55
pump, a Phenomenex Degassex model DG-440 inline degasser unit and Shimadzu SPD-M10Avp PDA detector (254 nm). The chromatographic analyses were carried out using gradient elution with an initial mobile phase composition of 100% water with 0.1% formic acid running to a final mobile phase composition of 100% methanol with 0.1% formic acid, at a rate of 2% min-1 rate was set to 2 mL min-1 volumes were 20 µL.
. The flow and injection 2.2.4 RF - Phenol Detection 2.2.3 RF - ABTS Detection
ABTS detection was carried out in RF mode on the same instrument in section 2.2.2 Conventional UV-Vis Detection, with the same chromatographic conditions. The flow ratio between the peripheral port and central port was set at 50:50. Figure 1a illustrates the ABTS detection instrumental set up.
The ABTS and potassium persulfate solutions were both pumped at a flow rate of 0.5 mL min-1
. The two solutions were
mixed at a zero dead volume t-piece before the resultant mixture was passed through a 1000 µL mixing loop (note: this mixing loop was employed to mix reagents only, prior to the introduction to the sample and column, i.e., at no time did the sample that was
The chromatographic experiments for phenol detection were undertaken on a Waters 600E Multi Solvent Delivery LC System, equipped with a Waters 717 plus auto injector, two Waters 600E pumps, two Waters 2487 series UV-Vis detectors and two Waters 600E pump controllers. An additional Shimadzu LC-10ATvp pump was used for the delivery of PCD reagents. Chromatographic separation was carried out using gradient elution with an initial mobile phase composition of 100% 0.1 M ammonium acetate buffer (pH 9) running to a final mobile phase composition of 100% methanol, at a rate of 2% min-1 rate was set to 2 mL min-1
. The flow and the injection
volume was 20 µL. The flow ratio between the peripheral port and central port was set
3. Results and Discussion
The lemon myrtle leaf extracts were analysed for their bioactivity with respect to antioxidant and phenolic content. Water and methanol extracts of the leaves were analysed in RF using ABTS•
and phenol
specific detection using 4-aminoantipyrene and potassium ferricyanide as PCD reagents. The phenol specific analysis was also multiplexed with a UV-Vis detector connected to the central port of the column to monitor the underivatised eluent, however, the ABTS•
multiplexed. Since, the phenol detection method utilised a slightly different mobile phase compared to the ABTS•
analyses, Figure 1a
a conventional UV-Vis analysis on the leaf extracts was carried out to account for the chromatographic difference between the two RF-PCD techniques. Figure 2 illustrates the chromatographic profile for the leaf extracts with conventional UV-Vis detection at 254 nm. A significant number of peaks were observed for each extract, with the majority of peaks eluting before 30 minutes. However, some peaks were still observed up to 50 minutes. The chromatograms of the lemon myrtle water and methanol extracts (Figure 2a and 2b, respectively) showed that the highest intensity peaks occurred after 5 minutes, with the intensity of the peaks being greater in the methanol extract, especially for the latter eluting components.
Figure 1b 3.1 ABTS• Detection
eluting from the column pass through this loop). The solution was then pumped into 1 peripheral port of the RF column. A second Peripheral port was connected to a PDA detector with the analysis wavelength set to 734 nm to monitor the excess ABTS•
within
the eluent. The third peripheral port was blocked while the flow from the central port was allowed to flow to a collection vessel.
to 50:50. Figure 1b illustrates the phenol detection instrumental set up.
Phenol detection was achieved by introducing the 4-aminoantipyrene and potassium ferricyanide reagents into two of the peripheral ports of the RF column. The flow rate of the 4-aminoantipyrene solution was set to 0.5 mL min-1
and the flow rate of
the potassium ferricyanide solution was set to 0.4 mL min-1
. The third peripheral port
of the column was connected to a UV-Vis detector set to 500 nm to monitor the derivatised eluent. The central port of the column was connected to a second UV-Vis detector set to 254 nm to monitor the native (underivatised) effluent stream.
2.3 Data processing
Data analysis was undertaken using Origin and Microsoft Excel. For the reagent based detection methods (i.e. ABTS•
and phenol
detection) the blank chromatographic profile was subtracted from the sample chromatographic profile.
analysis was not
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