Capillary Electrophoresis is important tech.

1. Date :- 01/10/2012
By
Sachin Kuhire
Junior Research Fellow
National Chemical
Laboratory, Pune

2. Ref. –www.directindustry.com

3.  Capillary electrophoresis is a separation method
based on the differential rates of migration of charged
species in an applied dc electric field
 Electrophoresis was first developed by Swedish
chemist Arne Tiselius in the 1930‟s(serum proteins)
 He was awarded the Nobel prize for his work (1948)
 The speed of movement or migration of solutes in CE
is determined by their charge and size ratios. Small
highly charged solutes will migrate more quickly then
large less charged solutes.

4. Types of Molecules that can be
Separated
by Capillary Electrophoresis
•
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•
•
•
•
•
Proteins ,vitamins
Peptides
Amino acids
Nucleic acids (RNA and DNA)
Inorganic ions
Organic bases
Organic acids
Enzymes
etc.

5. .
• A buffer filled fused-silica capillary
10-100 µm in internal diameter & 40-100 cm
long
• Two electrode(platinum)
• High voltage supply (5 to 30 kv)
• Sample injector (by pressure or vacuum)
• Detector
• Buffer solution (like sodium dihydrogen
phosphate,NaH2 PO4)

6. Generic diagram of a capillary electrophoresis system
Ref.science vol 142

7. • It is the process in which sample ions move under the
influence of an applied voltage.
• The ion undergoes a force that is equal to the product of
the Electrophoretic mobility and the electric field
strength.
• The flow of ions is toward the opposite charged
electrode.
µEP =
q
& VEPF = µ .E
EP
6ηπr
Where,
µEP
q
η
r
E
= Electrophoratic Mobility
= Charge on ions
= Viscosity
= Radius
= Electric field strength.

8. The heart of capillary electrophoresis
is electroosmotic flow(EOF).
This is the mobile phase „‟pump‟‟ in
capillary electrophoresis.
The rate of EOF is generally greater
than the electrophoretic migration
velocities
This flow occurs when buffer pH
greater than 3 and the SiOH groups
lose a proton to become SiOIncrease the pH intensity of EOF also
increases.
V electroosmatic (EO)
V electrophoretic (EP)
V total = VEO + VEP

9. Cross sectional flow profile Due to electro osmotic flow
Cross sectional flow profile Due to Hydrodynamic flow
Electroosmotic
flow does not
contribute
significantly to
band broadening
like pressuredriven flow in LC
and related
techniques

10. ● The speed of EOF can be adjusted by
changing the buffer pH
● Bulk movement of solutes is caused by EOF
● EOF is usually sufficient to sweep all
+ve, neutral, -ve species towards the same
end.
μ = C.ζ & VEOF= μEOF.E
4πη
EOF
The velocity of ions is sum of velocity of EOF and EPF
VTotal=VEOF+VEPF
Where,
μEOF =Electro osmotic mobility.
C= Dielectric constant
E= Electric field strength
ζ= Zeta potential.
η= Viscosity
VEOF=Velocity

11. .
• If the analyst wants the EOF in
opposite Direction then the
capillary can be coated with a
cationic Surfactant or added to
the buffer.
• Ex. Trimethylchlorosilane.
• Coated capillary also available
in market.
• This flow is toward the
positively charged electrode.

12. Ref- camis.sr.unh.edu

13. .
• Hydrodynamic injection
• By applying pressure
• By applying vacuum.
• By gravitation
• Electrokinetic injection
• By using Electric supply

14. .
• Detectors similar to those used in GC,HPLC
• majority of instruments have UV detectors
available.
• Alternative detector modes include commercially
available fluorescence, laser induced
fluorescence, conductivity and indirect detection.
• The mass spectrometers is frequently used to
give structural information on the resolved peaks.
• Sensitive detectors are needed for small
concentrations in CE

15. .
Electropherogram is like a chromatogram
A plot of the time from injection on X-axis Vs The
detector signal on Y- axis.
The general layout of an electropherogram
Neutral
Cation
Detector
Response
Anion
Time

16. Capillary Zone electrophoresis (CZE)
Capillary gel electrophoresis (CGE)
Capillary electrochromatography (CEC)
Capillary isoelectric focusing (CIEF)
Capillary isotachophoresis (CITP)
Micellar electrokinetic capillary chromatography (MEKC)

17. Capillary Gel Electrophoresis (CGE
Capillary Gel Electrophoresis (CGE) is the adaptation of
traditional gel electrophoresis into the capillary .
CGE uses separation based on the difference in solute size
as a particle migrate through the gel.
Gels prevent the capillary walls from absorbing then solute
Capillary Isoelectric Focusing (CIEF)
Capillary Isoelectric Focusing (CIEF) is a technique
commonly used to separate peptides and proteins
These molecule are called zwitterionic compounds.
So, each molecule has a specific isoelectric point (pI).
If pH = pI then molecule become a neutral.

18. Capillary Isotachophoresis (CITP)
• Capillary Isotachophoresis (CITP) is a focusing technique
based on the migration of the sample components between
leading and terminating electrolytes.
• Micellar Electrokinetic Capillary
Chromatography
• MEKC is a separation technique that is based on solutes
partitioning between micelles and the solvent
• Without micelles neutral molecule will migrate with the
electroosmotic flow and no separation occurs.
• The aggregates have polar negatively charged surfaces and are
attracted to the positively charged anode.

20. From
P.Jandik, W. R. Jones, O.
Weston, and
P. R. Brown, LCGC, 1991, 9, 634.
Detection:UV,214 nm.Peaks:
1 = rubidium (2 ppm), 7 = lanthanum (5 ppm), 13 = gadolinium (5 ppm),
14 =terbium (5 ppm),
2 = potassium (5 ppm), 8 =cerium (5 ppm),
3 = calcium (2ppm), 9 = praseodymium (5ppm), 15 = dysprosium(5ppm)
4 = sodium (1 ppm), 10 = neodymium (5 ppm) 16 =holmium (5 ppm)
11
17 =erbium (5 pprn),
5 =magnesium (1 ppm), =samarium (5 ppm),
18 = thulium (5 ppm),
6 = lithium (1ppm), 12 = europium (5ppm)
19 = ytterbium (5 ppm).

21. Here UV detector used (254 nm)
1-thiosulphate
2-bromide
3-chloride
4-sulfate
5-nitrite
6-nitrate
7-molybdate
8-azide
9-tungate
10-monoflorosulphate
11-chlorate
21-ethanesulphonate
12-citrate
22-propionate
13-fluoride
23-propanesulphonate
14-formate
24-butyrate
15-phosphate 25-Bu-sulphonate
16-phosphite 26-varalate
17-chlorite
27-benzoate
18-galactarate 28-l-glutamate
19-carbonate 29-pn-sulphonate
20-acetate
30-d-gluconate
Ref.
W.A.Jones andP.Jandik,J.Chromatogr.,1991,546,445

22. From
P.Jandik, W. R. Jones, O.
Weston, and
P. R. Brown, LCGC, 1991, 9, 634.
Detection:UV,214 nm.Peaks:
1 = rubidium (2 ppm), 7 = lanthanum (5 ppm), 13 = gadolinium (5 ppm),
14 =terbium (5 ppm),
2 = potassium (5 ppm), 8 =cerium (5 ppm),
3 = calcium (2ppm), 9 = praseodymium (5ppm), 15 = dysprosium(5ppm)
4 = sodium (1 ppm), 10 = neodymium (5 ppm) 16 =holmium (5 ppm)
11
17 =erbium (5 pprn),
5 =magnesium (1 ppm), =samarium (5 ppm),
18 = thulium (5 ppm),
6 = lithium (1ppm), 12 = europium (5ppm)
19 = ytterbium (5 ppm).

23. peak
A= unknown impurity;
B=labeled lysine;
C= dilabeled lysine;
D= leucine;
E= serine;
F= glycine;
G and H =unknown impurities;
I= dilabeled cystine;
J= glutamic acid;
K= aspartic acid;
L= cysteic acid.
Ref.
Science,Vol-222, Pg.266

24. .
• Advantages
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Offers new selectivity, an alternative to HPLC
Easy and predictable selectivity
High separation efficiency (millions of theoretical plates)
Small sample required (1-10 nl)
Fast separations (1 to 45 min)
Can be automated
Easily coupled to MS
Different “modes”
• Disadvantages
• Can not do preparative scale separations
• Low concentrations and large volumes difficult

25. .
• “Principles of instrumental analysis”, 6th
edition, by holler skoog & crouch, page 10031013.
• Manuel J. Gordon,Xiaohua,Stephen L.,science
Vol.242 page 224-228.
• James W Jorgenson science Vol.222, page
266-272