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liquid chromatography column - Scott R.P.W.

Scott R.P.W. liquid chromatography column - John Wiley & Sons, 2001. - 144 p.
Download (direct link): liquidchromatographycolumntheory2001.djvu
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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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toxicity' However, it must be remembered that the elution characteristics of methanol and acetonitrile, although similar, are certainly not identical. Furthermore, it should be emphasized that the solvent optimization procedure must take place after the individual solvents have been chosen and, if subsequently changed again, then the optimization procedure must be repeated. The elective variables can be summarized as follows,
Elective Variables
1/ The separation ratio of the critical pair.
2/ The capacity ratio of the first solute of the critical pair.
3/ The capacity ratio of the last eluted solute.
4/ The viscosity of the solvent
5/ The diffusivity of the first solute of the critical pair in the mobile phase
Column Specifications and Operating Conditions
Employing the conditions defined in the three data bases and the appropriate equations derived from the Plate and Rate Theories the physical properties of the column and column packing can be determined and the correct operating conditions identified. The precise column length and particle diameter that will achieve the necessary resolution and provide the analysis in the minimum time can be calculated. It should again be emphasized that, the specifications will be such, that for the specific separation carried out, on the phase system selected and the equipment available, the minimum analysis time will be absolute No other column is possible that will allow the analysis to be carried out in less time.
The optimum mobile phase velocity will also be determined in the above calculations and, as the minimum radius will also be calculated in order to achieve minimum solvent consumption and maximum mass sensitivity, the optimum flow rate can also be identified. The column specifications and operating conditions can be summarized as follows.
Column Specifications and Operating Conditions
1/ The optimum column length. 2/ The optimum column radius.
3/The optimum particle diameter.
4/ The optimum mobile phase linear velocity.
5/Mobile phase flow rate.
Analytical Specifications
The analytical specifications must prescribe the ultimate performance of the total chromatographic system, in appropriate numerical values, to demonstrate the performance that has been achieved. The separation of the critical pair would require a minimum column efficiency and the number of theoretical plated produced by the column should be reported. The second most important requisite is that the analysis should be achieved in the minimum time and thus the analysis time should also be given. The analyst will also want to know the maximum volume of charge that can be placed on the column, the solvent consumption per analysis, the mass sensitivity and finally the total peak capacity of the chromatogram. The analytical specifications can be summarized as follows.
Analytical Specifications.
1/ Column efficiency in theoretical plates
2/ Analysis time.
3/ Maximum volume of charge.
4/ Solvent consumption per analysis.
5/ Overall mass sensitivity.
6/ Total peak capacity.
It is obvious that such a protocol would not be employed to design a column for a single analysis or even for a few dozen analyses. The optimization procedure entails a considerable amount of work and therefore, would only be justified for a routine analysis that was repetitive and would be carried cut regularly many times day. Under such circumstances, the use of the protocol to construct the fully optimized column could be economically advantageous, increasing the sample throughput dramatically and significantly reducing operating costs.
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Unfortunately, some of the data that is required to calculate the specifications and operating conditions of the optimum column, involves instrument specifications which are often not available from the instrument manufacturer. In particular, the total dispersion of the detector and its internal connecting tubes is rarely given. In fact, under some circumstances the dispersion or volume of the sensing cell alone is reported, implying that the overall detector dispersion results solely from the detector cell. This is particularly misleading as, in many detectors, the major dispersion takes place in the internal connecting tube to the cell not in the cell itself. In a similar manner, a value for the dispersion that takes place in a sample valve is rarely provided by the manufactures. The valve, as discussed in a previous chapter, can make a significant contribution to the extra column dispersion of the chromatographic system which, as has also been shown, will determine the magnitude of the column radius. Unfortunately, it is left to the analyst to measure this data and a procedure for measuring the extra column dispersion has already been given in the previous chapter.
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