Preparative Chromatography
Preparative chromatography, as opposed to analytical chromatography, is used to isolate specific quantities of a particular substance contained in a mixture. The basic difference between the two techniques is that the mixture is not merely monitored or analyzed but the individual solutes of interest are actually isolated, collected and recovered for further use. Preparative chromatography is not necessarily involved with large samples, or large columns (although very large columns are often used in preparative chromatography) it is, however, always associated with component isolation and collection. The forensic chemist may use preparative chromatography to isolate a few micrograms of a component for spectroscopic examination, a biochemist may employ preparative chromatography to provide a few milligrams of material for assessment of biological activity. At the other extreme kilograms of an intermediate or final product may be prepared or purified for industrial use. It follows, that in preparative chromatography column diameters can range from a few millimeters to a meter or more, and mobile phase volumes may range from a few ml to hundreds of liters. Preparative chromatography is generally an expensive method of isolation and purification and, thus, can only be used on a large scale if the intrinsic value of the materials being collected is very high.
Author: RPW Scott
Book:Preparative Chromatography
Section:Preparative Introduction
the technique of chromatography,
originally invented by Tswett in the latter part of the nineteenth century, 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
Preparative Introduction
Author: RPW Scott
Book:Gas Chromatography
Section:YES Preparative-Gas-Chromatography
Preparative Gas Chromatography
Gas
chromatography has not been used extensively for preparative work although its
counterpart, liquid chromatography, has been broadly used in 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
YES Preparative-Gas-Chromatography
Author: RPW Scott
Book:Preparative Chromatography
Section:Preparative Apparatus Detectors
is quickly distributed across the plate surface and then onto
the column through the slots. In this way the sample is injected across the top
surface of the packing.
Preparative Detectors
Preparative
chromatography detectors can have very limited specifications, compared with
their analytical counterparts. They need not be particularly sensitive (in fact
too great a sensitivity is a distinct disadvantage) as the sample size and the
eluent solute concentrations are very large. Preparative chromatography
detectors can have large sensor volumes and, as the detector is required only
to monitor the separation, they need not have a linear response. They do need
to tolerate high flow rates and thus, must have low flow impedance. Analytical
detectors can be used for preparative purposes but a portion is usually split
from the column eluent, diluted with more mobile phase and then passed through
the detector. In practice this becomes a rather clumsy procedure.
The most
commonly used
Preparative Apparatus Detectors
Author: RPW Scott
Book:Principles and Practice of Chromatography
Section:Principles Introduction
Introduction X
Chromatography, although primarily a separation technique, is mostly
employed in chemical analysis. Nevertheless, to a limited extent, it is also
used for preparative purposes, particularly for the isolation of relatively
small amounts of materials that have comparatively high intrinsic value.
Chromatography is probably the most powerful and versatile technique available
to the modern analyst. In a single step process it can separate a mixture into
its individual components and simultaneously provide an quantitative estimate
of each constituent. Samples may be gaseous, liquid or solid in nature and can
range in complexity from a simple
Principles Introduction
Author: RPW Scott
Book:Preparative Chromatography
Section:Preparative Apparatus
is far less than that of the front. The reduction in retention is by far
the greatest for the over loaded peak. It is also clear that in chromatography,
column over load is a very effective way of increasing the throughput and by
adjusting the selectivity (using temperature, selected stationary phases, or
gradient elution) very large sample loads can be tolerated. This approach
should always be considered first for moderate loads before contemplating large
scale column design.
Preparative Chromatography Apparatus
Very large
sample loads will necessitate the use of large scale chromatographic equipment.
However, the conventional preparative chromatograph, although certainly more
massive, is generally less complex than the analytical chromatograph. Although
gradient elution has been used in preparative chromatography, it should be
avoided, if possible, due to the cost of solvents and the complication involved
in solvent recovery
Preparative Apparatus
Author: RPW Scott
Book:Preparative Chromatography
Section:Preparative Apparatus Fraction-Collectors
plate
surface. A reference photo cell (not shown) is placed close to the lamp to
compensate for changes in light intensity that may arise from variations in
lamp emission. Due to the very short path length (the thickness of the film of
column eluent) the detector has the required low sensitivity and low flow
impedance. The detector can operate very satisfactorily at concentrations as
great as 10-2 g/ml (1% w/w), which is ideal for preparative
chromatography. Another advantage of the device for preparative work, is its
very low flow impedance and thus can easily cope with the high flow rates used
in preparative LC. The film thickness does depend, among other things, on the
column flow rate and thus fairly precise flow control is necessary for the
satisfactory performance of this detector.
Fraction Collectors
Fraction
collection in preparative chromatography is usually achieved using a multi-port
valve and a number of collection vessels. The selector valve should be
programmable
Preparative Apparatus Fraction-Collectors