Anal. Chem. 1991, 63, 823-827
Society: Washington, DC, Jones, T. L.; Betowski, L.
1990; pp 48-61. D.; Yinon, J. In Li9u/d Chromtographyl Mess Spectrometry: Apphtkm In Agvfcuthual, pharmaceutical and Envkmmental Chemistry; Brown, M. A., Ed.; ACS Symposium Series 420; American Chemical Society: Washington, DC, 1990; pp 62-74. (7) Brown, M. A.; Kim, I . S.; Sasinos, F. I.; Stephens, R. D. Environ. Scl. Technol. 1990, 24, 1832-1636. (8) Brown, M. A.; Kim, I. S.; Sasinos, F. I.; Stephens, R. D. In Li9uM ChromatographylMass Spectrometry: Application in Agricultural,
(6)
823
pharmaceutical and Envkmmtal Chemistry; Brown, M. A., Ed.; ACS Symposium Series 420 American Chemical Society: Washington, DC, 1990; pp 198-214.
R E C E ~for D review October 15, 1990. Accepted January 7, lggl*M. A*Brown Qatefu1b' financial SUPPofi from the U.S. EPA EMSL, Las Vegas, NV.
Micellar Electrokinetic Capillary Chromatography of Illicit Drug Substances Robert Weinberger*-' Applied Biosystems, Inc., 170 Williams Drive, Ramsey, New Jersey 07446
Ira S.Lurie
Drug Enforcement Administration, Special Testing and Research Laboratory, 7704 Old Springhouse Road, McLean, Virginia 221 02
Micellar electrokinetic capillary chromatography (MECC) was found to give slgnlfkantly greater efficiency, selectivity, peak symmetry, and speed compared to high-performance liquid chromatography (HPLC) for the determination of llllcit drug substances. For a complex mixture consisting of acldlc and neutral Impurities present In an illicit heroin seizure sample, MECC resolved at least twice as many peaks as HPLC. MECC permitted the analysis of heroin and Its basic impurities, the common adulterants phenobarbital and methaqualone, In approximately one-third the analysis time of HPLC with superior resolution. IlHcIt cocaine, and Its basic impurities, were analyzed by MECC without the significant tailing that Is found with reversed-phase liquid chromatography (LC) using bonded-phase columns. Other drugs investigated via MECC include opium alkaloids, amphetamines, hallucinogens, barbiturates, benzodiazepines, and cannablnoids. All of these separations were accompkhed with 25100-cm caplliarles (length to detector) by using a hydroorganic buffer consisting of 85 mM sodium dodecyl sulfate, 8.5 mM phosphate, 8.5 mM borate and 15% acetonitrile at a pH of 8.5. Detection was by uttravioiet (UV) absorption at 210 nm. Due to its speed, high resolving power, and the probability that all compounds must elute at or before t, (micellar aggregate mlgration tlme), MECC is well suited for general drug screening.
INTRODUCTION Illicit drug substances invariably consist of compounds that are polar, thermally degradable, or nonvolatile and thus can be difficult to analyze via gas chromatography (GC). Highperformance liquid chromatography (HPLC) has been employed (1-4) but in general lacks the resolving power of capillary GC (5, 6). Forensic drug substances that are clandestinely manufactured such as heroin, methamphetamine, Current address: CE Technologies, P.O. Box 140,Chappaqua, NY 10514. 0003-2700/91/0363-0823$02.50/0
and fentanyl can be highly complex. For example a heroin sample can consist of heroin, its basic, acidic, and neutral manufacturing impurities, and various adulterants. Natural products such as opium and psilocybin contain exogenous plant and processing matrix impurities. In still other examples, matrix complications such as dyed blotter paper or parsley are characteristic of the dosage forms for LSD and PCP. For a myriad of forensic drug samples HPLC affords adequate resolution but gives fair to poor peak shape for many basic compounds. Many of these drug determinations require gradient elution LC to speed the analysis yet the time of separation can still be quite long ( 4 ) . Micellar electrokinetic capillary chromatography (MECC) is a subclass of capillary electrophoresis (CE). First described by Terabe et al. in 1984 (7, 8), the technique brings reversed-phase and ion-pairing mechanisms to CE as additional tools in developing separations. MECC provides the opportunity to separate in a single run, both neutral and charged molecules in an electroosmotically driven system, an application that was not possible before its inception. A great variety of molecular types are amenable to MECC including phenols (7),chlorinated phenols (9,101,phenylthiohydantoin amino acids ( I l ) , nucleosides and oligonucleotides (12),nucleic acids (13),catechols (14),vitamins (15,16), antibiotics (17), chiral substances (18,19), isotopic substituents (20),peptides (21,22),barbiturates (22),and porphyrins (23). The goal of this paper is to assess the applicability of MECC for the separation of illicit drugs. Comparisons are made with HPLC separations currently being employed in forensic laboratories.
EXPERIMENTAL SECTION Instrumentation. A Model 270A capillary electrophoresis system (Applied Biosystems, Inc., San Jose, CA) was employed for all MECC studies. A chromatography data system (Model 7500 Perkin-Elmer, Norwalk, CT) or ChromJet integrator (SpectraPhysics, San Jose, CA) was used for data handling. A prototype fluorescence detector adaptation to the CE instrument was previously described (23). Four different sized capillaries (Polymicro Technologies, Scottsdale, AZ) were used: 122 cm X 50 gm i.d., 72 cm X 50 pm 0 1991 American Chemical Society
824
ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991 I
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Figure 1. HPLC separation of acidic and neutral Impurities in an illicit heroin sample. Conditions: injection size; 50 pL; column, 11.0 cm X 4.7 mm Partisil 5-ODs-3; phosphate buffer (pH 2.2). Initial conditions: 13% methanol, 8.9% acetonitrile,6.7% tetrahydrofuran (THF), 7 1.1% phosphate buffer. Final conditions: 21.7 % methanol, 14.5% acetonitrile, 10.8% THF, 53% phosphate buffer; gradient, 15min linear gradient, hold for 5 min at final conditions: flow rate, 1.5 mL/min: detector Wavelength, 210 nm.
i.d., 72 cm X 25 pm i.d., and 47 cm X 50 pm i.d.. The lengths to the detector were 100,50, and 25 cm, respectively, and are so specified in the figure captions. The polyimide coating at the detector window was removed by flaming followed by a methanol wash. The capillaries were conditioned by aspirating with 1M sodium hydroxide for 10 min, water for 10 min, and the run buffer for 10 min. A Model Series 4 liquid chromatograph (Perkin-Elmer, Norwalk, CT) equipped with a Model 1040M photodiode array detector (Hewlett Packard, Waldbronn, FRG)and a Partisil5-ODs-3 column (11.0 cm x 4.7 mm i.d.) (Whatman, Clifton, NJ) was used for liquid chromatography. Reagents. Sodium dodecyl sulfate (Aldrich Chemical, Milwaukee, WI) was used as received. Sodium borate, sodium phosphate (monobasic), phosphoric acid, hexylamine, and sodium hydroxide were reagent grade. Deionized water (Milli-Qor Hydro) was used to prepare all buffers. Methanol, acetonitrile, and tetrahydrofuran were LC grade solvents. The drug standards were supplied by the Special Testing and Research Laboratory, Drug Enforcement Administration (McLean, VA). The run buffer was prepared by combining 85 parts of 100 mM SDS/10 mM borate/lO mM phosphate adjusted to pH 8.5 with 15 parts acetonitrile. The HPLC mobile phases were mixed internally from solvent reservoirs containing methanol, acetonitrile, tetrahydrofuran, and either phosphate buffer or phosphate buffer containing hexylamine. The phosphate buffer consisted of a mixture of 3480 mL of water, 120 mL of 2 M sodium hydroxide, and 40.0 mL of phosphoric acid. Procedures. For the HPLC analysis of acidic and neutral impurities in heroin, an acid extract of a 10 pg/mL solution of a heroin seizure sample was dissolved in a starting mobile phase prior to a 50-pL injection. For MECC analysis, the sample at a starting heroin concentration of 200 pg/mL was dissolved in 50% methanol and 50% run buffer. Heroin and cocaine were illicit seizure samples. The cocaine was highly purified and used as is. The working concentration was 1 mg/mL. The heroin sample was approximately 50% pure. Two additional adulterants, methaqualone and phenobarbital, which were not found in this particular sample, were added by blending 100 mg of heroin sample with 10 mg of each of the adulterants. The working concentration was 1mg/mL as heroin. An injection solvent consisting of acetonitrilewater-acetic acid (10:89:1) pH 3.7 was used for the HPLC analysis of heroin and cocaine samples. For MECC analysis, the cocaine sample was dissolved in the run buffer while the heroin sample as well as other drugs of forensic interest were dissolved in run buffer diluted 1:4 with water. Vacuum-assisted injections of 1 s were used for all MECC studies. The individual MECC run parameters, capillary length, and diameter, voltage, detector wavelength,temperature, etc., are noted in each figure caption.
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Figure 2. MEW separation of acidic and neutral heroin impurities using an aqueous buffer. Conditions: capillary, 100 cm X 50 pm i.d.; voltage, 25 kV; temperature, 50 OC; buffer, 100 mM SDSllO mM phosphate/lO mM borate, pH 8.5: detector wavelength, 210 nm. 0.300YI
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lustrated in Figure 3, where the run buffer contained 15% acetonitrile. The peaks are now well distributed throughout the electropherogram making good use of the available peak capacity. Approximately twice as many peaks are resolved via MECC than HPLC. Other modifiers such as THF or methanol did not give as uniform a peak distribution pattern and resolved fewer peaks (data not shown). In this regard, it appears that two adjustable buffer parameters are very powerful. The hydrophobic nature of the pseudophase can be adjusted by varying the micelle concentration or type. This is analogous to varying either the carbon loading or polarity of an HPLC stationary phase. The addition of an organic modifier to the buffer can then alter the partition coefficient much like the modifier in HPLC. While the adjustment of micelle and modifier affects other factors than described above, it appears that optimization of these two buffer additives can yield a uniform peak distribution for complex mixtures. The use of gradient elution might further facilitate employing the available peak capacity (26). The reproducibility is shown in Figure 4 for five repetitive injections of the same sample. For peaks eluting in under 40 min, the relative standard deviation (% RSD) for migration time is about 0.5%,peak area, 4-7%, and peak height, 4-8%. The greatest variations were found for the later eluting peaks that have migration times above 40 min. The % RSD increases to migration time 1.3%, peak area 11.9%, and peak height 13.3% for the peak at 52.4 min. The cause for this variation is not obvious but may be due to inconsistent evaporation of organic modifier from the open run buffer reservoir. The later eluting peaks are more hydrophobic, and their migration times are more sensitive to the organic solvent content of the buffer. The day-to-day and capillary-to-capillary reproducibility is yet to be determined, although initial data indicate that this is satisfactory. In addition, an injection solvent to fully solubilize the sample needs to be developed. The use of an internal standard would be expected to improve the peak area and peak height % RSD. Further resolution of this complex sample can be achieved by using a 25-pm capillary. The improved resolution is a consequence of less Joule heating. Cutting the capillary diameter in half results in a 4-fold reduction in the current passed through the capillary. In addition, the narrower capillary diameter facilitates heat transfer, resulting in a smaller temperature gradient in the cross-sectional area of the capillary. The improved separation is shown in Figure 5. The major disadvantage of the narrower capillary is the loss of sensitivity due to the shorter optical path length.
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Figure 5. MECC separation of acidlc and neutral k d n hnpwities with a 25-pm capillary. C o n d i i s : capi#aty,50 cm X 25 pm i.d.; voltage, 25 k V temperature, 50 'C; buffer, 85 mM SDS/8.5 mM phosphate/8.5 mM borate/l5% acetonitrile, pH 8.5; detector wavelength, 200 nm.
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TIME (mln.) Figure 6. MECC separation of acidic and neutral heroin impurities with fluorescence detection. Conditions: capillary, 50 cm X 50 pm i.d.; voltage, 30 kV; temperature, 60 O C ; buffer, 85 mM SDS/8.5 mM phosphate/8.5 mM borate/l5% acetonitrile, pH 8.5;excitation
wavelength, 257 nm; emission wavelength; 400-nm band-pass filfer (70-nm band-pass).
To determine if the electropherogram could be further simplified, fluorescence detection, optimized for phenanthrene compounds was attempted. This selective separation is shown in Figure 6. Only 14 major peaks are detected. In addition, the signal-to-noise ratios are improved by a factor of 20. Since the organomiceh buffer described above was shown capable of providing high resolution for a complex mixture, separations of a wide variety of illicit drug substances were attempted with the identical buffer system. Heroin. Comparisons between LC (Figure 7) and MECC with a 25-cm capillary (Figure 8) illustrate a superior separation via the latter technique both in terms of overall resolution and speed. The separation is about 3 times faster by MECC. This is remarkable considering the comparison is between a fully optimized LC separation (4, 27, 28) and an MECC separation where little optimization was performed. Possible variables for future optimization studies include
ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991
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Flgure 7. HPLC of bulk heroin, heroin impurities, degradation products, and adulterants. Conditions: column, 11.0 cm X 4.7 mm Partisil 5-ODs-3; mobile phase A, phosphate buffer (23 mM hexylamine, pH 2.2); mobile phase B, methanol; gradient, 5-30% B over 20 min, 30% B for 6 min, 3 0 4 0 % B over 10 min, 80% B for 4 min, then reequilibrate over 5 min; flow rate, 1.5 mL/min; detector wavelength, 210 nm. Key: (a)acetic acid, (b) morphine, (c) 03-monoacetylmorphine, (d) 0'-monoacetylmorphine, (e) acetylcodeine, (f) heroin, (g) phenobarbital, (h) noscapine, (i) papavarine, (j)methaqualone.
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Figure 9. HPLC of cocaine. Condltions: column, 11.0 cm X 4.7 mm
Partisil 5-ODs-3; mobile phase A, phosphate buffer (23 mM hexylamine, pH 2.2); mobile phase B, methanol; gradient, 5-30% B over 20 min, 30% B for 6 min, 30-80% B over 10 min, 80% B for 4 min, then requilibrate over 5 min; flow rate, 1.5 mL/min; detector wavelength, 228 nm. Key: (a) cocaine, (b) cis-cinnamoylcocaine, (c) trans -cinnamoylcocaine. 0.454-
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Flgure 8. MECC of bulk heroin, heroin impurities, degradation products, and adulterants. Conditions: capillary, 25 cm X 50 pm i.d.; voltage, 20 kV; temperature, 40 O C ; buffer, 85 mM SDSl8.5 mM phosphate/8.5 mM borate/l5% acetonitrile, pH 8.5; detector wavelength, 210 nm; Key: as per Figure 7.
micellar type and concentration, buffer type, buffer concentration and pH, organic modifier type, concentration, and temperature. The major disadvantage of MECC is concentration sensitivity, where HPLC has an 80-fold advantage. For bulk drug analysis, the limit of detection in CE is sufficiently low to detect impurities in heroin down to levels of 0.2% relative to heroin. Lower levels of detection might be possible by assuming that sample sizes larger than 1.0 mg/mL heroin will still give satisfactory electrophoretic performance. It is of interest to note the considerable selectivity differences between HPLC and MECC. Among these changes are reversals in the elution order of 03/06-monoacetylmorphine,heroin/acetylcodeine, and noscapine/papaverine. It is also significant that unlike HPLC the sample which consists of approximately 10% phenobarbital and 10% methaqualone completely dissolves in the MECC injection solvent. Cocaine. The most striking difference between the HPLC run (Figure 9) and MECC (Figure 10) is the peak symmetry of cocaine. The low surface area of the fused-silica capillary precludes the effect of the peak tailing commonly found in LC. Shorter run times with superior resolution are found with MECC. Similar selectivities are observed for both techniques except for benzoylecgonine,which via HPLC elutes just before cocaine. By MECC, benzoylecgonine (which contains a car-
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Figure 10. MECC of cocaine. Conditions: capillary, 25 cm X 50 pm i.d.; voltage, 20 kV; temperature, 40 O C ; buffer, 85 mM SDS/8.5 mM phosphate/8.5 mM borate115 % acetonitrile, pH 8.5; detector wave-
length, 233 nm. Key: (a) cocaine, (b) cis-cinnamoylcocaine, (c) trans -cinnamoylcocaine, (d) benzoylecgonine.
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Figure 11. MECC forensic drug screen. Conditions: capillary, 25 cm X 50 pm i.d.; voltage, 20 kV; temperature, 40 O C ; buffer, 85 mM
SDSl8.5 mM phosphate/8.5 mM borate/l5% acetonitrile, pH 8.5; detector wavelength, 210 nm; sample concentration, 250 pg/mL of each drug. Key: (a) psilocybin, (b) morphine, (c) phenobarbital, (d) psilocin, (e)codeine, (f) methaqualone, (g)LSD, (h) heroin, (i) ampha tamine, (j)librium, (k) cocaine, (I) methamphetamine, (m) lorazepam, (n) diazapam, ( 0 ) fentanyl. (p) PCP, (4)cannabidiol, (r) Ae-THC.
boxyl group) is negatively charged at the pH of the mobile phase. Because of electrostatic repulsion from the negatively
ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991
Table I. Migration Times of Compounds of Forensic Interest Relative t o Heroin compd
re1 m i g r a t i o n t i m e
benzoylecgonine psilocybin morphine dilaudid phenobarbital psilocin OB-monoacetylmorphine 03-monoacetylmorphine codeine methaqualone m esc a1ine
0.38 0.46 0.51 0.54 0.58 0.62 0.66 0.72 0.74 0.78 0.92 0.93 1.00 1.00 (8.91 min) 1.14 1.24 1.27
LSD LAMPA heroin acetylcodeine papaverine
MDA amphetamine
1.28
librium
1.34 1.49 1.55 1.56 1.56 1.69 1.76 1.91
cocaine
MDMA methamphetamine phentermine noscapine cis-cinnamoylcocaine lorazepam diazepam trans-cinnamoylcocaine fentanyl flurazepam
PCP pyrene" cannabidiol cannabinol
A'-THC
" A d d e d as a p o t e n t i a l t,,
2.21
2.24 2.36 3.07 3.28 3.72 3.90 4.25 4.25
marker. See t e x t f o r discussion.
charged micelle, the compound elutes near to. Not all carboxyl compounds exhibit this problem. In many cases, the hydrophobic interaction with the micelle is sufficient to overcome the electrostatic repulsion. Due to tailing, benzoylecgonine was not observed in the HPLC chromatogram. Applicability to General Forensic Analysis. In order to test the applicability of MECC to other forensic drug substances, the electrophoretic performance characteristics (migration time, peak shape, and stability) were examined for a broad spectrum of compounds. The separations were accomplished by using the same electrophoretic system as was described previously for the analysis of heroin and cocaine. All compounds exhibited excellent peak shape via MECC with no apparent breakdown in the buffer system employed. A representative electropherogram of these drugs as well as others previously discussed is shown in Figure 11. The relative migration time data of these compounds plus selected drug impurities is shown in Table I. The MECC system is excellent considering that only modest optimization schemes were applied. It is applicable to a large variety of forensic samples, especially those which are difficult to analyze via GC such as phenethylamines, benzodiazapines, ergot alkaloids,
827
psilocybin, and PCP. It should be noted that the excellent separation between LSD and LAMPA (Table I) is superior to anything attainable via HPLC (29). From the data presented, and the property of MECC that all compounds must elute at or before t,,, it appears that this separation technique is excellent for drug screening. Pyrene, which is practically insoluble in the aqueous mobile phase, was used as a probe for .t, However the data in Table I indicate the cannabinoids elute later than pyrene. The amount of acetonitrile present in the buffer provides sufficient solvating power such that pyrene has some solubility in the bulk solution. The same probably holds true for Sudan B, another t,, marker. The method of Bushey et al. (20) is probably suitable for determining t,, in this system.
ACKNOWLEDGMENT We gratefully acknowledge Sam Cooper for providing technical assistance. LITERATURE CITED (1) Wittwer. J. D. I n High-ferfmnce LiquM Chmsfcgmphy; Lurie, I. S., Wittwer, J. D., Eds.; Marcel Dekker: New York, 1983; pp 53-160. (2) Lurie, I.S. I n High-Perfwmance Liquid Chromsfogi?phy; Lurk, I . S., Wittwer, J. D., Eds.; Marcel Dekker: New Ycfk, 1983; pp 161-264. (3) Krull, I.S.; Lurie, I.S. I n Forensic Scknce; Davles. G., Ed.; Amecican Chemical Society: Washington, DC, 1986; pp 153-200. (4) Lwie, I.S. LC-GC 1990, 8 , 454-466. (5) Verpoorte R.; Svendsen A. B. Chromatography of Alkaldds, Part 8 : gas -liquid chrom8tography and high - p e r f m n c e liquid ahromafogra phy ; Journal of Chromatography Library; Elsevier: Amsterdam, 1984; Vol. 238. (6) Lurie, I . S.; Moore, J. M.; Cooper, D. A. I n Ulhefrace Analysis of Pharmaceuticals and Other Compounds of Interesf; Ahuja, S.. Ed.; John Wiley and Sons: New York, 1986; pp 319-352. (7) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A,; Ando, T. Anal. Chem. 1984, 5 6 , 111-113. (8) Terabe, S.; Otsuka. K.; Ando, T. Anal. Chem. 1985, 5 7 , 834-841. (9) Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1985, 348. 39-47. (10) Otsuka, K.; Terabe, S.; Ando, T. J. chromafog. 1987, 396, 350-354. 1985, 332, 219-226. (11) Otsuka,K.; Terabe, S.; Ando, T. J. chroma-. (12) Cohen, A. S.; Terabe, S.; Smith, J. A.; Karger, 9. L. Anal. Chem. 1987, 59, 1021-1027. (13) Row, K. H.; @lest, W. H.; Maskarinec, M. P. J . Chromsfogr. 1987, 409, 193-203. (14) Walllngford, R. A.; Ewing, A. G. J . Chromafogr.1988, 447, 299-309. (15) Fujiwara, S.; Iwase, S.; Honda, S. J . Chromafogr. 1988, 447, 133- 140. (16) Nishi, H.; Tsumagari. N.; Kakimoto. T; Terabe, S. J. Chromafogr. 1989, 465. 331-342. (17) Nishi, H.; Tsumagarl. N.; Kaklmoto, T; Terabe, S. J . Chromstogr. 1989, 477, 259-270. (18) Dobashi, A.; Ono, T.; Hara, S.; Yamaguchi, J. Anal. Chem. 1989. 67, 1984-1986. (19) Nishi, H.; Fukuyama, T.; Matsuo, M; Terabe, S. J. Mlcrocol. Sep. 1989, 7. 234-241. (20) Bushey. M. M.; Jorgenson. J. W. Anal. Chem. 1989, 81, 491-493. (21) Swedberg, S. A. J. Chromafogr. 1990. 503, 449-452. (22) Wainwright, A. J . Microcol. Sep. 1990, 2 , 166-175. (23) Weinberger, R.; Sapp, E.; Moring, S. J . Chromafogr. 1990, 576, 27 1-265. (24) Lurie, I.S.; Cooper, D. C. Unpublished results. (25) Grose, J.; Balchunas, A. T.; Swaile, D. F.; Sepaniak, M. J. HRC & CC, J . High Resoluf. Chromatogr. Chromatogr. Common. 1988, 7 7 , 554-559. (26) Balchunas, A. T.; Sepaniak, M. J. Anal. Chem. 1988, 60, 617-621. (27) L u r k I . S.; Carr S. M. J. Liq. Chromafogr. 1988, 9 , 2485-2509. (28) Lurle, I. S.; McGuinness, K. J . Llq. Chromefogr. 1987, 70. 2189-2204. (29) DeRuiter J.; Noggle, F. T.; Clark, C. R. J . Liq. Chromatogr. 1987, 70, 3481-3488.
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RECEIVED for review October 23,1990. Accepted January 29, 1991.
