The Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
J Chromatogr A. 2011 Sep 16;1218(37):6448-53. doi: 10.1016/j.chroma.2011.07.030. Epub 2011 Jul 20.
High performance size-exclusion chromatography (HPSEC) is useful for the molecular size separation of soil humic acids (HAs), but there is no method available for various HAs with different chemical properties. In this paper the authors propose a new preparative HPSEC method for various soil HAs. Three soil HAs with different chemical properties were fractionated by a Shodex OHpak SB-2004 HQ column with 10mM sodium phosphate buffer (pH 7.0)/acetonitrile (3:1, v/v) as an eluent. The HAs eluted within a reasonable column range time (12-25 min) without peak tailing. Preparative HPSEC chromatograms of these HAs indicated that non-size-exclusion effects were suppressed. The separated fractions were analyzed by HPSEC to determine their apparent molecular weights. These decreased sequentially from fraction 1 to fraction 10, suggesting that the HAs had been separated by their molecular size. The size-separated fractions of the soil HA were mixed to compare them with unfractionated HA. The analytical HPSEC chromatogram of the mixed HA was almost identical to that of the unfractionated HA. It appears that the HAs do not adsorb specifically to the column during preparative HPSEC. Our preparative HPSEC method allows for rapid and reproducible separation of various soil HAs by molecular size.
高效体积排阻色谱（HPSEC）可用于土壤腐殖酸（HA）的分子大小分离，但对于具有不同化学性质的各种 HA 却没有可用的方法。在本文中，作者提出了一种新的用于各种土壤 HA 的制备性 HPSEC 方法。使用 Shodex OHpak SB-2004 HQ 柱，以 10mM 磷酸钠缓冲液（pH 7.0）/乙腈（3:1，v/v）作为洗脱液，对具有不同化学性质的三种土壤 HA 进行了分级。HA 在合理的柱时间范围内（12-25 分钟）洗脱，没有峰拖尾。这些 HA 的制备性 HPSEC 色谱图表明，非尺寸排阻效应得到了抑制。分离的级分通过 HPSEC 进行分析，以确定其表观分子量。这些级分的分子量依次从第 1 级到第 10 级降低，表明 HA 已按分子大小进行了分离。土壤 HA 的大小分离级分混合后与未分级 HA 进行比较。混合 HA 的分析性 HPSEC 色谱图与未分级 HA 的色谱图几乎相同。这表明在制备性 HPSEC 过程中 HA 不会特异性地吸附到柱子上。我们的制备性 HPSEC 方法可通过分子大小快速且可重复地分离各种土壤 HA。
