c) A mixture of four compounds as shown in Figure 1 was analyzed using supercritical fluid chromatography (SFC) with carbon dioxide as the supercritical fluid mobile phase. 0 0 CH CH .. .. HN " NH 0 N 0 0 0 H CH, CH CH, Caffeine Theophylline Theobromine Uracil Figure 1 Compounds that present in the sample mixture 1 The compounds were separated using column 4.6 mm x 150 mm, packed with 3.5 um RX-SIL (silica column). A temperature of 50 C and a pressure of 150 bar was used. The flow was set at 4 mL/min. i) Poor separation with longer elution time was observed using these conditions. Thus, to improve the separation, an organic modifier was added to the mobile phase. A series of methanol of different percent compositions (%) was added and the chromatograms obtained are shown in Figure 2. Based on the results, briefly discuss the polarity of the compounds in the mixture. (3 marks) Chromatogram A Methanol Composition 9% 120 12 80 3.4 40 0 0 2 3 4 5 6 Chromatogram B 120 1 7.5 % 80 2. 3 4 40 0 0 2 3 5 6 Chromatogram C 40 2 5 % 20 3 4 0 0 6 8 10 12 Retention time [min] Figure 2 Chromatograms obtained using methanol of different compositions percent added to the CO, supercritical fluid ii) Explain why methanol was used to improve the separation. (3 marks) iii) State a parameter that can be used to optimise the above separation (to shorten the analysis time without demolishing the resolution). Explain how it works. (3 marks) iv) Based on chromatograms in Figure 2, which condition of organic modifier percentage (%) should be chosen to start-off for optimising the separation. (2 marks)