Programmed primary parameters

Of course, the simultaneous optimization of different (primary) program parameters (initial and final composition, slope and shape of the gradient) and secondary parameters (nature and relative concentration of modifiers) may involve too many parameters, so that an excessive number of experiments will be required to locate the optimum. This problem may be solved by a separate optimization of the program (primary parameters) and the selectivity (secondary parameters) based on the concept of iso-eluotropic mixtures (see section 3.2.2). This will be demonstrated below (section 6.3.2.2). However, the transfer of... 

In some cases we may speed up the selection of appropriate primary parameters with the help of programmed analysis, i.e. temperature programming in GC or solvent programming in LC. Another useful scouting technique may be thin layer chromatography (TLC). Possibilities for establishing the appropriate values of the primary parameters will be discussed in section S.4. 

Ideally, the eventual chromatogram is arrived at under constant conditions (i.e. isothermal in GC, isocratic in LC). Nevertheless, it may be impossibble to achieve a signal (peak) for each component in the sample under constant conditions. In that case it may be necessary to use programmed analysis methods, in which one of the (primary) parameters is varied ( programmed ) during the separation. Programmed analysis will be discussed in chapter 6. 

We have seen in chapter 3 (table 3.10 b-d) that the composition of the mobile phase is a primary parameter in various forms of LC (LLC, RPLC, LSC). Gradient elution is only relevant for the latter two techniques, because the LLC system is not compatible with mobile phase gradients. Figure 6.6a shows a typical gradient program for LC. The complete program can be divided into a number of segments. 

The situation in programmed analysis is similar to the one described above for chromatographic elution under constant conditions, in that retention and selectivity may be optimized more or less independently. However, under constant elution conditions the optimization of the retention only involves adapting the primary parameters such that all capacity factors fall into the optimum range (1 programmed analysis the optimization of the retention involves optimizing the characteristics of the program (initial and final composition, slope and shape) in conjunction with the physical parameters (e.g. flow rate and column dimensions, see section 3.6). 

Parameters for the optimization of programmed analyses in various chromatographic techniques. Primary parameters may be used to optimize the program parameters (initial and final conditions, slope and shape). Secondary parameters may be used to optimize the selectivity. 

Method Primary parameter(s) (program) Secondary parameter(s) (selectivity)... 

Optimization of the primary parameters of the gradient program may only lead to sufficient separation if the selectivity is sufficiently large. If the a values between one or more pairs of solutes are low, resolution may be enhanced by a reduction of the programming rate and by increasing the number of plates. However, this will be at the expense of increased analysis times and the resolution will never be better than under constant elution conditions. 

For the interpretive optimization of the primary (program) parameters in the programmed analysis of complex sample mixtures it may well be sufficient to optimize for the major sample components. This may be done if it is assumed that the primary parameters do not have a considerable effect on the selectivity, so that if the major sample components are well spread out over the chromatogram, the minor components in between these peaks will follow suit automatically, and if it is assumed that the minor peaks are randomly distributed over the chromatogram. The major chromatographic peaks can be separated to any desired degree if optimization criteria are selected which allow a transfer of the result to another column. 

Simplex optimization of the primary parameters in programmed temperature GC analysis is possible. 

For programs where the primary assay is a binding type assay, it is important to continue to evaluate the functional response of newly synthesized compounds. Changes in functional potency should correlate with changes in binding potency. Other parameters that may need to be tracked are efficacy and agonism vs antagonism. 

The basic SFC system comprises a mobile phase delivery system, an injector (as in HPLC), oven, restrictor, detector and a control/data system. In SFC the mobile phase is supplied to the LC pump where the pressure of the fluid is raised above the critical pressure. Pressure control is the primary variable in SFC. In SFC temperature is also important, but more as a supplementary parameter to pressure programming. Samples are introduced into the fluid stream via an LC injection valve and separated on a column placed in a GC oven thermostatted above the critical temperature of the mobile phase. A postcolumn restrictor ensures that the fluid is maintained above its critical pressure throughout the separation process. Detectors positioned either before or after the postcolumn restrictor monitor analytes eluting from the column. The key feature differentiating SFC from conventional techniques is the use of the significantly elevated pressure at the column outlet. This allows not only to use mobile phases that are either impossible or impractical under conventional LC and GC conditions but also to use more ordinary... 

None of the programs can predict kinetics, that is, the rate of reaction, the activation energy, or the order of the reaction. These parameters can only be determined experimentally. Except for CHETAH, the primary use of the programs is to compute the enthalpies of decomposition and combustion. In fact, acid-base neutralization, exothermic dilution, partial oxidation, nitration, halogenation, and other synthesis reactions are not included in the programs except for CHETAH, which can be used to calculate the thermodynamics of essentially any reaction. 

In this chapter we will take a look at some aspects of programmed analysis, particularly those which bear relation to the chromatographic selectivity. The parameters involved in the optimization of programmed analysis will be divided into primary or program parameters and secondary or selectivity parameters. These parameters will be identified for different chromatographic techniques and procedures will be discussed for the optimization of both kinds of parameters. 

An important characteristic of primary optimization parameters is that whereas they have a large effect on the capacity factors of the solutes, they have a relatively minor effect on the selectivity (a). This implies that the factors involved in optimizing an analysis program (the initial and final conditions, programming rate and shape of the program) do not affect the chromatographic selectivity to a large extent. This will be even more true... 

The primary and secondary parameters that may be used for the optimization of the program and the selectivity in programmed analysis, respectively, are listed in table 6.3 for the various chromatographic techniques. 

It can be seen in table 6.3 that the optimization of selectivity in programmed temperature GC involves variation of the (nature or composition) of the stationary phase. To vary this parameter, a different column and re-optimization of the (primary) program parameters will be required. This is clearly not a very attractive proposition and therefore the optimization of programmed temperature GC is usually restricted to optimizing the program. 

Simplex optimization of the primary (program) parameters in programmed temperature GC analysis has been demonstrated [612]. A systematic sequential search [613] may be used as an alternative. The Simplex method may be used to optimize a limited number of program parameters, whereas the latter approach was developed for the optimization of multisegment gradients. The use of interpretive methods has so far only been suggested [614,615]. 

The (primary) program parameters may be used to optimize the separation in programmed solvent LC in a non-selective way. Since this involves optimization of the... 

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