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Capillary Electrophoresis: Technique and Application

Capillary Electrophoresis: Technique and Application. By Vanessa Hobbs. Capillary Electrophoresis. Development of Capillary Electrophoresis. 1803 F.F. Reuss Clay Slab 1886 O. Lodge Zone Electrophoresis 1937 A. Tiselius Electrophoretic Cell 1967 S. Hjerten Rotating tubes (300 um)

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Capillary Electrophoresis: Technique and Application

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  1. Capillary Electrophoresis: Technique and Application By Vanessa Hobbs

  2. Capillary Electrophoresis

  3. Development of Capillary Electrophoresis • 1803 F.F. Reuss Clay Slab • 1886 O. Lodge Zone Electrophoresis • 1937 A. Tiselius Electrophoretic Cell • 1967 S. Hjerten Rotating tubes (300 um) • 1970 V. Neuhoff PAG filled tubes • 1979 Mikkers, Everaerts, Verheggen FZE • 1981 Jorgenson and Lukags 75 um cap. • 1983 Micellar Electrokinetic Chrom. • 1988 Commercial introduction

  4. Electroosmotic Flow http://www.electrokinetic.co.uk/images/tech1.gif

  5. Movement of Analyte • Analyte • ν = µ E • 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

  6. Injection of Sample Current Analytical Chemistry. 2005, 1 http://www.calstatela.edu/dept/chem/gomez/pubs-pdf/flow-injection.pdf

  7. 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

  8. Injection of Samples Anal. Chem.2001, 73,1974-1978

  9. Injection of Sample Current Analytical Chemistry. 2005, 1 http://www.calstatela.edu/dept/chem/gomez/pubs-pdf/flow-injection.pdf

  10. 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

  11. Increasing Path Length http://www.chem.agilent.com/Library/technicaloverviews/Public/5989-9808EN.pdf

  12. MicellarElectrokinetic Chromatography UV Neutral compounds Comprable to HPLC

  13. Capillary Electrochromatography Packed column with no pressure applied, only electroosmotic pressure.

  14. Capillary Gel Electrophoresis • Crosslinked vs. non crosslinked • DNA sequencing • Protein analysis • Chirality possible • EOF less desirable

  15. Capillary Gel Electrophoresis http://www1.qiagen.com/Images/Catalog/2134.jpg

  16. Capillary Isoelectric Focusing http://www.targetdiscovery.com/~tdidocs/App_Note_5_200405.pdf

  17. Capillary Isoelectric Focusing • pH gradient • Sample focusing and detection • Movement of gradient towards the detector • Zone broadening • Not useful for chiral compounds

  18. Capillary Isotachophoresis • Two buffers form ionic zones • Anions and Cations seperately • Neutral compounds • Used for concentration • EOF less desirable

  19. Applications CE and Analysis of Illicit Drugs

  20. HPLC Heroin Analysis

  21. 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) 6-MAM; (E) heroin and heroin-d6; (F) = methadone and methadone-d9; (G) EMDP; (H) cocaine; (I) benzoylecgonine. DIODE ARRAY AND TRIPLE MS 5 ng/ml

  22. First Published Analysis of Illicit Drugs

  23. 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

  24. LSD Analysis with Laser Fluorescence

  25. LSD Analysis with Laser Fluorescence (0.2 ng/ml)

  26. Methamphetamine Analysis 50 μm Capillary with length of 40 cm UV Detector Electrophoresis 2006, 27, 4711–4716

  27. 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

  28. 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

  29. Analysis (liquid Liquid Extraction) Electrophoresis 2008, 29, 4078–4087

  30. Analysis of Hair Electrophoresis 1998, 19, 42-50

  31. Analysis of Blood Biomed. Chromatogr. 19: 737–742 (2005)

  32. 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 – 158. • Chankvetadze, B. Capillary Electrophoresis in Chiral Analysis. 1997: pgs 43- 46.

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