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17


a gradient from 5% to 40% methanol in 7 minutes. Flow rate 60 ml/min, back pressure 100 and 150 bar, oven temperature 40°C. Detection at 230 nm.


Analytical Columns: CHIRALCEL OD-H, CHIRALPAK AD-H (Chiral Technologies, West Chester, PA), GreenSep Basic (ES Industries, E Berlin, NJ) and DAISOGEL SP-100 silica (Daiso Fine Chem USA, Inc., Torrance, CA), all 150 x 4.6 mm


Warfarin was purchased from Sigma-Aldrich. Cloves were purchased locally.


Figure 3. Conventional injection of warfarin (12 mg), 210 nm. Other conditions as in experimental section.


DAISOGEL 50 micron C-1 phase was kindly donated by Daiso Fine Chem USA.


Empty Column blanks (AmChemTeq, Port Matilda, PA, 100 x 10 mm and 50 x 21.2 mm) were used as extractors, being dry-packed with material coated on 50 µm C-1 silica or with fi nely ground cloves.


LC-MS experiment


HPLC Equipment: Agilent 1200 LC with LC/ MSD SL


Column: Gemini-NX C18, 3 micron; 30 x 4.6 mm


Mobile Phase: 30% Acetonitrile in 50:50 (v:v) water : methanol containing 0.1% formic acid; fl ow rate 2 ml/min


Temperature: Ambient. Injection Volume: 10 µl UV detection 220 nm.


Figure 4. Plot of peak area vs injection #. 250 nm. Key shows peak retention times.


Results and Discussion Warfarin


Figure 5. Collection of impurities from warfarin. The numbers indicate the fraction number. Injection time = 20 seconds, detection 220 nm.


extension of the fi ndings into the fi eld of natural product isolation.


Experimental


SFC equipment: SFC-PICLab Hybrid 10-100 analytical/preparative SFC system (PIC Solution Inc, Media, PA).


Preparative Column: CHIRALCEL OD-H,


150 x 21.2 mm (Chiral Technologies, Inc, West Chester, PA}.


Mobile phase (warfarin analysis): 15% or 20% methanol in CO2


at a fl ow rate of 60 ml/min;


back pressure 100 bar, oven temperature 40°C.


Mobile phase (Cloves analysis): extraction at 0% methanol for 20 seconds, followed by


Previous work on extraction-injection for preparative separations was carried out by coating a sample on a suitable stationary phase and loading this into an extraction cartridge which replaced the injection loop in the standard injector on the Hybrid system. The design of the injector allows pressurisation of the extractor with the supercritical mobile phase during the separation process and making sequential injections by a time-based opening of the injection valve to introduce a certain volume of the extractor content into the column. An initial experiment designed to develop suitable separation conditions for the preparative separation of Warfarin enantiomers was run. A sample of warfarin (750 mg) was coated on the C-1 support (4.2g) using acetone as solvent and evaporating to dryness in a rotary evaporator. The resulting free-fl owing material was packed into a column 100 x 10 mm used as an extraction cartridge. The small void at the top of the cartridge was fi lled with fresh C-1 material. The chromatogram


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