Hi folks,
can anyone please tell me what exactly the difference between ion exchange chromatography and chromatofocusing is ?
I've heard that in chromatofocusing you first establish a (linear) pH gradient. Does it mean that i'm having for example pH 6 at the inlet and pH 3 at the outlet of the column ?
thx for any kind of help !
:D
In answer to your query I first describe briefly the major improvement to chromatofocusing we have invented and are marketing. Below that I give a short history and description of classic CF, then return to pISep. Full details are to be had in our publications: Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases , Journal of Chromatography A, 1200 (2008) 166–182; Application of Well-Controlled pH Gradients at Variable Isocratic Salt Concentrations to IEX Chromatography, American Biotechnology Laboratory October 2008.
I hope this helps.
We have developed what amounts to a new form of chromatofocusing IEX that we have called pISep: IEX chromatography with fully controllable wide range pH gradients. The pISep buffers consist of a mixture of a small number of polyionic organic buffering molecules that are specifically characterized by possessing reliable overlapping buffering capacities (pKas) covering a broad pH range. The overlap of pKas is robust over a wide pH range from pH 2 to pH 12 (the extended pISep) or from pH 2.4 to
pH 10.9 (the standard pISep). Over these wide pH ranges, the pISep
buffering compounds do not bind strongly to either the charged
groups of proteins or to the charged groups of anionic and cationic
ion exchangers at those pHs where they are fully charged. Strong
ion exchangers are the preferred stationary phases for performing
pISep chromatography since they remain completely charged over
a wide pH range allowing exploitation of the full capability of the
method. The lack of interaction between pISep buffers and totally
charged stationary phases permits a controlled formation of pH gradients
of virtually any desired shape over a wide pH range as easily
as the formation of variable slope salt gradients by either a dual or a
single ternary or quaternary pump LC gradient system.
In the late 1970s Sluyterman et al. developed a pH gradient
LC technique called chromatofocusing (CF). In this method,
one equilibrates a specially designed weak anion exchanger such
as PBE94, PBE118 or Mono P with an initial buffer at a pH
high enough to allow the binding of the target proteins to the
exchanger. In general, after application and binding of the proteins,
followed by washing of the CF stationary phase with several
column volumes of the initial buffer, an elution is performed
with a second (final) buffer containing Polybuffer (a mixture
of ampholytes) at a final pH lower than the pH of the initial
buffer. As the final buffer descends through the column the various
ampholyte components bind differentially to the stationary
phase developing an internally retained pH gradient. This gradient
dynamically evolves from the higher pH of the initial buffer to
the lower pH of the final buffer as the final buffer moves down the
column. During this process proteins detach sequentially from
the stationary phase as their ionized side groups are neutralized
and begin to move down the column with the bulk flow at a pH
usually near the pH of their electro-neutrality as measured by isoelectric focusing (IEF).
CF has several serious shortcomings. First, for each pH range
over which one might wish to create a gradient, it is necessary
to prepare different initial and final buffer solutions. Second, for
any given pH range, the slope of the gradient inside the column
is proportional to the width of that pH range, thus precluding
variations in the slope during the separation. Third, no single formulation
of the ampholytes can maintain good buffering capacity
over more than three pH units, requiring three different Polybuffer
formulations (G. E. Healthcare) which cover the pH ranges
11–8, 9–6, and 7–4. Finally, ampholytes are expensive and some of
them bind to purified proteins requiring their removal by size exclusion chromatography (SEC). It is therefore not practical to use CF in mid and large scale preparative protein purifications. Yet, despite these drawbacks, CF has
sparked renewed interest as the first dimension LC technique of
the Beckman-Coulter ProteomeLabTM PF 2D Protein Fractionation
System, because even uncontrolled retained pH gradients deliver significantly higher chromatographic resolution than salt gradients at
isocratic pH when proteins from complex mixtures are separated.
In the intervening years since the initial work of Sluyterman
et al., numerous attempts have been made to address CF’s disadvantages
. Generally, those attempting to improve on CF have continued to assume, as Sluyterman did, that complex interactions between the buffers and the preferred stationary phase, a weak anionic exchanger, are the key to successful pH gradient formation, even though this
inevitably results in an inability to accurately predict and control
the development of the pH gradient. As a consequence, it has been
widely accepted that controlled pH gradients over very wide pH
ranges are not achievable. In a few instances the buffers forming
the pH gradient have been mixed externally to the CF column
. However, these efforts have failed to achieve: (1) the
formation of multi-step, multi-variable slope linear or non-linear
pH gradients over a very wide pH range of up to 10 pH units
under software-enabled algorithmic control, (2) a single low ionic
strength buffering composition allowing the controllable formation
of pH gradients on both AEX and CEX stationary phases with arbitrary
starting point and length over the entire pH range or (3) the
use of strong ion exchangers as the preferred stationary phase. A
more recent effort, using a buffer chemistry very similar to our
pISep, but at a much higher ionic strength, was able to achieve
uncontrolled, descending, nearly linear wide ranging pH gradients but without the accompanying software-enabled algorithmic control of the
pH gradient formation, varying the gradients was trial and error. Because of the high ionic strength of the mobile phase in that study, the AEX stationary phase failed at pH10 to strongly bind proteins with electrophoretic pIs in the neutral (pH 6–8) range causing the elution of all these proteins at around pH 9 independent of their pI.
As a consequence of the lack of progress in achieving accurately controlled pH gradients, chromatographers face a number of shortcomings of LC that remain unresolved and which formed the impetus for our development of pISep. The more obvious needs are for: (1) software-driven, multi-step ascending or descending pH gradients with changing slopes limited only by the accuracy of modern LC gradient pumps; (2) software-driven capability to vary the slope of a pH gradient arbitrarily throughout a LC separation independent of the initial and final pH and without changing the chemistry of the mobile phases in the source reservoirs with each slope change; (3) generation of controlled pH gradients on both anionic and cationic stationary phases using the same mobile phase; (4) software driven control of pH gradients formed in the presence of isocratic additives such as nonionic detergents, organic solvents, salts, etc. as well as controllable development of simultaneous independent gradients of pH plus gradients of one or more additives on the same stationary phase; (5) relatively cheap, simple, mobile phase
compositions with few buffering components allowing solution of
problems 1–4 above; (6) a buffering mobile phase with nearly constant
composition and nearly constant buffering capacity over a
very broad pH range with very low ionic strength; (7) a mobile
phase composed of buffer components that do not bind to proteins,
nor to the stationary phases and, (8) providing mobile phases that
have simultaneously hydrophobic, polar, and electrostatic properties
allowing formation of efficient, controllable, independent and
simultaneous gradients of their constituents throughout LC separation
on the same chromatographic column.
