Preparative Chromatography - Criteria for the Successful Operation of Preparative Chromatographs. > Page 74

2. It is far more effective, less expensive, and in most cases faster, to over load a column to the maximum possible level and run a repeat series of separations, than to attempt to prepare large columns to handle the same sample in a single run..

The use of column over load has been discussed in detail in the early chapters of this book. Mass over load is to be preferred to volume over load, although the latter may well be preferable, if the solutes of interest have a limited solubility in the mobile phase that provides the optimum selectivity. Mass overload also produces asymmetric peaks with a sharp front and a sloping tail which can be advantageous for the production of high purity fractions by peak cutting. This advantage is illustrated in the last example given. The first peak can be collected as a fraction up to a point just before the sharp front of the second peak starts. This will produce a very pure fraction of the first peak The second peak is re-run in an identical manner and as the first peak is now an impurity (and therefore present at a low concentration) it is not overloaded and, therefore, will be eluted as a symmetrical peak in front of the second main peak. If required, the second peak can be collected just after the trace of the first peak is eluted and will also be extremely pure. The first fraction of the second separation, still containing a mixture of both peaks, although containing a very small percentage of the total mixture can be recycled if considered appropriate.

3. If large sample loops are employed the injection must be cut so that tail of sample left in the loop does not cause serious peak dispersion and loss of resolution

Sample cutting is a common technique in preparative separations and, as shown by the example earlier in the chapter, can eliminate significant peak dispersion which can result in either low product purity or reduced yield.

4. Under certain circumstances the column dimensions can be increases to improve both load and product yield.

There are two ways of increasing the size of the preparative column, by increasing its length and by increasing its diameter. Increasing the diameter increases the loading capacity and maintains the same separation time. It does not, however, increase the resolution. Increasing the column length increases the separation time and the resolution but does not increase the loading capacity, if the maximum efficiency is needed. If the sample consists of a simple pair of substances (e.g., the separation of a pair of enantiomers) then increasing the column length will allow multiple samples to be separated in the column at one time and, thus, will increase throughput and effectively reduces the separation time.