Column evaluation efficiency

A discussion of column evaluation may seem inappropriate to biochemists who have traditionally packed gel SEC columns themselves and run them without testing. Such columns would eventually be unpacked when the performance degraded, and the packing material would be cleaned or discarded. The quality of the column was not significant in relationship to the time necessary to evaluate it. HPSEC columns are usually more expensive than their classical counterparts, and consequently, users are concerned about quality assurance, column longevity, sample recovery, and extension of column lifetime. Because there are major differences in columns from different manufacturers, users must have a qualitative basis for column comparison. Columns should also be evaluated periodically during their use so that loss of efficiency can be monitored and columns cleaned, when necessary, to restore resolution. 

A quantitative means of evaluating column efficiency that treats the column as though it consists of a series of small zones, or plates, in which partitioning between the mobile and stationary phases occurs. 

With soft gels, column packing has often been plagued with such problems as inferior reproducibility and excessive time requirements. These problems are alleviated with physically stable Toyopearl HW media. However, an improperly packed column can have significantly reduced efficiency. The two key variables for the successful packing of Toyopearl HW media, packing velocity and column size, have been evaluated to determine the optimal packing conditions. 

Many operating variables, such as sample volume, flow rate, column length, and temperature, must be considered when performing any separation. The relative importance of these variables for Toyopearl HW-55F resin columns has been specifically evaluated. For example. Fig. 4.47 shows the relationship between column efficiency, or height equivalent of a theoretical plate (HETP),... 

Each SynChropak column is tested chromatographically to assure that it has been packed according to specifications. For SynChropak GPC columns, a mixture of a high molecular weight DNA and glycyltyrosine, a dipeptide, is used to evaluate internal volume and efficiency. The mobile phase used for the test is 0.1 M potassium phosphate, pH 7, and the flow rate is 0.5 ml/min for 4.6-mm i.d. columns. Minimum plate count values and operational flow rates are listed in Table 10.4 for 4.6-mm i.d. columns of all supports and the various diameters of the SynChropak GPC 100 columns. 

Column efficiency (number of theoretical plates) As in batch chromatography, one needs to determine the efficiency of the column in order to evaluate the dispersion of the fronts due to hydrodynamics dispersion or kinetics limitations. The relationship of N proportional to L can be expressed in terms of the equation for height equivalent to a theoretical plate (HETP) as ... 

Note that with this procedure, the effect of the number of theoretical plates arailable can be determined. In an existing column where the number of trays are fixed, the theoretical trays can be obtained by evaluating an efficiency for the system. 

The separation nuaber is the only column efficiency par2uaeter that can be deterained under teaperature progr2uued conditions [45,46]. The critical parameters that aust be standardized to obtain reproducible SM values for coluans of different length are the carrier gas flow rate and the temperature program. The SN is widely used as part of a standardized test method to evaluate the quality of open tubular columns for gas chromatography (section 2.4.3). 

Retrospective validation uses historical information gathered in actual process runs to evaluate the process. For example, batch records can provide extensive data on column performance and analytical data of fractions and final product can provide valuable information on the efficiency of the chromatographic steps in removing contaminants. Chapman67 cautions that while retrospective validation is a valid and valuable approach, it is not meant to be retroactive — validation must be done before product is released to market. 

Analysis was performed on an ES-Ovomucoid column for stereoselectivity assessment, and for MS/MS, an X-Terra MS C18 column (2.1 x 100mm, 5 fan) was used. Figure 1.17 shows the wash and elution fractions from the SPE in a 384-well plate. The SPE conditions evaluated are listed in the table below the figure. The binding of the drug to the affinity sorbent in a 96-well plate was less efficient than the 384-well plate because the sorbent formed a disk on the former and a column on the latter. The efficiency is reflected in the >95% recoveries achieved with the 384-well format. 

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