Capillary Electrophoresis: Technique and Application

1
Capillary Electrophoresis Technique and
Application

• By Vanessa Hobbs

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Capillary Electrophoresis
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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

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Electroosmotic Flow
http//www.electrokinetic.co.uk/images/tech1.gif
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Movement of Analyte

• Analyte
• ? µ E
• ? velocity µ electrophoretic mobility E
  Electric field
• Electrophoretic mobility
• µ q/6p?r
• q charge ? solution viscosity r radius
• Electroosmotic flow
• ?EOF e/4p??E
• e dielectric Constant ? Zeta potential
• Flow of migration
• ? (µEO µe)V/L
• V potential L length of capillary

Forensic Science International 77 (1996) 211 - 229
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Injection of Sample
Current Analytical Chemistry. 2005,
1 http//www.calstatela.edu/dept/chem/gomez/pubs-p
df/flow-injection.pdf
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Injection of Sample

• Injection is difficult due to sample size
• Electrokinetic Injection
• Differs by analyte
• Hydrodynamic
• Many parameters

Anal. Chem., 1997, 69 (15), pp 29522954
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Injection of Samples
Anal. Chem.2001, 73,1974-1978
9
Injection of Sample
Current Analytical Chemistry. 2005,
1 http//www.calstatela.edu/dept/chem/gomez/pubs-p
df/flow-injection.pdf
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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

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Increasing Path Length
http//www.chem.agilent.com/Library/technicaloverv
iews/Public/5989-9808EN.pdf
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Micellar Electrokinetic Chromatography
UV Neutral compounds Comprable to HPLC
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Capillary Electrochromatography

• Packed column with no pressure applied, only
  electroosmotic pressure.

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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
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Capillary Isoelectric Focusing
http//www.targetdiscovery.com/tdidocs/App_Note_5
_200405.pdf
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Capillary Isoelectric Focusing

• pH gradient
• Sample focusing and detection
• Movement of gradient towards the detector
• Zone broadening
• Not useful for chiral compounds

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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

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HPLC Heroin Analysis
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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
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First Published Analysis of Illicit Drugs
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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
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LSD Analysis with Laser Fluorescence
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LSD Analysis with Laser Fluorescence (0.2 ng/ml)
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Methamphetamine Analysis
50 µm Capillary with length of 40 cm UV Detector
Electrophoresis 2006, 27, 47114716
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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
28
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
29
Analysis (liquid Liquid Extraction)
Electrophoresis 2008, 29, 40784087
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Analysis of Hair
Electrophoresis 1998, 19, 42-50
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Analysis of Blood
Biomed. Chromatogr. 19 737742 (2005)
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