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)

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