A technique in chemical and molecular analysis : reaction flow chromatography

Abstract

High performance liquid chromatography (HPLC) coupled with post column derivatisation (PCD) is a powerful technique available to the analytical chemist. However, traditional HPLC-PCD techniques require the use of large volume reaction coils in order to provide time for the column effluent to mix with the PCD reagent(s) and the reaction to take place. These high volume reaction coils are counter-productive to the performance gains obtained from modern HPLC columns. During reaction flow (RF) chromatography no reaction loops are required as the mixing of effluent and PCD reagent(s) occurs in a highly efficient process under pressure within the outlet frit of the column. In addition, RF chromatography has the ability to multiplex multiple detection modes both derivatised and underivatised with no detection delay making peak matching easier. The FRAP-PCD reaction was converted for use as a RF chromatography method. RF chromatography was found to be advantageous over traditional PCD techniques in terms of signal to noise ratio, limits of performance, precision and linear range. The FRAP RF chromatography was then utilised for the analysis of complex samples represented by various teas using a modern HPLC column only 30 mm in length as well as high throughput screening analysis. A second RF chromatography assay for the analysis of phenolics using 4-aminobenzoic acid and potassium ferricyanide was performed on the same tea samples using the same column as used with the FRAP reagent. Since many antioxidants are phenolics it would be expected that similar information would be obtained using both assays. However, GAFA analysis showed correlation between the data series in the ballpark of results obtained for many two-dimensional separations showing how users need to be aware of differing information that is gained from differing derivatisation reactions. In addition, the RF-PCD analysis for phenolics was successfully employed for analysis using narrow-bare columns. RF chromatography can therefore be used with modern HPLC columns that are short in length as well as narrow bore. In addition, high throughput screening utilising RF chromatography can be performed. The thesis concludes that by utilising RF chromatography, PCD techniques can be performed with modern HPLC columns and separation techniques.

Date of Award	2021
Original language	English