Transcript Capillary Electrophoresis: Technique and Application

CAPILLARY ELECTROPHORESIS:
TECHNIQUE AND APPLICATION
By Vanessa Hobbs
CAPILLARY ELECTROPHORESIS
DEVELOPMENT OF CAPILLARY
ELECTROPHORESIS
1803
 1886
 1937
 1967
 1970
 1979
 1981
 1983
 1988
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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
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Analyte
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ν=µE
ν = velocity
µ = electrophoretic mobility E = Electric field
Electrophoretic mobility
µ = q/[6πηr]
 q = charge η = solution viscosity r = radius
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Electroosmotic flow
νEOF = [ε/4πη]ζE
 ε = dielectric Constant ζ = Zeta potential
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Flow of migration
 ν = [(μEO + μe)V]/L
 V = potential L = length of capillary
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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
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Differs by analyte
Hydrodynamic
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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
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Separated by mass to charge ratio
Based on Electroosmotic Flow
Detectors:
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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
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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
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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
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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
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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
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Garvin, D and Ajuhar, S. Handbook of Isoelectric Focusing and Proteomics. 7th Volume. 1995: pgs 14-15, 181 191.
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Tiselius, A. Electrophoresis of Serum Globulin. Biochem. J. 1937, 31: 313 – 317.
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Van Oss, C. Interfacial Forces in Aqueous Media. 1994: pg 145.
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Hjerten, S. Free Zone Electrophoresis. Chromatogr. Rev. 1937, 9: 122 – 219.
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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.
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http://www.chemistry.or.jp/gakujutu/bcsj/bc-cont/b98nov_gif/kea1009con.gif
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Altria, K. Capillary Electrophoresis Handbook: Principles, Operations, and Applications. Version 52. 1996: pgs 158