Column overloading, chromatography

Contemporary development of chromatography theory has tended to concentrate on dispersion in electro-chromatography and the treatment of column overload in preparative columns. Under overload conditions, the adsorption isotherm of the solute with respect to the stationary phase can be grossly nonlinear. One of the prime contributors in this research has been Guiochon and his co-workers, [27-30]. The form of the isotherm must be experimentally determined and, from the equilibrium data, and by the use of appropriate computer programs, it has been shown possible to calculate the theoretical profile of an overloaded peak. 

In all modes of chromatography, high sample loads distort peak shapes and cause an overall decrease in efficiency due to column overload. Sample loads may be increased by using organic solvents to enhance the solubility of the sample or by using higher column temperatures to lower the viscosity of... 

Lor a particular analytical separation, each biosolute will have an optimal k] value for maximum resolution with a designated column, flow rate, and mobile phase composition. Similar criteria apply in preparative (overload) chromatography with multicomponent mixtures, where resolution is similarly enhanced following optimization of chromatographic selectivity and zone bandwidth. The conventional approach to process purification with low molecular weight solutes has frequently been based on linear scale-up of the performance of an analytical column system. In these cases, high-resolution separations can be achieved often without the burden of conformational or... 

In analytical applications of liquid chromatography the most common causes of peak asymmetry are mixed mechanisms of retention, incompatibility of the sample with the chromatographic mobile phase, or development of excessive void volume at the head of the column. In preparative applications of liquid chromatography and related techniques, column overload can also contribute to peak asymmetry. The causes of severe peak asymmetry in analytical applications should be identified and corrected because they are frequently accompanied by concentration-dependent retention, non-linear calibration curves and poor precision. In addition, peak asymmetry can significantly compromise column efficiency leading, in turn, to reduced resolution and lower peak capacity (see sections 2.5 and 2.6). 

While an examination of the chromatogram, shows that the 10-mm diameter column was overloaded at the 50-mg sample the data in Table 10.1 indicate excellent recovery independent of sample or column size. In the preparative chromatography nonlinear effects caused by column overload are often observed, " and this affects the separation resolution as sample... 

The solutes can access the whole volume of the stationary phase, not only the surface of a solid stationary phase as in most other chromatographic techniques. Column overload is much more difficult with a CCC column than with a classical preparative liquid chromatography (LC) column of similar volume. Large amounts of a substance can be processed in a single run. Countercurrent chromatography is truly a preparative technique. 

The injector is an interface that achieves reproducible and accurate transfer of a sample volume between the system operator and the chromatographic column in a front (band) as narrow as possible. Usually, for analysis by high-performance liquid chromatography (HPLC), samples are prepared in a liquid state (as a solution in an appropriate solvent or mixture of solvents, obtained as a result of sample preparation of solid or liquid samples). For practical reasons (avoiding column overloading), even for analytes in a liquid state under ambient conditions, dilution in appropriate solvents is more often used. 

Courtesy of the Journal of Chromatography [ref. 1] Figure 1. Theory of Volume Column Overload... 

Preparative chromatography involves theory of some sort in almost every aspect, whether it is for calculation of the performance of a column to determine if it is sufficiently well packed for prolonged use or for a full optimisation. Very often calculations are made to determine the column and particle dimensions which allow operation of a separation at the maximum operating pressure of the equipment to maximise the production from the column. Overloading usually is more controllable if one knows the saturation capacity of the column, since this can allow at least approximate calculation of the appropriate load to try in a separation. Once the sample... 

Preparative chromatography involves the collection of individual solutes as they are eluted from the column for further use, but does not necessarily entail the separation of large samples. Special columns can be designed and fabricated for preparative use, but for small samples the analytical column can often be overloaded for preparative purposes. Columns can be either volume overloaded or mass overloaded. Volume overload causes the peak to broaden, but the retention time of the front of the peak... 

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