Analysis of Fulvic Acids Using HPLC/UV Coupled to FT-IR Spectroscopy

Abstract

Reversed-phase high performance liquid chromatography (RP-HPLC) coupled with an ultraviolet (UV) detector was used to separate three fulvic acid samples into two major fractions which contain several components based on the polarity of the samples. The chromatographic effluent was then directed into the LC-Transform, an interface between HPLC and Fourier transform infrared spectroscopy (FT-IR). The LC-Transform used a heated ultrasonic nozzle to evaporate all the HPLC mobile phase and deposit the eluates onto a rotating sample collection disk. The collection disk with the sample was subsequently analyzed by FT-IR. The UV chromatogram and the corresponding FT-IR chromatogram (Gram-Schmidt reconstruction) agreed with each other quite well. Spectral comparisons between the unseparated fulvic acid and separated fractions were done. Most of the typical functional groups found in fulvic acid, such as carboxylic acids, alcohols and phenols, were present in all fractions separated. Significant amounts of the aliphatic groups were detected in the least polar fractions which responded only to the FT-IR measurement. The carboxyl groups in the early eluting fractions were determined to be present in the form of carboxylate anions. Carboxylic acid anhydrides were found in the soil fulvic acid extracted from Amherst, MA. Second-derivative spectroscopy was used to confirm the anhydride absorbance band.

Keywords:

• Fulvic Acid
• HPLC
• FT-IR
• Spectroscopy
• UV