Solvatochromic Studies on Reversed-Phase Liquid Chromatographic

Our group has utilized two π* dyes, N,N-diethyl-4-nitroaniline and N-methyl-2-nitroaniline, to measure π* values in reversed-phase thin-layer chroma...
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Anal. Chem. 1996, 68, 1387-1393

Solvatochromic Studies on Reversed-Phase Liquid Chromatographic Phases. 2. Characterization of Stationary and Mobile Phases Huiyan Lu and Sarah C. Rutan*

Department of Chemistry, Box 2006, Virginia Commonwealth University, Richmond, Virginia 23284-2006

UV-visible spectra of three solvatochromic dyes, 4,6dichloro-2-[2-(1-methyl-4-pyridinio)vinyl]phenolate, Nmethyl-2-nitroaniline, and 2-nitroaniline, are employed to characterize the octadecyl-derivatized silica (ODS2) stationary phase and the corresponding mobile phases used in reversed-phase liquid chromatography. The mobile phases studied are methanol-water, acetonitrile-water, and tetrahydrofuran-water, with composition ranges from 100% to 5% of organic modifier. Across the whole mobile phase composition range, the ODS2 stationary phase shows three distinct behaviors upon variation of the mobile phase composition, which are described as nonretentive, solvated, and collapsed. Reversed-phase liquid chromatography (RPLC) has been widely used as a separation and analysis method. Retention is based on the differential interactions of the solutes with the stationary and mobile phases, but the properties of these phases have not been fully elucidated. Johnson and co-workers1 have investigated the relationship of retention with solvent properties and found that the logarithm of the capacity factor (k′) for many solutes has a linear relationship with the ET(30) parameter of the mobile phase, which is a comprehensive parameter reflecting the dipolarity/polarizability and hydrogen-bond-donating ability of a solvent.2 These retention relationships have stimulated the investigation of mobile phase properties. In addition to the singleparameter ET(30) scale,1,3,4 the Kamlet-Taft solvatochromic scales5-8 have also been used to characterize RPLC systems.9-11 Recently, Carr’s group reported a series of π*,12 ET(30), and R12,13 values probed by different dyes for the most commonly used mobile phases in RPLC. Plots of these values versus mobile phase composition reveal some important information about the retention mechanism of the solutes. (1) Johnson, B. P.; Khaledi, M. G.; Dorsey, J. G. Anal. Chem. 1986, 58, 2354. (2) Reichardt, C. Solvents and Solvent Effects in Organic Chemistry, 2nd ed.; VCH: Weinheim, 1988; pp 364-371. (3) Johnson, B. P.; Khaledi, M. G.; Dorsey, J. G. J. Chromatogr. 1987, 384, 221. (4) Dorsey, J. G.; Johnson, B. P. J. Liq. Chromatogr. 1987, 10, 2695. (5) Kamlet, M. J.; Abboud, J. L.; Taft, R. W. J. Am. Chem. Soc. 1977, 99, 6027. (6) Taft, R. W.; Kamlet, M. J. J. Am. Chem. Soc. 1976, 98, 2886. (7) Kamlet, M. J.; Taft, R. W. J. Am. Chem. Soc. 1976, 98, 377. (8) Kamlet, M. J.; Abboud, J. L.; Abraham, M. H.; Taft, R. W. J. Org. Chem. 1983, 48, 2877. (9) Sadek, P. C.; Carr, P. W.; Doherty, R. M.; Kamlet, M. J.; Taft, R. W.; Abraham, M. H. Anal. Chem. 1985, 57, 2971. (10) Carr, P. W.; Doherty, R. M.; Kamlet, M. J.; Taft, R. W.; Melander, W.; Horva´th, Cs. Anal. Chem. 1986, 58, 2674. (11) Cheong, W. J.; Carr, P. W. Anal. Chem. 1989, 61, 1524. (12) Cheong, W. J.; Carr, P. W. Anal. Chem. 1988, 60, 820. (13) Park, J. H.; Jang, M. D.; Kim, D. S.; Carr, P. W. J. Chromatogr. 1990, 513, 117. 0003-2700/96/0368-1387$12.00/0

© 1996 American Chemical Society

Recent work has suggested not only that selectivity in RPLC is affected by the mobile phase but also that the environment of a solute within the stationary phase has strong effect on selectivity.14 To probe the stationary phase polarity, fluorescence spectroscopy has been used very often. Two of the most common probes are pyrene and the dansyl moiety. Under the conditions used for C18-bonded silica reversed-phase chromatography, the surface polarity detected by pyrene for various mobile phase compositions shows an interesting trend. At the middle range of solvent composition, the surface polarity decreases with decreasing organic modifier in the mobile phase for methanol-water, acetonitrile-water, and THF-water solvent mixtures.15-17 However, with higher water concentrations, the surface polarity increases when the amount of organic modifier is decreased in the mobile phase.16,17 Another class of fluorescence probes contains a dansyl group. Men and Marshall18 used two dansyl derivatives, n-propanedansylamide and n-decanedansylamide, to characterize the C18derivatized silica surface in methanol-water mixtures with compositions ranging from 100% to 50% methanol. It was concluded that the solvent composition in the partitioning zone of the stationary phase differs from that of the bulk mobile phase and is different at different depths within the zone. Another fluorescence technique that has been used is to covalently bond the dansyl group to the surface-bound alkyl chains. Unlike the sorbed fluorophor, the bonded fluorophor can detect the environment at a specific position within the stationary phase. Lochmu¨ller et al.19 reported that the polarity experienced by the probe is affected by the solvent. Surfaces solvated with THF, acetonitrile, methanol, and water are all more polar than the dry surface. Rutan and Harris have given a detailed discussion of these studies in a recent review.20 The fluorescence investigations described above characterized the overall surface polarity, which is a nonspecific parameter. To have a more detailed understanding of the surface chemistry, the Kamlet-Taft parameters have been used to study the heterogeneous chromatographic stationary phases by using UV-visible spectroscopy. Lindley et al.21 used a series of dyes as solvatochromic probes. These authors have indicated that, in comparing the silica surface to ordinary solvents, its dipolarity/polarizability, (14) Carr, P. W.; Li, J.; Dallas, A. J.; Eikens, D. I.; Tan, L. C. J. Chromatogr. A 1993, 656, 113. (15) Carr, J. W.; Harris, J. M. Anal. Chem. 1986, 58, 626. (16) Carr, J. W.; Harris, J. M. Anal. Chem. 1987, 59, 2546. (17) Ståhlberg, J.; Almgren, M. Anal. Chem. 1985, 57, 817. (18) Men, Y.-D.; Marshall, D. B. Anal. Chem. 1990, 62, 2606. (19) Lochmu ¨ ller, C. H.; Marshall, D. B.; Wilder, D. R. Anal. Chim. Acta 1981, 130, 31. (20) Rutan, S. C.; Harris, J. M. J. Chromatogr. A 1993, 656, 197. (21) Lindley, S. M.; Flowers, G. C.; Leffer, J. E. J. Org. Chem. 1985, 50, 607.

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as measured by the π* scale, gives the most important contribution to the overall polarity of the silica surface. The second most important factor is the hydrogen-bond-donating ability, as measured by the R value, and the least important component is the hydrogen-bond-accepting ability. Our group has utilized two π* dyes, N,N-diethyl-4-nitroaniline and N-methyl-2-nitroaniline, to measure π* values in reversed-phase thin-layer chromatography (TLC)22 and RPLC23,24 with methanol-water and acetonitrilewater solvent mixtures as mobile phases, with compositions ranging from 100% to 50% of organic modifier. The results showed that the π* values for the C18 phase slurries are higher than those for bulk alkanes because of stationary phase solvation and that the values are less variable than those for the mobile phase due to the protection of the solute by the C18 chains. The R values of the stationary phase slurries detected by ET-3325 are generally higher than those for the bulk mobile phase in this range of mobile phase composition for acetonitrile- and methanol-water mixtures.23,24 Dallas and Carr used n-hexadecane as a model for the stationary phase to evaluate the feasibility of a bulk partition mechanism.26 There are significant differences between the solvatochromic values for the bulk alkane and the bonded alkyl phase. Due to the heterogeneity of the surface region serving as the stationary phase, the values detected by different probes may be quite variable. Li and Rutan27 have demonstrated differences in the R values determined using two different dyes for an underivatized silica surface in contact with various mobile phases. Different dyes may reveal different environments at different positions within the stationary phase environment, possibly due to steric constraints. In this work, RPLC systems with octadecyl-derivatized silica stationary phases are further characterized by using UV-visible spectroscopy. The mobile phases are methanol-water, acetonitrile-water, and THF-water mixtures, with compositions ranging from 100% to 5% in organic modifier. Three solvatochromic dyes are used as probes: N-methyl-2-nitroaniline (NM2NA) as a π* value indicator, 4,6-dichloro-2-[2-(1-methyl-4-pyridinio)vinyl]phenolate (DCMPVP) as an R dye, and 2-nitroaniline (2NA) as the β dye. DCMPVP is a new merocyanine dye, synthesized in our laboratory,28 that has lower basicity than ET-30 and other stilbazolium betaines. The structure of this compound is shown below.

EXPERIMENTAL SECTION Chemicals and Materials. DCMPVP was synthesized in our laboratory.28 NM2NA and 2NA were purchased from Lancaster (22) Jones, J. L.; Rutan, S. C. Anal. Chem. 1991, 63, 1318. (23) Helburn, R. S.; Rutan, S. C.; Pompano, J.; Mitchem, D.; Patterson, W. T. Anal. Chem. 1994, 66, 610. (24) Hayashi, Y.; Helburn, R. S.; Rutan, S. C. In Proceedings of the 4th Symposium on Computer-Enhanced Analytical Spectroscopy; Wilkins, C. L., Ed.; Plenum Press: New York, 1993; p 257. (25) Kessler, M. A.; Wolfbeis, O. S. Chem. Phys. Lipids 1989, 50, 51. (26) Dallas, A. J. Ph.D. Dissertation, University of Minnesota, Minneapolis, MN, 1995. (27) Li, Z.; Rutan, S. C. Anal. Chim. Acta 1995, 312, 127. (28) Lu, H.; Rutan, S. C. Anal. Chem. 1996, 68, 1381.

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Laboratory. Methanol, acetonitrile, and THF were purchased from Baker Chemical Co., Baxter Chemical Co., and Fisher Scientific Co., respectively. All purchased reagents were used as received without further purification. Spherisorb silica (S5W) and octadecyl-bonded Spherisorb silica (ODS2) were purchased from Alltech. The average particle size is 5 µm in diameter, and the surface area is 220 m2/g for both stationary phases. The ODS2 is an endcapped, monomeric C18bonded silica with a total carbon content of 12%. Sample Preparation. The solvent mixtures and the dye solutions of the solvent mixtures were prepared by mixing measured volumes of the individual components. The solution concentrations of NM2NA and 2NA were ∼2 × 10-4 M for all systems studied. The concentrations of the DCMPVP solutions were 2 × 10-4 M for methanol- and acetonitrile-aqueous solutions, acetonitrile-ODS2 slurries, and silica slurries, 1 × 10-4 M for ODS2 slurries with 100%-60% methanol, 5 × 10-5 M for 50%-20% methanol, and 2 × 10-5 M for 10%-5% methanol. Each sample of the ODS2 and silica slurries was prepared by mixing 150 mg of stationary phase with 6 mL of the designated liquid to keep a constant ratio of liquid and stationary phase. The precipitated slurry was collected, and a sample spectrum was obtained a few hours after sample preparation. Procedure. All absorption spectra were obtained from a Shimadzu UV-265 spectrophotometer. An integrating sphere was attached to the instrument for collecting the diffusely reflected light. BaSO4 was used as a standard in the reference beam and as a reflectance backing for the sample cell. Each sample was placed in a quartz cell with a 0.1 cm path length, and a slit width of 5 nm was used. Data were collected and transferred to an IBM PC through an IEEE-488 interface using software supplied by Shimadzu. All data treatment was done with programs written with Turbo Pascal (Borland) and in Lotus spread sheets on IBMcompatible PCs. Background contributions were subtracted from the sample signal by matching intensities in a horizontal region of the spectrum. To reduce the noise, the spectral curves were smoothed using a quadratic polynomial smoothing filter with a 21-point window width.29 The peak position was found at the zero point of a regression line fit to the first derivative of the smoothed curves in the peak region. The π* values determined using this approach are good to about (0.02, R values are good about (0.05, and the β values are precise within (0.10. The retention data were collected on a Hewlett Packard Model 1050 liquid chromatograph equipped with a variable wavelength UV-visible detector. A 75 mm × 4.6 mm column packed with Spherisorb ODS2 was purchased from MetaChem. The dead volume marker was uracil, detected at 254 nm. The retention times for 2-nitroaniline and N-methyl-2-nitroaniline were measured at 400 nm. RESULTS AND DISCUSSION To understand the interactions of solutes with the stationary phase and mobile phase in RPLC, it is necessary to examine the environments in the stationary phase and the mobile phase that a solute experiences during the procedure of chromatography. Three basic parameters can describe the abilities of the stationary and mobile phases to interact with a solute: the π* value, used to describe the dipolarity/polarizability; the R value, used to describe the hydrogen-bond-donating ability; and the β value, used (29) Savitzky, A.; Golay, M. J. E. Anal. Chem. 1964, 36, 1627.

Figure 1. π* values versus percent methanol for solution, bare silica slurries, and ODS2 slurries.

to describe the hydrogen-bond-accepting ability of the environment of a solute, i.e., the stationary phase or mobile phase. The dyes used, 2NA, DCMPVP, and NM2NA, can be used either as probes to detect environments using UV-visible spectroscopy or as solutes to measure the retention in chromatography. In this work, slurry spectra are measured. A slurry spectrum is a mixture of contributions from dye in stationary phase and dye in solution that remains with the slurry. The higher the retention, the higher the concentration of dye in the stationary phase, and the lesser the contribution from solution. The slurry spectrum, as well as the resulting λmax, will reflect more of the stationary phase characteristics. Previous studies have established the relative concentration required to adequately assess the stationary phase characteristics.23 Dipolarity/Polarizability. The simplest measurement of the solvent properties is the dipolarity/polarizability, which can be quantitatively described by using the Kamlet-Taft π* parameter. The UV-visible spectral shift of the NM2NA dye was used to measure this value. The spectrum of this dye reflects only the dipolarity/polarizability of its environment,30 and this dye is therefore called a π* dye. The Kamlet-Taft standard is used here, which is to set the π* value of cyclohexane equal to zero and the value for dimethyl sulfoxide (DMSO) equal to unity.5 Figure 1 shows how the π* values change with the ratio of methanol in a methanol-water mixture solvent system. Since water is more dipolar (π* ) 1.09)8 than methanol (π* ) 0.60),8 increasing the amount of this component should increase the overall dipolarity of system. Therefore, it is reasonable that the solvent curve goes up with a decrease in the methanol ratio in the bulk solvent mixture. The silica curve and bulk solvent curve are very similar. This similarity reflects the fact that NM2NA has little retention on silica when methanol-water mixtures are used as mobile phases. This has been confirmed by chromatographic retention measurements.31 However, these measurements are useful in (30) Yokoyama, T.; Taft, R. W.; Kamlet, M. J. J. Am Chem. Soc. 1976, 98, 3233. (31) Helburn, R.; Rutan, S. C. Unpublished results, Virginia Commonwealth University, Richmond, VA, 1993.

Figure 2. Retention data on ODS2 for 2-nitroaniline and N-methyl2-nitroaniline with methanol-water mobile phase.

confirming that the presence of light-scattering particles does not affect the position of the absorbance peak maximum. The third curve in Figure 1 shows the π* values for the ODS2 slurries. This curve can be separated into three regions, corresponding to composition ranges of 100%-70%, 70%-30%, and 30%5%, respectively. With 100% methanol, the ODS2 slurry has a π* value of 0.58, which is essentially the same as that of the bulk solvent (0.59). This is because there is no significant retention of these aniline dyes on the ODS2 surface when a 100% methanol mobile phase is used (see Figure 2).23 As the amount of water in the solvent increases (methanol decreases from 100% to 70%), the π* value of the ODS2 slurries increases. But the ODS2 curve gradually deviates from the bulk solvent curve as retention increases. In the literature, the results obtained after elimination of the mobile phase contribution showed that the π* values of stationary phases probed with N,N-dimethyl-4-nitroaniline are between those for pure methanol and 90% methanol for this methanol composition range (∼0.7).23 Retention of the analyte gradually increases as the methanol content decreases within this composition range, as shown in Figure 2. Since the NM2NA dye has very small retention in this composition range, the mobile phase makes a significant but variable contribution to the observed π* values of the ODS2 slurries. It is likely that the “true” π* of the stationary phase surface is approximately constant in this range, with a π* similar to that for a 90% methanol environment (∼0.7). The second range to be discussed here corresponds to the methanol percentage varying from 70% to 30%. Within this range, the π* values of ODS2 slurries decrease as the water content increases, even though the π* value of bulk solvent continues to increase. Since the alkyl chains are less polar than the mobile phase, this gradual decrease in the π* values (which is a totally different trend from that observed for the mobile phase) is probably caused by increasing retention of the dye and decreasing the solvation of the C18 chains. As partitioning is thought to be the major mechanism for retention, the dye NM2NA experiences surroundings that are less characteristic of the mobile phase and more characteristic of the octadecyl chains. Analytical Chemistry, Vol. 68, No. 8, April 15, 1996

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Some other results from literature work are consistent with the interpretation given above. McCormick and Yonker and their co-workers measured the solvent content in an ODS phase using a gas chromatographic method.32-34 Carr and Dallas used hexadacane as a model for the stationary phase and measured the mobile phase content in bulk hexadacane.26 These results also showed that the degree of solvation of the stationary phase was affected by the bulk mobile phase composition. The description above is also supported by observations made during the sample preparation procedure. The ODS2 slurries with mixtures containing 100%-70% methanol can be easily prepared by simply mixing the ODS2 material with the corresponding solvent mixture, but with methanol percentages below 70%, the same method does not work at all. To obtain slurries of these solvent mixtures, we had to prewet the ODS2 material with a small amount of pure methanol before mixing with the appropriate solvent mixture. This method worked acceptably for solvent compositions down to 50% methanol, but as the methanol composition is decreased further, a higher extent of prewetting is required. Thus, the true composition of the mobile phase may not be controlled, especially for very low methanol percentages. This problem prompted us to develop another method for preparing the samples. The method used here is that of mixing the ODS2 material with a calculated volume of methanol first and then carefully adding the appropriate amount of water and gently stirring. At solvent compositions above 30%, the C18 chains are solvated by the mobile phase. The solvated C18 chains are “organized and extended away from the surface”.20 Partitioning is the predominant retention mechanism. The alkyl chains can protect the retained solute from exposure to bulk solvent. Therefore, the change in π* values in ODS2 slurries versus solvent composition is much smaller than that for the bulk solvents. The third range to be discussed here is for methanol concentrations below 30%. In this composition range, virtually all of the dye can be considered to be within the stationary phase, as dye retention is very high (seen Figure 2). Here, the π* values for the ODS2 slurries increase again as the methanol concentration decreases further, and the rate of the increase is even higher than that for the bulk solvents over the same range. From 30% to 5%, the π* values increase from 1.19 to 1.23 in the bulk solvent and from 1.23 to 1.30 in the silica slurry. The net increases are 0.04 and 0.07, respectively. But in ODS2 slurries, the value increases from 0.74 to 0.97. The 0.23 increment in the ODS2 slurry value is almost 5 times greater than that observed for the bulk solvent. The ODS2 slurry curve appears to gradually approach the bulk solvent curve. This trend indicates that the environment of the NM2NA molecule now is becoming more and more similar to that of the bulk solvent. A plausible explanation for this phenomenon is that the extent of solvation has an influence on the condition of the stationary phase surface. As the methanol ratio decreases below 30%, the extent of chain solvation decreases rapidly. The alkyl chains on the surface gradually bend, and the ends of the chains aggregate together through hydrophobic attractions. This chain configuration is termed as a “collapse” as described by Carr and Harris.15 More direct evidence for this collapse was given recently by Pemberton et al. using Raman (32) McCormick, M. M.; Karger, B. L. Anal. Chem. 1980, 52, 2249. (33) Yonker, C. R.; Zwier, T. A.; Burker, M. F. J. Chromatogr. 1982, 241, 257. (34) Yonker, C. R.; Zwier, T. A.; Burker, M. F. J. Chromatogr. 1982, 241, 269.

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Figure 3. π* values versus percent organic solvent for solution and ODS2 slurries in acetonitrile- and tetrahydrofuran-aqueous systems.

spectroscopy.35 Such a configuration prevents the solute from completely partitioning into the alkyl chains. Thus, some of the dye (solute) molecules remain on the outside of alkyl chain layer and are exposed to bulk solvent. As the methanol concentration decreases further, the collapse becomes more extensive, leaving more solute exposed to bulk solvent. Thus, adsorption becomes the predominant mechanism in this composition range. Figure 3 shows the π* curves for acetonitrile- and THFaqueous systems. The trends exhibited by these curves are very similar to those observed for the methanol-water system shown in Figure 1. The ODS2 curves in Figure 3 can be separated into three regions, as was done for the methanol system. The similarities of these curves indicate that the description given above for the methanol system is suitable for the acetonitrile and THF systems as well. The first range starts at 100% and ends at 70% for methanol, 60% for acetonitrile, and 50% for THF. The range is extended in acetonitrile and even more so in the THF system. The second range is also extended to a lower ratio of organic modifier, i.e., 20%, for both acetonitrile and THF. This range extension indicates the stronger solvation ability of acetonitrile and THF relative to methanol. The slope (∆π*/∆C) in the third range for THF (0.33) is smaller than those for both methanol (0.92) and acetonitrile (1.07). A larger slope indicates a larger change in π* values of the ODS2 slurries toward the solution values. This slope difference implies that the C18 chain collapse occurs more extensively in the methanol- and acetonitrileaqueous systems than in the THF-aqueous mixtures and shows that THF has a stronger solvation ability for C18 chains than the other two solvents. Note that for all three solvents, the π* of the ODS2 phase is never less than that of the pure organic solvent (even when retention is high), indicating that the mobile phase plays a critical role in determining the dipolarity/polarizability of the stationary phase. Hydrogen Bond Acidity. Beside the π* value, the R value, which measures the hydrogen-bond-donating ability of a solute (35) Pemberton, J. E.; Cai, M.; Thompson, W. R. Abstracts of Papers, 208th National Meeting of the American Chemical Society, Washington, DC, Fall 1994; ACS: Washington, DC, Abstract No. 16.

Figure 4. R versus vs percent organic solvent for solution, bare silica slurries, and ODS2 slurries in methanol- and acetonitrileaqueous systems.

environment, is also an important parameter that reflects the conditions of a system. The new stilbazolium betaine dye, DCMPVP, was employed to determine the R value of these phases. Its solvatochromism was described in a previous article.28 To determine an R value, the dipolarity/polarizability effect on the absorbance shift must be eliminated, so a reference dye which is sensitive only to dipolarity/polarizability has to be used. For convenience, the dye NM2NA is used here. The π f π* transition energies of DCMPVP in solvents without hydrogen-bond-donating ability, such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), THF, ethyl acetate, and N,N-dimethylacetamide, are plotted against the corresponding values for NM2NA. The regression line for these data is the standard line, i.e., the zero R effect line. The deviation from this line is considered to be a quantitative measurement of the R effect for the solvent.6 The R value of methanol is normalized to 1.00. The resulting R values versus the percentage of organic solvent in methanol- and acetonitrile-aqueous systems are plotted in Figure 4. In the methanol system, as shown by solid lines in Figure 4, the R values in bulk solvents display unusual behavior. At compositions near 40%, the R values detected by DCMPVP reach a minimum. This trend is in agreement with data for the methanol-water system probed with another zwitterionic dye, 2,6diphenyl-4-(2,4,6-triphenyl-1-pyridino)phenoxide (ET-30), which was studied by Cheong and Carr.12 The formation of a complex via hydrogen-bonding between methanol and water is one possible explanation for this minimum. The R value, i.e., hydrogen-bonddonating ability, of this complex is lower than those for either pure methanol or water. Similar to the π* values, the R data in Figure 4 for the ODS2 slurries of methanol aqueous solvents can also be separated into three solvent composition ranges, i.e., 100%-70%, 70%-30%, and 30%-5% of methanol. This behavior can be discussed by using the bulk solvent data as the reference. In the first composition range, the R values for both the ODS2 slurries and the bulk solvents decrease when the methanol ratio decreases, and the R values for the two phases are similar. An attempt to get retention

data for the DCMPVP dye on the Spherisorb ODS2 stationary phase failed, since we never saw the dye elute from the HPLC column using any mobile phase. This dye seems to have a very strong interaction with the stationary phase, but the R values of the ODS2 slurries are very similar to those of the mobile phase in the high methanol composition range, indicating that the dye experiences an environment similar to that of the bulk mobile phase. As the composition drops below 70%, these two curves begin to deviate from one another. The bulk solvent values decrease further, and values for the ODS2 slurries begin to increase. The bulk solvent curve reaches a minimum at 40%, and the ODS2 slurry curve shows a plateau from 50% to the end of the second range (30%). The hydrogen-bond-donating ability and dipolarity of residual silanols in the ODS2 phase are higher than those in the solvated C18 chain region. The very high R values probably indicate that the DCMPVP molecules interact with the residual silanols and are highly oriented so that the O atom in the molecule points toward the silica surface. For ODS2 used here, the C18 coverage of silica surface is about 2.72 µmol/m2.36 Compared with 8.0 µmol/m2 of silanol groups37 on silica surface, about 66% of the silanol groups on the silica surface are underivatized. The residual silanol groups have a significant contribution to both the retention and the spectral shifts of the DCMPVP over this second solvent composition range. The 30% composition point represents the end of the second and the beginning of the third composition range. After this point, the R value of the ODS2 slurries starts to decrease. The values rapidly approach the bulk solvent values and even go below these values at about 25% methanol. This trend reflects the collapse of the C18 chains, and the dye DCMPVP is gradually exposed to bulk solvent, which causes the ODS2 slurry curve to gradually approach the bulk solvent curve. After the C18 chains have completely collapsed, a significant amount of the dye molecules are exposed to bulk solvent and adsorbed on the hydrophobic alkyl surface. Due to the very low solvation (almost nil) of the C18 chains with the solvent, the dye DCMPVP must “feel” both the hydrophobic environment of the alkyl surface and the mostly hydrophilic environment (dipole-dipole and hydrogen-bonding) of the bulk solvent. The fact that the R values of the slurries are less than those for the bulk solvent is the result of averaging the values for both interactions described above. The silica slurry curve in Figure 4 can give us some clues about the nature of the silica surface. First, the very high R values indicate that the silanol groups have very high hydrogen-bonddonating ability. Second, this hydrogen-bonding ability is strongly affected by the properties of the mobile phase. The R value of silica slurries detected by DCMPVP for the whole mobile phase composition range cannot be obtained, since the absorbance of DCMPVP in a high-water-content solvent slurry does not give a satisfactory spectrum; the reasons for this behavior are described below. The stilbazolium betaine dye, DCMPVP, was synthesized in our lab,28 and this is first time that it has been used as a probe to detect properties of reversed-phase liquid chromatographic systems. DCMPVP is successful in probing both the mobile phase and the ODS2 stationary phase for RPLC. However, in silica slurries of aqueous solvent mixtures, DCMPVP is less successful as a probe because of protonation of this dye. Satisfactory spectra (36) MetaChem Technologies Inc. 1995 Catalog, p 33. (37) Nawrocki, J.; Buszewski, B. J. Chromatogr. 1988, 449, 1.

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of the silica slurries can be obtained only using high methanol ratios such as 100% and 90%. Any silica slurry with a methanol ratio below 60% does not give a reliable spectrum that allows determination of the peak maximum position for the π f π* transition of the unprotonated form. An increase in acidity with an increase in water content may cause this situation, since the spectrum of the protonated form does not exhibit a solvatochromic band in this spectral region. For silica slurries with acetonitrilewater, no satisfactory spectra can be obtained over the entire solvent composition range. The R values versus solvent composition for the acetonitrilewater system are also shown in Figure 4 (dotted lines). The 100% bulk solvent has an extremely low R value compared to the others. There is then a sudden rise from 100% to 90% and almost no change afterward. The low R value at the 100% point can be easily rationalized from the structure of acetonitrile. This molecule has only three hydrogens directly attached to the carbon atom, and these hydrogens do not have strong hydrogen-bond-donating ability. The rest of the curve indicates that water may aggregate near the O atom that is the hydrogen bond functional group of the DCMPVP molecule. The structure of acetonitrile-water mixtures was explored by Marcus and Migron38 on the basis of thermodynamic data and by Rowlen and Harris using Raman spectra of the CN stretching band.39 These authors pointed out that there exists a strong microheterogeneity in this solvent mixture, i.e., preference for neighbors of the same kind of molecule, and this is consistent with the relatively constant behavior of the acetonitrile curve throughout most of the composition range. The data in the high acetonitrile content range of this curve are in good agreement with R value trends detected by ET30, as done by Cheong and Carr12 and Marcus and Migron,38 and by ET-33, as done by Helburn and Rutan.23 But in the high water ratio range, as the water content increases, the R value measured here does not show an increase as does the R value determined by using the ET-30 dye. The curve for the R values of the ODS2 slurries of acetonitrilewater solvent mixtures in Figure 4 shows some interesting phenomena. The value for 100% acetonitrile seems to be an implausible point. In contrast to the very low R value in bulk solvent, the ODS2 slurry has a very high R value relative to the other data on the plot. This pronounced difference of the R values for 100% acetonitrile also appeared in the values detected by ET33 shown in the earlier literature.23 At this point, the residual silanol groups must make a significant contribution to the hydrogen bond acidity of the stationary phase environment. Due to the extremely low R value of pure acetonitrile, the dye molecules have a hydrogen-bonding ability that is unsaturated. This hydrogen-bonding ability forces the dye molecules to interact with residual silanol groups and to form hydrogen bonds. The hydrogen bonds donated from the surface silanols to DCMPVP contribute to the very high R value of the ODS2 slurry. The rest of the ODS2 curve can be explained similarly to the methanolODS2 curve. The three regions are 90%-60%, 60%-20%, and 20%5%, distinguished in the same way as for the π* curve for the acetonitrile system shown in Figure 3. If the bulk solvent curve is still used as the reference, there is a tremendous similarity between the acetonitrile-ODS2 curve and the methanol-ODS2 (38) Marcus, Y.; Migron, Y. J. Phys. Chem. 1991, 95, 400. (39) Rowlen, K. L.; Harris, J. M. Anal. Chem. 1991, 63, 964.

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Figure 5. β values versus percent organic solvent for solution and ODS2 slurries in methanol-, acetonitrile-, and tetrahydrofuranaqueous systems.

curve, except for the starting and end points of each composition range. Hydrogen Bond Basicity. The third parameter to be discussed here is the β value, which is a measurement of the hydrogen-bond-accepting ability. The method for obtaining the β values is similar to the method for obtaining R values. The dye 2NA is used as the β value indicator, and NM2NA is employed to eliminate the dipolarity/polarizability contribution to the 2NA spectrum. The solvents benzene, methylene chloride, toluene, trichloroethylene, carbon tetrachloride, cyclohexane, and hexane were used as standard solvents to determine the non-β effect line. The deviation from this line is the β measurement, which is normalized so that the β value for hexamethylphosphoramide (HMPA) is equal to 1. The β data versus percentage of organic modifier in methanol-, acetonitrile-, and THF-aqueous mixture systems are plotted in Figure 5. These curves have some similarities to the data plotted in the π* graphs and support the model described above. Overall, the β values for the bulk solvents decrease as the concentration of organic modifier decreases, since water has a very weak β effect (β ) 0.18 with this particular probe) compared with those of THF (β ) 0.56), methanol (β ) 0.62), and acetonitrile (β ) 0.31). Compared to the bulk solvent curves, the ODS2 slurry does not show big changes in β values over the entire composition range. An evaluation of the β values for the ODS2 phases is consistent with the model described above. Three distinctive regions can be clearly seen in the methanol-ODS2 curve in Figure 5 (solid line), which are 100%-70%, 70%-30%, and 30%-5%. The first range (100%-70%) is nonretentive. In this composition range, the major contribution to the spectrum is from dye present in the mobile phase. The second range (70%-30%) is moderate solvation and high retention. The β values are higher than those for any other solvent composition, even for pure methanol or silica slurry, but lower than those for the dry silica. Based on the β values for dry silica reported by Lindley et al. (β ) 0.66-0.92 for the 4-nitroaniline probe at low dye concentrations),21 it is likely that 2NA molecules stay within the C18 chains near the silica surface. In the third range (30%-5%), the bonded chains are collapsed. This chain collapse makes the β value decrease quickly (toward

the solution curve). Because the dye 2NA is less solvent-sensitive than the R dye, DCMPVP, the precision of the β values is poorer than that of the R parameters. This higher imprecision makes us hesitant to give a more detailed discussion here, especially for the acetonitrile and THF systems, although the same general trends are observed with these data. Problems with Hydrogen-Bonding Probes. There are some problems which can affect the R and β measurements in heterogeneous media. When solutes are retained on an ODS2 stationary phase, they may reside in different environments, especially if a partitioning mechanism is operative. It is obvious that DCMPVP and 2NA stay deep within the C18 chains, as they can detect some residual silanol groups on the ODS2 surface for some mobile phase compositions. But NM2NA seems to stay away from the residual silanols. Even in a bulk solvent, the probe pair may also have different solvation spheres because of their differential preference for each solvent component. This microenvironmental difference may cause some errors in the R and β values. Another factor is that the two dyes may have different retentions. This retention difference may cause a difference in the solution contribution to the slurry spectra of the two different dyes. The third factor is that different R and β dyes may give different R and β values. A very obvious example is the R value of methanol-water mobile phase detected by bis[R-(2-pyridyl)benzylidine-3,4-dimethylaniline]bis(cyano)iron(II) complex.13 This dye shows a totally different trend of R values versus solvent composition from that observed for the zwitterionic dyes. Conclusion. According to the results described above, a description of the retention mechanism of a solute in RPLC must take into account the properties of both the stationary phase and the mobile phase, as supported by earlier discoveries. The (40) Lu, H.; Rutan, S. C. Presented at Pittcon ’95. New Orleans, LA, March 9, 1995; Abstract No. 1260.

properties of the stationary phase are affected by the properties of the mobile phase, i.e., composition. Over the composition ranges studied here, the C18 chains of the ODS2 stationary phase can have three different behaviors. One is the very high solvation and low retention with a high organic modifier mobile phase. In this range, the π* and β dyes used here are not retained on the octadecyl alkyl layer of stationary phase. The second range is a solvation-controlled range with intermediate composition. The solvation extent may decrease with increasing water ratio in the mobile phase. The decrease in the π* values in the ODS2 slurries with increasing water ratios indicates that the dye partitioned in the C18 chains can “feel” the effects of both the C18 chains and the mobile phase partitioned within the stationary phase. Significant retention occurs in this range. The third range is a low solvation range. In this range, chain collapse becomes a major configuration of the octadecyl alkyl chains. The retention mechanism changes from partitioning to adsorption, and the solute is gradually exposed to the bulk solvent. Some of our more recent results with the addition of propanol to the mobile phase also show that solvation can affect the solvatochromic parameters of ODS2 slurries in mobile phases with medium to high water contents.40 The above rationalization of the experimental data gives an overview of the stationary phase as well as the mobile phase and provides some information on choosing appropriate conditions for chromatographic procedures. ACKNOWLEDGMENT This work was supported by a grant (CHE-9318484) from the National Science Foundation. Received for review August 2, 1995. Accepted January 17, 1996.X AC9507810 X

Abstract published in Advance ACS Abstracts, March 1, 1996.

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