Humic acid molecular weight estimation by high-performance size-exclusion chromatography with ultraviolet absorbance detection and refractive index detection

High-performance size-exclusion chromatography (HPSEC) with ultraviolet absorbance detection (UVAD) has been widely utilized to estimate the molecular weight (MW) and MW distribution of humic acids (HAs). The MW estimated by UVAD was inherently inaccurate, however, because UVAD set at 254 nm only detects limited HA components, and the molar absorptivities of different HA constituents are not equal. The objective of this study was to evaluate the refractive index detection (RID)-based HPSEC method for quantifying the MW of HAs. Five HA samples were quantified with both UVAD and RID. The chromatograms obtained on the two detectors showed that the RID/UVAD response ratios were consistently > 10, indicating that RID is more sensitive for the detection of HAs. The chromatograms obtained with RID had three peaks compared with two peaks shown on UVAD chromatograms because RID detected a late-eluting peak (F₃) that was not shown on the UVAD chromatograms. Comparison of the RID/UVAD response ratio showed that the highest MW HA fraction (F₁) and lowest MW HA fraction (F₃) have higher RID/UVAD response ratios, whereas the medium HA fraction (F₂) had a lower RID/UVAD response ratio. These suggested that F₁ and F₃ may have relatively lower contents of UV-sensitive bonds such as C=C double bonds than F₂. Compared with HPSEC-UVAD chromatograms, the HPSEC-RID chromatograms resulted in higher weight-averaged MWs, lower number-averaged MWs, and higher polydispersivity for the tested HA samples. This study indicated that RID is less selective than UVAD for detection of structurally highly heterogeneous HA molecules and is thus better for characterizing the MW distribution of HA molecules.