CAPILLARY

ELECTROPHORESIS:
TECHNIQUE AND
APPLICATION
By Vanessa Hobbs

CAPILLARY ELECTROPHORESIS

DEVELOPMENT OF CAPILLARY
ELECTROPHORESIS
1803
1886
1937
1967
1970
1979
1981
1983
1988

F.F. Reuss
Clay Slab
O. Lodge
Zone Electrophoresis
A. Tiselius
Electrophoretic Cell
S. Hjerten
Rotating tubes (300 um)
V. Neuhof
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

Analyte

=E
= velocity

= electrophoretic mobility E = Electric field

Electrophoretic mobility

= q/[6r]
q = charge = solution viscosity r = radius

Electroosmotic flow
EOF

= [/4]E
= dielectric Constant = Zeta potential

Flow of migration
= [(EO + e)V]/L
V = potential L = length of capillary

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

Difers

by analyte

Hydrodynamic
Many

parameters

Anal. Chem., 1997, 69 (15), pp 29522954

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

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

INCREASING PATH LENGTH

http://www.chem.agilent.com/Library/technicaloverviews/Public/5989-

MICELLAR ELECTROKINETIC
CHROMATOGRAPHY

UV
Neutral compounds
Comprable 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 bufers form ionic zones
Anions and Cations seperately
Neutral compounds
Used for concentration
EOF less desirable

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 m Capillary with length of 40

cm
UV Detector

Electrophoresis 2006, 27, 47114716

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, 39994007

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, 39994007

ANALYSIS (LIQUID LIQUID

EXTRACTION)

Electrophoresis 2008, 29, 40784087

ANALYSIS OF HAIR

Electrophoresis 1998, 19, 42-50

ANALYSIS OF BLOOD

Biomed. Chromatogr. 19: 737742 (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.

Neuhof, 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. Of 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.