Chromatographic analysis plate number

The checkers found that a fraction, b.p. 45-71° (18 mm.), had the following spectral properties infrared (carbon tetrachloride) no absorption in the 3300-1600 cm.-1 region attributable to OH, C=0, or C=C vibrations proton magnetic resonance (chloroform-d) <5, multiplicity, number of protons, assignment 3.1-4.2 (multiplet, 4, CH—Cl, CH—O, and C//2—O), 1.0-2.5 (multiplet, 7, GH3 and 2 x C//2)-Thin layer chromatographic analysis of this fraction on silica gel plates using chloroform as eluent indicated the presence of a major component (the cis- and fraus-isomers), Rf = 0.60, and a minor unidentified component, Rf = 0.14. 

For all chromatographic techniques the time needed for an analysis can be predicted from plate number N, reduced plate height h, characteristic length d, reduced velocity v, diffusion coefficient D, and retention factor of the last eluted peak ust ... 

The linear velocity of the mobile phase is an important determinant of both speed of analysis and the chromatographic plate number, which affects peak resolution. Both are critical features of the values of chromatographic analysis. 

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. 

Thin-layer chromatography (TLC), sometimes also called planar chromatography, employ a stationary phase immobilized on a glass or plastic plate and an organic mobile phase. It is a rather old technique whose application in residue analysis has been limited in the past by poor chromatographic resolution, inadequate selectivity, and insufficient sensitivity (49). This was due to inherent problems in the quality of the available stationary phase materials and in the uniformity of the layers prepared. Today, the availability of affordable, precoated plates with acceptable performance and consistency has led to the general acceptance of TLC as an efficient procedure for residue analysis (50). The method is used preferentially when analysts must process large numbers of samples in a short period of time (51). 

Packed columns are still used extensively, especially in routine analysis. They are essential when sample components have high partition coefficients and/or high concentrations. Capillary columns provide a high number of theoretical plates, hence a very high resolution, but they cannot be used in all applications because there are not many types of chemically bonded capillary columns. Combined use of packed columns of different polarities often provides better separation than with a capillary column. It sometimes happens that a capillary column is used as a supplement in the packed-column gas chromatograph. It is best, therefore, to house the capillary and packed columns in the same column oven and use them routinely and the capillary column is used when more detailed information is required. 

Another factor that contributes to Rs is the plate count N. However, we have seen in section 1.5 that optimization through an increase in N is expensive, not only in terms of equipment and columns, but also in terms of analysis time. Therefore, as long as the shape of the peaks and the plate height (length of the column divided by N) are satisfactory, we should not rely on the number of plates for optimization, unless as a last resort. Methods which may be used to optimize the chromatographic system with respect to the required number of plates will be described in chapter 7. 

The second field of application involves the occasional analysis of wide range samples. In this category we find samples which only occur in the laboratory occasionally and in small numbers, so that only a small number of chromatographic analyses have to be performed. For samples of this kind it is usually sufficient to realize a separation and it is not rewarding to try and optimize the selectivity, not even if the analysis time is rather long and the required number of plates high. 

The optimization of the efficiency of the chromatographic system involves the selection of a column with a sufficient but not excessive number of plates. If a column is used with twice the required number of plates, then the observed resolution would exceed the required value by 40%, but both the analysis time and the pressure drop over the column would be double the required value. Moreover, the sensitivity of the detection would be decreased by 40% (see section 7.3). It is clear that we should aim to use a column with the optimum (i.e. the required) number of plates. 

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