Characterization and analysis of soil humic acids by off-line combination of wide-pore octadecylsilica column reverse phase high performance liquid chromatography with narrow bore column size-exclusion chromatography and fluorescence detection

Liquid chromatography method arranged around RP-HPLC using step-wise gradients of dimethylformamide (DMF) in pH 3.0 aqueous phosphate buffered mobile phase and a wide-pore (30 nm) 4 mm inner diameter (I.D.) octadecylsilica column was applied to the fractionation separation of soil and peat humic substances (HS), respectively. Combination of acceptable DMF solvating properties for HSs together with wide pore RP sorbent improved surface interactions of the analytes and suppressed influence of size-exclusion effects in RP-HPLC. Individual fractions collected from the RP-HPLC were off-line analyzed by size-exclusion chromatographic method (SEC) using 99/1 DMF/aqueous phosphate buffer pH 3.0 with Spheron HEMA 100 stationary phase filled in a 2.2 mm I.D. column. Both methods provided reproducibility of characterization profiles and robustness resulting from excellent reproducibility of HSs fraction retention times (±0.5% RSD) of peaks enforced by the step gradient shape. Obtained results indicate that the methods can be combined in a compatible, compact, automatic, 2D orthogonal separation system for fluorimetric characterization of such complicated natural substances as are examined humic acids and obtain so more information about their character.