54 May / June 2017
can also be varied, and depends upon the relative pressure differential between the radial central exit port and the peripheral ports, and the characteristics of the outlet frit. In this column design it is important to emphasise that the wall flow and radial central flows are separated by the impermeable barrier in the annular frit design. An understanding of this basic function is important since post column derivatisation reagents can be added into the flow stream through one or two of the outlet ports that align with the outer porous region of the frit, rather than using Tee pieces in post column configurations [10-12]. These reagents then mix with the mobile phase and sample that leaves the HPLC column inside the frit. The mixing process is very efficient, often negating the use of reaction coils that are normally required to mix reagents with the sample. Furthermore, since the radial flow stream is separated from the wall flow region, the central flow is not subjected to the post column derivatisation reagents and thus ‘native’ sample leaves the column through this single port and can be detected using a detection process separate from the derivatisation method. Therefore, multiplexed detection enables a greater understanding of the sample characteristics [7]. Since no reaction coil is required, or at least the volume of the reaction coil can be greatly reduced, separation efficiency is not compromised through the band broadening that takes place in large reaction coils [13]. The diagram above illustrates the application using the antioxidant detection process employing DPPH reagent. Peaks are therefore sharper and depending on the type of chemical derivatisation and detection process, the signal intensity is often increased, even despite the shorter period of time required to undergo the derivatisation process and the fact that only a small portion of the sample is subjected to the post column derivatisation. More often than not, the background noise is decreased, so that even when, or if, the signal intensity is decreased, the actual specific sensitivity (factoring in S/N) is increased [13-17].
The combination of RF and multiplexed detection has the potential to yield a significant amount of information about the nature of the sample of interest in half the time it would otherwise take if the analyses were undertaken separately in the conventional manner [7]. RF chromatography has also been found to minimise the complexity in PCD preparation time as well
as instrumental setup [11,12,16], such as in the case of PCD reagent, fluorescamine, for the analysis of amino acids [11]. The use of a RF column reduced the number of pumps, coils and mixing devices that are otherwise required in conventional HPLC-PCD analyses using fluorescamine [11]. Ultimately, the RF column enables the complete removal of post column reaction coils, providing high efficiency separation with specific detection.
In this study, RF was utilised to analyse the bioactivity of NAFPs (specifically, lemon myrtle (LM) (Backhousia citriodora)) using ABTS• and phenol reagents – 4-aminoantipyrene and potassium ferricyanide, to provide insight to its antioxidant and phenolic content of leaf extracts.
2. Experimental 2.1 Chemicals and Reagents
HPLC grade mobile phase solvents (Themo Fisher Scientific, North Ryde NSW Australia.) were used. Milli-Q water was prepared in house using a 0.2 µm filter (Ultrapure, Millipore, Kilsyth, VIC Australia.). Ammonium acetate, 2,2-azino- bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and potassium persulfate were purchased from Sigma Aldrich (Castle Hill, NSW Australia.). 4-aminoantipyrine was purchased from ACROS Organics (Geel, Antwerp, Bel.) and potassium ferricyanide was purchased from AnalaR (Poole, Dorset, UK).
Solutions containing 0.3 mg/mL of ABTS and 10 mg/mL sodium persulfate were prepared in Milli Q water and sonicated for 10 minutes
to dissolve. The 4-aminoanitpyrene and potassium ferricyanide reagents were both prepared at a concentration of 1.5 mg/ mL in 0.1 M ammonium acetate (pH 9) and sonicated for 10 minutes to dissolve.
Mature lemon myrtle (LM) leaves were harvested from established trees grown in an uncontrolled environment at Muru Mittigar culture centre gardens in Castlereagh, NSW Australia. The freshly harvested leaves were pounded separately in either Milli-Q water or methanol using a ceramic mortar and pestle. A total of 20 mL of solvent was added per gram of sample. The samples were sonicated for 5 min at room temperature and filtered using a 0.22 µm nylon filter [7].
2.2 Instrumentation and Chromatographic Conditions
2.2.1 Column
Separations for RF were conducted using a HyPURITY C18 50 × 4.6 mm, 3 µm column with a 4-port outlet head-fitting (Thermo Scientific,Runcorn, Cheshire, UK). Conventional HPLC separations were conducted using a column of the same phase and dimensions with a standard column end-fitting.
2.2.2 Conventional UV-Vis Detection
The leaf extracts were analysed via UV-Vis detection using the conventional column. The chromatographic separations were carried out on a Shimadzu HPLC equipped with a Shimadzu Prominence LC-20AD
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