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CAPILLARY ELECTROPHORESIS: TECHNIQUE AND APPLICATIONBy 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) 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
ELECTROOSMOTIC FLOW
http://www.electrokinetic.co.uk/images/tech1.gif
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 International77 (1996) 211 - 229
INJECTION OF SAMPLE
Current Analytical Chemistry. 2005, 1http://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, 1http://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/5989-9808EN.pdf
MICELLAR ELECTROKINETIC CHROMATOGRAPHY
UVNeutral compoundsComprable 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
APPLICATIONSCE 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) 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
FIRST PUBLISHED ANALYSIS OF ILLICIT DRUGS
FIRST PUBLISHED ANALYSIS OF ILLICIT DRUGS
Relative Standard DeviationMigration 0.5%Peak Area 4 – 8%
Twice as many peaks observed in Heroin analysis with MEKCHPLC more sensitiveSmaller 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 urinemixing by a vortex mixer for about 1 min. 1000 mL of ethyl acetate was pipetted incontinued 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 addedevaporated to dryness at 60oCresidues 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 urinemixing by a vortex mixer for about 1 min. 1000 mL of ethyl acetate was pipetted incontinued 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 addedevaporated to dryness at 60oCresidues 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 –
158. Chankvetadze, B. Capillary Electrophoresis in Chiral Analysis. 1997: pgs 43- 46.
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