Anal. Chem. 1880,62,2606-2612
2606 T a b l e VIII. D e t e r m i n a t i o n Formulationso** sample no.
1
2 .‘3
of Human I n s u l i n
concn
(SD)‘
3.94 (0.08) 3.83 (0.07) 3.88 (0.05)
RSD,70 3.0 1.9 1.2
a Claims of manufacturers: 3.79 f 5% mg/mL. *See Figure 8 for conditions. (Concentration is in ma/mL; n = 3.
protein and amino acids. Three different spiked concentrations were analyzed for each compound with triplicate injections for each concentration, having good reproducibility. The relative errors for the determinations were within 6%, also suggesting good accuracy of the method. Application of the method for the determination of Phe in urine has been reported, in which the advantages of selectivity and sensitivity of the method for analysis of a complex matrix such as urine have been shown (17). The determination of insulin formulation samples using gradient reversed-phase chromatography and photolytic electrochemical detection are summarized in Table VIII. Human insulins from three different sources w2re analyzed with good precision. The results agreed with the claims of manufacturers (3.79 mg/mL f 5%) and also agreed with the results obtained by using different HPLC methods with UV detection, as suggested by the FDA (25).
ACKNOWLEDGMENT Helpful and constructive discussions were provided by Professor B. Karger and numerous colleagues, especially C. M. Selavka, X.-D. Ding. M. Lookabaugh, and J. Mazzeo. LITERATURE CITED (1) Moore, S.; Spackman, D. H.; Stein, W. H. Anal. Chem. 1958, 30, 1185. (2) Spackman, D. H.; Stein, W. H.: Moore, S. Anal. Chem. 1958, 30, 1190. (3) Schuster. R Anal. Chem. 1980, 52, 617.
Banson, J. R.; Hare, P. E. Roc. Natl. Acad. Sci. U.S.A. 1975, 72, 619. Cronin, J. R.; Hare, P. E. Anal. Blochem. 1977. 87, 151. Hill, D. W.; Walters, F. H.; Wllson, T. D.; Stuart, J. D. Anal. Chem. 1979, 57, 1338. Engelhardt, H.; Asshauer, J.; Neue, U.; Weigand, N. Anal. Chem. 1974, 46, 336. Bayer, E.; Grom, E.;Kaltenegger, B.; Uhman, R. Anal. Chem. 1976, 48, 1106. Tapuhi, Y.; Schmidt, D. E.; Lindner, W.; Karger, 9. L. Anal. Biochem. 1981, 775, 123. Einarsson, S.; Josefsson, E.; Lagerkvist, S. J. Chromatogr. 1983, 282, 609. Kruii, I.S.; Seiavka, C. M.; Lookabaugh M.; Childress. W. R. LC-GC 1989, 7, 758. Selavka, C. M.; Jiao, K. S.; Kruii, I.S. Anal. Chem. 7987, 5 9 , 2221. Seiavka, C. M.; Krull, I . S.; Lurle, I.S. J. Chromatogr. Scl. 1985, 2 3 , 499. Selavka, C. M.; Krull, I.S.; Anal. Chem. 1987, 59, 2699. Seiavka, C. M.; Krull, I. S.; Anal. Chem. 1987. 59, 2704. Dou, L., Krull, I. S. J. Chromatogr. 1990, 499. 885. Dou, L., Krull, I.S. J. P h r m . Biomed. Anal., in press. Hare, P. E.; St. John, P. A. Detection Limk for Amino Acids in Environmental Samples. I n Detection in Analytical Chemishy; Cwrie, L. A,, Ed.; American Chemical Society: Washington, DC, 1988 Chapter 15. Iriyama, K.; Yoshiura, M.; Iwamoto, T. J. Li9. Chromatogr. 1986, 9 , 2955. Rabenstein. D. L.; Saetre, R. Anal. Chem. 1977, 49, 1036. Saetre, R.; Rabenstein, D. L. Anal. Chem. 1978, 50, 276. Bergstrom, R. F.; Kay, D. R.; Wagner, J. 0. J. Chromatogr. 1981, 222, 445. Allison, L. A.; Shoup, R. E. Anal. Chem. 1983, 55, 8. Hearn, M. T. W., Ed. Ion-Pair Chromatogrephy; Chromatographic Sclence Series; Marcel Dekker: New York, 1985; Voi. 31. Lookabaugh, M.; Krull, I. S. Unpublished work.
RECEIVED for review May 10,1990. Accepted August 23,1990. This research was supported, in part, by a grant to Northeastern University from Bioanalytical Systems, Inc., through the generosity and assistance of P. T. Kissinger. Additional funding was provided, in part, by an NIH Biomedical Research Support Grant to Northeastern University (RR071431, Department of Health and Human Resources (DHHS). Funding was also provided, in part, by a grant from P f i i r , Inc. (Groton, CT). This is contribution no. 436 from The Barnett Institute of Chemical Analysis and Materials Science at Northeastern University.
Relationship between Surface Polarity, Retention of Moderately Polar Solutes, and Mobile-Phase Composition in Reversed-Phase High-Performance Liquid Chromatography Yue-Dong Men and David B. Marshall* Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300 The interfacial pdarity of a RP-18 statlonary phase In methanol-water slurries has been measured by a fluorescence probe technlque. The fluorescence spectra of solvent-sllca siunles were compared to spectra of analogous damyl freesdutlon probe compounds h the same solvents to obtain an assessment of the acceseibHHy to solvent of the surface-retalned probe. I t was found that the interfacial -on sensed by the probe has an effective polarity comparable to that of 10 % -20 % water-methanol and 5 % -10 % water-methanol for n-propanedansylamlde (F-I ) and n-decanedansyiamlde (F-II), respectlvely. No change In effective swface polarity wlth changes In mobile-phase composltlon or k’of the probe was observed over the 2%-50% water compooitlon range. Impllcatlons for the mechanlsm of retentlon In RP-HPLC (reversed-phase high-performance llquld chromatography) (C,8) are discussed.
INTRODUCTION Reversed-phase high-performance liquid chromatography (RP-HPLC or RPC) has become a powerful and valuable analytical tool. Recent research has shown that the nonpolar stationary phase not only acts as a passive solute receptor but also plays an active role in the mechanism of retention in RP-HPLC. Selectivity and solute retention in RP-HPLC vary with the length, shape, and orientation of bonded chains (1-5). The separation mechanism on chemically bonded nonpolar phases has long been a widely debated topic. In 1977, Colin and Guiochon discussed three possible retention mechanisms (6): (A) RPC is a kind of liquid-liquid chromatography. (B) RPC is similar to classical liquid-solid Chromatography, but interactions between solute and stationary phases are weaker than in adsorption so that the solute behavior in the mobile phase is dominant. (C) Partition of the solutes takes place
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
between the mobile phase and a "mixed" stationary phase formed by adsorption of the organic modifier on the stationary phase (7). The preponderance of more recent experimental evidence and theoretical developments support a mechanism for RPC that is largely partitioning, particularly for c18 stationary phases. Wise et al. (8)have proposed that the selectivity of RPC for polycyclic aromatic hydrocarbons (PAHs) depends on the length-to-breadth ratio of the PAH. Martire et al. (9) have developed a unified molecular theory based on a lattice model to describe the solute distribution process in RPC. Dill has developed another statistical-mechanical theory that accounts for bonded-chain reorganization energy (10). Both of these theories indicate that retention energies are dominated by partitioning of eluites into the bonded-phase mass. However, important questions remain concerning the composition of the stationary phase and how that composition changes with mobile-phase composition. Another question is how the stationary-phase composition sensed by an eluite varies with retention: that is, do highly retained eluites encounter the same stationary-phase environment as weakly retained eluites? In the last several years, a number of researchers have used fluorescence probes to study the surface environment of reversed-phase stationary phases and have gained a better understanding of the influence of stationary-phase structure and composition on selectivity and the mechanism of retention. The first such use was a covalently attached fluorescent probe, (a dansylamide), used to study the polarity and microheterogeneity of a reversed-phase surface environment (11,12). Covalently attached pyrene has been used to measure the local structure and distribution of the bound pyrene groups (13, 14). The fluorescence of physisorbed pyrene excimers has been used to measure the viscosity at stationary-phase surfaces (15). Pyrene has also been employed as a probe for measuring the polarity of the surface on RP-HPLC (16-18). In the fluorescnece vibronic fine structure of pyrene, the ratio of the emission intensity of the third band to the first band, III/I, can be used as an indicator of surface polarity. Stahlberg and Almgren studied the fluorescence of pyrene physisorbed to silica. Mixed solvent suspensions with a high molar fraction of water were used to maintain a high concentration of pyrene on the surface for determining the polarity of the surface (16). Their results show a decrease in stationary-phase polarity with decreasing mobile-phase polarity (decreasing water). Carr and Harris utilized a capillary flow cell packed with chromatographic silica to study the fluorescence of physisorbed pyrene at higher organic modifier fractions (17). An increase in the polarity of the C18 surface was observed with decreasing mobile-phase polarity (decreasing water). A later study by Carr and Harris over the entire mobile-phase composition range covered in the two previous studies confirmed that the stationary-phase polarity of polymeric cl8 silica first decreases then increases with decreasing mobile-phase polarity (decreasing water) (18). In this work, two fluorescent probes, n-propanedansylamide (F-I) and n-decanedansylamide (F-11)are employed to determine the stationary-phase polarity of an end-capped, monomeric silica. The fluorescence spectra of the dansyl probes in solventsilica slurries are compared to free solution spectra of the dansyl compounds in the same solvents. The difference between the emission spectra of the physically adsorbed and free solution fluorophores is used to assess the accessibility of solvent to the physisorbed probes. In addition, the emission intensity ratio of the two fluorescence spectra peak maxima of the dansyl compounds is shown to be a slightly more sensitive measure of changes in polarity than emission maxima wavelength shifts of either peak. The relationship between surface polarity and probe partition
2607
coefficient is also determined. Implications of the results for the mechanism of retention on RP-HPLC (C18)are discussed.
EXPERIMENTAL SECTION Chemicals. Spherical silica gel (octadecyldimethylchlorosilane (ODs), Nucleosil; 100-5,C18, Macherey-Nagel)of average particle diameter 5 wm, average pore size 100 A, and nominal surface area of 350 m2/g was used as supplied. n-Propylamine (98%) and n-decylamine were obtained from Aldrich Chemical Co. (Milwaukee, WI) and fractionally distilled. Solvents used for fluorescence measurements and chromatographic measurements were all of HPLC/spectral grade quality. All other chemicals used in the experiment were of reagent grade except dansyl chloride (5-(dimethy1amino)naphthalenesulfonyl chloride) (Aldrich Chemical Co., Milwaukee, WI), which was recrystallized prior to use. Carbon and hydrogen values, expressed as percentages (by weight), were determined to be 13.26% C and 2.56% H for the octyldimethylchlorosilane-modifiedsilica gel by Atlantic Microlabs (Norcross,GA). This gives a nominal C18coverage of 550 pmol/m2 for this stationary-phase material. The weight of C18silica in the packed 25 cm X 2.5 mm i.d. column was 0.786 g. Synthesis of Dansyl Probes. The synthetic procedure was based on a procedure previously described (11). n-Propanesulfonamide derivative (F-I): Dansyl chloride (0.76 g) was dissolved in 17 mL of acetone, and then the solution was added to 0.7 mL (0.49 g) of n-propylaminein 30 mL of 0.1 N NaHC03. The mixture was reacted with stirring at 50 "C for 24 h. After termination of the reaction, the product was extracted twice with ether and the ether fractions dehydrated by slurryingwith MgSOI. The ether was then evaporated by stirring at 50 "C and the product purified by column chromatography (silica gel, 20% hexane-acetate). n-Decanesulfonamidederivative (F-11): Dansyl chloride (1 g) was dissolved in 50 mL of acetone, and then the solution was added to 1 mL (0.785 g) of n-decylamine (99% Aldrich) in 35 mL of 0.1 N NaHC03. The mixture was reacted with stirring at 50 "C for 50 h under nitrogen. After termination of the reaction, the product was extracted twice with ether (50 mL/each), dehydrated with MgSO,, and then the ether removed by rotary evaporation at 50 "C. The compound was purified by column chromatography (silica gel, 30% hexane-ethyl acetate) and recrystallized from n-hexane. The purity of the two compounds was verified by TLC and NMR spectroscopy. Only one elution band for each compound was observed in the chromatographic measurements. Chromatographic Measurements. Chromatographic data were obtained with a Waters Model 6000A liquid chromatograph and a Perkin-Elmer Tridet fluorescence detector. Samples were introduced into the system with a Rheodyne 7010 injector with a 20-qL injection loop. The stationary phase (C18) (0.78 g) was packed into a stainless steel column blank at 6700 psi, using the upward slurry method with methanol as the slurry solvent and driving solvent. Chromatographic retention times were measured as the average of triplicate runs using the peak maxima of the recorder traces. Average relative standard deviation values for the retention time values were better than 1%.The dead volume (V,) was obtained by averaging the retention times of the unretained solute, uracil, in a series of water-methanol mixtures. The stationary-phase volume (V,) was calculated by the method of Sentell et al. (19). Chromatographic measurements involved evaluation of test solute elution bands as exponentially modified Gaussian curves. Fluorescence Measurements. The fluorescence spectra were obtained by using a Fluoro IV spectrofluorometer (Gilford). Emission and excitation bandwidths were 5 nm. The excitation wavelength used throughout the experiment was 350 nm. All spectra were recorded at ambient temperature. The location of Rayleigh scattering peaks, Raman peaks, and higher order bands were observed to be well removed from the emission envelope of the dansyl fluorophore in the solvent-silica slurries. The fluorescence spectra of both F-I and F-I1 M) in a series of free solvent mixtures (from 100% methanol to 50% watermethanol) were recorded as a function of emission wavelength. for both compounds in free solution is 527 nm in The ,A, methanol and 541 nm in 50% water-methanol. A 1-mm quartz cuvette (Wilmad Glass Co.) with the excitation beam at a grazing incidence angle was used in the acquisition of fluorescence spectra
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
2608
Table I. Chromatographic Parameters of n -Propanedansylamide (F-I) vs Volume Percent of Water in Methanol-Water (Flow Rate = 0.50 mL/min) 70 H2O
to, s
V,, mL
0 1 2
96.6 97.8 99.7 99.0 94.5 94.0 91.7 90.0 89.6
0.805 0.815 0.831 0.825 0.787 0.783 0.764 0.750 0.746
5 10 20 30 40 50
tR,
s
k'
103.9 0.075 104.5 0.068 106.0 0.063 111.5 0.126 121.8 0.289 160.4 0.706 272.1 1.969 687.3 6.633 1995.7 21.29
V,/V,
Kp
4.74 4.79 4.89 4.85 4.63 4.61 4.49 4.41 4.39
0.355 0.326 0.308 0.611 1.34 3.25 8.84 29.3 93.4
t .-+-(A: c
\ i
> .-+ 07
W O K
2
Table 11. Chromatographic Parameters of n -Decanedansylamide (F-11) vs Volume Percent of Water in Methanol-Water (Flow Rate = 0.50 mL/min) V,, mL
% H20 to, s
0 1 2 5
10 20 30 40 50
96.6 97.8 99.7 99.0 94.5 94.0 91.7 90.0 89.6
0.805 0.815 0.831 0.825 0.787 0.783 0.764 0.750 0.746
tR, s
-
k'
131.9 0.365 136.0 0.390 0.451 144.6 174.2 0.760 1.82 266.4 1020.0 9.85 60.8 5668.9 36540 -404
VdVm
KP
4.735 1.78 4.794 1.87 4.888 2.20 4.853 3.69 4.629 8.43 4.606 45.3 4.494 273.0 4.412 1790 4.388
in solvent-silica slurries. Smooth, reproducible emission spectra were obtained. The slurries were introduced into the 1-mm cuvette by gravity feed, and the cuvette was held in place by a plastic holder at an angle of 4 5 O to the excitation beam. Probe emission maxima in solvents and slurries did not vary with probe concentration over the range 10-8-10-3M, indicating that dimer formation is not likely over this range. All subsequent studies M for ease were therefore done at probe concentrations of of detection.
'
420
440
460
480
500
520
540 5 6 0
580
600
Wavelength (nm)
Figure 1. Representative fluorescence emission spectrum of the alkanedansylamide probes.
Table 111. Emission Maxima (nm) of n -Propanedansylamide (F-I) and n -Decanedansylamide (F-11) in Pure Solvents (Excitation at 350 nm)
solvent
F-I
F-I1
hexane to1u ene ethyl acetate pyridine acetone acetonitrile methanol
443 513 515 517 525 525 527
442 513 515 517 525 525 527
Table IV. Emission Maxima (nm) and Mole Fractions of n -Propanedansylamide (F-I) and n -Decanedansylamide (F-11) vs Volume Percent of Water in Methanol-Water Solutions and CI8Silica Slurries (Excitation at 350 nm)
RESULTS AND DISCUSSION
F-I
F-I1
Chromatographic Parameters. Chromatographic measurements of the CI8 phase were made to determine the partition coefficient, K , of the sorptive system. Tables I and I1 list the chromatographic parameters for F-I and F-11, respectively. The retentbn time of a nonretained solute, uracil, was used to estimate the dead time (to)and dead volume (V,) in each water-methanol mobile phase. No systematic variations in the uracil retention time with mobile-phase composition were observed, so all measurements were averaged to provide a single value of the dead time and volume. The chromatographic phase ratio (V,/V,) is a necessary quantity for obtaining thermodynamic information such as the partition coefficient and changes in entropy and Gibbs free energy for solute partitioning between mobile and stationary phases. In this study, V , was approximated by the retention time of a "nonretained" solute and V, was calculated according to the following equation (19):
9713 527 527 83/17 527 527 82/18 9416 527 528 79/21 9416 5 9416 527 528 71/29 10 8713 528 528 51/49 20 72/28 531 528 15/85 30 528 3.2196.8 50150 534 40 535 528 0.5199.5 22/78 50 b b -0/100 10/90 541 is the mole fraction of solute in the mobile phase, and f s p is the molar fraction of solute in the stationary phase (fmp= 111 + k', k' was adjusted to account for the difference in phase ratio in the fluorescence cuvette vs the chromatographic column). ConcentraM. *Probe did not dissolve enough tion of the probes was 1 x to be detected.
The % C (grams of carbon per 100 g of bonded silica) is 1370, as obtained from elemental analysis, M (molecular weight of the bonded-phase alkyl group for molar volume) is 311, W , (the weight of the bonded packing contained in the column) is 0.786 g, n, (number of carbon atoms in the bonded dimethyloctadecyl group) is 20, and p (density of the bonded alkyl groups) was taken as 0.8607 (20). The volume of the stationary phase is thus 0.145 mL. From this result, the phase ratio for F-Iand F-I1 can be determined (see Tables I and 11). The values of the partition coefficients were calculated from
K = k'(V,/V,) and are shown in Tables I and 11. Fluorescence Emission Maxima. Fluorescence spectra were obtained for both of the dansyl probes in pure solvents varying in polarity from hexane to methanol and in multicomponent water-methanol solvents varying in the percentage of water from 0% to 50%. The emission spectrum of both dansyl probes in solution shows two maxima (see Figure 1). Earlier work with this probe was done a t lower spectral resolution (excitation and emission bandwidths of 9 vs 5 nm), and the lower peak maximum at longer wavelengths was observed as a shoulder on the more intense, shorter wavelength peak (11). The shift of either emission maximum can serve
7'0 H 2 0 0 1 2
hmx,
Amam
soln
slurry
527 527 527 527 529 532 534 535 541
527 527 527 528 529 530 530 530
Amam fmp/fspa
soln
slurry
fmp/fsp
Ofmp
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990 2809 1.27
hexane 440
i
1
0
2
4
3
5
6
51 0
polarity Index
Figure 2. Plot of fluorescence probe maxima vs solvent polarity index.
520
530
wavelength Flgure 5. Plot of height ratio of fluorescence probe maxima vs Wavelength of the longer wavelength maximum in various solvents. The height ratio is defined as the ratlo of the longer wavelength peak height to the shorter wavelength peak height.
c
c
u l S
-QQ >
z
520 -t 0 0
I
I
I
02
0.4
0.6
0.6
mole fraction water Figure 3. Plot of fluorescence probe maxima vs mole fraction of water in water-MeOH solvent mixtures: F-I = n-propanedansylamide; F-I I
= ndecanedansylamide. 1.2
1 .o
11
1
methanol Q
o,61
acetonitrile
I
0.6
0.01
0
ethyl acetate
.
, 1
.
I
2
.
1
3
.
, 4
.
I
5
. 6
polarity index Figure 4. Plot of height ratio of fluorescence probe maxima vs solvent polarity index. The height ratio is defined as the ratio of the longer Wavelength peak height to the shorter wavelength peak height.
as a measure of solvent polarity. For example, the wavelength maximum of the longer wavelength peak for both dansyl probes varies from 443 nm in pure hexane to 541 nm in 50% water-methanol (see Tables I11 and IV).
520
530
540
550
wavelength Flgure 6. Plot of height ratio of fluorescence probe maxima vs wavelength of the longer wavelength maximum in water-MeOH solvent mixtures. The height ratio is defined as the ratio of the longer wavelength peak height to the shorter wavelength peak height.
A plot of the, A values for the two dansyl probes in various pure solvents versus polarity index (21)and in water-methanol solvent mixtures versus mole fraction water are shown in Figures 2 and 3, respectively. From Figure 2, spectral shift data can be expressed as a function of the polarity index or molar fraction of solvents used in the experiment. The polarity index or molar fraction as used here serves as a convenient parameter for correlation of general solvent properties. It can be seen to give a reasonable correlation for all solvents. A comparison of solvent shift data of F-I and F-I1 can thus be used to characterize differences of solvent polarity, as noted earlier (II 1. Height Ratio of Emission Maxima. The emission spectrum of dansyl probes in solution shows two emission maxima, and there is an interesting additional feature that can be obtained from the emission spectrum. It was found that the intensity ratio of these two emission maxima also correlate with solvent polarity. We label the maxima at shorter wavelengths as peak I and the maximum at longer wavelengths as peak 11. The II/I ratio was found to be 0.18 in hexane and 1.6 in 50% water-methanol. From Figures 4-6, it can be seen the II/I ratio gives a good linear relationship
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ANALYTICAL CHEMISTRY, VOL. 62, NO.
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with the solvent polarity index, mole fraction of water, and emission wavelength shift of peak 11. The ratio is seen to give a reasonable correlation for all solvents. This effect is phenomenologically very similar to the effect of the ratio of the emission intensity of the third band to the first band, III/I ratio, in the fluorescence vibronic fine structure of pyrene that was found by Kalyanasundaram and Thomas (22). There is presently no quantitative explanation for this effect. Qualitatively, however, the ratio of the emission intensities in pyrene is sensitive to the extent of interaction between the solvent dipoles and the excited singlet state (22)and a similar effect may be operable here. (Polarization studies of pyrenyl compounds in frozen glasses indicate that the solvent interaction perturbs the transition moment direction, inducing a symmetry-lowering mixing of the Lb and La states (23).)The relative magnitude of the change in ratio is larger than the relative magnitude of the wavelength shift and is thus a more sensitive measure of changes in polarity. Unfortunately, interference from silica scattering under some solvents conditions (low dansyl quantum yields) makes determination of the II/I ratio less reliable than determination of the wavelength maximum of peak 11. If the scatter from silica could be reduced, the II/I ratio would serve as a very good measure of solvent polarity. Surface Polarity of RP-HPLC.A comparison of solvent shift data of the dansyl probes was further used to characterize the polarity of the silica surface environment in RP-HPLC in water-methanol solvent mixtures. The fluorescence spectra of the solvent-silica slurries were compared to free solution spectra of the dansyl compounds in the same solvents. The difference between the emission spectra of the physically adsorbed and free solution fluorophores provides an assessment of the accessibility of solvent to the physisorbed group. From Table IV, it can be seen that the emission spectra of F-I in solvent-silica slurries from 20% to 40% water-methanol solvent mixtures have a constant emission maximum wavelength of 530 nm. In the solvent mixtures from methanol to 10% water-methanol, the emission maxima of F-I in solvent-silica slurries are the same as the free solution spectra of F-I in the same solvents. The emission spectra of F-I1 in the solvent-silica slurries show no significant change in the emission maximum position (528 nm). The total fluorescence intensity of the solventsilica slurries is given by the following: F = f m g m p + fspFsp = f m p F m p + (1 - f m p ) F s p withim,= l / ( l + k ? a n d f , , = k ’ / ( l + k ? . f,,andf,,arethe mole fractions of solute in the solvent (“mobile”) and on the silica surface, respectively. The k’ values were adjusted for the difference in phase ratio between the fluorescence and chromatographic experiments. Given the values of the ratio of molar fraction (fmP/fep), the total emission intensity should be dominated by the mobile-phase contribution for F-I in o%, l % , 2%, 5%, and 10% water-methanol mixtures and for F-I1 in 0% and 1% water-methanol. The observed emission maxima for the corresponding slurries are indeed the same as the free solution maxima of F-I and F-I1 in same solvents. However, the stationary-phase contribution should dominate the total emission intensity for the remaining solvent compositions. An additional factor indicating that the stationary-phase contribution to the total intensity is dominant is the higher quantum yield of dansyl probes with decreasing solvent polarity, as shown by the relative intensity data in Table V. A remaining unknown factor affecting the emission intensity contributions from the two phases is the unknown geometric light collection factors, which should be somewhat different for the two phases. However, we have employed front-surface detection
Table V. Relative Intensities of n -Propanedansylamide (F-I) and n -Decanedansylamide (F-11) Emission Peaks in Various SolventCIBSilica Slurries F-I
solvent hexane n-propylamine acetonitrile methanol 5% water-MeOH 10% water-MeOH 20% water-MeOH 30% water-MeOH 40% water-MeOH
peak I
peak I1
peak I
0.29 0.46 0.45 0.45 0.44 0.41 0.32 0.26
0.51 0.49 0.44 0.42 0.40 0.34 0.23
1.00
1.00 0.53 0.49 0.45 0.42
0.40 0.34 0.24 0.19
F-I1 peak I1 0.27 0.45 0.44 0.44 0.43
0.39 0.30
in thin cells containing a dense pack of silica slurry. These collection conditions should further enhance the solid surface contribution to the total intensity relative to the bulk solution. A quencher technique was used to obtain unequivocal emission maxima from the stationary phase. When KI is used as a quencher, iodides are excluded by the ODS-bonded phase and stay in the mobile phase (18). Thus, the emission from the ODS-bonded phase dominates the total intensity because the intensities of emission maxima of the mobile phase are reduced by iodides and dansyl probes in ODS-bonded phase have a higher quantum yield. The data from the dansyl probes with quencher in solvent-silica slurries for F-I IN 10%-50% water-methanol and for F-11 in 2%-50% water-methanol are in agreement with the corresponding results from the dansyl probes without quencher. Thus, the total fluorescence of the slurries in these solvent compositions is dominated by the contribution from the fraction of the probes retained in the stationary phase and represents the polarity of the stationary-phase environment sensed by the probes. In the solventsilica slurries with 10%-50% water, the, X for F-I remains constant at 528 nm. The maximum for F-I1 remains constant at 530 nm over the 2%-50% water composition range. The effective surface polarity of the ODSbonded phase in mixed methanol-water solvents thus remains constant (within the sensitivity of the probes used) over a t least the 2%-50% water composition range. The effect of probe size and polarity results in a higher effective surface polarity for F-I compared to F-11. Other researchers have noted that the amount of organic modifier extracted by CIS phases changes over this same range of solvent composition (24-27). McCormick and Karger have noted that the amount of methanol extracted from methanol-water mobile phases by a Cls stationary-phase changes over the 0%-40% methanol range. The distribution isotherm could not be reliably determined at higher methanol levels (those of this study) due to imprecision in the data, although the amount extracted seems to plateau at the higher methanol levels (24). Indirect measurements of the amount extracted via solute displacement experiments seem to indicate a small change in the amount of methanol extracted at higher methanol levels (24, 25). Yonker, Zwier, and Burke measured a change in the amount of methanol extracted by ClS from 0.37 to 0.25 mL of methanol/g of bonded phase, over the mobile-phase composition range 0%-40% water. The amount of water extracted changed from zero to 0.09 f 0.03 mL/g over this same range (26). The free-solution data of Table IV show that the probes used are not sensitive to changes in polarity due to changes in water content of less than a few volume percent. Apparently, the change in stationary-phase polarity due to changes in amount of organic modifier and water extracted is less than this. Changes in the amount of methanol extracted may not significantly change the polarity of the solvated stationary phase but instead change the stationary-phase
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
volume by promoting greater extension of the C18chains from the silica surface. The dependence of the emission maximum on excitation wavelength, previously shown to be a measure of environmental heterogeneity (12), was measured for some of the slurries. The excitation wavelength was changed from the 350-nm value used throughout this study to 400 nm. F-I and F-I1 on ODS showed little or no emission maximum shift (ca. 1nm) in 5% water-methanol slurries. Increasing the fraction of water to 40% caused a 3-nm shift for F-I. A smaller increase in percent water, to 30%, caused a larger 4-nm shift for the less polar, more highly retained F-11. Solvent Composition near t h e Interfacial Region. The data in Table IV show that changes in the water-methanol mixtures cause almost no change in A,, for solvent-silica slurries of F-I or F-11. If these values are taken to be representative of the surface environments of solvent-silica slurries, then the effective surface polarity for each F-I and F-I1 on the ODS-bonded phase remains constant and is equivalent to that of 10%-20% and 5%-10% water-methanol mixtures, respectively. A reasonable explanation for these results is that the organic modifier in the mixed organic-water mobile phase is preferentially adsorbed (27,28). The solvent composition of the stationary-phase zone is different from that of the mobile phase and varies as a function of distance from the ODS silica surface. Partitioning of the solutes takes place between the mobile phase and this zone. The percentage of the organic modifier in the zone increases as the distance from the solid surface decreases because water is at least partially excluded by the ODS-bonded phase. This mixed solvent/ stationary phase zone varies in composition from nearly pure organic modifier at the surface to the composition of the bulk organic modifier-water solvent mixture used as the mobile phase. The difference between the results for F-I and F-I1 is the anticipated result if one considers the difference in their polarities arising from the difference in alkyl chain lengths (3 carbons for F-I, 10 for F-11). F-I is more polar than F-I1 because of its shorter carbon linkage, thus it will be retained in a zone with a higher average polarity. Therefore, partitioning of F-I and F-I1takes place in zones with different mean distances from the solid surface. Partitioning of F-I should thus takes place at a mean distance further from the solid surface than F-11. One would expect that the effective surface polarity might increase with decreasing polarity of solute, and this is indeed observed with A,, = 530 and 528 nm for F-I and F-11, respectively. The partitioning of F-I takes place in a zone with an average polarity equivalent to that of 10%-20% water-methanol, and F-I1 partitions in a zone with a polarity equivalent to 5%-10% water-methanol. Also, the excitation-wavelength-dependent results show that the heterogeneity of the average stationary-phase zone occupied by the solute increases with decreasing distance from the solid surface. Relationnhip between t h e S u r f a c e Polarity and Partition Coefficient. The relationship between the surface polarity and partition coefficient can also be determined. From the data in this study, it can be seen that changes in the partition coefficient cause almost no change in surface polarity for both of the dansyl probes. The surface polarity sampled by these probes under the range of mobile-phase conditions studied here is thus independent of the magnitude of probe retention. Comparison with Previous Results. In 1981, Lochmuller and co-workers were the first to use a fluorescent probe to study a liquid chromatographic stationary phase. Dansyl chloride was reacted with a partially animated, exhaustively octadecylated silica to study the polarity of the surface environment of a reversed-phase silica (11). A microheterogeneity of the reversed-phase surfce was observed as a de-
2611
pendence of the emission maximum on the excitation wavelength (12). The results indicated that the change in polarity of a monomeric cl8 surface with methanol as organic modifier from 50% to 100% is small and the surface polarity is close to that of pure acetonitrile. The difference with the present study may be that covalently attaching the fluorescent probe prevents the probe from entering the lowest energy site. The heterogeneity experienced by the probe may well be greater when the probe is constrained by covalent attachment nearer the silica surface. Stahlberg and Almgren (16) observed a decrease in stationary-phase polarity with decreasing mobile-phase polarity (decreasing water) in suspensions with high molar fractions of water. Carr and Harris (17,18) studied the environment of the surface over an extended range of solvent composition. As noted above, they observed that the stationary-phase polarity of polymeric C18silica first decreases then increases with decreasing mobile-phase polarity (decreasing water). First, the results described here show that the surface polarity on CISis always lower than that in the overlying solvent because of the intercalation of organic modifier into the bonded phase, in agreement with previous experimental results. When organic modifier intercalates into the bonded phase, water will be excluded by the alkyl chains due to the hydrophobic effect and thus make the surface polarity of C18 lower than that of the mobile phase. However, the value of the polarity measured in the present study is higher than that determined previously, and shows no variation with solvent composition, in direct contrast to the previous studies. This difference may be due to differences in the molecular nature of the probes used. Pyrene is a planar, nonpolar PAH molecule but the dansyl compounds are moderately polar, nonplanar molecules. Partitioning of the solutes takes place between the mobile phase and a mixed zone formed by adsorption of organic modifier on the stationary phase. A number of researchers have reported that planar PAH molecules are retained on C18 phases much longer than corresponding nonplanar PAH molecules (3,8,9). This effect is predicted by Dill to occur due to differences in the energy required to reorganize the alkyl chains to accommodate the solute (10). Therefore, when pyrene is used as a probe, it may penetrate deeper into the mixed zone and form a more direct, adsorptive contact with the alkyl chains, because the hydrophobicity of pyrene would tend to keep it in the stationary phase and far away from the aqueous environment. Thus, the apparent surface polarity measured by pyrene is lower than the surface polarity measured by dansyl compounds. At low concentrations of water, pyrene is shielded by the alkyl chains. The surface polarity of c18 depends on the degree of solvation of the alkyl chains by the mobile phase. An increase of the concentration of organic modifier increases the degree of solvation and the surface polarity. At high concentrations of water, the collapse of the alkyl chains exposes pyrene to the surrounding solvent (17, 18), and the surface polarity measured by pyrene will be higher than that measured by the dansyl compounds. Early studies suggested that the selectivity of reversed phases for PAH is based on differences in the solubility of each PAH in the mobile phase (29, 30). These results suggested a partitioning mechanism in reversed phases. A number of other studies on the relationship between retention and the shape of PAH have suggested an adsorption mechanism (€431-33). This and other more recent experimental studies (16-18) and theoretical developments (9,lO) are resulting in a more complete albeit more complicated picture of the separation mechanism in reversed-phase chromatography. The stationary-phase environment sensed by an eluite is a function not only of the mobile-phase composition, but of the molecular
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Anal. Chem. 1990, 62. 2612-2616
nature of the eluite itself. Data from a wide variety of probes will be needed to formulate a more complete picture of the separation mechanism in reversed-phase liquid chromatography.
ACKNOWLEDGMENT We thank a reviewer of the first version of this manuscript for stimulating the discussion comparing our results to those of refs 24-27.
LITERATURE CITED Lochmuller, C. H.; Wilder. P. R. J. Chromatogr. Sci. 1979, 777, 574. Lochmuller, C. H.: Hangac, H. H.: Wilder, D. R. J . Chromatogr. Sci. 1981, 79, 130. Tanaka. N.; Sakagami, K.; Araki, M. J. Chromatogr. 1981, 799, 327. Gilpln. R. K. Am. Lab. 1982, 74, 104. Scott, R. P. W.; Kucera, P. J. Chromawr. 1977, 742, 213. Colin, H.; Golochon, G. J. Chromatogr. 1977, 747. 289. Knox, J. H.; Pryde, A. J . Chromatcgr. 1975, 772, 171. Wise, s. A.; Bennett. W. J.; Guenther, F. R.; May, W. E. J. Chromatogr. Sci. 1981, 79, 457, Martire. D. E.; Boem, R. E. J. Phys. Chem. 1983, 87, 1045. DiiI, K. A. J. Phys. Chem. 1987, 9 7 , 1980. LochmuUer, C. H.; Marshall, D. B.; Wilder, D. R . Anal. Chim. Acta 1981, 730, 31. Lochmuiier, C. H.: Marshall, D. E.; Harris, J. M. Anal. Chim. Acta 1981, 737. 263. Lochmblier, C. H.; Colborn, A. S.: Hunnicutt, M. L.; Harris, J. M. Anal. Chem. 1983, 55, 1344. Lochmukr, C. H.; Colborn. A. S.; Hunnicutt, M. L.; Harris, J. M. J. Am. Chem. SOC. 1984. 706, 4077.
(15) Boaar. R. G.: Thomas. J. C.: CaHls, J. B. Anal. Chem. 1984, 56, 1080. Stahlberg, J.; Aimgren, M. Anal. Chem. 1985, 5 7 , 817. Carr, J. W.; Harris, J. M. Anal. Chem. 1986, 58, 626. Carr, J. W.: Harrls. J. M. Anal. Chem. 1987, 59, 2546. Senteil, K. B.; Dorsey, J. G. J. Liq. Chromatogr. 1988, 7 7 , 1875. Cheng, W. Anal. Chem. 1985, 57, 2409. Miller, J. M. Chromatography Concepts and Contrasts; Wiley: New York, London, 1987; p 10-12. Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. SOC. 1977, 9 9 , 2039. Langkiide, F. W.; Thulstrup, E. W.; Michl, J. J. Chem. Phys. 1983, 78, 3372. McCormick, R. L.; Karger, 8.L. Anal. Chem. 1980, 52, 2249. McCormick, R. L.; Karger, 8.L. J. Chromatogr. 1980, 799. 259. Yonker, C. R.; Zwier, T. A,; Burke, M. F. J. Chromatogr. 1982, 247, 257. Yonker, C. R.; Zwier, T. A.; Burke, M. F. J. Chromatogr. 1982, 247, 269. Knox. J. H.; Kaiiszan, R.; Kennedy, G. J. Chem. Soc Faraday Symp. 1980, 75, 113. Sleight, R. B. J. Chromatcgr. 1973, 83, 31. Locke, D. C. J. Chromatogr. Sd. 1974, 72, 433. Popl, M.; Fahnrich, J.; Stejskal, M. J. Phys. Chem. 1976, 74, 537. GLddlngs, J. C.; Kucera, E.: Russell, C. P.; Myers, M. N. J. Phys. Cham. ia68. 72. 4397. Edstrom, T.; Petro, B . A. J. Polym. Sci., Part C : Polym. Symp. 1968, 27, 171. .1
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RECEIVED for review June 15,1990. Accepted September 5, 1990. This research was supported by NSF Grant CHE8719266.
Suppression of Boron Volatilization from a Hydrofluoric Acid Solution Using a Boron-Mannitol Complex Tsuyoshi Ishikawa* and Eizo Nakamura* Institute for Study of the Earth's Interior, Okayama University, Misasa, Tottori 682-02, Japan
Volatlliratlon and mass fractionation of boron during evapor a t h of the hydrofhwk and hyckocMorlc acid sdutions were investigated wRh varying mann#ol/boron ratlos. The degree of vdafllzation and mass fractlonatlon decreases with increasing mannltoVboron mole ratio, and the boron volatlllration Is completely suppressed when the ratio Is more than unlty. These resutts indicate that the flnai stable compound is an equimolar complex of boron and mannltol. The formation of this complex in the acid solutions allows the use of hydrofluorlc and hydrochloric acids for the dissolution of sllicate rock samples and for the subsequent chemical separation of boron from the samples adopting anion-exchange chromatography In F- form.
INTRODUCTION Boron in acidic solutions is easily volatilized during evaporation to dryness relative to neutral or alkaline solutions. This property of boron prohibits the use of acids in the separation of boron from natural samples. Boron in hydrofluoric and hydrochloric acids produces gaseous boron fluoride (BFJ and chloride (BClJ, which have boiling points of -101 and +12.5 "C, respectively. Hence they easily escape from the solutions and cause boron isotopic fractionation even at room temperature. Due to the above reasons, the most widely used techniques to separate boron for isotopic analysis are methyl 0003-2700/90/0362-2612$02.50/0
borate distillation (1-4), ion-exchange chromatography (3, 5-7), and pyrohydrolysis (5). None of these techniques employ acid treatments and/or evaporations without rendering the solution alkaline, usually by using sodium hydroxide to form borax. It is widely known that boric acid reacts with many hydroxy compounds such as alcohol or phenol to form stable complexes. In particular, mannitol has been adopted commonly to the quantitative determination of boron (491,because it forms the mannitol-boric acid complex by the reaction with boric acid and drastically raises the degree of electrolytic dissociation. Furthermore, some previous studies have revealed that mannitol suppressed the volatilization of boron from solutions of water, hydrochloric acid, and nitric acid during evaporation (IO, 11). In these studies the experiments were carried out at pH 3-11 with a small amount of boron (around 1 pg) or with concentrated acids with considerably large amounts of boron (>500 pg). However, the volatilization of boron and resulting isotopic fractionation have not been examined for the boron-mannitol complex in the hydrofluoric acid and concentrated hydrochloric acid solutions with small amounts of boron. In this paper, we report that the boron-mannitol complex traps even small amounts of boron (
