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Encyclopedia of Physical Science and Technology EN014J-683 July 30, 2001 20:3

Separation and Purification of Biochemicals 657

For conventional porous stationary phases the van 6. Resolution
Deemter plot runs through a minimum. At very low flow
Resolution is a measure of the quality of the actual sepa-
rates (B-term region), molecular diffusion is the predom-
ration achieved between two peaks. In quantitative terms,
inant reason for zone spreading. At comparatively high
the resolution between two peaks, Rs, is equal to the dif-
flow rates (C-term region), intraparticular mass transfer
ference in retention time, divided by the average of peak
resistance and non-equilibria become decisive. The mini-
widths at the base of the peaks (see Fig. 3). As it is in
mum value of H should be around 2–3 times the particle
practice easier to measure widths at half peak height, Rs
diameters for a well-packed column and the correspond-
is usually calculated according to
ing flow velocity is termed optimum velocity. The study
of the van Deemter curve is a good method for deter- Rs = t/(0.85 · (w 0.5 + w 0.5 )). (16)
2
1
mining the optimal flow velocity to be used to achieve
A resolution of 1.0 corresponds in this context to a nearly
highest efficiency and allows optimization of a given sep-
baseline separation. If resolution is poor, it is possible to
aration in terms of various goals (resolution, run time,
improve it either by (a) improving the efficiency or (b) the
etc.). The resolving power of a column will be highest
selectivity of the column.
when operated at the optimum reduced flow velocity. Due
to the low effective diffusion coefficient of high molecu-
lar mass molecules, however, this will often be impractical
B. Separation by Stationary Phase Interaction
for biopolymers.
Separations in liquid chromatography are based upon dif-
ferences in the strength of the interaction between the dif-
4. Peak Asymmetry ferent types of molecules in the sample and the stationary
phase. Thus, molecules will be eluted in increasing order
Under nonoptimal conditions, separations often yield non-
of affinity to the chromatographic medium. In order to
symmetrical peaks, i.e., other than Gaussian in shape.
separate a target biomolecule from the impurities, condi-
Asymmetry can be measured in terms of the band asym-
tions that favor the interaction of the wanted product with
metry factor As, that is calculated from measures taken at
the stationary phase, while decreasing that of the impu-
10% of the peak height (see Fig. 3) by dividing the width
rities, or vice versa, have to be established. Resolution is
segment after the peak maximum projection and the width
achieved by selectively retarding the target component or
segment before the peak maximum projection.
the impurities to different extents while keeping the dis-
As = CB/CA. (14) persion of solute bands as small as possible.
The several types of chromatography exploit differ-
A “perfect” Gaussian peak is symmetrical and As is hence
ences in simple physicochemical parameters to achieve
equal to 1.0. Adequate symmetry is considered achieved
separation. Examples include the hydrophobicity of the
as long as bands have asymmetry factors between 0.9 and
molecules as in hydrophobic interaction chromatography
1.2. Tailing (As > 1.5) and fronting (As < 0.7) can cause
(HIC) and in reversed-phase chromatography (RPC) or
poor separations, and indicate possible problems of the
their charge density as in ion exchange chromatography
column or the chromatographic system.
(IEC) or to some extent in hydroxyapatite chromatogra-
phy (HAC). Although the components of a given (protein)
mixture will vary in a number of these parameters, chro-
5. Selectivity
matographic conditions, e.g., in regard to the choice of
The separation quality of two components is strongly stationary and mobile phase, are usually chosen in such a
affected by the proximity of the adjacent peaks in the way, that the separation is clearly governed by one of the
chromatogram. An important measure of band proximity possible interaction mechanisms.
is the selectivity, or separation factor, α, given by the ration
of retention factors of two adjacent bands (see Fig. 3).
1. Ion Exchange Chromatography

α = k /k . (15)
2 1
IEC is at present the most widely applied technique in
For good separations, values of α must usually be at least preparative protein chromatography both at the laboratory
between 1.05 and 1.10. These values depend on the nature and the production scale; the ion exchange principle is
of the solutes as well as on the chemical composition of shown in Fig. 5.
both stationary and mobile phase. The temperature is also Compared to most other stationary phases for biochro-
of influence. The concept of selectivity is more relevant matography, IEC matrices are characterized by a rela-
in isocratic than in gradient elution. tively large binding capacity and hence allow purification