Liquid Chromatography - Liquid Chromatography Applications 9

The column was 50 cm long and 7.6 mm wide giving a total column volume of about 22.7 ml. This conflicts with the value for the dead volume which appears to be about 22 ml (the retention time of 22 min. at a flow rate of 1 ml/min.). Consequently, it would appear that the resin occupied no volume in the column. This paradox could be explained on the basis that the separation was not achieved solely by exclusion. The dispersive and polarizable nature of the resin was contributing dispersive and possibly slight polar interactions with the solutes increasing their retention beyond that expected from exclusion alone. This would be supported by the fact that the mobile phase contained no solvent and consisted of a buffer solution containing 0.1 M sodium phosphate and 0.3 M sodium chloride (pH 7.0) which would have minimum dispersive interactions with the solute. In contrast, the hydrocarbon matrix of the styrene gel would be capable of exerting strong dispersive interactions with the dispersive groups present in the proteins. The use of mixed retention systems to achieve the separation is perfectly acceptable and occurs in most distribution systems. The stationary phase was GFA-50, the number 50 referring to the columns length. The pore size was defined in terms of the minimum molecular weight of a totally excluded solute which was given as 300,000 and the material was specifically prepared for exclusion chromatography.

The analyst should be aware that generally, when working with large molecules (particularly proteins), column efficiencies may, under some circumstances, be as little as 10% of the expected value and this must be taken into account when choosing the column and the phase system.