Chromatographic theory resolution

Gasparrini, R, Misiti, D., and Villani, C., Chromatographic optical resolution on frani-l,2-diaminocyclohexane derivatives theory and applications, Chirality, 4, 447, 1992. 

The practical application of HPLC is aided by an awareness of the concepts of chromatographic theory, in particular the measurement of chromatographic retention and the factors which influence resolution. 

A general account of chromatographic theory was presented in volume 2 of Encyclopedia of Pharmaceutical Technology.Therefore, the following discussion will focus specifically on GC theory. The separation of the component of a mixture depends upon the column performance (efficacy) and the relative retention capability of the stationary phase (selectivity). The former determines the width of the peaks relative to the length of time a component spends in the column, while the latter determines the relative position of each emerging component (resolution). 

While extremely large numbers of theoretical plates are possible with larger diameter columns (22, 23), calculations from chromatographic theory of the Internal diameters and column lengths necessary to achieve relatively high efficiencies in reasonable analysis times Indicate that column diameters of 50 to 100 ym l.d. are necessary for high-resolution SFC (23). For example, more than 10 effective theoretical plates are possible In less than two hours on 30-m long columns of 50 ym l.d. 

As a rule, chromatographic optimizations are based on the trial-and-error approach, relying on experimentation, and the basic relationships of chromatographic theory. In HPLC [58-62] is used the well-known expression for the resolution... 

The experimental optimization procedures outlined above can be replaced with others based on computer simulations [64,65], which make use of the chromatographic theory and of one or two prior experiments intended to define critical parameters such as the sample, mobile phase, column, temperature, flow-rate and pressure. Simulated chromatograms are obtained for different experimental conditions (column dimensions, particle size, mobile phase composition, flow-rate, temperature, etc.) until the required resolution is achieved. In essence, the procedure is similar to experimental optimization, although the chromatograph functioning is replaced with programming. The information obtained can be checked experimentally or be used for designing new approached to experimental optimization. 

At this point, it is important to note that it is, in fact, misleading to discuss theoretical plates in electrophoresis. The concept is a carry-over from chromatographic theory, where a true partition equilibrium between two phases is the physical basis of separation. In electrophoresis, separation of the components of a mixture is determined by their relative mobilities in the apphed electric field, which is a function of their charge, mass and shape. The theoretical plate is merely a convenient concept to describe the analyte peak shape, and to assess the factors that affect separation. Refer to Appendix 1 for examples on calculating resolution and efficiency. 

The primary objective in any chromatographic separation is always the complete resolution of the compounds of interest, in the minimum time. To achieve this task, the most suitable analytical column (dimension and stationary phase type) has to be used, and adequate chromatographic parameters must be applied to limit peak enlargement phenomena. A good knowledge of chromatographic theory is, indeed, of great support for the method optimization process, as well as for the development of innovative techniques. 

Highspeed SEC Columns The pore volume of the column packing has been shown to be one of the major factors influencing peak resolution in SEC. The reduction of the column length conld, in theory, reduce the time requirements of the separation substantially. However, several limitations predicted by chromatographic theory have to be considered. A study of the influence of column dimensions on fast SEC separations has been reported [9]. It has been... 

In a chromatographic separation, the individual components of a mixture are moved apart in the column due to their different affinities for the stationary phase and, as their dispersion is contained by appropriate system design, the individual solutes can be eluted discretely and resolution is achieved. Chromatography theory has been developed over the last half century, but the two critical theories, the Plate Theory and the Rate Theory, were both well established by 1960. There have been many contributors to chromatography theory over the intervening years but, with the... 

Recalling that a separation is achieved by moving the solute bands apart in the column and, at the same time, constraining their dispersion so that they are eluted discretely, it follows that the resolution of a pair of solutes is not successfully accomplished by merely selective retention. In addition, the column must be carefully designed to minimize solute band dispersion. Selective retention will be determined by the interactive nature of the two phases, but band dispersion is determined by the physical properties of the column and the manner in which it is constructed. It is, therefore, necessary to identify those properties that influence peak width and how they are related to other properties of the chromatographic system. This aspect of chromatography theory will be discussed in detail in Part 2 of this book. At this time, the theoretical development will be limited to obtaining a measure of the peak width, so that eventually the width can then be related both theoretically and experimentally to the pertinent column parameters. 

The elaboration of the most efficient chromatographic systems for the optimization of velocity and resolution of the chromatographic process is necessary for solving different analytical problems. The most important factor in the TLC optimization is the mobile phase composition. Taking into consideration the similarity in the retention mechanism between TLC and PLC, the optimized TLC mobile phase can be transferred to the preparative chromatographic system. There are different accepted models and theories for the separation and optimization of chromatographic systems [19,20,61]. 

After concentration, high-resolution chromatographic purification is usually undertaken. A variety of different chromatographic techniques are available, which separate proteins from each other on the basis of differences in various physiochemical characteristics (Table 3.18.) Detailed description of the theory and practice underlining these systems go far beyond the scope of this text, and are freely available in the scientific literature. 

For any given chromatographic system, there is a limiting charge that can be placed on a column before the resolution is impaired. Loss of resolution from column overload can arise from two causes, either excessive sample feed volume or excessive sample mass. The theory of moderate sample volume overload has already been considered in the applications of the Plate Theory. The theory of excessive sample volume overload will now be discussed. 

In a chromatographic separation procedure the parameters of the chromatographic system (stationary phase, flow, temperature, etc.) have to be selected respectively optimized with respect to some criterion (resolution, time, etc.). In gas chromatography retention data series are published and used for the sttidy of solvent/solute interaction, prediction of the retention behaviour, activity coefficients, and other relevant information usable for optimization and classification. Several clKmometrk techniques of data anal s have been employed, e.g. PCA, numerical taxonomic methods, information theory, and j ttern recognition. 

G. R. Asbury and H. H. Hill, Jr., Evaluation of Ultrahigh Resolution Ion Mobility Spectrometry as an Analytical Separation Device in Chromatographic Terms, J. Microcolumn Sep. 2000,12, 172 H. E. Revercomb and E. A. Mason, Theory of Plasma Chromatography/Gaseous Electrophoresis, Anal. Chem 1975,47,970. 

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