Routine with programmed elution

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. 

The direct coupling of HPLC with NMR spectroscopy has waited for a number of technological developments to make it a feasible routine technique. Since then, major advances have made the routine use of on-line NMR detection of HPLC fractions a useful adjunct to the armoury of analytical methods. " Experiments can be carried out in one of three modes, direct on-line NMR detection of the HPLC eluent (on-flow), a stopped-flow approach or finally the eluted fractions can be stored in capillary tubes for later recall for detailed NMR spectroscopic studies. No compromise needs to be made in the chromatographic conditions, and programmed gradient elution profiles can be accommodated. The extension of the method to nanolitre scale separations has also been discussed. ... 

A number of alternative techniques have been developed to tackle the general elution problem, such as flow and temperature programming, and especially column switching techniques. Coupled column chromatography involves the use of three and sometimes four different stationary phases. The sample is introduced to the primary column for a preliminary separation and then the various polar fractions are switched to the appropriate secondary columns and then to the detector. Coupled column chromatography provides enhanced resolution, and as isocratic elution is used the technique is particularly useful for the routine assay of samples with a wide range of capacity factor values. Further details on these various techniques may be found in a review by Koenigbauer and Majors [96]. 

Capillary columns of the Carbowax type are the standard in most laboratories for routine analysis of fatty acids, but they are of limited value for the analysis of CLA. The two main components, 9-cis, -trans- and 0-trans,l2-cis- >-2 (standards are available from commercial sources), are well separated from each other, but not from other positional isomers. They elute after 18 3n-3, but can overlap with 18 4 (in fish oils). Figure 1.1 illustrates a typical GC separation on a Carbowax phase. More problematically, other geometrical isomers can elute with 20 0 and 20 1 fatty acids if care is not exercised in devising an appropriate temperature program. The various types of geometrical isomers give distinct peaks, but within these groups, positional isomers are not fully resolved, unfortunately. For reasons that are not at all clear, with all polar phases, cis,trans- elute before cis,cis-... 

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