Page 259 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

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

Separation and Purification of Biochemicals 667

acceptable only for research purposes and will not be con- rating of the pump or of the chromatographic medium.
sidered for large-scale production. In a production en- In that case a decrease in column length may be a bet-
vironment the separation (column) has to be scaled up. ter solution. The injection of the sample and the detec-
The scaling up of chromatographic separation processes tor capacity may also have their limits. In some cases
admittedly remains a challenging problem for biosepara- the problem of combining high flow rates with accept-
tion engineering. However, columns with inner diameters able pressure drops can be circumvented by using novel
of 3.6 m and heights of 12 m have been built and operated, stationary phases characterized by lower mass transfer re-
mainly for separations in the oil and sugar industries and sistance and low backpressure (see also Section III, Sta-
more recently also for the large-scale separation of biolog- tionary Phases for Biochromatography). When scaling up
icals such as recombinant human insulin and blood plasma the chromatographic process, the quality of the separa-
fractionation. Sephadex gel filtration columns with inner tion ought to be maintained, which means in the simplest
diameters of up to 180 cm (custom built by Pharmacia, case that the column efficiency, i.e., the number of plates,
Sweden) have been used to separate milk proteins, amino should be about the same. This can be accomplished rel-
acids, technical enzymes, and penicillins. Three parame- atively easily when only the diameter of the column is
ters are usually considered for the optimization of prepara- increased and the column length and the stationary phase
tive chromatography—namely, yield, purity, and through- material remain the same. Whereas the mobile phase com-
put. In preparative and process scale chromatography position and linear flow velocity are kept constant, the vol-
the throughput, or amount of sufficiently pure product umetric flow rate of the eluent and the sample load may
obtained per unit time and column volume, is of major be increased in proportion to the cross-sectional area of
importance. the column. Columns having inner diameters greater than
Linear elution chromatography does not use the full 5 cm should be equipped with some sort of compression
capacity of either the stationary or the mobile phase. device to maintain bed stability throughout the separation.
Whenever the sample concentrations are in the nonlinear Several designs, for the static or dynamic compression of
range of the respective adsorption isotherms, the sample the column packing, have been patented; dynamic axial
molecules compete for the binding sites on the chromato- compression by a piston being currently perhaps the most
graphic surface and interfere with each other’s migration. suitable method. Dynamic compression is generally pre-
In the case of a single component Langmuirian isotherm, ferred to static compression, as the former allows for the
the zone will have an increasingly sharp front and a diffuse adjustment of the applied pressure as the particles swell
rear boundary or “tail” due to the self-interference of the or shrink. Resolution and peak shape should be indepen-
molecules (triangular zones). A simple procedure to max- dent of the column, keeping everything else constant. In
imize the throughput in elution chromatography (“over- practice, however, a certain decrease in efficiency should
loaded” elution chromatography), while still maintaining be expected because of difficulties in attaining uniform
baseline separation, is the so-called touching-band opti- column packing at large scale, due to transcolumn nonuni-
mization. The sample load is increased until the second formities and the consecutive irregular flow distribution.
component just touches the rear of the first. If the col- Radial temperature gradients may also be produced with
umn is severely overloaded, separation becomes largely concomitant decrease in column efficiency.
a matter of the sample composition. In certain cases, the Evidently, a dynamic similarity must be maintained
competition of the two components will cause the zone between the functions of the smaller column for which
of the less strongly bound component to be pushed ahead the separation has been developed and optimized, and the
by the zone of the more strongly bound one; “sample dis- larger column to be used for production. Most experience
placement”occursoftenwithsomefelicitousresultsonthe has been gained in scaling up elution chromatography on
separation. The opposite, a “tag-along” effect, may also the basis of similitude relationships. Some data for the de-
be observed. Especially in the case of smaller and chem- sign of the large-scale column, for example in regard to the
ically/mechanically more stable molecules, a solution to flow rate, the sample load, and the gradient conditions, are
the optimization of the throughput may be to operate the available in the literature. For the calculations of produc-
column under conditions of pronounced overloading and tion rates and column utilization, the expressions thus de-
simply recycle the mixed zone, i.e., add it to the fresh feed. rived can be consulted. The length-to-diameter ratio of the
To scale up the column, the diameter may be increased, large-scale columns will often be smaller and the station-
and higher flow rates used. The limitations to this approach ary phase particles larger in preparative than in analytical
are usually technical; for instance, even though the sep- liquidchromatography.Thechoiceoftheparticlediameter
aration factor is large enough to allow for a decrease in of the stationary phase will be influenced by most oper-
separation time, the pressure drop over the packed bed at ating parameters when the length of the bed is changed
elevated mobile phase velocities may exceed the pressure upon scaling up, or the specifications for the throughput