Chromatographic processes resolution

The separating power of a chromatographic process arises from the development of many theoretical plates to achieve adsorption equiUbrium within a column of moderate length. Even though the separation factor between two components may be small, any desired resolution may be achieved with sufficient theoretical plates. 

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]. 

At present, the purification by chromatographic processes is the most powerful high-resolution bioseparation technique for many different products from the laboratory to the industrial scale. In this context, continuous simulated moving bed (SMB) systems are of increasing interest for the purification of pharmaceuticals or specialty chemicals (racemic mixtures, proteins, organic acids, etc.).This is particularly due to the typical advantages of SMB-systems, such as reduction of solvent consumption, increase in productivity and purity obtained as well as in investment costs in comparison to conventional batch elution chromatography [1]. 

The ability of a chromatographic process to separate or resolve two similar compounds (Figure 3.10) is measured as the resolution index (Rs). 

The advent of multichannel detection methods, such as photodiode array detection (DAD), for monitoring chromatographic processes has opened up new prospects for the resolution of overlapping peaks. 

The major advantage of this technique over the one-dimensional system approach is the increased resolution and higher spot capacity. The two-dimensional technique has the potential of separating 150-300 components [35]. This is because the whole area of the plate is used, which increases the resolving power by almost the square of that obtained by onedimensional development. This procedure can also be used for determining whether decomposition occurred during the chromatographic process. Usually one sample per plate (never more than four samples per plate) can be analyzed. 

The advantages of HPLC over classical chromatographic methods stem from the employment of a precision instrument that utilizes high-performance columns with concomitantly high analytical speed and resolution and affords total control over the chromatographic process and sensitivity of analysis. In a way, the recent emergence of capillary electrophoresis (CE) follows the same patterns electrophoresis, a well-established and widely used method of biopolymer analysis, is carried out... 

Band broadening. The dilution of the sample with mobile phase during the chromatographic process. For example, a 10-ju.L volume is injected, and by the time it reaches the detector, the sample is spread throughout a 200-//.L volume. Band broadening in some cases can affect resolution of... 

The goal of the chromatographic process is the separation of one component of a mixture from another. The measurement of the degree of separation between two components is resolution, defined as the distance between the... 

The ideal chromatographic process is one in which the components of a mixture form narrow bands which arc completely resolved from one another. The narrowness of a band or peak is a measure of the efficiency of the process whilst resolution is assessed by the ability to resolve the peaks of components with similar r or R, values. 

The final area of concern in the chromatographic process is the size of the fractions. Decreasing the fraction size does not increase the resolution of the column, but does allow realization of the full resolution capability of the column to be expressed. 

From the practical point of view, it is not important which mechanism controls the chromatographic process during a gradient elution, but how the changes in experimental conditions affect the resulting retention times of separated compounds. Typically, conditions that support a strong retention are applied at the start of the run, and conditions enhancing an elution are applied more and more over the course of the separation. This allows for sufficient resolution of the early eluted. 

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