56 February / March 2019
the sensitivity of the main analyte, a large injection volume is used. For the impurity analysis the main analyte being detected is the impurity not the main component within the sample.
Conclusion
Equation 3 Where
M – mass loaded onto column K – retention or capacity factor
N – chromatographic efficiency of the system (column) V0 – column dead volume
Prep Chromatography
In some forms of chromatography, column overloading is acceptable and indeed the columns are specifically run in an overloaded state to ensure that an optimum yield is obtained. The obvious example here is the field of preparative and process chromatography where the separation technique is used to allow for isolation of one or more of the analytes. The aim of this form of chromatography is to isolate specific analytes and although resolution is important as this will affect the final purity of the isolated analyte, it is typically the amount of analyte that can be separated per unit time that is the critical component. This means that resolution and amount that is separated have to both be considered, whereas most analytical separations it is the speed of analysis and the resolution that are critical parameters.
Other areas of chromatography where overloading may routinely occur is in trace and also impurity analysis, where in order to achieve
Reducing column diameters to reduce sample consumption, reduce solvent consumption and also to improve sensitivity needs to be done with care as scaling has to be done on the amount of sample injected as well as the sample volume. For columns that are equivalent in the type of stationary phase that is being used, and in length, a simple squared relationship exists between the different diameter columns and the injection volume. Thus reducing the diameter of the column by a factor of two will require a reduction in the injection volume of a factor of 4. Where different stationary phases and column lengths are being used the relationship is more complicated.
Other important variables to consider when looking at reducing overloading effects are the injection solvent, the mass loading and also the nature of the molecule that is being separated. Care has to be taken with ionisable compounds to ensure that peaks are not excluded from the pore structure. It is also useful to know that highly elutropic solvents can be used but only in small measure.
References
1. K.K. Unger. Porous Silica: Its Properties and Use as Support in Column Liquid Chromatography. Elsevier. Amsterdam. 1979. D. 170.
2. Handbook of Analytical Separations Vol 4 Bioanalytical Separations ed. Ian D. Wilson p. 287
3. Chromatography Today, 20, Nov 2014
Versatile 96-well Multi-Tier Microplate System for High Throughput Chromatography
The patented 96-well Multi-Tier Microplate System (MTP) from Porvair Sciences and sister company JG Finneran is a unique device providing analysts with the convenience of an ANSI/ SLAS microplate footprint but loaded with inert glass vials.
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More information online:
ilmt.co/PL/l30k
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