Preparative Column
Chromatography is mostly used for analysis, the preparation of pure samples for synthetic purposes or subsequent biological testing or, perhaps, to obtain thermodynamic data. Columns used for preparative work are called ‘preparative columns’ and, in general, have larger diameters than analytical columns to handle larger samples. The size of the column, however, depends on the amount of material required in pure form. In the extreme, samples required for subsequent mass spectrometric analysis may need only a few micrograms of material for the test and, thus, the preparative sample could be obtained from an analytical column 4 mm I.D. Samples of 1 to 100 gram, or even more, may be necessary for synthetic purposes and this could require the use of columns 2 cm, 5 cm, 50 cm or even 1 m in diameter. The problem associated with large diameter columns is the difficulties involved in packing them. Large diameter GC columns are usually packed by vibration. Large diameter LC columns are usually slurry packed. Experience and considerable care is necessary to produce preparative columns of high efficiency. There are several unique types of preparative column such as the moving bed column in gas chromatography that operates on a continuous basis and the simulated moving bed column used mostly in LC which is normally slurry packed. There is also the radial column, where the packing takes the form of a cylinder and the flow of mobile phase passes from the outside of the cylinder, through the packing to the inside of the cylinder, the separation taking place on the way through. This type of column has low resolution but high loading capacity; it is slurry packed.
Author: RPW Scott
Book:Preparative Chromatography
Section:Preparative Introduction
, was
not initially developed for analytical purposes, but for the isolation of some
specific pigments from plant extracts. In fact, all the early applications of
chromatograph were exclusively for preparative purposes and it was not until
gas chromatography (GC) was introduced by Martin and Synge (1) was the
technique used for analytical purposes. Even after the introduction of GC,
liquid chromatography (then called column
chromatography) was still used largely for preparative work. Liquid
column chromatography evolved from a preparative procedure into an analytical
technique during the late nineteen sixties, largely provoked by the development
of high performance liquid chromatography
(HPLC), which, in turn, was largely
sparked off by the successful development of GC. Initially, column loads were
increased for preparative purposes by increasing the dimensions of the column
both in GC and in HPLC. However, this approach has distinct limitations.
If the column
radius is
Preparative Introduction
Author: RPW Scott
Book:Preparative Chromatography
Section:Preparative Packing-Preparative-Columns LC-Columns
LC Columns
If particle
sizes in excess of 20 mm are used, then
the column can often be dry packed, with appropriate tapping, or, even better,
with longitudinal and radial sonic vibration. The variance per unit length
obtainable from a preparative LC column should be less than 2 particle
diameters (determined using analytical scale samples). It is worth remembering
that (as already discussed) when designing preparative columns, it is better to
obtain the necessary efficiency using a longer column packed with larger
particles, than the converse. The long column will permit much larger charges
and, if pertinent for the sample concerned, will also allow multiple sample
development techniques. In addition, the larger particles will provide greater
column permeability, and thus lower pressures can be used. Lower pressures
will, in turn, allow lighter and less expensive materials to be used in the
construction of the preparative system
Preparative Packing-Preparative-Columns LC-Columns
Author: RPW Scott
Book:Preparative Chromatography
Section:Preparative Packing-Preparative-Columns LC-Columns
are other problems
that need addressing, once packed, the practical lifetime of a column is also
uncertain. The changes in performance of a preparative HPLC column that occurs
with time depends upon the stability of the packed bed. Frequently, the bed
settles after operation for even a short time and the top of the column needs
to be repacked. Sometimes channels are formed in the bed, in which case the
entire column has to be repacked. The rate of settling again depends upon the
diameter of the column. This bed instability arises because there is a
significant change in wall support as the column diameter increases. In
analytical columns the walls are relatively close to the center of the column
and 'bridges' of packing particles can be formed across the bed, as shown in
Figure 16. These bridges allow the longitudinal forces acting on the packing
within the column to be dissipated to the walls. When a column is packed, it is
never in its optimal configuration and there are always areas
Preparative Packing-Preparative-Columns LC-Columns
Author: RPW Scott
Book:Gas Chromatography
Section:YES Preparative-Gas-Chromatography
the pharmaceutical
industry for the isolation and purification of physiologically active
substances. There are a number of unique problems associated with preparative
gas chromatography. Firstly, it is difficult to recycle the mobile phase and
thus large volume of gas are necessary. Secondly, the sample must be fully
vaporized onto the column to ensure radial distribution of the sample across
the column. Thirdly, the materials of interest are eluted largely in a very
dilute form from the column and therefore must be extracted or condensed from
the gas stream which is also difficult to achieve efficiently. Finally, the
efficient packing of large GC columns is difficult. Nevertheless, preparative
GC has been successfully used in a number of rather special applications; for
example the isolation of significant quantities of the trace components of
essential oils for organoleptic assessment.
The layout of a
preparative gas chromatograph is shown in figure38
YES Preparative-Gas-Chromatography
Author: RPW Scott
Book:Preparative Chromatography
Section:Preparative Loading-Capacity
Equation (1),
although, apparently simple in form, has some very significant implications on
preparative column design. It is clear that increasing radius and length of the
column increases both the maximum sample volume and the maximum sample mass. It
is also seen that increasing the column length will also increase the column
efficiency (unless it is accompanied by an corresponding increase in the
particle diameter).
However,
increasing the column efficiency will have the opposite effect, as seen by
equation (1), it will reduce the maximum sample load. Consequently, if
the necessary efficiency to achieve the required separation has been obtained,
then if the column is lengthened to increase the loading capacity for optimum
performance, either the flow rate will need to be increased to reduce the
efficiency and thus maintain the maximum loading, or the particle size will
need to be increased to reduce the efficiency to its required
Preparative Loading-Capacity
Author: RPW Scott
Book:Preparative Chromatography
Section:Preparative Apparatus Columns
and tailing on
the column. This procedure may entail sample recovery from the residue
contained in sample valve.
Preparative Columns
Preparative
columns (GC or LC) are usually made of glass or stainless steel the latter
being used for high pressure systems. Preparative columns must be designed to
accommodate the inlet pressure necessary to obtain the required flow rate
through the packed bed which is determined by the size of the particles
selected for the packing. The larger the column diameter, the stronger must be
the column and the thicker the walls. Large column operating at high pressures
with relatively small particles can become extremely bulky and heavy. In addition,
the construction of wide columns (3 in. O.D. and greater), irrespective of the
packing, can be extremely expensive to both construct and to pack and it is
essential to take cost into all design considerations. Columns having diameters
greater than 0.5 in. need to have the frit supported on a
Preparative Apparatus Columns