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High Efficiency Post Column Derivatisations of Natural Products using Reaction Flow High
Performance Liquid Chromatography by E. Janaka R. Rupesinghe1
Food Science and Technology Research Group, School of Science and Health, Western Sydney University, Hawkesbury Campus, Londonderry Rd, Richmond, NSW 2753, Australia. 2
1
Australian Centre for Research on Separation Sciences (ACROSS), School of Science and Health, Western Sydney University, Parramatta North Campus, Victoria Rd & Pemberton St, Parramatta, NSW 2150, Australia.
Native Australian Food Plants are well known for their high antioxidant activity. High performance liquid chromatography (HPLC) coupled with post-column derivatisation (PCD) is a useful tool for the fast and efficient screening of antioxidants in complex mixtures. However, PCD usually involves the use of large reaction coils that jeopardise the separation performance gained on the HPLC column. Thus, a new and alternative technique for HPLC-PCD assays was employed, known as Reaction Flow (RF) chromatography, for the analysis of antioxidants and phenolic compounds in extracts from Backhousia citriodora leaves using 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) for the analysis of antioxidants and a dual component reagent for the analysis of phenols comprising of potassium ferricyanide and 4-aminoantipyrene. RF chromatography not only provides a platform for efficient mixing to take place but does so without compromising separation performance. In this study, a number of antioxidant and phenolic compounds were detected using RF-PCD.
1. Introduction
There are countless known Native Australian Food Plants (NAFPs) that have served the indigenous community of Australia as food and medicine for thousands of years [1-3]. As interest in the health benefits of NAFPs has grown in the past decade, the nutritional composition of many plants have been investigated [4]. However, there are still many NAFPs commonly used by indigenous people where very little is known about the nutritional composition [5,6]. A range of NAFPs have been found to contain greater antioxidant content than common fruits, such as, blueberries. The antioxidant and phenolic content of twelve NAFP fruits was investigated by Netzel et al. [1,2], five of which exhibited 3 to 5 fold greater Trolox equivalent antioxidant capacity (TEAC) than that of blueberries, and six of which totalled Folin-Ciocalteu phenolic levels 2.5 to 4 times greater than blueberries [2,7]. Thus, it is evident that NAFPs are a rich source of antioxidant and phenolic compounds [7,8].
Most antioxidant and phenolic assays involve complex sample preparation,
derivatisation reagents, such as, Oxygen Radical Antioxidant Content (ORAC), Ferric Reducing Ability of Plasma (FRAP), DPPH• (2,2-diphenyl-1-picrylhydrazyl) and ABTS• (2,2’-azino-bis(3-ethylbenzothiazoline- 6-sulphonic acid) and analysis by UV absorbance. Such methods when applied at the bench on the bulk sample can only give information about the total antioxidant content and no information gained is about the chemical composition of the samples. For the determination of the presence of antioxidants and their identification other analytical techniques, such as, HPLC with post column derivatisation and HPLC-MS must be employed. These methods can be labour intensive and time consuming, thus, efficient and rapid methods for the screening of antioxidant and phenolic content in complex mixtures is always sort [7].
Post-column derivatisation (PCD) methods of detection are useful for the natural product chemist because they enhance the information that is gained from separations performed in HPLC. Not only do they provide detection, but the derivatisation
process can be specific to functional aspects of a molecule, which then can be related to its bioactivity. Despite these benefits, PCD is hindered by large post-column dead volumes arising because of the need to the use reaction coils, subsequently causing band broadening and loss of separation power [9]. A new technique for efficient PCD analyses has been developed using Active Flow Technology (AFT) columns in Reaction flow (RF) mode [9-12].
Reaction flow chromatography columns employ a special purpose-built four-port end-fitting and a three piece annular frit containing a central porous region, separated from an outer porous region by an impermeable ring. The radial central flow region of the eluent exits the column via a radial central exit port, while the flow near the wall region exits the column via any of the three ports that align with the outer peripheral porous region of the frit.
Therefore the wall flow region and the central flow region are isolated from each other [10]. The portion of flow in the wall region, relative to the central flow region
, Andrew Jones2 , R. Andrew Shalliker2 , and Sercan Pravadali-Cekic2 *
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