CAPILLARY ELECTROPHORESIS: TECHNIQUE AND APPLICATION By Vanessa Hobbs CAPILLARY ELECTROPHORESIS DEVELOPMENT OF CAPILLARY ELECTROPHORESIS 1803 1886 1937 1967 1970 1979 1981 1983 1988 F.F. Reuss Clay Slab O. Lodge Zone Electrophoresis A. Tiselius Electrophoretic Cell S. Hjerten Rotating tubes (300 um) V. Neuhoff PAG filled tubes Mikkers, Everaerts, Verheggen FZE Jorgenson and Lukags 75 um cap. Micellar Electrokinetic Chrom. Commercial introduction ELECTROOSMOTIC FLOW http://www.electrokinetic.co.uk/images/tech1.gif MOVEMENT OF ANALYTE Analyte ν=µE ν = velocity µ = electrophoretic mobility E = Electric field Electrophoretic mobility µ = q/[6πηr] q = charge η = solution viscosity r = radius Electroosmotic flow νEOF = [ε/4πη]ζE ε = dielectric Constant ζ = Zeta potential Flow of migration ν = [(μEO + μe)V]/L V = potential L = length of capillary Forensic Science International 77 (1996) 211 - 229 INJECTION OF SAMPLE Current Analytical Chemistry. 2005, 1 http://www.calstatela.edu/dept/chem/gomez/pubs-pdf/flow-injection.pdf INJECTION OF SAMPLE Injection is difficult due to sample size Electrokinetic Injection Differs by analyte Hydrodynamic Many parameters Anal. Chem., 1997, 69 (15), pp 2952–2954 INJECTION OF SAMPLES Anal. Chem.2001, 73,1974-1978 INJECTION OF SAMPLE Current Analytical Chemistry. 2005, 1 http://www.calstatela.edu/dept/chem/gomez/pubs-pdf/flow-injection.pdf CAPILLARY ZONE ELECTROPHORESIS Separated by mass to charge ratio Based on Electroosmotic Flow Detectors: UV Detector – Beer’s Law Laser Fluorescence – Deriv. MS - electrospray Chemiluminescence Diode Array Detector Indirect Refractive Index Compare with HPLC and GC Neutral Compounds Chiral Compounds INCREASING PATH LENGTH http://www.chem.agilent.com/Library/technicaloverviews/Public/5989- MICELLAR ELECTROKINETIC CHROMATOGRAPHY UV Neutral compounds Comprable to HPLC Capillary Electrochromatography Packed column with no pressure applied, only electroosmotic pressure. CAPILLARY GEL ELECTROPHORESIS Crosslinked vs. non crosslinked DNA sequencing Protein analysis Chirality possible EOF less desirable CAPILLARY GEL ELECTROPHORESIS http://www1.qiagen.com/Images/Catalog/2134.jpg CAPILLARY ISOELECTRIC FOCUSING http://www.targetdiscovery.com/~tdidocs/App_Note_5_200405.pdf CAPILLARY ISOELECTRIC FOCUSING pH gradient Sample focusing and detection Movement of gradient towards the detector Zone broadening Not useful for chiral compounds CAPILLARY ISOTACHOPHORESIS Two buffers form ionic zones Anions and Cations seperately Neutral compounds Used for concentration EOF less desirable APPLICATIONS CE and Analysis of Illicit Drugs HPLC Heroin Analysis HPLC Analysis of Heroin (SPE) Fig. 2. (a) Representative total ion chromatograms of all quantifiable analytes spiked at LLQ level in human plasma (5 ng/mL). The intensity of the deuterated analytes was above 2500 [cps]. (b) Representative total ion chromatograms of random chosen patient’ plasma sample. (c) Total ion chromatogram of a plasma sample of a non-drug using volunteer. (A) M3G and M3G-d3; (B) morphine and morphine-d3; (C) M6G; (D) 6MAM; (E) heroin and heroin-d6; (F) = methadone and methadoned9; (G) EMDP; (H) cocaine; (I) benzoylecgonine. DIODE ARRAY AND TRIPLE MS 5 ng/ml FIRST PUBLISHED ANALYSIS OF ILLICIT DRUGS FIRST PUBLISHED ANALYSIS OF ILLICIT DRUGS Relative Standard Deviation Migration 0.5% Peak Area 4 – 8% Twice as many peaks observed in Heroin analysis with MEKC HPLC more sensitive Smaller capillary did not help analysis with MEKC LSD Analysis with Laser Fluorescence LSD Analysis with Laser Fluorescence (0.2 ng/ml) METHAMPHETAMINE ANALYSIS 50 μm Capillary with length of 40 cm UV Detector Electrophoresis 2006, 27, 4711–4716 METHAMPHETAMINE ANALYSIS 50 mL of 0.1 mol/L NaOH was added to 100 mL of urine mixing by a vortex mixer for about 1 min. 1000 mL of ethyl acetate was pipetted in continued mixing for 30 min. centrifuged for 5 min at 5000 rpm. the upper organic layer was carefully transferred to another polyethylene tube, 20 mL of 1.0 mol/L HCl was added evaporated to dryness at 60oC residues were then dissolved in 100 mL of doubly distilled water Electrophoresis 2008, 29, 3999–4007 METHAMPHETAMINE ANALYSIS 50 mL of 0.1 mol/L NaOH was added to 100 mL of urine mixing by a vortex mixer for about 1 min. 1000 mL of ethyl acetate was pipetted in continued mixing for 30 min. centrifuged for 5 min at 5000 rpm. the upper organic layer was carefully transferred to another polyethylene tube, 20 mL of 1.0 mol/L HCl was added evaporated to dryness at 60oC residues were then dissolved in 100 mL of doubly distilled water Electrophoresis 2008, 29, 3999–4007 ANALYSIS (LIQUID LIQUID EXTRACTION) Electrophoresis 2008, 29, 4078–4087 ANALYSIS OF HAIR Electrophoresis 1998, 19, 42-50 ANALYSIS OF BLOOD Biomed. Chromatogr. 19: 737–742 (2005) REFERENCES Garvin, D and Ajuhar, S. Handbook of Isoelectric Focusing and Proteomics. 7th Volume. 1995: pgs 14-15, 181 191. Tiselius, A. Electrophoresis of Serum Globulin. Biochem. J. 1937, 31: 313 – 317. Van Oss, C. Interfacial Forces in Aqueous Media. 1994: pg 145. Hjerten, S. Free Zone Electrophoresis. Chromatogr. Rev. 1937, 9: 122 – 219. Neuhoff, V., Wolf-Bernhard, S., and Sternbach, H. Micro-analysis of Pure Deoxyribonucleic-dependant Ribonucleic Polymerase from E. Coli. Biochem. J. 1970, 117: 623 – 631. Camilleri, P. Capillary Electrophoresis: Theory and Practice. 2nd Edition. CRC Press. 1997: pgs 5-6. Jorgenson, and Lukags. Zone Electrophoresis in Open Tubular Glass Capillaries. Anal. Chem. 1981, 53: 1298 – 1302 Otto, M., Valcarcel, M. and Widmer, H. M. Analytical Chemistry. 2nd edition. Wiley. 2004: pgs 616-618. Wallingberg, R. and Ewing, A. Capillary Zone Electrophoresis with Electrochemical Detection. Anal. Chem. 1987, 59(14): 1762 – 1766. Rejtar, T. et. al. Off line coupling of High Resolution Capillary Electrophoresis to MALDI-TOF and TOF/TOF MS. J Proteome Res. 2002, 1(2): 171 - 179 Hashimoto, M. et. al. Microchip Capillary Electrophoresis using on-line chemiluminesce. J. Chrom. A. 2000, 867:271 – 279. Heiger, D. et. al. Diode Array Detection in Capillary Electrophoresis. Electrophoresis. 2005, 15:1234 – 1247. Paez, X. and Hernandez, L. Biomedical Applications of Capillary Electrophoresis with Laser Induced Fluorescence Detection. Biopharm. Drug Dispos. 2001, 22:273 – 289. Terabe, S. et. al. Electrokinetic seperations with Micellar Solutions and Open Tubular Capillaries. Anal. Chem. 1984, 56: 111 – 113. http://www.chemistry.or.jp/gakujutu/bcsj/bc-cont/b98nov_gif/kea1009con.gif Altria, K. Capillary Electrophoresis Handbook: Principles, Operations, and Applications. Version 52. 1996: pgs 158