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(2) Hovingh, M. E.; Thompson, G. H.; Myers, M. N.; Giddings, J. c. Anal. Chem. 1970, 42, 195. Giddings, J. C.; Yang, F. J. F.; Myers, M. N. Sep. Sci. 1975, 10, 133. CaMwell, K. D.; Nguyen, T. T.; Glddlngs, J. C.; Mazzone, H. M. J. Virol.
Methods l980, 1 , 24 1. Kirkland, J. J.; Yau, W. W.; Doerner, W. A.; Grant, J. W. Anal. Chem. 1980, 52, 1944. Caldwell, K. D.; Karaiskakis, G.; Glddlngs, J. C. Colloids Surf. 1981, 3 , 233. Caldwell, K. D.; Karaiskakis, G.; Myers, M. N.; Giddlngs, J. C. J. Pharm. Sci. 1981, 70, 1350. Giddlngs, J. C.; Karaiskakis, G.; Caldwell, K. D. Sep. Scl. Techno/. 1981, 16, 607. Karaiskakis, G.; Myers, M. N.; Caldwell, K. D.; Giddings, J. C. Anal. Chem. 1981, 53, 1314. Kirkland, J. J.; Rementer, S. W.; Yau, W. W. Anal. Chem. 1981, 53, 1730. Glddlngs, J. C.; Karaiskakis, G.; Caidweli, K. D.; Myers, M. N. J. Colloid Interface Sci. 1983, 92, 66. Yana. F.-S.:Caldwell. K. D.: Giddlnos. J. C. J. Colloid Interface Sci. 1985, 92, 81. Yang, F.4.; Caldwell, K. D.; Myers, M. N.; Giddings, J. C. J. Colloid Interface Sci. 1983, 93, 115. Kirkland, J. J.; Yau, W. W. Anal. Chem. 1983, 5 5 , 2165.
.
(15) Kirkland, J. J.; Dllks, Jr., C. H.; Yau, W. W. J. Chromatogr. 1983, 255,255. Caldwell, K. D., University of Utah, personal communication, 1985. Davis, J. M.; Giddings, J. C. J. Phys. Chem. 1985, 89, 3398. Davis, J. M., Ph.D. Dissertation, University of Utah, Salt Lake City, UT, 1985. Benton, G. S.;Bayer, D. J. Fluid Mech. 1966, 26, 69. Dean, W. R. Philos. Mag. 1928, V , 673. Dean, W. R. Philos. Mag. 1927, I V , 208. McConalogue, D. J.; Srivastava, R. S. Proc. R . SOC. Ser. A 1968, 303, 37. Bird, R. B.; Stewart, W. E.; Lightfoot, E. N. "Transport Phenomena"; Wiley: New York, 1960. Kreyszlg, E. "Advanced Engineering Mathematics"; Wiiey: New York, 1979. Mathews, J.; Walker, R. L. "Mathematical Methods of Physics"; Benjamln/Cummings: Menlo Park, CA, 1970. Giddings, J. C. J. Cbromtogr. 1960, 4, 11.
RECEIVED for review June 17,1985. Accepted August 16,1985. This work was supported by National Science Foundation Grant No. CHE-8218503.
Xenon, a Unique Mobile Phase for Supercritical Fluid Chromatography Scott B. French and Milos Novotny*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Due to Its optlcal transparency, supercritlcal xenon is shown to be a hlghly suitable mobile phase for supercrltlcal fluld chromatography/FourEer transform Infrared spectrometry. Its satisfactory chromatographic propertles were also verlfled by comparlng the solute diffuslon coefficients and retentlon data of xenon and carbon dloxlde. Potentlal uses of supercrltlcal xenon In addltlonal chromatographic measurements are Indlcated.
A recent renewal of interest in supercritical fluid chromatography (SFC) is primarily due to improved ways to utilize a solute's favorable (compared to liquids) mass transfer properties and certain advantages of supercritical fluids in detection procedures. While the use of small-bore open tubular columns (I,2) is primarily pursued for reasons of high column efficiencies and, consequently, superior component resolution, the unique properties of some supercritical fluids have also been exploited in the combinations of SFC with mass spectrometry (3, 4 ) , flame detection (5, 6), and Fourier transform infrared (FTIR) spectrometry ( 7 , 8 ) . Yet another, relatively unexplored advantage of small-bore columns in SFC is the possibility of using "exotic" mobile phases for the sake of improved separation and/or detection. Xenon occupies a unique position among the noble gases due to its convenient critical parameters (T,= 289.8 K, P, = 58.0 atm). Although its critical density is relatively high (p, = 1.105 g/mL), for being a monatomic gas, xenon could offer acceptable transport properties for chromatographic measurements. It has an appreciable polarizability (9),permitting relatively large and moderately polar molecules to be dissolved in liquid xenon (10-12). The most intriguing property of xenon is, however, its spectral transparency, virtually, from the vacuum UV up to the NMR region (11). Consequently, some of the most unique spectral studies could be carried out in the medium.
We have recently suggested (8) supercritical xenon as a potentially convenient mobile phase in SFC/FTIR spectrometry investigations. Although xenon is an expensive mobile phase, the flow rates on the order of microliters per minute are typical of the capillary SFC systems. Preliminary results reported in this communication verify our assumption (8) of the suitability of xenon in capillary SFC/FTIR spectrometry. In addition, chromatographic properties of xenon (solute diffusivity and relative retention) were briefly compared to those of the commonly used carbon dioxide.
EXPERIMENTAL SECTION Capillary SFC/FTIR Spectrometry. The experimental setup used was similar to that described in our previous work (8). A Brownlee Labs Micropump (Brownlee Labs, Santa Clara, CA) was substituted for the previously used larger syringe pump (8). The Brownlee pump was ideally suited for the experiments with xenon because of the small volume (20 mL total) of its syringes. It allowed an external liquefaction of xenon (research grade, 99.995%,Air Products, Allentown, PA),which was first transferred from a 1500-mL cylinder into a 7.6-mL loop (8 f t long, 0.2 mm i.d.) external to the pump. This loop was cooled by immersing it in liquid nitrogen, thus condensing the xenon into the loop. The loop was then shut off from the cylinder and opened to the pump and allowed to warm to room temperature. The pump syringes were wrapped with plastic tubing through which ice water flowed. This cooling provided a more stable pressure during pumping. While the samples were initially introduced into a 0.2-pL injection loop (a Valco valve no. EXI4U.2, Valco Instruments, Houston, TX), a further 2:l splitting was accomplished through a splitting device constructed by SGE, Inc., Austin, TX. The column used was a 17 m X 150 pm i.d. fused silica capillary (SGE, Inc., Austin, TX), coated with a 1.0 fim thick film of SE-54 silicone phase, which was immobilized by the use of azo-tert-butane (13). Detection was based solely on the Gram-Schmidt reconstruction of interferometric data collected and processed through an IBM/85 FTIR spectrometer (IBM Instruments, Danbury, CT). Retention measurements comparing xenon and carbon dioxide (Coleman grade, 99.99%, Air Products, Allentown, PA) were performed at the same reduced density of 1.10. Xenon mea-
0003-2700/86/0358-0164$01.50/0@ 1985 American Chemical Society
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Figure 1. Gram-Schmidt real-time chromatogram of test mixture (isobaric 1059 psi, 27 “ C , p = 1.21 g/mL): (1) benzaldehyde (5 pg), (2) 2,6-diitert-butylphenol(5 Kg), (3) phenol (5 pg), (4) 2-naphthaldehyde (5 pg), (5) 2-naphthol (5 pg), and (6) 9-anthraldehyde (10 1.19).
surements were performed at 27 “C and a 1059 psi inlet pressure (density of 1.21 g/cm3) and a linear velocity of 3.5 cm/s. Carbon dioxide at 41 “C and 1300 psi had a density of 0.51 g/cm3; the mobile-phase yelocity was 4.7 cm/s. In both cases, a sample mixture consisting of model aldehydes and phenols (between 5 and 10 pg each) was, introduced into the column. Band Dispersion Measurements. An established chromatographic band-broadening procedure (14) was used for evaluation of diffusion coefficients. On-column detection (15)was performed by using a Kratos 950 spectrofluorometric detector (KratosSchoeffel Instruments, Westwood, NJ), while the rest of the chromatographic setup was similar to that mentioned above. When phenanthrene was used as the solute to determine its diffusivity, supercritical xenon appeared to quench phenathrene fluorescence,and a Jasco UVIDEC 100-11UV absorbance detector (Jasco, Inc., Easton, MD) had to be substituted for the spectrofluorometer. The first and second moments of the phenanthrene peak were calculated by the use of a BASIC program written in our laboratory for the IBM personal computer (IBM Corp., Boca Raton, FL); diffusion coefficients were evaluated as described previously by our group (16). For the diffusion constants determined in this work, a 10 m X 200 pm i.d. uncoated fused silica capillary was used. A 4 1 split ratio was maintained throughout all measurements. Both mobile phases were operated at the same reduced density of 1.49.
RESULTS AND DISCUSSION The results obtained in this work verify our earlier assumption (8)that supercritical xenon provides a nearly ideal mobile phase for an on-line acquisition of IR spectra in the SFC/FTIR spectrometry combination. Figure 1demonstrates a Gram-Schmidt chromatogram obtained from a model mixture of IR-absorbing compounds together with a representative spectrum of one of these components. While the spectral features are solely due to the sample molecules, the overall spectral sensitivity was not greater than our previously obtained figures (8) with carbon dioxide as the mobile phase. We believe this is attributable to greater pressure fluctuations with the present pump as compared to the large syringe pump used earlier. Yet, the Brownlee micropump has been quite unique among the commercially available pressure sources in that it provided US with the external loop capability convenient for xenon handling. At any rate, this minor problem with pressure regulation of supercritical xenon is of a technological
165
rather than fundamental nature, and it could potentially be overcome by a better design. Because xenon is more transparent than the previously used carbon dioxide, a redesign of the present high-pressure cell (8)to provide a greater path length may be in order. If sensitivity increase is realized, narrower columns may be feasible together with smaller samples. Obviously, as seen in Figure 1, some column overloading now causes higher than expected plate heights. Spectroscopic properties aside, the next most crucial question has been related to chromatographic properties of supercritical xenon. Since the mass-transfer kinetics in chromatographic columns (and, consequently, their efficiency) are diffusion controlled, we have briefly compared the diffusion coefficients of phenanthrene in both xenon and carbon dioxide at the identical value of reduced density. The plate height w. linear velocity measurements (16)yielded the values cm2/s (f10.0%) and 1.26 X lo4 cmz/s (*5.2%) of 1.28 X for the diffusion coefficients in xenon and carbon dioxide, respectively. While no literature data are available for xenon, the value in carbon dioxide agrees favorably with the results of Lauer et al. (17) and Springston (18). Although the closeness of these values may be somewhat surprising in view of the greater absolute density of xenon (1.64 g/cm3) over carbon dioxide (0.70 g/cm3), in our experiments, the explanation lies, most likely, in the monatomic nature of xenon. These values are also in rough agreement with our tentative observation that the capillary SFC separations obtained with both mobile phases were roughly similar. Thus, from the kinetic point of view, supercritical xenon appears to be an adequate mobile phase. Yet another important question concerns the solubility of various compounds in supercritical xenon. Here, the availability of nonchromatographic data on the solubilities in liquid xenon (10-12) permits some projections to be made. Due to its high polarizability, liquid xenon was reported to readily dissolve various polymers (10, I I ) , “some biological molecules” (11), and picric acid, but not ions (12). The only literature information on the use of xenon as a supercritical solvent is a recent report by Krukonis et al. (9), whose careful P-T measurements indicate that it solubilizes naphthalene to a greater degree than supercritical alkanes, ethylene, and carbon dioxide. The values of the Hildebrand solubility parameter, proposed by Giddings et al. (19)as an approximate means of comparing the eluting power of various supercritical fluids, lie close to each other for xenon and carbon dioxide (in the lower middle part of the scale (19)). Recently, supercritical carbon dioxide was found to successfully migrate various oligomers (1,20) with molecular weights of up to several thousand. If supercritical xenon was equally effective, or more effective as the nonchromatographic measurements by Everett and Stageman (10) and Krukonis et al. (9) suggest, the scope of SFC measurements could be significantly enhanced. In order to indicate trends in chromatographic migration and selectivity, several model solutes were chromatographed in supercritical xenon and carbon dioxide, both adjusted to the same reduced density of 1.10. Table I provides a comparison of the relative retention values for both mobile phases. Differences in selectivity are indicated by these preliminary data. Although xenon is, admittedly, an expensive mobile phase, the very low flow rates that are typical of capillary SFC make its use feasible. Alternatively, attempts could be made to recycle this mobile phase for a greater number of measurements. The preliminary data reported here clearly show that supercritical xenon is a useful mobile phase for SFC/FTIR spectrometry. In addition, its fascinating physical properties
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Anal. Chem. 1986, 58,166-170
66-99-9; 2-naphthol, 135-19-3;9-anthraldehyde, 642-31-9.
Table I. Trends in Chromatographic Retention in Supercritical Xenon and Carbon Dioxide solute phenol 2,6-di-tert-butylphenol 2-naphthaldehyde 2-naphthol 9-anthraldehyde
LITERATURE CITED Novotny, M.; Sprlngston, S. R.; Peaden, P. A,; Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1981, 53,407A-414A. Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1984, 56,A618-A628. Smith, R. D.; Felix, W. D.; Fjeldsted, J. C.; Lee, M. L. Anal. Chem.
relative retention, a2ln in xenonb in carbon dioxide 2.60 1.94 4.39 9.58 12.39
1.52 3.59 4.79 7.11
17.30
= ( t ~ -2 t N ) / ( t R 1 - t N ) , where t N = retention time of a nonretained peak (solvent front) and tR1 = retention time of benzaldehyde. bConditions are as follows for xenon and COP, respecaa21
tively: inlet pressure, 1059 psi and 1300 psi; column temperature, 27 "C and 40 O C ; density, 1.21 g/cm3 and 0.51 g/cm3; and linear velocity, 3.5 cm/s and 4.7 cm/s. are likely to invite further explorations in coupling the chromatographic systems to other spectroscopic techniques and, possibly, ionization detectors.
ACKNOWLEDGMENT Thanks are due to Robert Brownlee (Brownlee Laboratories, Santa Clara, CA) for the generous gift of the micropump and to Ken Mahler (SGE, Inc., Austin, TX) for the inlet splitter. The continuous interest of Sharon Smith and Dennis Gerson (IBM Instruments, Danbury, CT) in our work is greatly appreciated. This study was supported by Grant No. CHE 82-00034 from the National Science Foundation and Grant No. N14-82-K-0561 from the Office of Naval Research. Registry No. Xe, 7440-63-3;benzaldehyde, 100-52-7;2,6-ditert-butylphenol, 128-39-2;phenol, 108-95-2;2-naphthaldehyde,
1982,5 4 , 18a3-ia85. Smith, R. D.; Kailnoski, H. T.; Udseth, H. R.; Wright, 6. W. Anal. Chem. 1984, 56, 2476-2480. Fjeldsted, J. C.; Kong, R . C.; Lee, M. L. J . Chromatogr. 1983, 279, 449-455. Chester, T. L. J . Chromatogr. 1984, 299,424-431. Shafer, K. H.; Grifflths, P. R. Anal. Chem. 1983, 55,1939-1942. Olesik, S.V.; French, S. B.; Novotny, M. Chromatographla 1984, 78, 489-495. Krukonis, V. J.; Hugh, M. A.; Seckner, A. J. J . Phys. Chem. 1984, 88,2687-2689. Everett, D. H.; Stageman, J. F. faraday Discuss. Chem. Soc. 1978, 65,230-241. Rentzepls, P. M.; Douglas, D. C. Nature (London) 1981, 293, 165-166. Marshall, D. B.; Strohbusch, F.; Eyring, E. M. J . Chem. Eng. Data 1981,26, 333-334. Rlchter, 6.E.; Kuei, J. C.; Park, N. J.; Crowley, S. J.; Bradshaw, J. S.; Lee, M. L. HRC CC,J . Hlgh Resolut. Chromatogr. Chromatogr. Commun. 1883,4,371-374. Giddings, J. C.; Seager, S. L. J . Chem. Phys. 1960,33, 1579-1580. Peaden, P. A.; Fjeldsted, J. C.; Lee, M. L.; Springston, S.R.; Novotny, M. Anal. Chem. 1982, 5 4 , 1090-1093. Springston, S. R.; Novotny, M. Anal. Chem. 1984, 56, 1762-1766. Lauer, H. H.; McManlgill, D.; Board, R . D. Anal. Chem. 1983, 55, 1370-1375. Sprlngston, S. R. Doctoral Thesis, Department of Chemistry, Indiana University, Bloomington, IN, 1984. Giddings, J. C.; Myers, M. N.; McLaren, L.; Keller, R. A. Science 1968, 762,67-73. Chester, T. L., Procter & Gamble Co., Cincinnati, OH, personal communication, June 1985.
RECEIVED for review July 25, 1985. Accepted September 4, 1985.
Capillary Zone Electrophoresis of Proteins in Untreated Fused Silica Tubing Henk H. Lauer* and Douglass McManigill Hewlett-Packard Laboratories, Chemical Systems Department, 1651 Page Mill Road, Palo Alto, California 94304
Capillary zone electrophoresis (CZE) of proteins In untreated fused silica caplllarles Is possible If Coulombic repulsion between proteins and the caplllary wail can overcome adsorptlon tendencies. Thls Couiomblc repulsion can be achleved elther by ralslng the pH of the buffer solution above the isoelectric polnt values of the sample protelns or by dynamlcally modlfylng the lnterfaclal double layer between the wall and the bulk sdutlon wtth selected ions. Separations of model protelns In the pH range 8-11 are shown and discussed. The low dispersion of sample zones In CZE is demonstrated with a theoretlcal plate number approaching 1 X 10'. A pH step gradlent and a slowdown of the ubiquttous electroosmotic flow are observed to Improve peak resolutlon.
Amino acids and small peptides are well-separated by CZE in untreated glass or fused silica capillaries (1, 2). When applied to large biomolecules like proteins, however, serious adsorption problems can prohibit proper separations (2).
These can be eliminated if the capillary wall is properly coated with a hydrophilic, nonionic phase, such as glycerol propylsilyl ( 1 , 2 ) . Although chemical deactivation of glass and fused silica walls has shown major breakthroughs in capillary gas chromatography, appropriate surface deactivations of capillaries for protein separations by liquid chromatography or CZE are still in an investigative phase. Two other methods to avoid wall adsorption of proteins in solution are based on Coulombic repulsions of species and surface. One utilizes the variation of solution pH relative to the isoelectric point (PI) of a protein ( 3 ) to change its net charge. The other method tries to change the charge characteristics of the wall by coating it dynamically with an ionizable phase ( 4 ) . In the first method a change in solution p H does not necessarily change the sign of charge on the wall. Silica gel in contact with aqueous solutions has a negative charge a t pH values above 2 (5-7). A protein bears a net negative charge if the p H of its solution is higher than its isoelectric point. It is therefore possible to adjust the p H of solutions to values
0 1985 American Chemical Society 0003-2700/86/0358-0166$01.50/0
