Step-Wise Fractionation and Recovery of Aquatic Fulvic Acid by Modified Supercritical Fluid CO2- Methanol Extraction at Near Critical Temperature

Modified supercritical fluid "SF" CO 2 -methanol mixture was used, under step-wise gradient conditions at near supercritical temperature, to fractionate Suwannee River reference fulvic acids (FA) into three fractions. The method was developed after a systematic study of the solubility of FA in SF CO 2 -methanol solvent system. Optimum supercritical fluid extraction (SFE) conditions were established at constant temperature of 70 °C and constant pressure of 2,500 psi; using modified CO 2 fluid mixed with methanol at 18, 24 and 100 percent, respectively. The dynamic extraction conditions were designed to achieve marcketable differences in the fluid solvent power. Fractions were characterized by total uv absorption and fluorescence emission. Under optimum conditions, fractions were collected and characterized by uv absorbance ratio at λ 400/λ 254 nm using non-column HPLC with uv PDA detector. The average total mass recovery of all three fractions was 102 % and coefficient of variation of 6.8%. The first fraction represented 21.5 of total FA and exhibited absorbance ratio of 0.11. The second fraction represented 55 % of total FA and exhibited almost twice the absorbance ratio. The third fraction represented 25 % of the total FA and exhibited absorbance ratio of 0.30. Total sample and fractions were analyzed using C-18 RP-HPLC with uv-vis PDA and fluorescence detection. RP-HPLC chromatograms of the SFE fractions showed the resolution of the same number of peaks as the total sample, but with different uv absorption and fluorescence emission intensities. The overall results provide some insight on the structural features of FA.