capillary electrophoresis, modes of operation note on microchip electrophoresis

1. Presenter Dr Anurag Yadav
Post-graduate, biochemistry
Father Muller Medical college
Dr Anurag yadav,Bio-FMMC1
ELECTROPHORESIS

2. Capillary electrophoresis
Dr Anurag yadav,Bio-FMMC2
Technique first described by- Jorgensen and
Lukacs (1980’s)
 Also referred as
 High performance capillary electrophoresis(HPCE)
 Capillary zone electrophoresis (CZE)
 Free solution capillary electrophoresis (FSCE)
 Capillary electrophoresis (CE)

3. Dr Anurag yadav,Bio-FMMC3
 The sensitivity has made it as one of the choice
for many biomedical and clinical analyses.
Application : used to separate
Amino
acids
Peptides Proteins
DNA
fragments
Nucleic
acid
Drugs /
even
metals.

4. Dr Anurag yadav,Bio-FMMC4
Other
clinical
applications
include
Multiple myeloma
testing (6bands).
Haemoglobinopathy
screening.
HbA1c
Monitoring chronic
alcoholism (GGT).

5. Components :
Dr Anurag yadav,Bio-FMMC5

6. Dr Anurag yadav,Bio-FMMC6
Small amount of
sample is required
(5-30 μm3)
Introduced into the
capillary with
appropriate buffer
at anode end.

7. High voltage injection Pressure injection
Dr Anurag yadav,Bio-FMMC7
 The buffer reservoir is
replaced by the
sample reservoirthe
high voltage is applied
(+ electrode)
buffer reservoir is
placed again and
voltage applied for the
separation.
 Anodic end of
capillary is removed
from buffer and
placed in air tight
sample sol with
pressure sample is
pushed into capillary
kept back in the
buffer sample and
voltage is applied.
Sample application is done by either of one method
High voltage
injection
Pressure
injection

8. Dr Anurag yadav,Bio-FMMC8
50μm – ID.
300 μm – ED.
Length – 50-100cm.
Fused silica capillary tube.
Polyimide coating external.
Packed with the buffer in use.
 As the name suggest, the separation is carried in
a narrow bore Capillary

9. Dr Anurag yadav,Bio-FMMC9
 High voltage is applied (up to 50 kV)
 The components migrate at different rate along
the length.
 Although separated by the electrophoretic
migration, all the sample is drawn towards
cathode by electroendosmosis.
Since this flow is strong, the rate of
electroendosmotic flow is greater than the

10. Dr Anurag yadav,Bio-FMMC10
Positively charged molecule reach the cathode first
(electrophoretic migration + electroosmotic flow).

11. Dr Anurag yadav,Bio-FMMC11
DETECTION:
near to cathode
end, viewing
window
- Detected by the
ultraviolet
monitor, transmit
signal and
integrated by
computer.
- Refractive index
- Fluorescence
- CE-MS

12. Dr Anurag yadav,Bio-FMMC12
Troubleshooting :
 Adsorption of protein to the wall of capillary – leading
to smearing of protein – viewed as peak broadening –
or complete loss of protein.
- Use of neutral coating group to the inner surface of
the capillary.

13. Dr Anurag yadav,Bio-FMMC13
Advantage over slab type:
 Reduce the problem of heating
effect.
 Large surface to volume ratio.
 Less diffusion of the separated
bands.

14. Dr Anurag yadav,Bio-FMMC14
 Variations in technique:
 Add of surfactant to buffer i.e., SDS (for Neutral
molecules).
 Micellar formation In MECC- electrophoresis +
chromatography.

15. Different modes of
operation
Dr Anurag yadav,Bio-FMMC15
 Capillary zone electrophoresis :
- Separation principle based on charge to mass
ratio of molecule.
- Separation is faster.
- Due to High EOF, the molecules regardless of the
charge, they are moved to cathode.

16. Different modes of operation
Dr Anurag yadav,Bio-FMMC16
 Micellar electrokinetic chromatography:
- It is an hybrid.
- Used for separation of the neutral and charged
solutes.
- The separation is accomplished by micelles
formation. (8-9mmol/L for SDS)
- During migration, micelle interact with analyte
as chromatographic manner and the separation
is brought about.

17. Dr Anurag yadav,Bio-FMMC17

18. Different modes of
operation
Dr Anurag yadav,Bio-FMMC18
 Capillary gel electrophoresis:
- Identical to the slab.
- Separation based on the sieving.
- The capillary is filled with “sieving matrix” or
“soluble polymer network”.
- Low viscosity, self entangling for formation of pore
size.
- Variety of polymeric matrices are available for DNA
and Protein.
- Cross linked polyacrylamide- choice of polymer.

19. Dr Anurag yadav,Bio-FMMC19
Advantage over
conventional
• Online detection.
• Improved quantification.
• Almost complete
automation.
• Reduced analysis time.
• Wider choice of gel
matrices.
• Linear polyacrylamide,
derivative of cellulose,
galactomannan, glucomannan,
polyvinyl alcohol,
polyethyleneoxide, agarose,
dextran, polymethylacrylamide,
and polyacryloylethoxyethenol.

20. Different modes of
operation
Dr Anurag yadav,Bio-FMMC20
 Capillary isoelectric Focussing
Electrophoresis:
- Is comparable to tube IEF.
- Carried out in the capillary.
- The focused zone migrate to the detector with the
separated sample.
- cIEF is completed in ~15 min.

21. Different modes of
operation
Dr Anurag yadav,Bio-FMMC21
 Capillary Isotachophoresis:
- Same feature as ITP.
- Except condition of pure ITP not achieved.
- Typically used for online sample
preconcentration.
- CZE, MEKC, CGE.

22. Dr Anurag yadav,Bio-FMMC22
a. Capillary Isotachophoresis
b. Capillary gel electrophoresis
c. Capillary isoelectric
Focussing Electrophoresis
d. Micellar electrokinetic
chromatography
summa
ry

23. Capillary Electrophoresis (CE) versus High
Performance Liquid Chromatography (HPLC)
Dr Anurag yadav,Bio-FMMC23
CE has flat flow, compared to pumped parabolic flow of HPLC.
Flat flow will have narrower peaks & better resolution.
CE has greater peak capacity.

24. Dr Anurag yadav,Bio-FMMC24
HPLC is more thoroughly developed.
HPLC is more complex than CE.
HPLC has wider variety of column length and packing
Both techniques uses similar modes of detection.
Can be used complementary to one another.

25. Dr Anurag yadav,Bio-FMMC25

26. Microchip electrophoresis
Dr Anurag yadav,Bio-FMMC26
 Current advanced method.
 Development in technique include
 Integrated microchip design
 Advanced detection system
 New application
 Protein and DNA separation can be done

27. Instrumentation
Dr Anurag yadav,Bio-FMMC27
Similar to the capillary
electrophoresis.
 Separation channel
 Sample injection (50-100pL)
 Reservoirs
 Voltage (1-4kV)
 sample preparation
 Precolumn or postcolumn
reactors.
 Classical Cross-T design.
 Time period of 50-200sec.

28. Dr Anurag yadav,Bio-FMMC28
Detector :
Laser induced
fluorescence
Electrochemical detectors
Pulsed amperometric
detector
Sinusoidal voltametry

29. Application
Dr Anurag yadav,Bio-FMMC29
An alternative for the DNA analysis.
 Herpes simplex virus DNA in CSF for diagnosing
encephalitis.
 Gene rearrangement correlative with
lymphoproliferative disorders.
 Polymorphisms in gene.
 Tetranucleotide associated with
hypercholesterolemia.
 Diagnosing fragile X syndrome.
 Muscular dystrophy.
 Anthracis specific PCR product.

30. Dr Anurag yadav,Bio-FMMC30

31. References
Dr Anurag yadav,Bio-FMMC31
 Keith Wilson- Principles and techniques of
biochemistry and molecular biology.
 Upadhyay- biophysical chemistry.
 Tietz- Text book of clinical chemistry.
 Kaplan- clinical chemistry.
 YouTube and Google images.