Elective variables

The choice of variables remaining with the operator, as stated before, is restricted and is usually confined to the selection of the phase system. Preliminary experiments must be carried out to identify the best phase system to be used for the particular analysis under consideration. The best phase system will be that which provides the greatest separation ratio for the critical pair of solutes and, at the same time, ensures a minimum value for the capacity factor of the last eluted solute. Unfortunately, at this time, theories that predict the optimum solvent system that will effect a particular separation are largely empirical and those that are available can be very approximate, to say the least. Nevertheless, there are commercially available experimental routines that help in the selection of the best phase system for LC analyses, the results from which can be evaluated by supporting computer software. The program may then suggest further routines based on the initial results and, by an iterative procedure, eventually provides an optimum phase system as defined by the computer software. 

Whether the optimum phase system is arrived at by a computer system, or by trial and error experiments (which are often carried out, even after computer optimization), the basic chromatographic data needed in column design will be 

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The selection of the mobile phase and the conditions of development. Having chosen the solvent(s) the following are defined from the reduced chromatogram, 

The capacity ratio of the first solute of the critical pair. 

Whether the optimum phase system is arrived at by a computer system, or by trial and error experiments (which are often carried out, even after computer optimization), the basic chromatographic data needed in column design will be identified. The phase system will define the separation ratio of the critical pair, the capacity ratio of the first eluted peak of the critical pair and the capacity ratio of the last eluted peak. It will also define the viscosity of the mobile phase and the diffusivity of the solute in the mobile phase. 

There remains little more for the operator to decide. Sometimes, alternative but similar solvent mixtures that have a lower viscosity or higher solute diffusivity could be selected. For example, a n-hexane/methanol mixture might be chosen as an alternative to the more viscous n-heptane/isopropyl alcohol mixture as it has similar elution properties. However, it will be shown later, that if a fully optimized column is employed the viscosity of the mobile phase does not seem to effect the column performance as it is taken into account in the optimization procedure. The operator would, under some circumstances, be free to choose less toxic or less costly solvents for example, in reverse phase chromatography the operator could select methanol/ water solvent mixtures as opposed to acetonitrile/water mixture on the basis of lower cost or less 

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The information contained in the three data bases provides the necessary information required to design the optimum column. In addition, once the column has been designed, and its properties defined, a complementary set of Analytical Specifications can also be calculated. Thus, the design protocol contains three data bases. Performance Criteria, Elective Variables and Instrument Constraints. 

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 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 (1). The four different components of the column design protocol will now be discussed in detail. 

The response time of the detecting system. Elective Variables... 

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