Capillary Electrophoresis Vanessa CHEM6200
Transcript of Capillary Electrophoresis Vanessa CHEM6200
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CAPILLARY
ELECTROPHORESIS:
TECHNIQUE AND
APPLICATIONBy Vanessa Hobbs
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CAPILLARY ELECTROPHORESIS
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DEVELOPMENT OF CAPILLARYELECTROPHORESIS
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, VerheggenFZE
1981 Jorgenson and Lukags 75 um cap.
1983 Micellar Electrokinetic Chrom.
1988 Commercial introduction
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ELECTROOSMOTIC FLOW
http://www.electrokinetic.co.uk/images/tech1.gif
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MOVEMENT OF ANALYTEAnalyte = E
= velocity = electrophoretic mobility E = Electric field
Electrophoretic mobility
= q/[6r] q = charge = solutio viscosity r = radius
Electroosmotic flowE!"= [#/$]%E
# = dielectric &ostat % = 'eta potetialFlow of migration = [()E!* )e+,]/-
, = potetial - = legth of capillary
Forensic Science International77 (1996) 211 - 229
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INJECTION OF SAMPLE
Current Analytical Chemistry. 2005,1http://www.calstatela.edu/dept/chem/gomez/pubs-pdf/flow-injection.pdf
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INJECTION OF SAMPLE
Injection is difficult due to sample size
Electrokinetic InjectionDiffers by analyte
HydrodynamicMany parameters
Anal. Chem., 1997, 69 (15), pp 29522954
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INJECTION OF SAMPLES
Anal. Chem.2001, 73,1974-1978
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INJECTION OF SAMPLE
Current Analytical Chemistry. 2005,1http://www.calstatela.edu/dept/chem/gomez/pubs-pdf/flow-injection.pdf
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CAPILLARY ZONEELECTROPHORESIS
Separated by mass to charge ratio Based on Electroosmotic Flow Detectors:
UV Detector Beers Law
Laser Fluorescence Deriv. MS - electrospray Chemiluminescence Diode Array Detector Indirect
Refractive Index Compare with HPLC and GC Neutral Compounds Chiral Compounds
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INCREASING PATH LENGTH
http://www.chem.agilent.com/Library/technicaloverviews/Public/5989-
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MICELLAR ELECTROKINETICCHROMATOGRAPHY
UVNeutral compoundsComprable to HPLC
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Capillary Electrochromatography
Packed column with no pressure applied, onlyelectroosmotic pressure.
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CAPILLARY GELELECTROPHORESIS
Crosslinked vs. non crosslinked
DNA sequencing
Protein analysis
Chirality possibleEOF less desirable
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CAPILLARY GELELECTROPHORESIS
http://www1.qiagen.com/Images/Catalog/2134.jpg
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CAPILLARY ISOELECTRICFOCUSING
http://www.targetdiscovery.com/~tdidocs/App_Note_5_200405.pdf
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CAPILLARY ISOELECTRICFOCUSING
pH gradient
Sample focusing and detection
Movement of gradient towards the detector
Zone broadeningNot useful for chiral compounds
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CAPILLARY ISOTACHOPHORESIS
Two buffers form ionic zones
Anions and Cations seperately
Neutral compounds
Used for concentrationEOF less desirable
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APPLICATIONSCE and Analysis of Illicit Drugs
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HPLC Heroin Analysis
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HPLC Analysis of Heroin (SPE)
Fig. 2. (a) Representative total ionchromatograms of all quantifiable
analytes spiked at LLQ level in
human plasma ( ng!mL). "he
intensity of the deuterated analytes
#as above 2$$ %cps&. (b)
Representative total ionchromatograms of random chosen
patient' plasma sample. (c) "otal ion
chromatogram of a plasma sample
of a nondrug using volunteer. ()
*+, and *+,d+- () morphine
and morphined+- (/) *0,- (1) 0**- () heroin and heroind0-
(F) 3 methadone and methadone
d4- (,) *15- (6) cocaine- (7)
ben8oylecgonine.
1791 RR: ;1 "R75L *