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chromatofocusing

chromatofocusing - Chromatography Forum

chromatofocusing - Discuss Chromatography techniques in this forum. Includes a HPLC forum, and other "liquid phase" separation techniques.


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Old 09-07-2006, 05:24 AM
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Question chromatofocusing



Hi
Please give the procedure and detail about chromatofocusing. and also what is difference between isoelectricfocusing and chromatofocusing?



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Old 05-26-2007, 08:47 AM
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Default Re: chromatofocusing

Chromatofocusing (CF) is a variant of ion e x change ch r o m a t o g ra p hy. CF invo l ves the elution of
ion exchangers solely by the mechanism of
pH. In anion CF, proteins are bound to an
anion exchanger at high pH. As the pH on
the column descends, protein positive ch a r g e
becomes stronger (more column-repellent)
and protein negative charge becomes weaker
(less column-attra c t ive). The opposite situation
p r e vails in cation CF. Either way, pH conditions
in the column eventually reach a point
where a given protein's net interaction with
the column becomes zero, and it elutes.
pH gradient elution partly defines CF, but the
full definition is more restrictive. First, CF
i nvo l ves generation of a linear pH gra d i e n t
*within* the column -- not an externally
applied gradient. With anion CF, this is done
by titrating the column initially to a pH high
enough to bind the protein of interest, then
applying a low pH titrating/elution buffer that
contains buffer species collectively embodying
a range of pKas. The range of pKas is
selected to provide level buffer capacity
across the entire pH amplitude of the intended
gradient. When the buffer capacity of the eluting buffer is properly matched to the
charge density of the column, a physical linear
pH gradient is created inside the column.
Proteins partition at the pH ostensibly representing
their isoelectric point (pI). As the
p hysical gradient migrates down the column,
d r iven by the continuing flow of titra t i n g / e l ution
buffer, the proteins move with it, eluting
when the pH of the buffer exiting the column
is equal to their pI.
A second defining ch a racteristic is that CF
be conducted at low conductiv i t y. This prevents
salt-elution effects from skewing the
s e l e c t iv i t y. A third defining ch a racteristic is
that the column should maintain level ion
e x change capacity over the pH range of the
s e p a ration. This is an essential determinant of
g radient linearity.
When all these conditions are met, some
proteins do indeed elute near their pI; sometimes
ve r y near their pI; but many miss it by a
wide mark. Discrepancies occur for seve ra l
reasons. One is that proteins are 3-dimensional
structures, while most ion exch a n g e r s
present a generally 2-dimensional binding
surface. This means that unless all of a protein's
charges are localized on one small part
of its surface, only a fraction of those ch a r g e s
will be in contact with the exch a n g e r. If the
distribution of charges is truly random, then
the protein's desorption behavior may mimic
its true pI. How e ve r, protein charges tend to
be distributed uneve n l y, and only the most
complementary surface interacts with the column.
This phenomenon is called preferential
orientation and its practical significance is
that a protein's "contact pI" may be significantly
different from its true pI. Elution behavior
will deviate accordingly.
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