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(1) R. W. P. Scott, J Chromatogr., 517( 1990)297,
(2) J. H. Purnell, Nature (London),184 (Suppl. 26)( 1959)2009,
(3) M. J. E. Golay,in "GasChromatography 1958", (Ed. D.A.Desty), Butterworths, London,(1958)36,
(4)" Handbook of Chemistry and Physics" ,The Chemical Rubber Company,Ohio, (197DF-24I,
(5) R. P W. Scott," Liquid Chromatography Detectors, Elsevier, Amsterdam, (1986)7
(6) R. P. W. Scott and E. Katz US Patent (1991)
Preparative Liquid Chromatography Columns
The use of the adjective, 'preparative', to describe a particular form of chromatography does not convey a specific meaning to most modern chromatographers, nor does it suggest a particular scale of separation. The term 'preparative' chromatography has been applied to sample loads ranging from a few milligrams, to grams or even hundreds of grams of material and to columns having diameters ranging from a few millimeters to hundreds of centimeters. Preparative chromatography can be employed to isolate a few milligrams of a substance for structural elucidation by appropriate spectroscopic techniques or, perhaps, to obtain 50 gram of a pure intermediate for subsequent synthesis. At the extreme, preparative chromatography can be carried out in columns over a meter in diameter to purify a biosynthetic product on the kilo scale.
In previous chapters, liquid chromatography column theory has been developed to explain solute retention, band dispersion, column properties and optimum column design for columns that are to be used for purely analytical purposes. The theories considered so far, have assumed that solute concentrations approach (for all practical purposes) infinite dilution, and, as a consequence, all isotherms are linear. In the specific design of the optimum preparative column for a particular preparative separation, initially, the same assumptions will be made.
The theory of preparative columns must show how the optimum preparative column should be constructed, and how it should be operated to provide the throughput required. However, preparative columns can be expensive to construct and often the operator has an already existing column with which to work. The theory must, therefore, be extendable to columns that are not specifically designed for a given preparative separation but for general preparative use. As a consequence, the theory must also help to determine the maximum possible volume and mass of charge that can be placed on an existing column ano stiii permit the isolation of a pure sample of the solute of interest. If the preparative column is not specifically designed for the separation then, when working efficiently from the point of view of
maximum column loading, it may operate under conditions where the isotherm is not linear and the column is overloaded. In fact, the theory must indicate the limits to which a column can be 'abused' to achieve the maximum sample load and satisfactory resolution for a particular separation.
In the first instance, the design of the optimum column for a given preparative separation will be considered .
Preparative Column Design
Many of the equations that have been previously employed in the design of analytical columns (1,2) are pertinent to preparative columns with certain provisos. Normally, there is only one component in the mixture that needs to be isolated. Thus, in preparative columns, the critical pair is no longer the pair of solutes most difficult to separate, but consists of the solute of interest and its closest neighbor; the resolution of other components of the mixture becomes irrelevant to the problem. In almost all cases, this means that the separation will be fairly simple as the critical pair will probably not be the most difficult in the mixture to resolve. Thus, the phase system can be chosen with greater flexibility, the operator focussing on the solute of interest and its neighbor and ignoring the proximity of all other solutes in the mixture. Furthermore, the column radius will no longer be determined by the extra column dispersion of the chromatographic system but will be the direct and major factor controlling the maximum sample load.
The Efficiency Required from the Preparative Column
The first equation to be employed will again be that of Purnell (1), which is used to calculate the efficiency required to separate the critical pair. The data used is the separation ratio of the critical pair and the capacity ratio of the first eluted peak of the critical pair. The critical pair now being the peak for the solute of interest and its closest neighbor. The Purnell equation is reiterated as follows,
(I + k’ )2
= 16 A— ....................................... (i)
A ~ '•
where (a) is the separation ratio of the critical pair, and (k') is the capacity ratio of the first eluted peak of the critical pair.
Optimum Particle Diameter
In the operation of preparative columns, it is necessary to obtain the maximum mass throughput per unit time and, at the same time, achieve the required resolution. Consequently, the column will be operated at the optimum velocity as in the case of analytical columns. Furthermore, the D'Arcy equation will still hold and the equation for the optimum particle diameter can be established in exactly the same way as the optimum particle diameter of the analytical column. The equation is fundamentally the same as that given for the optimum particle diameter for a packed analytical column, i.e. (18) in chapter 12, except that (a) and (Þ have different meanings.