Chromatography

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Chromatography is a separation process that involves partitioning a protein (or any other soluble analyte) between an insoluble stationary phase and a mobile phase that passes over its surface. A simple, but complete, chromatography system is shown in the diagram below.

Chromatogram: the record of a separation produced by a recorder or integrator based on the signal obtained from the detector.

tm: the time required for the mobile phase to travel the entire length of the column. For example, if your column has a total volume of 100 mL and the stationary phase occupies 40 mL, then the mobile phase occupies 60 mL. If the mobile phase were flowing at a rate of 5 mL/min, then it would take 12 min for the mobile phase to travel from the injector to the detector through the column. The tm can sometimes be determined by identifying a small peak early in the chromatogram resulting from a slightly different solvent in the mobile pause that comes from the injected sample. The tm can also be determined by checking the elution time of a sample that is not retained by the stationary phase at all. In the case of a cation exchange resin, a negatively charged protein could be used to determine the tm. It should also be noted that other factors, including the length and diameter of tubing that connect the injector and detector to the column could affect the tm values.

When an analyte of any kind is undergoing chromatography, it is moving at the same rate as the mobile phase when it is in the mobile phase and it is stationary when it is bound to the stationary phase. In other words, all analytes spend time equivalent to tm in the mobile phase. Differences in the amount of time they spend bound to the stationary phase enable them to be separated from each other.

tr: the retention time, or the time required for a protein to elute from a column.

tr': the adjusted retention time, which describes the amount of time the protein spent bound to the stationary phase during a separation.

k': the capacity factor, can be determined by the equation below. k' is used to compare chromatographic behavior of an analyte on the same column when it is attached to two different systems. In that case, the tm values may vary, but the k' values should remain the same. The capacity factor also indicates the number of column volumes of mobile pause that must be used to elute an analyte. A k' value of 1 indicates that 2 column volumes of mobile phase must be used, while a k' value of 5 indicates that 6 column volumes must be used.

Vr: retention volume is proportional to retention time, based on the constant flow rate of a chromatography system. If the retention time is 5 min and the volume flow rate (uv) is 5 mL/min, then Vr is 25 mL.

Vm: mobile phase volume (also known as void volume)

Relative retention (a): ratio of capacity factors (k) or adjusted retention times (tr) for two analytes. By convention, a > 1.

Resolution: the degree of separation between two analytes on a chromatography system. In the above chromatogram, compound A and compound B are said to be "baseline resolved" whereas compound B and compound C are not well resolved. Resolution can be calculated from the following equation.

where wave is the average of the two peak widths in units of time. A second equation for resolution is useful for its predictive value in designing separations:

where N is the number of theoretical plates for the separation.

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