Characterization of Reversed-Phase Liquid ... - ACS Publications

Multivariate curve resolution has been employed to resolve the spectra collected ... phases for both normal-15,16 and reversed-phase chromatography.17...
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Anal. Chem. 1999, 71, 5225-5234

Characterization of Reversed-Phase Liquid Chromatographic Stationary Phases Using Solvatochromism and Multivariate Curve Resolution Shalini Nigam, Anna de Juan,† Vickie Cui, and Sarah C. Rutan*

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

The solvatochromic method has been used to probe the solid/solution interface of bare silica and two modified silica surfacessphenyl bonded and C18 bondedsin mobilephase mixtures of methanol-water and acetonitrilewater. Spectral measurements of solvatochromic dye solutions in different mobile-phase compositions and of slurries wetted by these same solutions have been recorded and used to characterize the different solid/ solution interfaces. Multivariate curve resolution has been employed to resolve the spectra collected into the contributions due to the different solvated species of the dye, i.e., those related to the dye associated with the stationary phase and those related to the dye solvated by the different species present in the mobile phase. Spectral profiles of the dyes solvated by a methanol-water complex in the presence of stationary phase have been resolved for the first time. Chromatographic capacity factors (k′) have been measured, and they have complementary information about how the retention of the dye changes with the mobile-phase composition on the different silica surfaces. The validity of the spectral studies performed to characterize real chromatographic environments, which are usually under much higher pressures, has been investigated. In a wide range going from atmospheric pressure to values higher than 100 bar, no significant variations of the capacity factors were observed for the dyes used, and therefore, the information about retention mechanisms and solid/solution interface properties obtained from the spectral studies can be safely extrapolated to the real chromatographic systems. The results obtained indicate that the phenyl bonded silica shows a dipolarity/ polarizability very similar to that of bare silica. For these two silica surfaces, the interactions of the dye and the stationary phase are independent of the mobile-phase composition. The C18 bonded silica has a significantly lower polar character and evidences two retention mechanisms depending upon the mobile-phase composition. Reversed-phase high-pressure liquid chromatography (RPHPLC) is a commonly used separation technique for a wide variety † Departament de Quı´mica Analı´tica, Universitat de Barcelona, 08028 Barcelona, Spain.

10.1021/ac9904314 CCC: $18.00 Published on Web 10/09/1999

© 1999 American Chemical Society

of analytes. Retention is based on the differential interactions of the solutes with a relatively nonpolar stationary phase and a more polar mobile phase such as water mixed with an organic solvent. Although this separation system is widely used, the details of the solute interactions with the stationary phase are still not completely understood. The characterization of the interphase region of the stationary and mobile phases has been done using various spectroscopic methods. Infrared1 and Raman2 spectroscopies have revealed information about the conformations of the bonded chains at the surface of the stationary phase. ESR spectroscopy probes the molecular rotational motion and polarity of the solvent surrounding the solute,3 and NMR spectroscopy can determine the phase structures, anisotropies, and motions of both the chain and mobilephase components.4 Fluorescence spectroscopy has also been used to probe the polarity of the stationary phases.5-7 However, most of these techniques do not allow specific interactions such as dispersion, dipolarity/polarizability, and hydrogen bonding to be differentiated and only give the overall polarity of the probe environment. Here we use the term “polarity” as defined by Reichardt8sa global measurement of the intensity of all possible solute/solvent interactions in a system. As opposed to these approaches, the use of solvatochromic dyes can allow the identification of the different factors contributing to the overall polarity of the solvent medium. The solvatochromic comparison method was originally developed by Kamlet and Taft9-11 as a means to unravel and quantify the different solute/solvent interactions present in homogeneous solvent systems. By using different solvatochromic dyes and mathematical treatments, the overall solvent polarity can be described by three solvatochromic parameters, π*, R, and β, (1) Sander, L. C.; Callis, J. B.; Field, L. R. Anal. Chem. 1983, 55, 1068. (2) Thompson, W. R.; Pemberton, J. E. Anal. Chem. 1994, 66, 3362. (3) Miller, C.; Joo, C.; Roh, S.; Gorse, J.; Kooser, R. G. In Chemically Modified Oxide Surfaces; Leyden, D. E., Ed.; Gordon and Breach: New York, 1990; Vol. 3, p 251. (4) Albert, K.; Bayer, E. J. Chromatographia 1991, 544, 345. (5) Carr, J. W.; Harris, J. M. Anal. Chem. 1986, 58, 626. (6) Hansen, R. L.; Harris, J. M. Anal. Chem. 1995, 59, 492. (7) Rutan, S. C.; Harris, J. M. J. Chromatogr., A 1993, 656, 197. (8) Reichardt, C. Solvent and Solvent Effects in Organic Chemistry; VCH: Weinheim, 1988. (9) Kamlet, M. J.; Abboud, J. L.; Taft, R. W. J. Am. Chem. Soc. 1977, 99, 6027. (10) Taft, R. W.; Kamlet, M. J. J. Am. Chem. Soc. 1976, 98, 2886. (11) Kamlet, M. J.; Taft, R. W. J. Am. Chem. Soc. 1976, 99, 377.

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representing the solvent dipolarity/polarizability, the solvent hydrogen-bonding acidity, and the solvent hydrogen-bonding basicity, respectively. UV-visible spectroscopy is the technique most often used to follow this solvent-dependent behavior. The dyes shift the position of their spectral band maximums as a function of the environment surrounding them, and the frequency related to these maximums is used to calculate the value of the solvatochromic parameters. The underlying model related to the solvatochromic comparison method is represented by the following equation:

ν ) ν0 + sπ* + aR + bβ

(1)

where the magnitude of the π*, R, and β values depends on the solvent studied and ν0, s, a, and b vary, depending on the properties of the dye used. This method has been frequently used to characterize mobile phases used in RPLC by using different π*12 and R12-14 dyes and to characterize the stationary phases for both normal-15,16 and reversed-phase chromatography.17-19 These studies have revealed some important information about the chromatographic retention mechanisms. In complimentary studies using linear solvation energy relationships (LSERs), the retention of different solutes in RPLC has been correlated to their individual properties such as the molecular size, dipolarity, and hydrogen-bonding abilities.20-26 In the case of chromatographic retention, the appropriate equation takes the form25,26

log k′ ) log k′0 + mV2 + sπ2* + aR2 + bβ2

(2)

Here, k′ is the chromatographic capacity factor and V2 is the volume of the solute. The subscript 2 is used to denote that these properties are those of the solute and not the solvent. The m, s, a, and b coefficients and the log k′0 intercept are normally determined by using regression of the log k′ values using a diverse set of solutes. It can be rationalized that these coefficients should be related to the properties of the mobile and stationary phases as follows: m ) M(λm - λs), where λ is related to the Hildebrand solubility (a measure of the cohesiveness of the phase), s ) S(π*s - π*m), a ) A(βs - βm), and b ) B(Rs - Rm). Here M, S, A, and (12) Cheong, W. J.; Carr, P. W. Anal. Chem. 1989, 61, 1524. (13) Cheong, W. J.; Carr, P. W. Anal. Chem. 1988, 60, 820. (14) Park, J. H.; Jang, M. D.; Kim, D. S.; Carr, P. W. J. Chromatogr. 1990, 513, 107. (15) Park, J. H.; Dallas, A. J.; Chau, P.; Carr, P. W. J. Phys. Org. Chem. 1994, 7, 757. (16) Li, Z.; Rutan, S. C. Anal. Chim. Acta 1995, 312, 127. (17) Lindley, S. M.; Flowers, G. C.; Leffler, J. E. J. Org. Chem. 1985, 50, 607. (18) Helburn, R. S.; Rutan, S. C.; Pompano, J.; Mitchem, D.; Patterson, W. T. Anal. Chem. 1994, 66, 610. (19) Hayashi, Y.; Helburn, R. S.; Rutan, S. C. In Proceedings of the Fourth Symposium on Computer-Enhanced Analytical Spectroscopy; Wilkins, C. L., Ed.; Plenum Press: New York, 1992. (20) Park, J. H.; Carr, P. W.; Abraham, M. H.; Taft, R. W. Doherty, R. M.; Kamlet, M. J. Chromatographia 1988, 25, 373. (21) Abraham, M. H.; Rose´s, M. J. Phys. Org. Chem. 1994, 7, 672. (22) Carr, P. W.; Doherty, R. M.; Kamlet, M. J.; Taft, R. W.; Melander, W.; Horvath, C. Anal. Chem. 1986, 58, 2674. (23) Sadek, P. C.; Carr, P. W.; Doherty, R. M.; Kamlet, M. J.; Taft, R. W.; Abraham, M. H. Anal. Chem. 1985, 57, 2971. (24) Tan, L.; Carr, P. W.; Fre´chet, J. M.; Simgol, V. Anal. Chem. 1994, 66, 450. (25) Li, J.; Carr, P. W. Anal. Chim. Acta 1996, 334, 239. (26) Carr, P. W. Microchem. J. 1993, 48, 4.

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B are constants and the subscripts s and m refer to the stationary and mobile phases, respectively. Thus, the sign and magnitude of the coefficients give a measure of the effect of the differential properties of the stationary and mobile phases on the solute retention. It is clear from an examination of eqs 1 and 2 that the meaning of the s, a, and b coefficients obtained in both equations is not the same. In the first case, the coefficients are related to solute properties, whereas in the latter case, the coefficients account for the mobile- and stationary-phase properties. In previous work, solvatochromic studies on the RPLC phases with octadecyl bonded silica and commonly used mobile phases such as methanol-, acetonitrile-, and tetrahydrofuran-water mixtures were reported for a broad range of compositions.18-19,27 It was found that the mobile phase plays a critical role in determining the dipolarity/polarizability of the stationary phase. The π* values of octadecylsilylated silica exposed to the typical mobile phases are generally lower than the corresponding mobilephase values but much higher than the values for alkanes due to the mobile-phase solvation of the stationary phase. The R values for the stationary-phase slurries are generally higher than those for the bulk mobile phase in these studies. The addition of a small amount of a wetting agent such as propanol has been shown to dramatically affect the solvatochromic parameters of the octadecylsilylated stationary phases in mobile phases with medium to high water content.28 A problem that has not been addressed satisfactorily is that in order to reliably characterize the interphase region of the stationary phase under conditions relevant to liquid chromatographic separations, it is necessary that the stationary-phase particles be completely wetted by the surrounding mobile phase. In achieving this condition, it is possible that a substantial fraction of the probe dye remains solvated in the mobile phase. This is particularly likely for the mobile-phase/stationary-phase conditions where the dye is not strongly retained by the stationary-phase material, as evidenced by the relatively small chromatographic capacity factors (k′). In these instances, it is necessary to subtract the mobile-phase contribution from the measured spectroscopic response, which will be a composite signal with contributions from the dye molecules present in the mobile phase and in the stationary interphase regions. The retention mechanisms in RPLC are affected by the nature and change in the mobile-phase composition. Thus, the exact nature of organic modifier-water mixtures is a subject of theoretical and experimental interest.29-30 For binary solvent mixtures, there are reports of formation of aggregates of solvent molecules such as methanol and acetonitrile with water, usually called clusters.31,32 These studies indicate that such solvent-water mixtures contain several distinct chemical species with which a solute can interact.33 Even though there is experimental evidence for the existence of such species by liquid chromatography,29-31 fluorescence measurements,34,35 and infrared spectroscopy,36-37 no (27) Lu, H.; Rutan, S. C. Anal. Chem. 1996, 68, 1387. (28) Lu, H.; Rutan, S. C. Anal. Chim. Acta 1999, 388, 345. (29) Guillaume, Y. C.; Guinchard, C. Anal. Chem. 1997, 69, 183. (30) Guillaume, Y. C.; Guinchard, C. Anal. Chem. 1998, 70, 608. (31) Guillaume, Y. C.; Guinchard, C. Anal. Chem. 1996, 68, 2869. (32) Katz, E. D.; Ogan, K.; Scott, R. P. W. J. Chromatogr. 1986, 352, 67. (33) Katz, E. D.; Lochmuller, C. H.; Scott, R. P. W. Anal. Chem. 1989, 61, 349. (34) Kusumoto, Y.; Takeshita, Y.; Kurawaki, J.; Satake, I. Chem. Lett. 1997, 349. (35) Zana, R.; Eljebari, M. J. J. Phys. Chem. 1993, 97, 11134.

isolated UV-visible spectra related to dyes solvated by these mixed species are available. To obtain the spectral profiles of the dye representative of different environments such as solvent clusters and the stationary phase, we have applied the multivariate curve resolution-alternating least-squares (MCR-ALS) method to our data. This is an iterative method for mixture analysis which has been used earlier for a variety of analytical problems such as speciation in multiequilibria systems using spectroscopic titrations,38 chemical changes during process monitoring and control,39 and resolution of LC-DAD data.40,41 The MCR-ALS method decomposes the composite signals in a data matrix (e.g., the mixture spectra) into the product of two smaller matrices, C and ST, which contain the concentration profiles and the spectra of each species contributing to the recorded mixture signal, respectively. To do so, the method uses initial estimates of C or ST which are iteratively refined through an alternating least-squares process that takes advantage of the data structure and the application of chemically meaningful constraints. In this work, characterization of bare silica, octadecyl-modified, and phenyl-modified silica stationary phases was carried out using solvatochromic techniques. Aqueous mixtures of methanol and acetonitrile were used as mobile phases. Two π* dyes, N,Ndimethyl-4-nitroaniline (NDMNA) and N-methyl-2-nitroaniline (NM2NA) were used as the solvatochromic dyes. In another study, we focused the attention solely on mobile-phase interactions42 while the present study focuses on the dipolarity/polarizability parameters of the stationary phases. Information about the characteristics of the stationary phase are obtained by applying the multivariate curve resolution methods to the experimental data. A problem associated with correlating the results obtained from the spectroscopic measurements to those from the liquid chromatographic retention measurements is the different pressure conditions used in these two techniques. The spectroscopic studies done with the solvatochromic dyes are almost always carried out at atmospheric pressure, and often, little information is available as to the effect of pressure on the structure of the stationary phase. So the question arises of whether it is reasonable to compare the solvatochromic results obtained at atmospheric pressure with chromatographic measurements that are obtained at higher pressures. In this study, an answer to this question has been provided by carrying out retention measurements at varying pressures and observing the effect on the capacity factors. EXPERIMENTAL SECTION Chemicals and Materials. NDMNA and NM2NA, both 98%, were purchased from Lancaster Laboratory and Aldrich, respectively. Methanol and acetonitrile, both HPLC grade, were purchased from EM Science and used directly without further purification. Allsphere silica (a Spherisorb “look-a-like”), 10 µm in diameter, Waters phenyl bonded Spherisorb silica (S10P), 10 (36) Alam, M. K.; Callis, J. B. Anal. Chem. 1994, 66, 2293. (37) Zhao, Z.; Malinowski, E. R. Anal. Chem. 1999, 71, 602. (38) Tauler, R.; Izquierdo-Ridorsa, A.; Gargallo, R.; Casassas, E. Chemom. Intell. Lab. Sys. 1995, 27, 163. (39) Tauler, R.; Kowalski, B. R.; Fleming, S. Anal. Chem. 1993, 66, 2040. (40) De Braekeleer, K.; de Juan, A.; Massart, D. L. J. Chromatogr. 1999, 832, 67. (41) Barcelo´, D.; Tauler, R. Anal. Chem. 1993, 12, 319. (42) Nigam, S.; Stubbs, R. J.; de Juan, A.; Rutan, S. C., submitted to Anal. Chem.

µm in diameter, and Spherisorb octadecyl bonded silica (ODS2), 5 µm in diameter, were purchased from Alltech. S10P and ODS2 are end-capped materials. The S10P material has a carbon loading of 3% while the ODS2 material has a carbon loading of 12%. All the stationary phases have a surface area of 220 m2/g. The surface coverage for the phenyl phase was 1.3 µmol/m2 and for the octadecyl phase was 2.9 µmol/m2.43 Sample Preparation. Dye solutions in the solvent mixtures were made by mixing appropriate amounts of dye solutions prepared in the pure solvents. The concentrations of the dye solutions were kept at 8 × 10-5 M for all the experiments. The slurries were made by mixing 250 mg of the stationary phase with 10 mL of the dye solvent mixture to keep a constant ratio of the liquid- and stationary-phase volume. The slurry samples were allowed to equilibrate for a couple of hours after stirring gently. The settled slurry was then collected and transferred to a 1-mm path length quartz cuvette for spectral data collection. Note since the amount of dye sorbed to the stationary phase is not known, the concentration of the dyes in the mobile-phase part of the suspension is also unknown. Procedure. All the silica and phenyl phase diffuse reflectance absorption spectra were obtained on a Cary 1E UV-visible spectrophotometer. An integrating sphere was attached to the instrument for collecting the light reflected from the sample. Pure solvent was used as the reference, and a white paper surface was used as the reflectance backing for the sample cell. A slit width of 2 nm was used in all the experiments. The spectra of the dye in different mobile-phase compositions were recorded under the same conditions as the slurry spectra. Data were collected and transferred to an IBM PC through an IEEE-488 interface card supplied by Varian. Data were converted to ASCII format for data analysis. The octadecyl bonded silica data, measured on a Shimadzu UV-265 spectrophotometer, were taken from previous work from our laboratory.27 All data analyses were carried out with programs written using Matlab, version 4.2.c.1 (Mathworks). The peak positions were determined by smoothing the spectra using the Savitzky-Golay method with a quadratic polynomial smoothing filter with a 21-point window width.44 The precision of the resulting π* values is (0.02. When the diffuse reflectance attachment is used, the data are obtained as apparent absorbance values (-log R), where R is the reflectance. The Kubelka-Munk transformation of the reflectance data was also examined, as this transformation theoretically makes the data to be directly proportional to the “true” absorbance.45 All data were analyzed by the MCR-ALS method using both the apparent absorbance values and the Kubelka-Munk transformed values. For all systems, either of these approaches led to similar fit qualities and the same model complexity (number of components). Since the Kulbelka-Monk transform relies on certain assumptions (i.e., infinitely thick layer), which may not be upheld in our experiments, and the results were similar for both data representations, we chose to work with the untransformed data to avoid introduction of errors due to deviations from the Kubelka-Monk model.45 (43) Sentell, K. B.; Barnes, K. W.; Dorsey, J. G. J. Chromatogr. 1988, 455, 95. (44) Savitzky, A.; Golay, M. J. E. Anal. Chem. 1964, 36, 1627. (45) Frodyma, M. M.; Lieu, V. T. In Modern Aspects of Reflectance Spectroscopy; Wendlandt, W. W., Ed.; Plenum Press: New York, 1968; Chapter 6.

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Figure 2. MCR-ALS decomposition of a data matrix D into concentration (C matrix) and spectral profiles (ST matrix). Figure 1. Data set formed by combining the mobile-phase data matrix (MP) and stationary-phase data matrix (SP).

Chromatographic retention data were obtained with a liquid chromatographsa Hewlett-Packard model 1050 with an autosampler, a vacuum degassing device, and a variable-wavelength UVvisible detector. The different columns used were Allsphere silica purchased from Alltech (250 mm × 4.6 mm), Spherisorb S10P purchased from Waters (150 mm × 4.6 mm), and Spherisorb ODS2 purchased from Metachem (75 mm × 4.6 mm). Nitromethane was used as the dead volume marker for the silica and phenyl columns while uracil was used as the dead volume marker for the C18 column. The detection wavelength was 254 nm. Flow rates of 1 mL/min were used in all the experiments, except for the pressure-dependence studies. Each series of experiments was started by recording a chromatogram with a mobile phase containing 100% organic modifier. Successive chromatograms were recorded decreasing the percent of organic modifier in the working mobile phase. The columns were equilibrated for 25 min at each mobile-phase composition before injection. Data Analysis Method. Two data matrices were built for each dye-mobile phase-stationary-phase (dye-SP-MP) system. The first matrix consisted of the spectra of the dye in solvent mixtures at mobile-phase compositions ranging from 100 to 0% organic phase. Hence, a matrix of size (p × n) was obtained, where p is the number of solution spectra collected at different mobile-phase compositions and n is the number of absorbance readings per spectrum, measured at regular wavelength intervals (this is the MP matrix). The second data matrix contains the spectra collected for the stationary phase wetted by various mobile phases with different compositions. This data matrix, whose size is (q × n), has the q spectra of the slurries prepared as described above, that have been recorded under the same conditions as the solution spectra (this is the SP matrix). The two data sets are appended together to form a column-wise augmented matrix D sized (m × n) where m ) p + q (Figure 1). MCR-ALS analysis decomposes the data matrix D into the product of a column-wise augmented matrix of concentration profiles, C, and a matrix of spectral profiles, ST, which best fit the experimental data, i.e., giving the minimum residual error, E (see Figure 2). 5228

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D ) CST + E

(3)

Each concentration profile and the related spectrum belong to one of the components contributing to the original data matrix. In this example, the spectra collected experimentally are considered to be the weighted sum of the spectral contributions related to the dye surrounded by the different possible environments in the dye-SP-MP system. The following steps are employed to carry out the MCR-ALS method on the data matrix D: (1) The number of components or different dye species (r) is estimated using singular value decomposition (SVD) and factor analysis based methods, such as evolving factor analysis (EFA).46-49 Prior knowledge about the system is also taken into account, and models of various complexities (number of factors) were always explored in the subsequent MCR-ALS analyses. (2) Nonrandom initial estimates of the concentration or of the spectral profiles are generated. In this case, either a column-wise augmented matrix of concentration profiles obtained by appending the estimates obtained for each matrix by EFA51 or a matrix obtained by selecting the most representative spectra in the MP and SP matrices can be used. (3) Constraints are selected for the iterative optimization procedure.49 For the concentration profiles, non-negativity, unimodality (i.e., only one maximum per concentration profile), and selectivity (i.e., only one species is present at certain mobile-phase compositions) can be applied. Additionally, a mass balance (closure) constraint can be applied to the concentration profiles of the mobile-phase data matrix by setting the total sum of the dye concentrations associated with the different components to be equal to the total concentration of the dye in the mobile phase; i.e., the sum of the elements of each row in the C matrix should be equal to the closure constant. Spectral profiles can be constrained to be non-negative and unimodal (this second constraint is applicable in this case because in the relevant wavelength (46) Maeder, M.; Zuberbuehler, A. D. Anal. Chim. Acta 1986, 181, 287. (47) Maeder, M. Anal. Chem. 1987, 59, 1987 527. (48) Maeder, M.; Zilian, A. Chemom. Intell. Lab. Syst. 1988, 3, 205. (49) Keller, H. R.; Massart, D. L. Chemom. Intell. Lab. Syst. 1992, 12, 209. (50) Tauler, R. Chemom. Intell. Lab. Syst. 1995, 30, 133. (51) Guiochon, G.; Sepaniak, M. J. J. Chromatogr. 1992, 606, 248.

Figure 3. Capacity factors for NM2NA on the phenyl column in acetonitrile-water mixtures determined at different pressure conditions: 70% (2), 50% ([), and 30% ACN (9).

range only one absorption band is known to be present). In addition, spectra of perfectly known components, e.g., the dye in pure water and in pure organic solvent (methanol or acetonitrile), can be fixed during the iterative optimization. (4) Iterative optimization of the initial estimates is then carried out. At each iteration, the concentration and the spectral profiles matrices are evaluated by using a constrained alternating leastsquares procedure. Convergence is achieved when the difference in the relative residuals between iterations is lower than a preset value, here taken as 0.1. RESULTS AND DISCUSSION Pressure Effects on Chromatographic Retention. As discussed previously, when results obtained by spectroscopic measurements are correlated to those of chromatographic retention, a factor which might be important is that of pressure. The spectroscopic experiments are done at atmospheric pressure conditions, while the dye and the mobile and stationary phases in chromatographic systems experience pressures ranging up to as much as 200 bar.51 Though solid and liquid phases are normally assumed to be incompressible over this range, it is reasonable to question the validity of comparing the results obtained at such different pressure conditions. Thus, we carried out retention measurements for NM2NA on the phenyl stationary phase at different pressures by changing the flow rates of the mobile phase through the column. Figure 3 gives the plots of the capacity factors k′ determined at different pressure conditions. In 70% acetonitrile, the capacity factors are constant over a pressure range from less than 1 to 119 bar, which corresponds to a flow range from 0.1 to 3.0 mL/min. Even at pressures close to ambient levels, we did not see any change in the capacity factors as a function of pressure. In 50% acetonitrile, the capacity factors are held constant as the pressure changes from 6 to 200 bar (flow rates between 0.2 and 2.0 mL/min). Above 200 bar, the pressure was not stable and the capacity factors were not measured. At 30% acetonitrile, the capacity factors decreased very slightly when the pressures changed from 6 to 120 bar (flow rates between 0.2 and 2.5 mL/ min). Above 120 bar, the system was unstable and no measurements were performed. It is important to note that the retention measurements presented in this work were made at a flow rate of 1.0 mL/min, for which all the systems studied are shown to be stable. From the results obtained, it can be concluded that the

pressure changes in the chromatographic system for pressures ranging from 1 to 200 bar should not have any effect on the capacity factors, at least for the phenyl stationary phases. Hence, it should be reasonable to extrapolate the solvatochromic results to the real chromatographic systems and compare these spectral studies with the chromatographic retention results. MCR-ALS Analysis. Before performing a detailed analysis of the combined data matrices for each of the dye-SP-MP combinations, SVD was performed on each of the two matrices individuallysthe MP data matrix and the SP data matrix. This gives an initial idea of the rank of the data sets. The number of components ranged from two to four in the different pure mobilephase matrices and ranged from three to six in the stationaryphase slurry matrices. This is illustrated in more detail in our study that focuses on the mobile-phase behavior only.42 MCR-ALS was first applied to resolve the two matrices individually. The analyses were run using the estimates for the concentration profiles obtained from EFA.48 The results obtained helped us to decide the number of components of the dye present in each of the matrices and provided a first approximation of the concentration profiles in the MP and SP systems, though the ambiguities related to the decomposition of each data matrix will be solved by treating both of them together.50 For each of the dye-MP-SP systems, we started out by using three components to model the dye in the pure mobile phases and at least two in the stationary phases. The initial estimates of the three spectral profiles for the dye in mobile phase were the spectra obtained from the application of MCR-ALS to the MP matrix, i.e., those present in the ST matrix. The two spectral estimates for the dye in the stationary phase were the experimental spectra of the dye in the slurry at 100% water and 100% organic solvent. All these spectral profiles were put in the same matrix and used as the initial estimates for the resolution of the combined data matrix by MCR-ALS. The most often used constraints were non-negativity for both the concentration and spectral profiles and unimodality for the concentration and spectral profiles. Selectivity was applied to the concentration profiles at the extreme mobile-phase compositions (for example, at 100% organic phase, no water-solvated species should be present and vice versa). Closure was applied to the concentration profiles of the mobile-phase matrix in most systems by fixing the sum of concentrations of all the components of the dye in the mobile phase to be equal to the total concentration of dye (i.e., a mass balance relationship). The selection of the constraints is guided by examination of the fit quality and the plausibility of the profiles obtained; i.e., the goal is to obtain the lowest percent of lack of fit and chemically meaningful spectral and concentration profiles. Table 1 summarizes the results obtained for different components of the dyes obtained in mobile and stationary phases after the MCR-ALS method was applied to our experimental data as explained above. The fitting errors and the sets of constraints used in each result are also given. In some of the systems, we did see a convergence of the optimization process while in others the iterations diverged after reaching a minimum fitting error. In these latter cases, the result that is reported is the solution corresponding to the iteration with the optimal fit. Please note that this solution has been accepted because no important departures from the optimal profiles were detected in successive iterations. The Analytical Chemistry, Vol. 71, No. 22, November 15, 1999

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Table 1. Results of the MCR-ALS Method Applied to the Different Dye-Stationary-Phase/Mobile-Phase Systems dye NM2NA

NDMNA

stationary phase silica phenyl silica octadecyl silica silica phenyl silica octadecyl silica silica phenyl silica phenyl silica

solvent

mobile-phase components

MeOH MeOH MeOH ACN ACN ACN MeOH MeOH ACN

2 2 2 2 2 2 2 3 2

stationary-phase components

fitting error (%)

1 1 2 1 1 2 1 1 1

10.5a 4.6b 8.2b 5.3a 6.9b 6.4b 5.1b 4.2b 2.8a

constraints c, e, g c, e, g-i c, e, g c, e, g c, e, i c, e, g c, d, f, h c, e, g-i c, e, g, h

a Iterations diverged after reaching a minimum fitting error. b Iterations converged to stable fit of experimental data. c Selectivity in concentration direction. d Non-negativity in concentration profiles only. e Non-negativity in concentration and spectral profiles. f Unimodality in concentration profile only. g Unimodality in concentration and spectral profiles. h Closure of mobile-phase components. i Fixed spectra of pure components.

reliability of the solutions obtained is also supported by the fact that the addition or removal of a constraint does not modify significantly either the fitting error or the shapes of the spectra and concentration profiles. For example, in the case of NDMNA in the phenyl bonded silica in methanol-water solutions, upon applying the constraints of non-negativity, closure and fixing the spectra of methanol and water as known, the fitting error was 4.8%. After applying additional constraints of unimodality in both spectral and concentration profiles and selectivity for the presence or absence or certain components, the fitting error was lowered to 4.2%. For the silica and the phenyl bonded silica, the best results were generally obtained by considering only two components in the mobile phase. This is in apparent disagreement with the results obtained from the analysis of the mobile-phase data matrix alone,42 which usually reveal a third dye species solvated by a solvent-water complex. However, it must be taken into account that the intensity of the spectra in the MP matrix is generally much smaller than that of the spectra in the SP matrix. This means that the SP matrix has a larger effect on the resolution results. The missing MP species, which has a spectrum similar in shape to the spectra of the rest of the species in the system, is a minor contribution within the total dye-MP-SP system and, therefore, cannot always be properly modeled. In this case, although there is an error in the mobile-phase part of the model, it should be small, and the focus is on the characterization of the stationaryphase component(s). When we analyzed the combined data matrix formed by column-wise appending the MP and SP data matrices, only one stationary-phase component was obtained for these two phases. However, in the case of the octadecyl bonded silica, two components in the stationary phase were obtained in both the solvent systems. Upon resolving the different components of the dyes in mobile and stationary phases, the separate spectra and concentration profiles of the dyes surrounded by the different stationary- and mobile-phase environments can be obtained. Two representative examples are discussed in detail below. Figure 4 shows an example of these profiles for the dye NDMNA in the phenyl phase in the mobile phase of methanol-water mixtures; the results obtained had a lack of fit equal to 4.2%. In this case, three mobilephase components and one stationary-phase component were obtained; the estimated spectral profiles are represented in Figure 4A and the related concentration profiles for the SP matrix are 5230 Analytical Chemistry, Vol. 71, No. 22, November 15, 1999

Figure 4. MCR-ALS estimated spectra (A) and concentration profiles of the SP matrix (B) for NDMNA in phenyl phase solvated with a mobile phase of methanol and water mixtures: methanol (---), methanol-water cluster (‚‚‚), water (- ‚ -), and phenyl phase (s).

shown in Figure 4B. Table 2 compares the wavelength maximums for the spectra of the dye in pure methanol, pure acetonitrile, and pure water obtained by MCR-ALS method with the experimental maximums and shows a close agreement. The MCR-ALS spectral profiles obtained for the dye in pure solvents are very similar to the experimental spectra of the dye. This demonstrates the high degree of accuracy with which the different components have been resolved. The reconstructed spectra of the dye solvated by methanol-water clusters have been obtained for the first time,42 and the spectra of the dye in the stationary phase have been isolated. When the above system was resolved using just two mobile-phase components and one stationary-phase component, the fitting error became higher (5.2%) for the same set of constraints used, and the spectral profile for the stationary-phase component became unacceptable. This demonstrates that the system above is best described by three mobile-phase and one stationary-phase components. The results obtained for other dyeMP-SP systems are summarized in Table 1. As commented

Table 2. Comparison of the Experimental Wavelength Maximums of the Dyes in Methanol, Acetonitrile, and Water with Those Obtained by MCR-ALS in the Presence of the Stationary Phases λmax (nm)

NM2NA MeOH ACN water NDMNA MeOH ACN water

k′ solute-methanol (%, v/v)

exp

ALSa

ALSb

ALSc

425.5 429.0 444.5

425.0 428.0 445.0

426.0 430.5 442.0

426.0 429.0 446.0

393.0 396.0 424.0

395.0

393.0 393.0 426.0

422.0

Table 3. Capacity Factors of the Dyes Determined in the Different Stationary Phases in Methanol-Water Mixtures

NM2NA 100 90 80 70 60 50 40 30 20 10 NDMNA 100 90 80 70 60 50 40 30 20 10

a Wavelength maximums calculated by MCR-ALS in the presence of silica. b Wavelength maximums calculated by MCR-ALS in the presence of S10P. c Wavelength maximums calculated by MCR-ALS in the presence of ODS2.

above, no evidence of a dye species solvated by methanol-water or acetonitrile-water complexes were observed in any other system. The concentration profiles give valuable information about how the environment of the dye changes as a function of the mobilephase composition, i.e., how the differently solvated dye species concentrations increase or decrease as the solvent around them is modified and, consequently, the way they interact with the stationary phase (Figure 4B). Thus, for NDMNA in methanolwater in contact with the phenyl phase, the methanol-solvated component is present over much of the mobile-phase range. The methanol-water complex forms as soon as water is added to the mobile phase, but this complex component disappears when more than 70% water is present in the mobile phase. Again the water component remains over much of the total mobile-phase range. The stationary-phase component appears only when more than 30% water is added to the mobile phase. This observation is in good agreement with the retention measurements, which show that there is no significant retention of this dye in the stationary phase when less than 30% water is in the mobile phase (compare the shape of the concentration profile of the stationary-phase component in Figure 4B with the shape of the curve describing k′ vs mobile-phase composition in Figure 5 or with the retention values in Table 3). Figure 6 shows the estimated spectral and concentration profiles of NM2NA in a C18 phase with the mobile phase of methanol-water mixture. Two components in the mobile phase (the organic phase and water phase) and two components in the stationary phase are obtained, and the fitting error for this result was 8.2%. The MCR-ALS spectral profiles obtained for methanol and water are very similar to the experimental ones, and the results in Table 2 again demonstrate this. When this system was resolved for three mobile-phase components along with two stationary-phase components, the fitting errors became larger (9.3%) and the spectral profiles for two of the components became unacceptable. Again the concentration profiles provide very valuable information about the evolution and disappearance of the different components as the mobile-phase composition changes. Two components of the stationary phase are observed in the case of the C18 phase. The first stationary-phase component appears

a

silica

phenyl

C18

a a a 0.01 0.04 0.12 0.26 0.53 1.12 2.6

0.03 0.05 0.11 0.21 0.43 0.85 1.91 3.76 7.19 14.5

0.17 0.33 0.54 0.92 1.83 3.49 11.11 25.8 55.5 101

0.06 0.07 0.10 0.18 0.24 0.42 0.79 1.52 3.07 6.99

0.08 0.11 0.19 0.34 0.67 1.48 3.20 6.82 14.5 31.6

Unretained on the column.

Figure 5. Capacity factors k′ for NDMNA plotted as a function of the composition of methanol-water in the phenyl (9) phase.

as soon as water is added to the mobile phase, and the contribution of this component keeps increasing with the water content up to 80% water. Upon addition of more water, the relative concentration of this component drops sharply while at the same time another component of the stationary phase appears at 80% water and increases as the mobile-phase composition increases to 100% water. These concentration profiles also indicate that the retention of the dye on the C18 stationary phase starts at much lower water content. This is confirmed in the retention measurements, where the retention is very high on the octadecyl surface (Table 3). The dye is less retained on the phenyl phase and shows almost no retention on the bare silica. In general, retention of the solute dyes increases with increase in water content in the mobile phase, which is consistent with a hydrophobic retention mechanism. Similarly, we have resolved the dye components of mobile and stationary phases in other dye-MP-SP systems, the results of Analytical Chemistry, Vol. 71, No. 22, November 15, 1999

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Figure 6. MCR-ALS estimated spectra (A) and concentration profiles of the SP matrix (B) for NM2NA in the C18 phase solvated by mobile phase of methanol and water mixtures: methanol (---), water (- ‚ -), stationary phase (2) (- ‚‚ -) and stationary phase (1) (s).

Figure 7. π* values obtained using NM2NA plotted as a function of the composition of methanol-water mixtures: bulk solvent ([), silica (b), phenyl (9), and C18 (2).

which are summarized in Table 1. The MCR-ALS obtained wavelength maximums for the mobile-phase components from the combined data sets are compared to the experimental values and are given in Table 2. Dipolarity/Polarizability. The simplest measurement of solvent properties using the solvatochromic approach is the π* parameter, which measures the environmental dipolarity and polarizability experienced by the solute molecules. The dyes NDMNA and NM2NA were used for the determination of the π* parameter since spectral shifts of these dyes reflect primarily changes in the dipolarity/polarizability of their environment.10 Figure 7 shows the π* values calculated for the different stationaryphase slurries with the change in the ratio of methanol in the mobile phase before subtracting the mobile-phase contribution to the spectra. These π* values have been calculated by using 5232 Analytical Chemistry, Vol. 71, No. 22, November 15, 1999

the frequency of absorption maximums of the composite spectrum of the slurries; therefore, they must not be considered as “true” polarity values of the stationary phase. However, the comparison between these values and those of the bulk solvent can help us to better understand the retention mechanisms of the different stationary phases studied and to confirm the validity of the results previously presented. We do observe that the bulk solvent, silica, and phenyl phase curves are very similar to each other. Since water is more dipolar than methanol, increasing the water content in the mobile phase should increase the overall dipolarity of the system in the bulk solvent. Thus, it is reasonable to observe the increase in π* values as the water content is increased. The silica data are most similar to the data for the bulk solvent, and this agrees with the fact that the dye is poorly retained by the bare silica (see Table 3). Since the retention of the dye is so low over the whole range of mobile-phase compositions, the spectral contribution due to the dye retained by the stationary phase to the total slurry spectrum is very small. As a consequence, no drastic differences between the position of the absorption bands of the slurries and of the pure mobile-phase solutions are detected. In the case of the phenyl phase, the behavior is similar to that of the silica, but in the water-rich region, there is a clear departure from the data for the bulk solvent (∼30% methanol). This agrees again with the retention results presented in Table 3 and with the behavior of the MCR-ALS concentration profile of the dye in the stationary phase presented in Figure 4B. For both the silica and phenyl phases, the π* parameter monotonically increases as the water content increases. This simple behavior coincides with the variation of this parameter in the mobile-phase system and seems to indicate that the stationary-phase/solute interactions do not have a very complex mechanism. This is supported by the fact that in both systems only one component is needed to describe the spectral contribution of the dye retained by the stationary phase. Unlike silica and phenyl phases, the C18 curve shows a complete departure from the bulk solvent line. This is consistent with the fact that this stationary phase retains the dyes more strongly than the other phases (see Table 3), and therefore, the spectral contribution due to the dye associated with the stationary phase to the total slurry spectrum is clearly significant. The different trends in the π* values for different ranges of methanol compositions indicate that the C18 stationary-phase/solute interactions are more complex than those of the silica and phenyl stationary phases. Indeed, this behavior of the C18 data was originally interpreted as indicating the possibility that the solute dye might be surrounded by more than one environment in the C18 phase, depending upon the methanol-water content of the mobile phase.27 This conclusion has been confirmed by the MCRALS results, which required two components to explain the interactions between the dye and the C18 phase. Upon carrying out the MCR-ALS analysis on the spectroscopic data of the different dye-MP-SP systems and obtaining the wavelength maximums from each of the reconstructed spectral profiles, the π* values of the different mobile- and stationary-phase components were obtained. The two dyes gave very similar results. The π* values for the methanol-water and acetonitrile-water system are presented in Tables 4 and 5, respectively. The π* values for pure methanol vary from 0.67 to 0.80 and the π* values

Table 4. π* Values of the Different Components of the Mobile and Stationary Phases for the Methanol-Water System stationary phase dye-stationary phase NM2NA silica phenyl C18 NDMNA silica phenyl

methanol

methanol-water complex

0.67 0.70 0.70 0.81 0.77

water 1.35 1.23 1.34 1.28 1.35

1.21

2

1

0.70

1.44 1.62 1.12 1.43 1.42

Table 5. π* Values of the Different Components of the Mobile and Stationary Phases for the Acetonitrile-Water System stationary phase dye-stationary phase NM2NA silica phenyl C18 NDMNA phenyl

ACN

ACN-water cluster

water

0.77 0.85 0.81

1.38 1.41 1.37

0.77

1.28

2

1

0.78

1.46 1.50 1.01 1.52

for pure acetonitrile vary between 0.77 and 0.85 using different dyes and stationary phases. These values are somewhat higher than those reported in the literature (the π* for methanol is 0.59, and that for acetonitrile is 0.70).52 The π* values obtained for water are between 1.23 and 1.40 and are again higher than those reported in the literature (π* ) 1.09).52 Interestingly, for the first time we have been able to obtain the π* parameter for the methanol-water complex as 1.21, a value that lies between that of methanol and water. This value of π* is the same as the obtained for the methanol-water complex analyzing only the MP matrix.42 The complex formation in the presence of a stationary phase was observed only in the case of the dye NDMNA in the phenyl phase wetted by methanol-water mixtures even though this complex formation in pure mobile phase has been observed by both the dyes in both the solvent systems.42 In the case of the stationary phases, only one component was obtained for silica and phenyl phases over the total mobile-phase composition range for both the dyes in the two solvent systems. The π* values of both of these stationary phases are very similar, and the average values are around 1.45 in both solvent systems, showing a very high degree of dipolarity and polarizability for both phases. Similar values for silica have been reported by other authors, even though there is considerable dispersion among the π* values obtained from different π* dyes, ranging from 1.18 to 1.54.16 The phenyl phase shows π* values very similar to that of silica. The large difference in retention between the phenyl and silica phases (see Table 3) indicates that the reasons for these high π* values may be different in silica and phenyl systems. While the silica π* value is due mostly to interactions of the dye (52) Kamlet, M. J.; Abboud, J. L.; Taft, R. W.; Abraham, M. H. J. Org. Chem. 1983, 48, 2877.

with the surface silanols, in the phenyl phases, there are probably strong interactions from both the dipolar silanols and the polarizable phenyl groups. The linker between the silica surface and the phenyl rings in the case of the phenyl phase is a propyl group, and we can hypothesize that the solute dye is able to interact with the silanol surface in such a way that it undergoes π stacking interactions with the phenyl rings. In this manner, it is able to experience the environmental effects due to both the silanol surface and the phenyl. The behavior of the octadecyl phase is very different from that of the other two stationary phases, and two components resolved in the stationary phase for the two mobile phases used. One component was of lower dipolarity with π* values of 0.70 and 0.78 in the methanol and acetonitrile solvent systems, respectively, which are similar to the values observed for the pure organic solvents. This component is present over much of the mobilephase composition range (Figure 6B). The other component obtained had a higher π* value of ∼1.1. This component was observed only at very high water content. It is known in the literature that, at lower water content, solvated C18 chains are “organized and extended away from the silica surface”.27 Partitioning is the predominant retention mechanism. As the water content in the mobile phase is increased, the extent of chain solvation decreases rapidly. The alkyl chains on the surface gradually bend, and the ends of the chain aggregate together through hydrophobic attractions. This collapsed chain configuration has been described in the literature,5 and more direct evidence of this has been provided by Raman spectroscopy.53 In such a case, the solutes are exposed to the surface of the phase and are exposed to the high-water-content mobile phase and thus experience a much more dipolar environment. This is consistent with the observed concentration profiles, where this second component appears only at very high water content and agrees with the conclusions of our prior investigation of these data.27 It is also interesting to note that the π* values of both these components are much lower than those obtained for the phenyl bonded silica, probably indicating that the silanols on the C18 surface are less accessible to the solute dyes than those on the phenyl surface. CONCLUSIONS In using solvatochromic dyes to investigate heterogeneous systems, the greatest challenge is in dealing with the multiple environments experienced by the individual probe molecules. The spectroscopic signal is usually a mixture of responses of the dye in these different environments. Multivariate curve resolutionalternating least squares has been used to resolve the different species of the dye surrounded by these environments. We have been able to separate out the contributions of the dye, which enables us to directly characterize these environments. For the first time, the evolution of the concentration of the different solvated species when the mobile-phase composition changes has been obtained on the basis of solvatochromic results. From this study, it can be concluded that the phenyl bonded silica shows dipolarity very similar to that of bare silica. But phenyl phases are considered to be more hydrophobic than silica phases. Thus, we propose that at the mobile-phase/phenyl bonded silica inter(53) Pemberton, J. E.; Cai, M.; Thompson, W. R. Abstracts of Papers; 208th National Meeting of the American Chemical Society, Washington, DC, Fall 1994; American Chemical Society: Washington, DC, 1994, Abstract 16.

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phase, the π* dyes are aligned such that they interact with the surface silanol groups, bulk mobile phase, and the phenyl rings of the stationary phase simultaneously. This anisotropy can result in an increase in the apparent polarizability experienced by the dye in this environment. This might be possible because of the size of the dyes, and the structure of the phenyl bonded silica permits this kind of stacking of the dyes. This could be confirmed by using other dyes with a structure that might not permit this kind of anisotropic alignment for steric reasons. The NM2NA dye also experiences two environments in the octadecyl bonded silica, as has been suggested in the literature previously. For the first time, these two environments have been resolved and their dipolarity characteristics determined. In mobile phases with low water content, the C18 chains remain in the extended configuration with the dye partitioned in the alkyl chains and the dipolarity of this environment is much lower with π* values of 0.7-0.8, very similar to the dipolarity of the pure solvents methanol and

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acetonitrile, indicating that the C18 phase is probably mostly solvated with the mobile-phase modifier. At high water content, the chain collapse becomes a major configuration of the octadecyl alkyl chains with the retention mechanism changing to adsorption. The dipolarity of this configuration of the stationary phase is higher at ∼1.1, which is lower than that of the bare silica and phenyl bonded silica surfaces. Thus, we see that these different functionalized silica surfaces do show different kinds of behavior. ACKNOWLEDGMENT This work was supported by Grant 9709437 from the National Science Foundation.

Received for review April 23, 1999. Accepted September 2, 1999. AC9904314