Chomatographic Separations

Raw copy from BASIC Biochemical Engineering. See student term project about chromatography.
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Chromatography is based on differential migration rates of components of a liquid or gas as it moves past adsorptive materials. Practically any soluble or volatile substance can be purified by chromatography. Some combination of adsorbent, conditions, and carrier fluid will allow you to apply a mixture of materials to a column of adsorbent and to flush so that differential migration rates separate the materials before they exit.

The flushing in conventional chromatography greatly dilutes the material, and the fractions usually require another step for concentration. A newer method called displacement chromatography elutes with some compound that has greater affinity for the adsorbent. Fractions of eluant can be considerably more concentrated than the original solution applied to the column.

Affinity chromatography uses ligands with high specificity for certain compounds based on antigen-antibody, enzyme-substrate, enzyme-cofactor, or special biochemical attractions such as the protein avidin for the vitamin biotin. Affinity chromatography is often able to isolate quite pure product from a very crude mixture. Expensive affinity agents are regenerated and reused many times. In some cases, the attraction is so strong that the adsorbent can be added batchwise and there is little relationship to chromatography. Batch techniques scale up well but add laborious steps for collection, elution, and regeneration of the affinity agent.

Scale up of chromatography has benefited greatly from improvements in the materials and from good engineering. A few years ago, the most product that could be expected from a single column operated over and over was only a few hundred grams per day. There were limits to the column diameter and to loading (Bungay, 1978). Today there are columns that provide several kilograms of product per day, an amount nicely matched to the needs for some pharmaceutical products but still far too small for others. For production-scale runs, it is essential to use larger columns to achieve reasonable yields at affordable labor costs. There are trade offs of resolution, yields, and costs.

Column chromatography can function with quite impure feed streams, but an undesired byproduct in high concentrations may give peaks that overlap with those of the desired product. Loading relates to capacity of the adsorbent, and overloading results in dispersion through the column and little or no purification. A run starts with concentrate at the top of the column, then a solvent (eluant) flushes materials through the column. Molecules have different attractions to the adsorbing material and move at different rates through the column to its exit. Concentrations at the exit from the column show peaks with tails. The peaks of two materials may be too closely spaced for resolution, and the tail of one may overlap with the rise of another. Fractions of high purity are combined and sent to a final step such as freeze drying. Low purity fractions may be discarded, but the fractions of intermediate purity from chromatography are reworked.

With light loading of small columns, resolution of peaks is often excellent so that very high purity is obtained. Calibration with known amounts of reference compounds allows chromatography in small columns or in thin layers or in paper to be of great value for analysis. The loading can be light when the detection scheme is sensitive. Heavier loading produces longer tails so that one material contaminates another. The dispersion of flow in large columns can lead to severe cross contamination of materials.