56 May / June 2017 ABTS• is a sterically hindered stable radical
reagent [18] that is commonly used for colorimetric assays of antioxidants [19]. Analysis of the lemon myrtle extracts using ABTS•
was performed in reaction flow mode,
meaning that the reagent was pumped directly into the end fitting of the column where mixing and reaction could take place within the end frit of the column. The increased mixing efficiency resulted in the ability to remove the reaction loop between the mixing of the reagents and the detector and therefore the negative effects of dead volume. Additionally, conventional methods of ABTS•
detection require lengthy reagent
preparation time, which involves a waiting period of up to 20 hours of reaction time before use [20]. In the process of RF-PCD, detection was achieved with the waiting period effectively eliminated by mixing the reagents in real time just prior to its introduction to column effluent. Although, the RF-PCD technique may require further investigation to optimise sensitivity, nevertheless a significant number of ABTS• peaks were observed.
Figure 3 shows the chromatographic profile of the ABTS•
detection response for each of
the leaf extracts (3a being the water extract, 3b being the methanol extract). Interestingly, all peaks that gave a response to ABTS• eluted within the first 21 minutes, and notably, the two major components in the UV chromatograms that eluted at around 25 minutes gave no ABTS•
), which can
Figure 2a
response. The lemon
myrtle water extract (Figure 3a) showed a cluster of peaks between 7 and 12 minutes (at least seven responding to ABTS•
also be seen in the UV-Vis chromatogram. The large peak at 8 minutes has four observable shoulders indicating the co- elution of multiple antioxidant compounds. The methanol extract (Figure 3b), shows a single large peak with a number of much smaller peaks appearing after 8 minutes.
The ABTS• response to the leaf extracts
indicates that there are a number of antioxidant compounds present in these leaves. Water extraction gave a greater number of observable peaks compared to methanol extraction. However, a number of peaks in the methanol extract may have co-eluted in the single large peak close to the void. Besides the single large peak in the methanol extract, the rest of the peaks gave a signal intensity that was similar to that in the water extract. Differences in the chromatographic profile of the water and methanol extracts indicate that each of the solvents extract a different range of antioxidant compounds indicating that
Figure 2b
no single solvent extraction procedure can provide a complete picture of the antioxidant profile of the sample. An obvious example of the difference in extraction efficiencies of each solvent could be seen in the colour of each solution. The methanol extracts showed an obvious intense green colour that was not present in the water extracts indicating that only the methanol was able to extract the chlorophylls from the leaves. The presence of the chlorophylls in the methanol extract may be the cause of the intense response around the void time in the methanol extract chromatograms as chlorophylls are organometallic molecules that may not be retained on the C18 stationary phase.
3.2 Phenol Detection
Detection using the phenol reagent was also carried out in RF mode. The central, underivatised flow was also monitored using a UV-Vis detector set to 254 nm. Figures 4 and 5 present the chromatograms, both derivatised and underivatised, collected using the phenol reagent method. All chromatographic profiles showed high correlation between the underivatised UV-Vis and phenol derivatised profiles suggesting that the extracts contain a number of phenol based compounds.
The lemon myrtle chromatograms (Figure 4 and 5) show a large underivatised UV-Vis response at the void time; however, the response of the phenol reagent was limited
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