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19


[2]. In addition, 21 other components of clove oil extracted by various procedures including SFE have been reported [3]. A study by Prado [4] indicated that the overall extraction curve for the extraction of clove


Figure 7. Clove extraction-injection 5% isocratic. For other conditions see the experimental section.


oil from clove buds with supercritical CO2 follows the usual form of constant extraction rate, falling extraction rate and diffusion controlled periods [5]. However, for the combined SFE/SFC experiment it was of interest to observe if the minor components would be extracted more effectively than the major ones. At the same time it was also an objective to evaluate the effectiveness of a combined extraction-injection separation of the clove components as a model for the purification and isolation of more valuable components from natural product mixtures.


Initial experiments to find a suitable column for the separation were carried out isocratically using four columns and commercial clove oil as sample. Two of these were enantioselective polysaccharide-based phases, not because the products from the cloves were expected to be chiral but because such columns have been observed to give excellent selectivity for other achiral natural products. The commercial product contained essentially two ingredients which were assigned to be eugenyl acetate and eugenol on the basis of the known composition of such oils [2]. In the event, an enantioselective phase (CHIRALPAK OD-H) was chosen for the preparative work on the basis of selectivity, retention and peak shape. Based on peak area at 230 nm, the clove oil was assigned a eugenol purity of 92%.


In order to extract the volatile oils while leaving less volatile, more polar components behind in the matrix, it was decided to run the extraction using pure CO2 maintained pure CO2


. This strategy than the co-solvent – CO2 Figure 8. Gradient chromatograms at 100 & 150 bar BPR. For other conditions see the experimental section.


no detectable peaks while fraction 3 was assigned the identity of acetone, the coating solvent. The reversed phase separation is largely orthogonal to the SFC separation and illustrates the utility of such two-dimensional analysis; some of the components collected are largely pure, while many of them show multiple peaks in HPLC despite being eluted as a single peak under the SFC conditions. The apparently identical (in terms of retention time and mass) major peaks in fractions 16 and 17 suggest that these two peaks are made up of at least two enantiomeric pairs, while fractions 21 and 22 are probably reasonably pure enantiomers. Table 1 summarises the results.


The separation was continued until the sample was exhausted, collecting the two enantiomers. The total recovery of warfarin from the experiment was 92%.


Cloves


Following the results of the warfarin experiments, it became of interest to find if a similar phenomenon would be found in the extraction of natural products from their organic matrix. Previously studies on the supercritical CO2


extraction of cloves


has been carried out, with the result that the extract contained mainly eugenol and eugenyl acetate under conditions not far from those that could be used in SFC


in the extractor rather mixture used for


the chromatographic separation. Extraction using a percentage of co-solvent would be expected to increase the extraction of polar components. The extractions were carried out at room temperature (i.e. 22 -23°C).


A sample of cloves was ground in a conventional kitchen blender and loaded into an extractor 100 x 10 mm. Using an injection time of 2 seconds (i.e. approximately 2 ml volume) under isocratic conditions at 5% methanol gave the chromatogram shown in Figure 7. In order to ensure elution of the more polar components, an elution gradient was developed. The extraction was carried out at two different pressures, modulated by the pressure at the back pressure regulator (100 and 150 bar) and the column pressure drop of 26 bar. The two chromatograms in


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