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Finally, the analyst is left with some choice in the strategy that can be used in the analysis by way of the chromatographic media selected, and in the level of some operating variables that may be considered appropriate or necessary. The range of variables left to the choice of the analyst constitutes the the third data base necessary for optimum column design and this will be termed the elective variables However, as most of the conditions that need to be specified will be defined under performance criteria and determined under instrument constraints, the analyst is not left with a very wide choice of variables from which to choose. This might be considered advantageous, however, as the fewer the decisions that are left in the hands of the operator, the less skill and experience will be required and fewer mistakes will be made.
The information provided by the three data bases allow the column to be designed and the column specifications to be calculated. Furthermore, once the column has been designed, and its properties defined, a complementary set of analytical specifications can also be determined.
The column design protocol, therefore, consists of three data bases, performance criteria, elective variables and instruments constraints These data bases will provide, firstly, the column specifications and finally, the analytical specifications. A diagram representing the overall design protocol is shown in figure (l). The four different components of the column design protocol will now be discussed in detail
Chromatography is a separation technique and, consequently, a satisfactory chromatographic analysis demands, a prion, that an adequate separation of the constituents of the sample is obtained. The separation must be such that an accurate quantitative measurement of each component is possible . In order to achieve this separation, an appropriate phase system must be chosen so that the individual components can be moved apart from one another in the column. The column must, therefore, be designed to have sufficient efficiency to separate all the components of the mixture. To do this, the concept of the Reduced Chromatogram must be introduced.
The Column Design Protocol
The Reduced Chromatogram
Any chromatogram that represents the separation of a complex mixture of solutes can be reduced to a relatively simple separation that will concisely and accurately represent the limits and extent of the separation problem. The simple separation can be depicted In the form of a reduced chromatogram, an example of which is given in figure (2).
The reduced chromatogram consists of four peaks, the first the dead volume peak, (which has been shown previously must be the fully excluded peak determined from the retention volume of a salt or solute of large molecular
weight), the pair of peaks in the mixture that are eluted closest together and thus, the most difficult to separate ê the critical pair), and the last peak that is eluted from the mixture which will define when the analysis is complete and determine the total analysis time The column must be constructed with sufficient efficiency to separate the critical pair and, if this is achieved, al! other peaks will also be resolved
The Reduced Chromatogram
However, it should be pointed out that any given column, operated at a specific flow rate, can exhibit a range of efficiencies depending on the nature of the solute that is chosen for efficiency measurement Consequently, under exceptional circumstances, the predicted conditions for the separation of the critical pair may not be suitable for another pair and the complete resolution of all solutes may not be obtained This could occur if the separation ratio of another solute pair, although larger, was very close to that of the critical pair but contains solutes, for example, of widely different molecular weight. However, the possibility of this situation arising, in practice, is extremely remote and will not be considered in this discussion. It follows, that the efficiency required to separate the critical pair, numerically defined, is the first performance criterion
The efficient laboratory manager will also require the maximum throughput of samples from the equipment and thus, the second criterion is that the analysis must be achieved in the minimum time. It should be pointed out
that separation is not just required to be separated rapidly but, that it should be achieved in the absolute minimum time that an optimized column can achieve. In fact, the column must be be designed such that with the apparatus available and the phase system chosen no other column can effect the separation more rapidly.
Another aspect of cost reduction would be the need to employ the minimum amount of solvent per analysis and this would be the third performance criteria. Finally, to conserve sample and to have the capability of determining trace contaminants, the fourth criterion would be that the the combination of column and detector should provide the maximum possible mass sensitivity. The performance criteria can therefore be summarized as follows,