Column maintenance

When reversed-phase columns are used for the analysis of enzymatic reactions, many of the components of the reaction may become bound to the packing material. As a result, the debris may alter the retention time, chromatographic profiles, or both of subsequently injected molecules. Types of column malfunction include peak splitting or the appearance of a shoulder, loss of baseline resolution, broadening of peaks, particularly at their base, or both and an increase in back pressure. To some extent, all these symptoms may be traced to material that adhered to the column and was not removed during the methanol wash. 

If divalent metals are suspected as the cause of the peak splitting, washing the column with 100 mL of 10 m M EDTA in 10 mM phosphate at pH 5.5 may help eliminate the problem by removing the metal. 

Many components that bind can be eluted by changing the pH of the mobile phase. Thus, a wash consisting of 200 mL total volume, at 2 mL/min, of 100 mAf phosphate solution ranging in pH from 2 to 8 is frequently useful. Finally, if an increase in back pressure is suspected to be a result of contamination from bound protein, washing the column with at least 100 mL of 6 M urea in 20 mM phosphate (pH 7.8) may eliminate the problem. Again, the return of back pressure to normal values can be taken as a sign of success of any one of these steps. 

We have found urea to be a poor wash solution on columns packed with reversed-phase packings. Dimethyl sulfoxide (DMSO) has been useful in some cases. 

In addition, a gradient progressing from 100% methanol through a series of less polar, more organic solvents such as carbon tetrachloride will serve to 

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The problem of column maintenance, particularly when the column is used for enzyme assays, is discussed, cleaning solutions are recommended, and a method for monitoring column performance described. 

Column maintenance will often require washing the column with chelators, denaturants, organics, or salt solutions of high concentration, all designed to remove column-bound debris that is not removed by routine washing. 

The column is arguably the most important component in HPLC separations. The availability of a stable, high-performance column is essential for developing a rugged, reproducible analytical method. Performance of columns from different vendors can vary widely. Separation selectivity, resolution, and efficiency depend on the type and quality of the column. Proper column maintenance is the key to ensure optimum column performance as well as an extended column lifetime. It ensures stability of column plate number, band symmetry, retention, and resolution. The major issues related to column performance and maintenance are discussed here. 

Proper column maintenance is very important to ensure optimal performance and extended column lifetimes. There are common procedures that apply to all columns, e.g., avoiding mechanical or thermal shock. There are procedures that are column specific, such as avoiding chloride-containing mobile phases to prevent halide cracking if the column tubing and frits are made of stainless steel (especially at low pH). Nonetheless, columns made with stainless steel tubing and packed with silica-based stationary phases are the most commonly used in HPLC. Thus problems associated with these columns and how to prevent such problems by proper column maintenance will be discussed here. 

Ender, C., and D. Laird, Minimize the Risk of Fire During Column Maintenance, Chem. Engr. Progress, 99, (9), 54 (September 2003). 

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