all cases less than 5 7%. All TI values are stated at a confidence level of plus or minus one standard deviation. Chemicals. Deuterium oxide (99.98%, Aldrich, Milwaukee, WI) and HPLC-grade methanol and acetonitrile (Fisher Scientific, Fair Lawn, NJ) were used without further purification. HPLC-grade water was obtained in-house using a Nanopure (Sybron Corp., Boston, MA) water purification system. Two CIS stationary phases were used in this work. The first, designated LT1, is a 10-pm monomeric non-end-capped phase with very high octadecyl ligand bonding density (4.4 pmol/mZ),which was synthesized in our laboratory under conditions which have been previously described.a*a The second stationary phase, designated ODs-1, is a commerciallyavailable (Spherisorb S5-ODs-1;Phase Separations, Norwalk, CT), 5-pm non-end-cappedphase with a much lower C18bonding density (1.41 pmol/m*). The octadecyl ligand is attached to the silica surface using a polyfunctional CIS (27) Bliesner, D. M.; Sentell, K. B. J. Chromatogr. 1993, 631, 23. (28) Sentell, K. B.; Barnes, K. W.; Dorsey, J. G. J. Chromatogr. 1988, 455, 95. (29) Sentell, K. B.; Henderson, A. N. Anal. Chim. Acta 1991,246,139.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
silane under anhydrous conditions. Because vertical polymerization of the silane under anhydrous reaction conditions is highly unlikely, the resultant phase is described as "monomeric" by the manufacturer. Subsequent to the initial attachment to the surface, some degree of horizontal polymerization between adjacent bonded ligands may take place, particularly upon exposureto aqueous or alcoholicsolvents.80 Therefore,the ODs-1 materialwas thoroughly washed in our laboratorywith methanol and dried prior to use in the NMR experiments, in order to ensure a reproducible phase morphology. Bonding densities for both phases were calculated as described by Berensen and de Galansl from elementalanalysisresults for percentcarbon and the surface area of the silica support. Percent carbon analysis for the LT1 phase was performed by Robertson Laboratories(Madison, NJ); all information about the commercial Spherisorbphase (ODs-1) was obtained directly from Phase Separations, Ltd. (Deeside, Clwyd, UK). Sample Preparation. Approximately 3% (by volume) deuterium oxide was added to pure water in a chromatographic mobile-phasereservoir. The labeled water and separatereservoirs of HPLC-grade methanol and acetonitrile were degassed by sonication under vacuum. The desired mobile-phase mixture (volume-to-volumepercent ratio of organic solvent to water) was delivered into a 10-mmNMR tube by two Waters (WatersAssoc., Milford, MA) Model 510 chromatographic pumps controlled by a Waters Model 680 automated gradient controller. For both the methanol/DzO and acetonitrile/DzO mixtures, samples were prepared in 10% or smaller volume/volume (v/v) organic/water incrementa ranging from 99:l to 0100 v/v %. 2H TIfor the deuterated water was then measured for each mobile-phase mixture. To ensure proper wetting of the stationary phase with the labeled mobile-phase solutions in the second half of the expermixtures were prepared iments, mobile-phase/stationary-phase in the following manner. First, the chromatographic stationary phase was dried under vacuum in excess of 12 h at 110 O C to ensure that all physisorbed solvents were removed. A 1.1-g portion of the dry stationary phase was hand-packed into an empty 10 cm X 4.6mm stainless steel chromatographic column. Mobile phase of the desired volume percent ratio was then pumped through the column at a flow rate which would generate at least 1600psi of backpressure. Approximately 50 mL of solvent was pumped through the column in one direction, at which point the column was reversed and an additional 50 mL was pumped in the opposite direction. Following the wetting procedure, one of the column end fittings was removed from the column and the wetted stationary phase was pumped as a smooth paste into an 8-mm NMR tube, resulting in a final sample height of 8 cm. This allowed irradiation of the entire sample by the transmitter coil, greatly enhancing signal to noise. The 21' experiment on the paste was then performed as described above for the solvent samples. It should be noted that at mobile-phase compositions containing a very low volume percent of organic solvent, it was necessary to increase the mobile-phase flow rate very slowly in order to prevent channeling of the solvent between the stainless steel columnjacket and the dry packing. In addition,considerably larger amountsof mobile phase (in excess of 800 mL) were needed to successfully wet the stationary phases with 100% water.
RESULTS AND DISCUSSION Mobile-Phase D20 2HTIMeasurements. The deuterium longitudinal relaxation times, 21' , for D2O in the acetonitrile/water and methanol/water mixtures are plotted versus volume percent organicsolvent in the mixture in Figure 1. Recall that a decrease in deuterium TIimplies molecular associationor decreased mobility. Over the compositionrange from 0 to 80 % acetonitrile in the acetonitrile/water mixtures, the D2O 2H 2'1's remain essentially constant, averaging approximately 0.55 s (Figure la). However,when the volume percent of acetonitrile is 90 % or greater, the DzO 21' increases rapidly, attaining a value of 1.2 s for the 99% acetonitrile solvent mixture. (30) Pfleiderer, B.; Albert, K.; Bayer, E. J.Chromatogr. 1990,506,343. (31) Berendsen, G. E.; de Galan, L. J.Liq. Chrornotogr. 1978,1,661.
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Marcus and Migrona2 have applied quasi-lattice quasichemical and inverse Kirkwood-Buff integral methods in studying the composition dependence of acetonitrile (ACN)/ water solution structure. They described the structure of acetonitrile/water mixtures as consisting of a number of composition-dependent regions.32 As acetonitrile is added to neat water, the water structure remains more or less intact, with acetonitrile entering cavities in the water structure. As the mole fraction of acetonitrile, XACN, approaches =0.30 ( ~ 5 5% 5 by volume), solution microheterogeneity seta in, wherein there are primary clusters of mutually hydrogenbonded water molecules as well as of acetonitrile molecules associated via dipole/dipole intera~tions.3~ At XACN 2 0.70, (2877% by volume), individual water molecules interact with individual acetonitrile molecules,with little disruption of the predominant acetonitrile structure (Le., dipole/dipole interactions). The water/acetonitrile mixed species are thought to exist as 1:l or 1:2 complexes surrounded by "bulk" acetonitrile solution struct~re.3~ Above XACN = 0.95 ( ~ 9 8 % by volume), there is evidence that these mixed complexes are no longer pre~ent.3~Katz et aZ.33 have reached similar conclusions. Their predictions and experiments indicate that in mixtures of acetonitrile and water, the proportion of associated acetonitrile/water complexes is very small, reaching a maximum volume percentage of -5% at a nominal acetonitrile volume fraction of 0.5.33 Rowlen and Harris's Raman spectroscopicstudies of the concentration dependence of the acetonitrile CN stretching band area and bandwidth also indicate that, at XACN > 0.3, large numbers of selfassociated acetonitrile species are present in solution, with the latter species greatly predominating at XACN 2 0.55 (20.78 volume fraction)." (32) Marcus, Y.; Migron, Y. J. Phys. Chern. 1991, 95,400. (33) Katz, E. D.; Ogan, K.; Scott, R. P. W. J. Chromatogr. 1986,352, 67. (34) Rowlen, K. L.; Harris, J. M. A d . Chem. 1991,63,964.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
If the above acetonitrile/waterbulk solution microstructure models are considered,3294it is reasonable that our measured DzO T I values for solution compositions containing up to 80 % acetonitrile are constant and approximately equal to the DzO T1 measured under purely aqueous conditions (0.64 s). In these microheterogeneous solutions, there is a very limited extent of hydrogen-bonding interactions between acetonitrile and water. Water interactions in solution over this composition range are dominated by a preponderance of self-associated water species; this is reflected by DzO T1values which are very close to that for the completely aqueous system. However, a t solution volume percent compositions containing greater than 80% acetonitrile, the dearth of water species and large preponderance of acetonitrile species will necessitate an increase in the number of water/acetonitrile mixed species. Since interactions between water and acetonitrile are comparatively much weaker than water/water interactions, the marked increase in the DzO 7'1 in this composition region reflects the disruption of waterlwater interactions by the weaker acetonitrile/water intera~ti0ns.l~ It should also be noted that the bulk solution DzO 2'1 behavior as a function of solution composition that was observed in the present study corresponds very well with that observed by Marshall and McKenna,14 although the T1 value that they measured for 1005% DzO (0.45 s) was, as expected, slightly smaller than was measured in our study (0.64 s), where 97% water with only 3% DzO enrichment was used. The plot of D2O T1 versus volume percent organic in the mobile phase for bulk methanol/water (Figure lb) exhibits quite different trends from the acetonitrile/water system. Primarily because of the presence of an additional pair of unpaired electrons on the methanol oxygen, there is a much greater extent of hydrogen bonding in methanoVwater systems than in acetonitrile/water systems.35 Katz et al.333 have also discussed the ternary nature of methanol/water systems, which have three distinct composition-dependent regimes of species distribution. For methanol volume fractions ranging from zero to ~ 0 . 4the , fraction of self-associated water species begins to decrease as water/methanol associated complexes are formed. Over the range of methanol volume fractions from ~ 0 . to 4 ~ 0 . 8 ,the water/methanol associated species attain their maximum distribution at a nominal methanol volume fraction of ~ 0 . 6 . At nominal methanol volume fractions above ~ 0 . 8 ,the volume fraction of free methanol begins to increase linearly.33~~~ As seen in Figure Ib, the measured T1values for DzO maintain a value of approximately 0.3 s from -30 to 100% methanol in the mobile phase, which corresponds to the composition region in which large numbers of water/methanol associated species are present. The decreased T1values measured in this composition region ( ~ 0 . 3 s) compared to those measured in the acetonitrile/water solutions (~0.5-1.2 s) over the same composition region are indicative of the more restricted mobility of water species in methanol solutions due to extensive hydrogen bonding. At nominal methanol concentrations less than 30%,the DzO 21' values approach the value measured for 100% water (0.64 9). This confirms that, in this composition region, as Katz et al. had predicted,33,36 water is primarily interacting with other water species. DzO TIMeasurements for Contact with the Stationary Phase. Although measurement of relaxation times at the bonded-phaselmobile-phase boundary might be expected to suffer from interface-induced inhomogeneities in the magnetic field, in practice T1 measurements are relatively insensitive to magnetic field inhomogeneitie~.~~ Therefore, if the above solutions are combined with stationary phase in the manner ~~
(35) Franks, F.; Ives, D. J. G. Q.Reo. 1966, 20, 1. (36) Katz, E. D.; Lochmtiller, C. H.; Scott, R. P. W. Anal. Chem. 1989, 61,349. (37) Glasel, J. A.; Lee, K. H. J.Am. Chem. Soc. 1974, 96,970.
described in the Experimental Section, and the D2O longitudinal relaxation times are remeasured, it becomes useful to compare the TI values for solutions in contact with the stationary phase to those observed in the solutions alone. This comparison in effect normalizes the relaxation data for the former case, because bulk solution phenomena that will affect the observed DzO 21' in both systems (e.g., hydrogenbonding interactions between the aqueous and organic solution species) are accounted for by the bulk solution measurements. The observed change in the D20 T1 upon combining the bulk solution with the stationary phase is therefore the result of the effects of DzO interactions with the stationary phase. It is reasonable to assume that rapid exchange of DzO species will occur between those in bulk solution and those associated with a solid ~urface3~ and that the resident lifetime of any associated D2O species will be much shorter than the T1 of the associated D20 ~pecies.l~n3~ This two-site rapidexchange model has been shown to be an appropriate model for solvent interactions with microporous silica gels14J6as well as similar solid materiak1.3~J8 In such systems, the observed relaxation rate i~l433~3
where Tl(oba)is the observed D20 2'1 value when the solution is placed in contact with the stationary-phase material, F b is the fraction of DzO molecules in the associated state, T l b is the relaxation time for the associated species, and T18is the T1 value in bulk solution. This equation is valid when the fraction of DzO molecules in the associated state that determines the observed T1 is small, and the relaxation rate of the associated DzO molecules is greater than for those in the bulk so1ution.l4J8 The TI values observed are a weighted average of those for free-solution D20 species and for those D20 species associated with the stationary phase.14 In practice, the overall fraction of aqueous species associated with reversed-phase stationary phases is expected to be relatively ~ m a l l . ~Additionally, ~J~ DzO makes up only 3% of the total volume of the aqueous portion of the bulk mobile phase and therefore makes up an even smaller fraction of the aqueous species associated with the stationary phase. Moreover, the DzO relaxation times measured for the solutions in contact with the stationary phases are in all cases smaller than those in the bulk solution. Therefore, the criteria for the validity of eq 2 38 are met by our experimental system. Due to the rapid exchange between the aqueous species in the bulk solution and the stationary phase, Tlb cannot be directly measured. Rigorous determination of F b is unavoidably accompanied by the experimental uncertainties inherent in attempting to accurately measure small amounts of sorbed mobile-phase species on RPLC stationary phase^.^ Therefore we are unable to obtain reliable quantitative information about the amount of DzO associated with the stationary phase from our TI measurements. However, via eq 2, the difference between the Tl(ob8) and TI, values a t each mobile-phase composition normalizes for the effects of bulk solution interactions on the observed DzO relaxation time. Examination of this difference allows qualitatiue trends in the relative degree of association of D20 with the stationary phase as a function of bulk mobile-phase composition to be gauged. If there is association of some fraction of the DzO with the stationary phase, it will be accompanied by reduction in diffusional freedom in going from a three-dimensional bulk fluid to a two-dimensional surface.14 Reduction in motional freedom of the associated aqueous species relative to those in the bulk solution will be manifested by a decrease in the (38)Woessner, D. E.; Snowden, B. S., Jr. J. Colloid Interface Sci. 1970, 34, 290.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
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TI for the former species compared to that for the latter,14 as per eq 1. Therefore the magnitude of the decrease in the DzO TI when the solution is placed in contact with stationary phase provides qualitative information about the relative fraction of DzO species associated with the stationary phase at that solution composition. Similarly,when all experimental variables except for the surface morphology of the stationary phase are held constant, comparison of DzO relaxation times for identical solutions in contact with different stationary phases is possible. A larger change in the DzO TI for one stationary-phase system compared to that for another implies that there is a larger degree of association of the DzO species with the former stationary phase. In Figure 2a, the relaxation time of DzO in the bulk mobile phase (TIJ minus the relaxation time of DzO when the same mobile phase is in contact with stationary phase TI(^^)) is plotted versus volume percent methanol in the bulk mobile phase for both stationary phases. This difference will hereafter be denoted as ATl. From the almost constant but very small ATl values for both ODs-1 and LT1 in contact with the methanol/water solutions, it can be concluded that there is only a very small degree of water association with either stationary phase throughout the mobile-phase binary composition range. In light of bulk solution behavior, this is a reasonable result. In these binary solvent mixtures, water is strongly associated with itself, and to a lesser degree with methanol, via hydrogen bonding. In comparison to the solution interactions, the dispersive interactions between water and the nonpolar stationary-phase alkyl ligands are extremelyweak;therefore, little to no attraction between water and the Cle moieties is expected or observed. The only stationary-phase sites with which the polar water molecules
1823
are expected to associateto any appreciable extent are residual surface hydroxyl (silanol) groups on the silica support. However, a greatly reduced number of silanols remain on the silica surface following derivatization with octadecylsilane reagents. There is ample experimental evidence that there is an increased extent of methanol association with the stationary phase for high methanol content mobile phases;2739,40this would be expected to result in some degree of C18 chain extension from methanol uptake. However in reality the degree of chain extension may be rather limited, particularly for highly aqueous methanoVwater mobile-phase compositions.3~1Under these "collapsed" conditions, the lengthy C18-bonded chains will be very effective a t shielding silanol groups from the bulk mobile phase and will thereby greatly restrict their accessibility for hydrogen bonding with water. Men and Marshall40 have used solute probes which intercalate into the bonded phase to measure the polarity of Cia-bonded phases in contact with methanol/water mobile phases and have found that the polarity of this interphase region was always less than the bulk solvent polarity. They attributed this to preferential intercalation of methanol over water into the bonded-phase structure.40 Therefore, regardless of the methanoVwater bulk solution composition,bondedphase surface structure and competing bulk solution interactions dictate that the degree of water association with both stationary phases will be small and remain essentiallyconstant over the methanol composition range. If the same sorts of experiments are performed with acetonitrile/DzO mixtures, distinctly different behavior is observed. In Figure 2b, the change in DzO relaxation time between the bulk mobile phase and that mobile phase in contact with both ODs-1 and L T l (AT1) is plotted versus percent acetonitrile in the bulk mobile phase. It is immediately evident that, unlike the methanol system, the DzO ATl's are not essentially constant over the entire composition range. In the bulk mobile-phase volume composition range from 0 to 80% acetonitrile, the degree of association of DzO with the stationary phase appears to be approximately constant. However, Figure 3 illustrates that the relative magnitude of the DzO AT1 in the acetonitrile/water mobilephase system is greater than that in the methanol/water mobile-phase system over virtually the entire composition range. As discussed in the previous section, in bulk acetonitrile/water mixtures acetonitrile-water hydrogen bonds are much less likely to be formed and are much weaker than methanol-water hydrogen bonds.323 The microheterogeneity of acetonitrile/water solutions allows water to preserve more of its native structure,92and this should allow water to more readily hydrogen bond with the residual stationary-phase surface silanols. In addition to much weaker solution interactions between water and acetonitrile, the microheterogeneity of acetonitrile/water solutions enables them to much more effectively solvate the stationary-phase alkyl chains than methanol/water mixtures. Alvarez-Zedpeda et aZ.42+43as well as Katz and co-workers33 have extensively discussed the effect of bulk solution structure of acetonitrile/ water mixtures on RP stationary-phase solvation. Because acetonitrile-rich microphases are formed even if acetonitrile is present in relatively small amounts,32#33acetonitrile/ water mobile phases are able to solvate the hydrophobic stationary-phase alkyl chains over virtually the entire mobilephase composition range.a~~2*~3 This results in increased stationary-phase chain extension4and therefore allows better (39) Carr, J. W.; Harris, J. M. Anal. Chern. 1987,59, 2546. (40) Men, Y.-D.; Marshall, D. B. Anal. Chem. 1990,62,2606. (41) Montgomery, M. E., Jr.: Green, M. A,: Wirth, M. J. Anal. Chern. 1992,64, 1176. (42) Alvarez-Zedpeda,A.; Barman, B. N.; Martire, D. E. Anal. Chern. 1992,64, 1978. (43) Alvarez-Zedpeda, A.; Martire, D. E. J. Chromatogr. 1991, 550, 285. ~
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
these stationary phases also agree with previously proposed models for stationary-phase solvation in acetonitrile mobile9 0.80 phase system^.^^,^^ a If the degree of water association for the two different L I 0.40 1 A stationaryphases studied are compared (Figure 2), it is evident that, for both mobile-phase systems, there is little difference 0 0 0.00 in the degree of water association between the two stationary phases. Water is slightly more associated with the lower .-E -0.40 tI I bonding density stationary phase (ODS-1) than with the 0 m higher bonding density phase (LT1) for the methanol/water -0.80 system at most compositions (Figure 2a). Figure 2b dem6 onstrates that, for the acetonitrileiwater mobile phases, the -1.20' ' ' ' ' ' ' ' ' degree of water association with the ODs-1 stationary phase 0 10 20 3 0 40 6 0 6 0 70 80 90 100 is approximately the same as for the LT1 phase for virtually % Organic Modifier in Bulk Mobile Phase all mobile-phase compositions. A qualitative prediction might have been made that there would be a significantly greater 1.20 I degree of water association with the ODs-1 phase than with 0 I LT1 phase, due to the much greater surface density of c18 0.80 alkyl ligands for the latter phase. However, as previously 1 r I-discussed, in methanol/water mobile phases the CIS alkyl groups are effective at shielding the silica surface from aqueous mobile-phase components even at relatively low alkyl ligand surface coverages. Any i n situ horizontal polymerization of ! the ODs-1 phase subsequent to the initial ligand surface -0.40 1 attachment would further shield the surface from contact with either mobile phase. Therefore in practice the degree of association of aqueous species with the silica substrate of both stationary phases is limited. However, recall that there -1.20 6 0 10 20 9 0 40 6 0 6 0 70 80 90 100 is a significant increase in the AT1 value for D20 for mobile phases with 180% acetonitrile. From the stability of the % Organic Modifier in Bulk Mobile Phase DzO chemical shifts measured in the mobile- and stationaryFlgure 3. Comparison of the change in 2H longitudinal relaxation time phase slurry samples compared to those in the neat mobile( Tl)for deuterium oxide in binary solutions with acetonitrile versus phase samples, the effect of mutual solubility of D20 in the methanol in contact with monomeric CIS stationary phases: (a, top) D20 in methanol (0)versus acetonitrile(A)mobile phases in contact acetonitrile-rich alkyl chain environment must play an with ODs-1. (b, bottom) D20 in methanol ( 0 )versus acetonitrile (A) important role in the association of water with either CISmobile phases in contact with LT1. bonded phase for the acetonitrile/water system over this composition range.14 penetration of mobile-phase components, including water, Comparisons to Organic Modifier 2H21' Studies. It to the stationary-phase surface silanols. should again be stressed that the AT1measurements discussed The water penetration (e.g., D2O association) for the most here are not quantitative measurements of the amounts of part increases only slightly as the volume percent of acetosorbed mobile-phase components in the stationary phase, but nitrile in the bulk mobile phase increases, up to = 80%. rather are qualitative measurements of the extent to which Beyond -80% acetonitrile, self-associated acetonitrile comthey are associated with the stationary phase under these plexes are the predominant species in bulk solution, and selfconditions. As shown in eq 2, T1 measurements for solvents associated water complexes are ~ c a r c e . ~ ~Within - 3 ~ this in contact with stationary phases will of necessity be an solution composition region, the disruption of water/water average of the bulk solvent and associated solvent T I~a1ues.l~ interactions in the mobile phase by the preponderance of Nonetheless, the T1 values measured in our laboratory for acetonitrile allowsthe water molecules to have greater freedom solvents in contact with the stationary phase are dominated to interact with the stationaryphase.14 The significant degree by the relaxation time of the bound solvent species, since T1 of stationary-phasealkyl chain solvation by the self-associated values for bound solvents are typically shorter than those for acetonitrile species also promotes a maximal degree of chain the bulk solution solvents.14 Examination of the difference extension.42 The resultant "extended" chain structure maxbetween the bulk solution T1 values and those measured for imizes stationary-phase accessibility to water. This is rethe solution in contact with the stationary phase is used to flected by the marked increase in the DzO AT1 over this normalize for the contributions from the relaxation of the concentration region (Figure 2b), which denotes a greater species of interest in the bulk solution. Finally, it should be degree of water association with both stationary phases. Since noted that spectroscopic measurement of any species' interthe chemical shifts that we observed for DzO in contact with actions with a RPLC bonded phase will be representative the bonded-phase materials are in all cases virtually identical of the average degree of interaction of that species with the to the corresponding chemical shifts in the bulk mobile-phase bonded-phase surface. For NMR T I studies of a n y solute in binary solutions, extreme changes in the chemical environcontact with RPLC bonded phases, it is expected that a ment of the DzO molecules, such as would be expected if the distribution of relaxation times would result from the varying majority of them were directly hydrogen bonded to surface degrees of solute association with such a heteroenergetic silanols, cannot be predominant.'* The association of water surface.'j with the CIS-bonded phases must therefore be due to a With these cautions in mind, it is useful to compare and combination of hydrogen bonding with residual silanols and contrast our 2H T1studies for D2O in the present work to the the formation of water/acetonitrilemixed species complexes32 results from our previous ZH TI studies of the methyl in the acetonitrile-rich alkyl chain environment.14 The results deuterons of methanol-d4 and acetonitrile-& in the same reported here for mobile phases containing in excess of 50% binary mobile-phase systems.27Such a comparison confirms by volume of acetonitrile are in excellent agreement with that organic modifier interactions with octadecyl stationary those of Marshall and McKenna from their similar D20 T1 phases are much more complex than D2O interactions and experiments.14 The general trends of water association with 1.20 1
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1003 6.00
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% Methanol in Bulk Mobile Pha8e Flgure 4. Cornpartson of the change In *H longitudinalrelaxation tlme (TI) for deuterkrm oxideand methyl deuteronsof m0than0I-d~In contact wlth monomeric Cle statbnaty phasee vmusvokune percent methanol In bulk methand/water moblle phase: (a, top) 0 2 0 (0)and methanold4 (0)In contact with ODS-1. (b, bottom) D20 (0)and methanol44 (0)In contact with LT1.
are distinctly different for methanol when compared to acetonitrile. In order to aid in the comparison, it is useful to overlay the results from our previous TI studies2' with the D2O results obtained in this study. In Figure 4, the change in deuterium 21' (AT1)for the methyl deuterons of methanold4 and for D2O are each plotted versus volume percent methanol in the mobile phase for both ODS-1 and LT1. Figure 4 illustrates that the degree of water associated with the stationary phase is almost constant and is quite limited for both ODS-1 and LT1, yet the degree of methanol association is considerably greater and is a function of the amount of methanol present in the bulk mobile phase. For methanol-d4 in contact with either stationary phase, there is relatively little disparity in the deuterium AT1's for bulk mobilsphase compositions ranging from -30 to 80% methanol and the degree of association of methanol with the stationary phase is less than for any other range of bulk methanoVwater compositions. This region correspondsto that in which water/ methanol associated complexes predominate in Katz et al.'s proposed solution structure of methanoVwater mixture^.^^^^ Hydrogen-bonding interactions are stronger in watedmethan01 associated species than in self-associated methanol species, and the former interactions are stronger and more directed than the predominantly dispersiveinteractions that take place between methanol and CISstationary-phaseligands. It is of course plausible that accessible silanol groups on the silica support could participate in hydrogen bonding with any of the methanol/water solution species. However, the extremely small AT1 values for DzO in this composition range (see also Figure 2a) indicate that aqueous association with the support surface silanola is likely minimal. Consequently, from the bulk solution characteristics, only a limited and relatively constant degree of methanol association with the stationary phase is expected and observed over this mobile-
1825
phase composition range. Solid/liquid interfacial tension measurements of CIS-modified silicas in contact with methanoVwater solutions in the composition range from 0 to 50 % methanol have demonstrated that methanol coverage of the Cla surface is essentially constant over the methanol volume composition range of 30-50 % .41 For mobile-phase compositions containing greater than 80% methanol, the methanol 2H AT1 increases with the volume percent of methanol in the bulk mobile phase (Figure 41, implying a significantly greater degree of methanol association with the stationary phase than is exhibited in the intermediate composition region. At nominal methanol volume fractions above -0.8, the volume fraction of the less polar self-associated methanol species increases linearly, commensurately with the decrease in water/methanol associated complexes.33@ Since the self-associated methanol solution species predominant in this composition region are not hydrogen bonded with water, they are much more likely to experience dispersive interactions with the CISmoieties of the stationary phase."' Uptake of methanol should enable the bonded alkyl chains to assume a more extended configuration.4@*uIf this extension is pronounced, the more open stationary-phase structure should allow a greater degree of penetration (and correspondingly,greater association)of both mobile-phase components into the stationary phase. However, examination of the D2O AT1 behavior in Figure 2a indicate that the degree of D2O association with the stationary phase is little changed for the low chain density phase (ODS1)and is virtuallyunchanged for the high-density phase (LT1) over this composition region. This signifies that any stationary-phase chain extension from methanol uptake is not so pronounced as to allow surface silanolgroups to effectively compete for hydrogen-bonding interactions with solution species. Therefore the much larger increase in methanol-d4 AT1 in this range is more logically attributable to more effective dispersive interactions between the bonded-phase alkyl ligands and the self-associated methanol species. A marked increase in methanol-d4 AT1 is also observed for mobile-phase compositions with less than 30% methanol. Contact angle measurements have confirmed that pure water does not wet Cu-bonded silica surfaces and that the addition of 20% methanol to pure water only brings about partial wetting." In predominantly aqueous mobile phases, the hydrophobic alkyl stationary-phase chains are thought to assume a highly collapsed or folded configuration in order to minimize their surface contact with the polar mobile phase.4p414This would cause methanol, the less polar mobilephase component, to become entrapped within the collapsed stationary-phasestructure (most likelywithinnarrow-necked silica pores) and thereby result in a distinct change in the methanold4 2HT I . Convincing chromatographic evidence for solvent entrapment under these conditions has been presented by Gilpin and Squires46from thermal studies on reversed-phasematerials. This description is also consistent with that of Maciel and Zeigler, who used CP/MAS solidstate 13C NMR and 2H solid echo NMR measurements of selectively deuterated C I alkyl ~ chain positions to study the mobility of these bonded phases in a variety of contact solvents.1B-18Bayer et al.lS2l and McNally and Rogers22have ale0found significantreductions in 13CTImeasurements (i.e., reduced mobility) for bonded-phase carbons in contact with highly aqueous systems. It is interesting to note the small increase in the D2O AT1 for both stationary phases in this region (Figure 2a), which is most likely due to increased accessibility to surface silanol groups which are adjacent to collapsed alkyl chain clusters. Figure 4 therefore illustrates (44)Lochtiller, C. H.; Hunnicutt, M. L. J. Phys. Chem. 1986, 90, 4318. (46) Gilpin,R. K.; Squires, J. A. J. Chrornatogr. Sci. 1981, 19, 195.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993 1
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phases changes rapidly in the solution composition region from 80 to 100% acetonitrile, and this cannot therefore be attributed solely to solution properties in the absence of the stationary-phase ~urface.'~ At low acetonitrile concentrations, acetonitrile is likely entrapped within silica pores by the more collapsed chain structure assumed by the hydrophobic alkyl stationary-phase chains in highly aqueous mobile phases,4 just as is the case for comparable methanol compositions. Comparison of our previous studiesz7with the present work illustrates that the association of mobile-phase components with the stationary phase that causes the formation of the solvation layer in RPLC is a complex phenomenon that is largely determined by the competition between the relative strengths of solvent/solvent and solventlstationary-phase interactions. This is illustrated by the distinct correlations between bulk solution structure and stationary-phase solvation by individual solution components.
CONCLUSIONS 1
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% Acetonitrile in Bulk Mobile Phase Comparison of the change In 2H longltudlnalrelaxation time (TI) for deuterlum oxide and acetonitrile-& in contact with monomeric CI8 stationary phases versus volume percent acetonitrile In bulk acetonlhlle/water mobile phase: (a, top) D20 (A)and acetonitrile-& (A)In contact with ODs-1. (b, bottom) D20(A)and acetonitrile-& (A) in contact with LT 1.
Flgure 5.
that when methanoLd4 and DzO are placed in contact with monomeric CISstationary phases, changes in TIfor both of these species are largely due to bulk solution microstructure and its resultant effects on bonded-phase solvation. A comparison between the degree of association with both stationary phases for acetonitrile and water can be ais0 be made by overlaying the changes in acetonitrile-& T1 values obtained in our previous studies27 with the D20 results presented here. From Figure 5 , it is evident that the degree of association with both stationary phases is much less for water than for acetonitrile. However, the degree of water association with the stationary phase is a function of bulk mobile-phase composition, whereas the degree of association of acetonitrile is relatively constant regardless of the mobilephase composition. This is opposite from the case with methanol/water mobile phases. These phenomena reflect the solution structure properties of acetonitrile-&/water mixtures and their concomitant effect on the RP stationaryphase surface. When acetonitrile is added to bulk aqueous solutions, it enters cavities in the well-defied water structure until these sites are occupied. As the amount of acetonitrile is further increased, it becomes increasingly self-associated in aggregates or loosely defined clusters.32~42Although this results in bulk solution microheterogeneity, the acetonitrile species experience a relatively homogeneous solution environment over a large binary composition range, due to their extensive self-association.32-34 As illustrated in Figure 5, it is therefore quite reasonable that acetonitrile-& exhibits a relatively constant degree of association behavior with both stationary phases throughout the composition range. The physical properties (viscosity, surface tension, dielectric constant) of acetonitrile/water mixtures change slowly and monotonically over the composition range.14 However, the association behavior for water in contact with both stationary
The work described here and in our previous studies2' confirms the usefulness of using deuterium NMR in the solution state for fundamental chromatographic research. Moreover,we have also shown that the technique can be made applicable to measurements at all mobile-phasecompositions despite problems encountered in previous studies.'* The results obtained give a qualitative picture of the degree to which both aqueous and organic mobile-phase components interact with the stationary phase as a function of mobilephase composition. They also show the complexity of these interactions and demonstrate how the final structure of the solvated stationary phase is not only greatly influenced by bulk mobile-phase solution structure but is also influenced by the degree of alkyl chain crowding on the surface of the silica. Finally, this study has again demonstrated the distinctly different RPLC solvation characteristics of methanoVwatermobile-phasesystems as compared to acetonitrile/ water systems.
ACKNOWLEDGMENT The authors thank Antony J. Williams,Anthony J. I. Ward, and Peter Can for helpful discussions. Jim Breeyear's superb technical assistance with the NMR experiments is greatly appreciated. The authors also thank Charles H. Lochmiiller for providing the Partisil silica for the LT1 stationary phase from the Duke Standard Collection established by a grant from Whatman-Chemical Separations. Grateful acknowledgement is made to the University Committee on Research and Scholarship at the University of Vermont, the Society for Analytical Chemists of Pittsburgh, and the donors of the Petroleum Research Fund, administered by the American Chemical Society,for partial financial support of this research. Some portions of this work have been presented at the NATO Advanced Study Institute on Theoretical Advances in Chromatography and Related Separation Techniques, held in Ferrara, Italy in August 1991; at the 1991Eastern Analytical Symposium;at the 1992 Pittsburgh Conferenceon Analytical Chemistry and Applied Spectroscopy; at the Sixteenth International Symposiumon Column Liquid Chromatography (HPLC '92); and a t the Fall 1992 National Meeting of the American Chemical Society. RECEIVEDfor review November 16, 1992. Accepted March 29, 1993.
