Heterogeneity of reversed-phase chromatographic surfaces

Jennifer L. Gasser-Ramirez and Joel M. Harris. Analytical Chemistry .... Shashikala Singh, Jürgen Wegmann, Klaus Albert, and Klaus Müller. The Journ...
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Anal. Chem. 1987, 59,2546-2550

Heterogeneity of Reversed-Phase Chromatographic Surfaces: Quenching of Sorbed Pyrene Fluorescence J. W. Carr’ and J. M. Harris* Department of Chemistry, University of Utah, Salt Lake City, Utah 84112

The influence of overlaying solvent OR the environment and structure of polymerk C18, alkylated silica svfaces Is studied by using the fluorescence of pyrene as a probe. IntensRy ratios of vibronic emlssion bands are used to determine changes in poiarlty around the probe sorbed to the C18 surface, whlle quenching of fluorescence by potasslum iodide is used to correlate those changes to exposure of pyrene to the mobile phase. Two dlstlnct regions of Stationary phase behavior are identlfied. At high concentrations of methanol in the mobile phase, pyrene Is partitioned or fully surrounded by alkyl chains wMch are apparently well solvated by methanol. At lower concentrations of methanol, the Increase in surface polarity and onset of quenchhg by sdutlon phase lons indicate that some fraction of sorbed pyrene is exposed to the surrounding solvent as the stationary phase volume collapses. Under these conditions, a stgnifkantly greater heterogeneity in the polarity of sorption environments Is observed, where a fractlon of the fluorescent probe molecules Is partltloned Into nonpolar domains which are inaccessible to a sorbed polar quencher.

In the past several years, there has been considerable interest in studying the surface microenvironment of reversed-phase chromatographic materials so that the contributions from the stationary phase to selectivity and retention might be better understood. Fluorescence spectroscopic studies pioneered by Lochmuller and co-workers (1-4) demonstrated the utility of covalently attaching fluorescent probe molecules to silica to determine the polarity of the environment and the organization and distribution of bound ligands. Two recent papers (5,6)utilized the fluorescence vibronic fine structure of pyrene as a polarity probe of alkylated silica surfaces, where the pyrene was sorbed to the stationary phase under solution conditions typical of chromatographic applications. The first of these two studies (5) was conducted with suspended slurries of silica where the stationary phase to mobile phase volume ratio was extremely small; the compositions of solution phase, with which the surfaces were equilibrated, were restricted to high mole fractions of water in order to maintain a significant concentration of the probe molecules on the surface. While higher water content regions of mobile phase composition produce excessive retention of large PAH molecules to be of direct interest to chromatographic resolution of these molecules, the spectroscopic data from pyrene can reveal useful information about the stationary phase environment under these conditions. The second study (6) utilized a capillary flow cell packed with chromatographic silica (3) where the ratio of stationary phase to mobile phase volume was large and allowed mobile phases of lower water concentration to be studied. This approach as thought to be inappropriate for measurements with Present address: IBM Watson Research Center, Yorktown Heights, NY 10596.

high water content conditions due to the large solution volumes which would be needed to equilibrate the pyrene concentration between mobile and stationary phases. As a result, the two studies explored entirely separate ranges of mobile phase compositional where significant differences in the behavior of the stationary phase were observed. Over a large range of solvent compositions, the apparent intercalation of organic modifier into the stationary phase produces an inverse relationship between the polarity of the C18 surface and that of the mobile phase (6). As the mole fraction of water in the mobile phase approaches unity, this relationship apparently reverses and a more polar stationary phase environment is observed (5). It has been argued that this change in behavior could be due to partial exposure of the sorbed probe to the mobile phase and/or residual silanols (6). In this study, these two distinct regions of stationary phase behavior are accessed in the same experiment to test the continuity of previous results, which were segregated in mobile phase composition. An ionic, unretained quencher is added to the aqueous methanol mobile phase to determine the accessibility of the sorbed fluorescent probe to the solvent, as a function of the solvent composition. The results indicate that the sorbed probe is in contact with the mobile phase only when the solution contains a high mole fraction of water. Quenching by a polar, nonionic, sorbed molecule produces large changes in the vibronic band ratio of the observed pyrene fluorescence; a distribution of sorption site polarities is inferred from these data which indicates that the bonded C18 phase is a chemically heterogeneous environment.

EXPERIMENTAL SECTION Chemicals. Pyrene was obtained from Aldrich and recrystallized from ethanol-water solution. Reversed-phase chromatographic analysis of the compound using a 10-cm column packed with a lO-pm, C18 stationary phase revealed no resolvable impurities. Reagent grade mercuric chloride (molecular quencher) and potassium iodide (ionic quencher), obtained from Baker and EM, respectively, were used without further purification. Small concentrations of potassium chloride (Mallinckrodt) were added to the solutions containing ionic quencher to maintain constant ionic strength. The uncertainty in composition of the water/ methanol solutions, prepared with class A burets, was less than 0.01%. Methanol was HPLC grade, obtained from MCB and Burdick and Jackson. Water was purified in-house with a Corning still (MP-1) in series with a Barnstead cartridge purification system. The polymeric stationary phase was ODs-2, a trifunctional, C18 material from Whatman. The carbon loading of the stationary phase, determined by Whatman, is 15.7%, on a 10-pm irregular silica support (Partisil-10) having a surface area of approximately 320 m2 g-’ and a mean pore diameter of 96 8, prior to derivatization. Retention Measurements. Retention of pyrene and the quenchers on the polymeric C18 stationary phase was evaluated by use of a 10 cm long column, slurry packed at 7300 psi with the same manufacturing lot of ODs-2 as used in the fluorescence studies. Deuterium oxide was used as the void volume marker (7). A linear correlation was established between the fluorescence intensity of an equilibrium concentration of pyrene sorbed to the C18 surface and its k’value. This relationship allowed convenient determination of k’ values in excess of 400 by measuring fluorescence intensity from a much smaller volume of stationary

0 1987 American Chemical Society 0003-2700/87/0359-2546$01.50/0

ANALYTICAL CHEMISTRY, VOL. 59, NO. 21, NOVEMBER 1, 1987

phase material equilibrated with a pyrene solution in a capillary spectroscopic cuvette (3, 6). Fluorescence Measurements. The apparatus and procedures for recording emission spectra of pyrene sorbed to reversed-phase surfaces using a spectrofluorometerhave been previously described (6).Sample solutions were deoxygenated by bubbling for 10 min with argon gas saturated with vapor from a solution of the same composition to avoid changing concentrations of solvent components in the sample. Typically seven spectra for each combination of solution phase and quencher concentration were acquired and averaged. Following a change in quencher concentration, the volume of solution phase required for the surface fluorescence to reach a steady state in the column was generally 0.5 mL or less. To assure homogeneous composition within the cell, at least 3.0 mL of solution was allowed to pass the column before collection spectra following changes in quencher concentration within a single mobile phase composition. After a change in solution phase composition, the equilibration volumes were between 10 and 45 mL. The larger volumes were necessary for more aqueous solutions, not only to allow the stationary phase structure to equilibrate but to allow the small changes in sorbed and solution pyrene concentrations to equilibrate when the capacity factor was large. Time-resolved fluorescence decay curves were recorded with a laser fluorometer consisting of a mode-locked argon ion laser (Spectra Physics, Model 171) synchronously pumping rhodamine 6G in a dye laser (Spectra Physics, Model 375). The pulse rate of the output beam was controlled at 8 kHz by a cavity dumper (Spectra Physics, Model 454). The 600-nm dye laser output was doubled with a KDP crystal (Quantum Technology), and the ultraviolet beam was filtered and directed onto the sample. Decay curves were recorded with a Tektronix sampling oscilloscope (Model 3S2, 3T2) controlled by a DEC 11/23 microcomputer. Lifetimes were sufficiently long that they could be determined from the slope of a plot of log intensity versus time. Uncertainty of fluorescence lifetime results was typically about *lo%, which was larger than the uncertainty in intensity measurements by more than a factor 2. Quenching mechanisms and changes in the unquenched fluorescence lifetime of the probe were therefore checked by time-resolved measurements, while changes in quenching efficiency were determined from intensity measurements due to their greater precision.

RESULTS AND DISCUSSION Stationary Phase Polarity. In order to test the polarity of the polymeric, C18 environment over a complete range of solvent compositions, spanning the previous two studies (5, 6), the stationary phase packing in capillary flow cell was equilibrated with the mobile phase containing pyrene a t a concentration C,. T o achieve an equilibrium concentration of probe molecules on the stationary phase following a change in mobile phase composition, one would need to pass a volume of solvent across the column (8), Vsorb= kVm,where k’is the capacity factor and V , is the mobile phase holdup volume in the packed cell. For high water content mobile phases where k’for pyrene can exceed 4 X 103,this expression predicta that the volume of solvent required to pass over the surface to achieve equilibrium would be excessively large, of the order of a liter, requiring about a day to pump through the cell. T o avoid the time delay and consumption of solvent, the surface was first preequilibrated with pyrene under solution conditions corresponding to lower values of k’. T o maintain a constant surface concentration, C,, following an increase in k’, the pyrene concentration in the mobile phase, C,, was reduced in proportion to llk’such that C, remains constant. As a result, the equilibrium surface concentration of pyrene does not change with changes in mobile phase composition, and the need to expose the surface to a large volume of mobile phase to adjust the surface concentration is eliminated. This simple strategy allowed the polarity of the polymeric C18 stationary phase to be measured over an extreme range of methanol/water solvent compositions and solute capacity factors, as shown in Figure 1. At each solvent composition,

1.304 0

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I

. do ’ 8’0 % MeOH Figure 1. Microenvironmental polarity of pyrene in a polymeric C18 stationary phase as a function of solvent composition. The pyrene fluorescence intensity ratio, III/I, of the third vibronic band and the vibronic orlgin is plotted; polarity increases up the yaxis or as the III/I ratio decreases. The Illo values indicate the quenching effect of 0.10 M potassium iodide. The capacity factor, k’,for pyrene retention on C18 is also shown on the scale at the top. ’

io .

40

the intensity of the third vibronic band of the pyrene emission spectrum is divided by the intensity at the vibronic origin. The resulting ratio is an inverse measure of polarity which ranges from 1.9 in perfluorinated alkanes to less than 0.6 in water (9). The results in the figure show a smooth transition between different behaviors exhibited in two regions of solvent composition. At high concentrations of methanol in the mobile phase, the stationary phase environment becomes more polar with increasing methanol concentration; this behavior is consistent with the intercalation of methanol into the bonded layer as previously determined from distribution isotherms (7). As the volume fraction of methanol in the mobile phase is reduced, the polarity of the stationary phase goes through a minimum at about 50% methanol by volume, followed by a sharp increase in polarity as the composition of the overlaying solution approaches pure water. I t was previously argued (6) that as the concentration of organic modifier is decreased, the stationary phase evolves to a collapsed state and decreases in volume due to loss of intercalated solvent, which could decrease the effectiveness of the stationary phase in shielding the hydrophobic probe molecule from exposure to the aqueous solution environment or to hydrated silanols on the silica surface. Fluorescence Quenching by Solution Phase Ions. To determine whether the upturn in stationary phase polarity at low concentrations of organic modifier relates to the exposure of sorbed molecules to the solvent or surface silanols, a series of fluorescence quenching measurements was carried out by using an ionic quencher, potassium iodide, having no measurable affinity for the alkylated silica surface. The capacity factor, k’, of KI was measured for the range of solvent conditions studied and was found to average --0.2, eluting slightly ahead of the D,O dead volume marker. The effect of a quencher on an observed fluorescence intensity, I , can be predicted by the Stern-Volmer equation (IO) where Io is the fluorescence intensity in absence of quencher, [Q] is the quencher concentration, and K , is the quenching constant. If the encounter of an excited state with a quenching

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species occurs by diffusion following the excitation event, the quenching process is dynamic and K, = ( k q T O ) , where the quenching constant is the product of k,, the second-order rate constant for quenching, and 70, the lifetime of the excited singlet state in absence of quencher. If the quencher and fluorescent species are preassociated before excitation, then the quenching process is static and the quenching constant is equal to the association equilibrium constant between the quencher and the ground state of the fluorescent probe, P

K q = K , = [P-*Ql/([pI[Ql)

(2)

The effects of the solution phase iodide quencher on the fluorescence intensity of the sorbed pyrene probe are summarized on Figure 1at four aqueous solution compositions, ranging from 10% methanol to 70% methanol by volume. The total intensity of the first, third, and fifth vibronic bands of pyrene fluorescence are added together to reduce the sensitivity of the measurement to relative intensity changes between vibronic bands. For each solution, the ratio, I o / I , the sorbed pyrene fluorescence intensity with 0.10 M iodide in the mobile phase divided into the fluorescence intensity in absence of quencher, is measured to determine the relative magnitude of the quenching constant according to eq 1. T o determine whether the changes in quenching constant were driven by changes in T~ or k,, the unquenched fluorescence lifetime of pyrene on the C18 surface was measured for the three solution compositions in which the quenching constant varied. To T~ values showed a weak dependence on solution composition, T~ = 138 ns, 130 ns, and 127 ns for l o % , 30%, and 50% methanol, respectively, which is insufficient to explain the decrease in Kq from 1.1 M-’ to 0.6 M-’ to 0.0 M-l over the same solution compositions. The results, therefore, indicate that at higher concentrations of methanol, the pyrene is well shielded from the solution phase quencher and that the probability of encountering iodide within its excited-state lifetime is less than 1% under these conditions. As the concentration of methanol is lowered in solution which also reduces the amount of methanol intercalated in the stationary phase (7),iodide begins to quench the pyrene fluorescence verifying that the sorbed probe is becoming partially exposed to the solution. Up to a 0.10 M iodide concentration, time-resolved fluorescence measurements showed that the change in fluorescence lifetime, within the error of measurement, was inversely proportional to the change in fluorescence intensity, indicative of a dynamic quenching mechanism. This result is consistent with the k ’measurements showing that the iodide is not associated with the silica surface and that encounters with the fluorescent probe are collisional. The polarity increase which the probe experiences at low methanol concentration is therefore likely to be due to exposure to the solvent. By use of time-resolved fluorescence techniques, the second-order rate constant for quenching of pyrene fluorescence by iodide in free solution of methanol was measured to be k , = 7.5 x lo7 M-’ s-l. This result corrected for the 90% greater viscosity of a 10:90 methanol-water solution and combined with the measured unquenched lifetime of pyrene fluorescence on the alkylated surface ( T ~= 138 5 ns) sets an upper limit on the value of K , = 5.5 M-l, for the kinetic quenching of fluorescence from pyrene sorbed to C18. The largest observed value of the surface quenching constant with 10% methanol, was about a factor of 5 smaller, K, = 1.1 M-’. One can therefore infer from the lower surface quenching constant that the efficiency of quenching a sorbed molecule is smaller by a factor of 5. This result and the observed polarity of the environment indicate that the sorbed pyrene is only partially exposed to the mobile phase and that the stationary phase surrounds this rather large probe molecule to a significant extent.

*

5

4 5

4

35 3 2 5

2

1 5

Qusnshar Consentrotion. M

Flgure 2. Stern-Volmer plots for the quenching of pyrene by mercuric chloride, both sorbed to a polymeric C18 statlonary phase. The yaxis is the total intensity of the first, third, and fifth vibronic bands of pyrene fluorescence, divided into the intensity in absence of quencher; the x axis is the solution phase concentration of HgCl,. The families of points indicate different solution composition in the methanol-water volume fraction. Points plotted as V’s are for 5050;X’s are for 10:90,and A’s are for a 2:98 MeOH-H,O. The smooth lines are the best fit to eq 4 with the parameters listed in Table I.

Fluorescence Quenching by a Polar Sorbed Quencher. In order to better understand the sorption environment of pyrene in a polymeric C18 layer, pyrene fluorescence measurements were also carried out in the presence of a polar quencher which could partition to a limited extent in the stationary phase. Mercuric chloride was chosen for its large quenching efficiency to allow a wide variation of quenching rate. Unlike the fully ionic potassium iodide quencher, mercuric chloride is primarily in an uncharged, molecular form in aqueous solution giving it more access to the hydrophobic stationary phase; over the range of concentrations studied, dissociation of mercuric chloride in aqueous solution varies from only 0.2 to 1.0% (11). While the gas-phase form of this molecule is linear and nonpolar, mercuric chloride in solution is bent, producing a rather large dipole moment, 1.23 D in benzene (12) and 1.43 D in dioxane (13). Due to the large dipole moment of mercuric chloride, capacity factors for retention on the C18 surface were found to be small and to increase with decreasing reversed-phase elution strength; k ’ = 0.19,0.39, and 0.45 for 5050, 10:90, and 2:98 volume fractions of methanol-water, respectively. The quenching by mercuric chloride of the fluorescence from pyrene sorbed to a C18 polymeric stationary phase is shown in Figure 2. The total intensity of three vibronic bands is divided into the intensity measured in absence of quencher. Unlike the ionic quencher, the greatest quenching effect is observed when the solution phase contains the largest percentage of methanol. This is an interesting result since the solution containing the greatest methanol produces the smallest concentration of mercuric chloride in the C18 layer, according to the measurements of the capacity factor for the quencher, described above. Note that the concentration of quencher in the stationary phase layer, [Q],,is related to the product of the mobile phase quencher concentration, the capacity factor, and the volume ratio of the two phases

(3) Another curious feature of these quenching results is their nonlinearity, indicating a deviation from Stern-Volmer behavior described by eq l. While a mix of static and dynamic quenching mechanisms can produce nonlinear quenching plots, the deviation from linearity for such a case is positive (IO) unlike the data in Figure 2. These results could be explained by a nonlinear isotherm for the sorption of mercuric chloride a t high concentrations. As a check on this expla-

ANALYTICAL CHEMISTRY, VOL. 59, NO. 21, NOVEMBER 1, 1987

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Table I. Pyrene Sorption Site Heterogeneity Parameters from HgClz Quenching Parameters from Total Intensity Data Fit to Equation 4 mobile phase composition

(MeOH-H,O)

quenching constant, K,: M“

5050 10:90 2:98

fraction of

protected probe intensity, F

138 62 54

0.13 0.24 0.21

1.22

1 Lx

t

B. Parameters from Vibronic Band Ratios Fit to Equation 6

quenchmobile phase composition (MeOH-H20)

constant, K,: M-I

III/I ratio for quenchable pyrene, R,

50:50 10:90 2:98

3650 1290 1230

1.24 1.08 1.07

ing

> ‘ ” 126 III/I ratio for protected 1.26 1.23 1.13

pyrene, R,

Relative to the concentration of quencher in the stationary phase determined by using ea 3. (I

nation, elution of mercuric chloride on a C18 column was carried out over the range of solvent composition and HgClz concentration (at the detector) as shown in Figure 2. The k’ values of mercuric chloride did not change significantly (1% or less) over the concentration range used in Figure 2, indicating that the roll over in the quenching plot is not due to a reduction in the surface concentration of mercuric chloride relative to that in solution when the solution concentration is high. The nonlinearity of these results is consistent, however, with a postulate that some fraction of sorbed pyrene molecules is in a protected environment, inaccessible to the polar mercuric chloride quencher. If the fluorescence intensity includes a constant unquenchable contribution, then the expected dependence of total intensity on quencher concentration should follow the relationship given by

(4) where F is the fraction of the initial intensity from probe molecules that are inaccessible to mercuric chloride. The solid lines in Figure 2 are given by the best fit to eq 4 using the parameters listed in Table IA. The reported quenching constants, K,, are determined relative to the stationary phase quencher concentrations, [Q],,which were obtained by using eq 3 and the solution-phase concentrations, [Q],, which comprise the n axis of Figure 2. The results in Figure 2 and Table IA begin to describe a heterogeneous sorption environment for pyrene in a C18 stationary phase, where most probe molecules are sorbed into regions which can also be occupied by a polar molecules such as the mercuric chloride. A smaller fraction of pyrene is sorbed into more hydrophobic regions inaccessible to a polar quencher. The efficiency of quenching given by K , was observed to rise with methanol concentration in the solution phase. This result is consistent with intercalation of methanol into the hydrocarbon ligands (6, 7) which would decrease the stationary phase viscosity and increase the rate of diffusional encounters between quencher and the fluorescent probe. The fraction of pyrene molecules that are protected from encounters with mercuric chloride also decreases with increasing methanol concentration, which could be due to methanol becoming more widely dispersed within the stationary phase layer decreasing the residual volume of hydrophobic domains. If hydrophobic regions exist within the stationary phase which can protect pyrene fi-om encounters with polar quenchers, then the vibronic band structure of the pyrene fluorescence should reflect differences in the average envi-

0 02

0 04

0 06

0 08

01

Buonchar Concentration Y

Flgure 3. Microenvironmental polarity of pyrene fluorescence from a C18 layer as a function of quencher concentration. The x axis Is the solution phase concentration of HgCI,. The families of points indicate different solution composition in the methanol-water volume fraction. Points plotted as V’s are for 5050;X’s are for 10:90, and A’s are for a 2:98 MeOH-H,O. The smooth lines are the best flt to eq 6 with the parameters listed in Table I.

ronment of probe molecules as a function of quencher concentration. This behavior is indeed observed in results shown in Figure 3, where the intensity ratios of the third and first vibronic bands of pyrene fluorescence are plotted as a function of mercuric chloride concentration. For all of the solution compositions, a similar pattern emerges where quenching by mercuric chloride extinguishes fluorescence from pyrene in polar domains of the stationary phase, leaving residual emission from pyrene having a smaller III/I ratio and residing in nonpolar domains which are less accessible to the quencher. A simple model for these data can be proposed where the III/I vibronic band intensity ratio, R, has a value, R,, for the quenchable fraction of sorption sites and a value, R,, representing the less polar sorption sites which are protected from quenching. The difference between these limiting values, AR = R, - R,: is a measure of the heterogeneity of the stationary phase envlronment. To determine the limiting values of these band ratios, the variation of R with quencher concentration was fit to an intensity weighted average of R, and R, where a Stern-Volmer model was used to describe the loss of intensity from the quenchable sites. The observed III/I ratio, R, is predicted to arise from the sum of a fraction of intensity, F, having a band ratio, R,, and the quenchable fraction which changes with [Q],[ ( l - F)/(1 + K,[Q])],having a band ratio, R,. Dividing this sum of intensity weighted band ratios by the total intensity, [F + (1 F)/(l + K,[Q])],yields the following expression for the observed III/I ratio:

+

which can be simplified by multiplying numerator and denominator by (1 + K,[Q]) to yield

This model was fit to the data in Figure 3, and the parameters which were determined by the fit are listed in Table IB. Since the best fit values of F , R,, and K q are interdependent and cannot be determined uniquely from the data, the fraction of fluorescence intensity protected from quenching, F, was fixed a t the values listed in Table IA, derived from the fluorescence intensity measurements of Figure 2. The data from this study bear a strong relationship to the results of the total intensity quenching experiments and the unquenched vibronic band ratios. For high concentrations of methanol in the solution phase, the polarities of the regions

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which are protected from quencher and accessible to quencher are nearly equal, indicating that methanol is widely distributed throughout the alkylated layer; this notion is consistent with the 2-fold smaller fraction protected from quenching and the roughly 3-fold larger value of the quenching constant which indicates an increased rate of diffusional encounters between quencher and probe. As the methanol concentration in the solution phase is lowered, the heterogeneity of pyrene sorption sites begins to increase dramatically. The change in polarity is greatest for the quenchable pyrene fraction, the environment of which increases in polarity as it becomes more exposed to the solution interface, while the protected domains remain comparatively nonpolar. The somewhat more polar environment observed for the protected pyrene fraction observed for 2% methanol may indicate an association between the probe and hydrated surface silanols (14) which increases as the stationary phase collapses under more aqueous solvent conditions. The only discrepancy between the results of Table I, parts A and E, is the 25-fold larger quenching constants found from the vibronic band ratios compared with the value found from the fluorescence intensities. The changes in the quenching constant as a function of solvent composition for the two experiments are proportional within 15%, which supports the existence of an underlying correlation between the two results. The most polar regions, perhaps at the solution interface, are apparently the most energetically favorable sorption sites for quencher, which also causes efficient quenching of pyrene sorbed within these domains. As a result, the vibronic band ratios change more sensitively a t small concentrations of quencher where the average intensity of emission is not strongly affected. This result provides some indirect evidence that sorption of small polar molecules, such as the mercuric chloride quencher, into C18 layers is also affected by heterogeneity of stationary phase environments.

CONCLUSIONS The fluorescence fine structure of pyrene sorbed to polymeric C18 silica provides convincing evidence for changes in environmental polarity as a function of overlaying solvent composition. At high concentrations of methanol in the solvent, the changes in polarity and homogeneity of the sorption environment and the high efficiency of quenching by mercuric chloride provide evidence for intercalation of the alcohol throughout the alkylated surface layer. Under these conditions, fluorescence of sorbed pyrene cannot be quenched by solution phase iodide ion, which is a strong indication that the probe is partitioned into the stationary phase rather than adsorbed to its surface (15). This model is consistent with studies of the retention selectivity of polymeric C18 stationary phases for polycyclic aromatic hydrocarbons (16,17) which indicated that planar and linear molecules were retained longer than corresponding nonplanar and nonlinear molecules presumably because of the greater tendency of the former to penetrate into extended (18)alkyl chains. The present results

point out the important role which intercalated organic modifier could play in allowing the partial extension and organization of bound ligands. As the concentration of methanol in solution is reduced below 50% by volume, the polarity of the sorption environment increases. Based on the onset of quenching by solution phase ions, some fraction of sorbed pyrene apparently becomes partially exposed to the surrounding solvent as the amounts of intercalated methanol and the stationary phase volume (19) are reduced. The stationary phase under these conditions also exhibits a greater degree of inhomogeneity in polarity, where a fraction of the fluorescent probe molecules are found in nonpolar domains inaccessible to a sorbed polar quencher. For both the quencher and PAH probe, spatially discrete distributions of retained molecules are inferred, whose populations do not mix, at least on the time scale of the 100-ns excitedstate lifetime of the fluorescent probe. The relationship between the observed environmental inhomogeneities and surface geometry factors (20) is currently under investigation.

ACKNOWLEDGMENT The authors acknowledge the generous help of M. Hunnicutt, who measured the solution-phase quenching constants and guided the surface lifetime studies, and A. Rauckhorst, who determined k’ values for quenchers on a C18 column.

LITERATURE CITED (1) Lochmuller, C. H.; Marshall, D. B.; Wilder, D. R. Anal. Chim. Acta 1981, 130, 31. (2) Lochmuller, C. H.; Marshall, D. B.; Harris, J. M. Anal. Chim. Acta 1981, 131, 263. (3) Lochmuller, C. H.; Colborn, A. S.; Hunnicutt, M. L.; Harris, J. M. Anal. Chem. 1983, 55. 1344. (4) lochmulier, C. H.; Colborn, A. S.; Hunnicutt, M. L.; Harrls, J. M. J. Am. Chem. SOC. W84, 106, 4077. (5) Stahlberg, J.; Almgren, M. Anal. Chem. 1985, 57, 817. (6) Carr, J. W.; Harris, J. M. Anal. Chem. 1988, 58, 626. (7) McCorrnick, R. M.; Karger, B. L. Anal. Chem. 1980, 52, 2249. (8) Knox, J. H.; Hartwlck, R. A. J. Chromafogr. 1981, 204, 3. (9) Dong, D. C.; Winnik, M. W. fhofochem. Phofobioi. 1984, 3 5 , 17. (10) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum: New York, 1983; Chapter 9. (11) Sfability Constants of Metal-Ion Complexes; Chemical Society: London, 1964; Special Publicatlon No. 17. (12) Handbook of Chemlstry and Physics, 63rd ed.; Weast, R. C., Ed.; CRC Press: Boca Raton, FL, 1982. (13) Nouveau Traife de Chemie Minerale; Pascal, P., Ed.; Masson: Paris, 1962; Vol. V. (14) Hunnlcutt, M. H.; Harris, J. M.; Lochmuller, C. H. J. Phys. Chem. 1985, 8 9 , 5246. (15) Dill, K. A. J. Phys. Chem. W87. 9 1 , 1980. (16) Wise, S. A.; Sander, L. C. HRCCC, J. High Res. Chromafogr. Chromatogr. Commun. 1985, 8 , 248. (17) Wise, S. A.; Sander, L. C.; May, W. E. I n Silanes, Surfaces, and Infeffaces; Leyden, D. E., Ed.: Gordon and Breach: New York, 1986. (18) Martire, D. E. Boehm. R. E. J. Phys. Chem. 1983, 8 7 , 1045. (19) Yonker, C. R.; Zwier, T. A.; Burke, M. F. J. Chromafogr. 1982, 241, 257. (20) Avnlr, D. J. Am. Chem. SOC. 1987, 109, 2931.

RECEIVEDfor review March 6, 1687. Accepted July 23, 1987. This research was supported in part by the Office of Naval Research and by fellowship support (to J.M.H.) from the Alfred P. Sloan Foundation.