Chromatographic principles optimization

The efficient utilization of most, if not all, systematic optimization strategies requires an understanding of basic chromatographic principles. Such an understanding also greatly facilitates the interpretation of the results. 

The parameters of preparative chromatography that can be adjusted by the chromatographer for optimization are listed in Table 16. Other parameters cannot be modified by the user but are rather related to the nature of the chromatography medium. When an optimization routine does not yield the expected results, it is best to switch to another medium, based on either a different mass transfer principle or another matrix material. The adsorption process that occurs at a chromatography surface is very complex and is poorly understood. This is especially true for non-specific adsorption. This is why it is necessary to carry out the optimization with the real solutions. Experiments with artificial samples often do not result in conditions that can be transferred to the real situation. The most important targets for optimization are purity and productivity. After the required purity has been achieved, the productivity can be optimized. Productivity includes costs, column size, and operation time. It also includes the lifetime of the column material. 

The latest trend is to smaller beads in smaller columns, as this saves eluent and shortens the time for a chromatographic analysis. This argument can be correct if only one suitable detector is used. However, these modern small columns are not optimal for a combination of detectors. So-called multiple detection is a combination of some detectors with different measurement principles (differential refractometer, spectral photometer, light-scattering detector, on-line viscometer) behind the last column, mostly in series, seldom in a branched ( parallel ) order. In this way, the tedious preparative fractionation of a polymer sample can often be avoided. 

FIGURE 6.14 (a) Principle of SBCD, elution with five interstitial volumes on 4-cm distance (5x4 cm) is faster than single development on 20-cm distance (thick line), (b) Rp values of sample components plotted as a function of modifier concentration. Optimal concentration (Y) for SBCD (5x4 cm) is lower than for development on the full distance of 20 cm (X). (Modified from Soczewinski, E., Chromatographic Methods Planar Chromatography, Vol. 1, Ed., Kaiser R.E., Dr. Alfred Huetig Verlag, Heidelberg, Basel, New York, 1986, pp. 79-117.)... 

In principle, the analytical results obtained by the GPC spin column/HPLC ESI-MS methodology described in this chapter should be similar to the results obtained using the tandem chromatographic method of GPC/reversed-phase HPLC ESI-MS described in Chapter 3. There are practical advantages for each method. Since each of the chromatographic and mass spectral steps are done serially for the GPC spin column/HPLC ESI-MS methodology, each of the steps can be performed and optimized individually. In the event of mass spectrometer failure, the production of spin column eluate samples can proceed and samples can be stored for future analysis. In contrast, the parallel methodology of tandem GPC/ reversed-phase HPLC ESI-MS requires the simultaneous optimization of multi-... 

The form of the isotherm need not be Langmuir in nature, but in any event, must be experimentally determined in order to identify the true profile of the overloaded peak. In practice, the determination of the adsorption isotherm of each compound to be separated by a preparative chromatographic procedure can be arduous and time consuming. A better alternative might be to design the fully optimized column from basic principles in the manner previously described. 

General aspects of the simplex method. Although the simplex algorithm can in principle be employed for the optimization of any kind or number of parameters of a particular process, for chromatographic applications it appears to be better suited for certain types of a limited number of variables. 

In principle, a longer dwell time leads to an improved signal-to-noise ratio (S/N). In practice, a compromise must be struck. The dwell time at the relevant m/z must be optimized to achieve 10-20 data-points over the chromatographic peak. 

Chapter 7, therefore, deals with model-based design and optimization of a chromatographic plant, where the already selected chromatographic system and concepts are applied. First, basic principles of the optimization of chromatographic processes will be explained. These include the introduction of the commonly used objective functions and the degrees of freedom. To reduce the complexity of the optimization and to ease the scale-up of a plant, this chapter will also emphasize the application of dimensionless parameters and degrees of freedom respectively. Examples for the... 

A. S. Rathore A. Velayudhan, Eds., Scale-Up and Optimization in Preparative Chromatography Principles and Biopharmaceutical Applications. In Chromatographic Science Series. 2003 Vol. 88. 

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