Reaction flow chromatography for rapid post column derivatisations: the analysis of antioxidants in natural products.

The analysis of antioxidants from complex samples is conveniently achieved using liquid chromatography, which provides sample fraction, coupled with an on-line antioxidant assay, which provides detection. One particularly useful on-line antioxidant assay that has routinely been coupled with HPLC involves the diphenylpicrylhydrazyl radical (DPPH), which provides a positive test for phenolic antioxidants through a decolorisation of the DPPH reagent. A limitation of this assay, however, is the need to employ a reaction coil, which is often large with respect to the peak volume, consequently adding substantial band broadening to the separation. In this study we introduce a new concept that can be employed for systems requiring post column derivatisations, such as the DPPH assay. We have termed this 'reaction flow' chromatography, whereby, the derivatisation reagent can be added directly into one of the outlet ports of a parallel segmented flow column. Subsequently, the mixing between the derivatising reagent and the solute is very efficient removing the need to employ reaction coils. The concept is tested here using the DPPH assay for the analysis of antioxidants in samples derived from natural origin.

Gradient elution chromatography with segmented parallel flow column technology: a study on 4.6 mm analytical scale columns.

A new column format known as parallel segmented flow has recently been introduced, whereby improvements in column performance are observed. These improvements are achieved via the separation of eluent from the column core from that of the column wall region. The segmentation of flow is accomplished immediately as the eluent exits the column through the use of a multi-channel end fitting. The ratio of flow exiting through the column central port relative to the peripheral ports, known as the segmentation ratio, can be tuned to optimise chromatographic performance. Investigations into the use of parallel segmented flow chromatography columns have demonstrated increased sensitivity and theoretical plates in analytical scale isocratic separations, but so far no studies have detailed the performance of these columns in gradient elution. The current study addresses the performance of parallel segmented flow columns in gradient elution, detailing the reproducibility of the gradient at various segmentation ratios and compares the performance to conventional columns. The study found that there was no observable difference in the gradient shape, or reproducibility of the gradient profiles generated at any segmentation ratio, tested on three different types of stationary phases. A separation of an 11-component test mixture verified that the primary advantage of parallel segmented flow columns was that the peak volume was reduced in proportion to the segmentation ratio.

Parallel segmented flow chromatography columns: conventional analytical scale column formats presenting as a 'virtual' narrow bore column.

Narrow bore columns find advantage in HPLC applications when volumetric flow is important, For example, for detection processes that are volume limited. Yet there are significant drawbacks to narrow bore columns. Due to their small column volume relative to analytical scale columns, narrow bore columns are more affected by system dead volume. In addition the wall effect and the variation in packing density from the centre to the wall are more significant in these columns relative to larger scale analytical columns. In this study we operate a 4.6mm i.d. parallel segmented flow column in such a manner that it emulates 2.1mm i.d. and 3.0mm i.d. columns. By using a parallel segmented flow column in this way, it was possible to combine the benefits of narrow bore and analytical scale columns.

Effects of acid treatment on the trace metal content of chromatographic silica: bulk analysis, surface analysis and chromatographic performance of bonded phases.

A series of studies has been carried out on the effect of refluxing silica chromatography particles for 0.5 h and 18 h in water, dilute hydrochloric acid and dilute hydrofluoric acid. The bulk and surface trace metal concentrations were measured by inductively-coupled plasma atomic emission spectroscopy, static secondary ion mass spectrometry (SSIMS) and X-ray photoelectron spectroscopy. Diffuse reflectance Fourier transform infrared spectroscopy was used to determine changes in 'isolated" and "bonded" silanol groups. The chromatographic behaviour of a series of weakly basic analytes was investigated on C8 and C18 bonded phases manufactured from the acid-treated silicas. The different reflux treatments all resulted in a reduction in the numbers of isolated silanols compared with the untreated silica and SSIMS analysis suggested that the HF-treated silicas had undergone a more efficient surface rehydroxylation. Bulk trace metals were removed most effectively by the HF treatment, with the multivalent elements (Ti and Al) being the most difficult to remove. Surface specific analysis suggested that trace metals were removed more rapidly from the surface of the silica compared to the bulk matrix and that the acid treatments resulted in halide contamination of the silica surface. Evidence is presented to suggest that the bulk metal content of the silica is not representative of the concentration of metals at the chromatographic surface. The chromatographic investigations showed that the HF-treated silica gave substantially better performance towards weak bases than the HCl-treated silicas.