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Chromatography

Chromatography

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Published by shiv_1987
continuation of chromatography.
continuation of chromatography.

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Categories:Types, Research, Science
Published by: shiv_1987 on Aug 05, 2009
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10/19/2011

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giving chromatogram as below

33

Mild, non-denaturing conditions - very suitable for separating proteins of different
molecular masses
.

Also used for:

De-salting or buffer exchange of, protein solutions
Determination of Molecular mass of biological macromolecules - calibrate
column with similar molecules of known molecular mass

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Quaternary structure usually remains intact.

Types of matrix for forming stationary phase:

•Cross-linked Dextran polymer (Sephadex G-10 to G-200)
•Cross-linked Polyacrylamide (Bio-gel P-2 to P-300)
•Agarose - the largest pore size

COLUMN CHROMATOGRAPHY

Column chromatography encompasses a number of techniques based around
utilizing a vertical glass column filled with some form of solid support, with the sample
to be separated placed on top of this support. The rest of the column is filled with a
solvent which, under the influence of gravity, moves the sample through the column.
Similarly to other forms of chromatography, differences in rates of movement through the
solid medium are translated to different exit times from the bottom of the column for the
various elements of the original sample.

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In 1978, W. C. Still introduced a modified version of column chromatography
called flash column chromatography (flash). The technique is very similar to the
traditional column chromatography, except for that the solvent is driven through the
column by applying positive pressure. This allowed most separations to be performed in
less than 20 minutes, with improved separations compared to the old method.

Modern flash chromatography systems are sold as pre-packed plastic cartridges,
and the solvent is pumped through the cartridge. Systems may also be linked with
detectors and fraction collectors providing automation. The introduction of gradient
pumps resulted in quicker separations and less solvent usage

.

The protein separation actually occurs on a chromatography column that contains
a charged stationary phase. Oppositely charged proteins bind to the stationary phase and
like charged or uncharged proteins wash through the column with the void volume. The
mobile phase travels through the column, carrying unbound proteins with it. The bound
proteins are then eluted from the column by increasing the salt concentration in the
mobile phase.

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The chromatography column is the heart of the separation process. For protein
separations the column usually contains porous beads of a hydrophilic polymer, such as
cellulose or some other type of carbohydrate polymer. The surface of the polymer beads
is chemically modified to give it properties that would make it suitable for various types
of chromatography: ion exchange, molecular exclusion, hydrophobic interaction or
affinity. The appropriate stationary phase is suspended in the desired mobile phase and
poured into the chromatography column.

Once the stationary phase has been fully equilibrated with the mobile phase, the
protein sample can be introduced onto the column. The separation then occurs based on
the attraction between the protein, the stationary phase and the mobile phase. For
example, a positively charged protein would bind to a negatively charged stationary
phase when the mobile phase has a low ionic strength (see Salts). An increase in the salt
concentration may displace the protein from the stationary phase when positive ions in
the mobile phase compete with the protein for binding sites on the stationary phase.

In column chromatography, the stationary phase, a solid adsorbent, is placed in a
vertical glass (usually) column and the mobile phase, a liquid, is added to the top and
flows down through the column (by either gravity or external pressure). Column
chromatography is generally used as a purification technique: it isolates desired
compounds from a mixture.

The mixture to be analyzed by column chromatography is applied to the top of the
column. The liquid solvent (the eluant) is passed through the column by gravity or by the

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application of air pressure. An equilibrium is established between the solute adsorbed on
the adsorbent and the eluting solvent flowing down through the column. Because the
different components in the mixture have different interactions with the stationary and
mobile phases, they will be carried along with the mobile phase to varying degrees and a
separation will be achieved. The individual components, or eluant, are collected as the
solvent drips from the bottom of the column.

Column chromatography is separated into two categories, depending on how the solvent
flows down the column. If the solvent is allowed to flow down the column by gravity, or
percolation, it is called gravity column chromatography. If the solvent is forced down
the column by positive air pressure, it is called flash chromatography, a "state of the
art" method currently used in organic chemistry research laboratories The term "flash
chromatography" was coined by Professor W. Clark Still because it can be done in a
“flash."

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