Capillary Gel Electrophoresis

has been used for protein separation for

more than two decades.

Due to the technology advancement, current

CGE methods are becoming more and more
robust and reliable for protein analysis,

and some of the methods have been

routinely used for the analysis of protein-
based pharmaceuticals and quality controls.
 Types of Gels
Polyacrylamide polymer formed by
polymerizing acrylamide (CH2=CH-CO-NH2)
in the presence of a cross linking agent.

The pore size of the polymer depends on the

ratio of monomer to cross-linking agent.

Increases in the amount of cross-linking agent

produce smaller pore size.
Other gels that have been used for CGE:
Agarose- is a polysaccharide extracted from a
marine alga.
Methyl cellulose
Polyvinyl alcohol
Polyethel leneoxide
dextran
Polydimethylaacrylamide
Polyethylene glycol
CE in DNA Sequencing

The major goal of the Human Genome Project

was to determine the occurence of the four
bases: Adenine (A), Cytosine (C), Guanine (G),
Thymine (T) in DNA molecules.

Capillary array electrophoresis

this technique, as many as ninety-six
capillaries are operated in parallel.

The capillaries are filled with a separation

matrix, normally a linear polyacrylamide gel.
The capillaries have inner diameters of 35-
75um and are 30-60cm long
Capillary Isotachophoresis
In all capillary isotachophoresis analyte bands
ultimately migratic at the same velocity, hence,
the name from iso for same and lach for speed.

In any particular application either cations or

anions can be separated, but not both on the
same time.
Application:
o Fast Separation technique
o Minute quantity of sample sufficient
o Simple to perform
o Sample pre-treatment not necessary
o Separation of small ions up to 1000Da is possible
Capillary Isoelectric Focusing
used to separate amphiprotic species such as amino
acids and proteins that contain a weak carboxylic acid
group and a weak base amine group.

It is a type of zone electrophoresis, usually performed

on proteins in a gel.
Separation of Amphiprotic species
In isoelectric separation of amplitude species,
the separation is performed in a buffer mixture
that continuously varies in ph along its length.

Ampholytes
are amphoteric compounds usually
containing carboxylic and amino groups.
mixture having different pH ranges can be
prepared or are available from several
commercial sources.
Mobilization of Focused Bands
To detect the focused bands in a capillary
isoelectric focusing separation

itis necessary to move, or mobilize the

contents of the capillary so that the bands
pass the detector located at one end.

can be accomplished by applying a pressure

difference, just as for sample loading, or by
simply changing the solution in the electrode
compartment.
Micellar Electrokinetic Chromatography
(MEKC) is a type of CE that offers several
unique features.

LikeCE, MEKC provides highly efficient

separations on microvolumes of sample
solution without the need for the high
pressure pumping system required for HPLC.

Micellar forms in aqueous solutions.

Packed Column Electrochromatography
is a hybrid of HPLC and CE that offers some of
the best features of the two methods.

Like
HPLC and MEKC, it is applicable to the
separation of neutral species or changed
species.

withpacked column is, however, the least

nature of the various electroseparation
techniques.
Field Flow Fractionation (FFF)

is a separation technique where a field is applied to a fluid

suspension or solution pumped through a long and narrow
channel

It was invented and first reported by J. Calvin Giddings.

The method of FFF is unique to other separation techniques due

to the fact that it can separate materials over a wide colloidal
size range while maintaining high resolution.

Although FFF is an extremely versatile technique, there is no

"one size fits all" method for all applications.
Sedimentation FFF
is by far the most widely used form.

In
this technique, the channel is coiled and
made to fit inside a centrifuge basket.

Components with the highest mass and

density are driven to the wall by the
sedimentation (centrifugation) force and elute
last.

Low-mass species are eluted first. There is

relatively high selectivity between particles of
different size in sedimentation FFF
Electrical FFF
-an electric field is applied perpendicular to the
flow direction. Retention and separation occur
based on electrical charge.

Thermal FFF
-a thermal field is applied perpendicular to the
flow direction by forming a temperature gra-
dient across the FFF channel.

-The temperature difference induces thermal

diffusion in which the velocity of movement is
related to the thermal diffusion coefficient of the
species.