Biomedical Engineering Reference
In-Depth Information
this branch of chromatography requires more discussion, which is
beyond the scope of this chapter. As a sole example, the analyti-
cal separation of carbohydrates by high-performance ion exchange
chromatography (HPIEC) is described at the end of this chapter.
The success of chromatographic separations depends on skillful
realization as well as proper selection of media, conditions, and
separation principles.
Biologic macromolecules are molecules bearing a lot of dif-
ferent properties. Mostly these properties are not distributed uni-
formly on the surface of the molecule. Each of these parts of the
molecules is able to interact with chromatographic media; so the
stationary and mobile phases produce a special interaction, but
other properties may also contribute to separation. Macroscopic
properties of a macromolecule as isoelectric point, amino acid
composition, posttranslational modifications, such as, for example,
glycosylation, acylation by fatty acids, or phosphorylation, may fa-
vor a type of chromatography, but mostly other kinds of interaction
have to be suppressed during a distinct chromatographic process.
Therefore, it is impossible to give a single protocol for a fast and
selective separation with high yield of the wanted macromolecule.
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But some general rules are possible:
If several chromatographic steps are planned, the same type
of chromatography should never follow the preceding, i.e., when
the first step was gel chromatography then ion exchange or affinity
chromatographyshouldfollow,orgelfiltrationshouldbeusedafter
ion exchange. The reasons are, for example, the concentration of
the sample in ion exchange after dilution by gel filtration and the
ionic strength caused by ion exchange is reduced by gel filtration,
respectively. So additional buffer exchanges can be avoided.
For selection of a chromatographic method it should be taken
into consideration whether the protein of interest may be dena-
tured (or if it can be renaturated) or some specific properties as
ligand binding or enzymatic activity must be conserved during
purification. These reflections are not relevant, if during analyti-
cal separation a signal produced by a covalently attached label is
measurable independent of the structure of the macromolecule.
The following checklist helps in planning a purification proce-
dure:
-
What is the source for preparation of the molecule of interest?
-
Which kind of tissue maceration is optimal with respect to
conservation of properties and yield?
-
Is it possible to start with non-chromatographic procedures,
e.g., centrifugation?
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Illustrative handbooks describing the different types of low-pressure
chromatography are found as .pdf files, e.g., at
www.chromatography.amershambiosciences.com.
