Koplík Richard, Pavelková Hana, Cincibuchová Jana, Mestek Oto, Kvasnicka Frantisek, Suchánek Miloslav
Institute of Chemical Technology, Prague, Czech Republic.
J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Apr 25;770(1-2):261-73. doi: 10.1016/s1570-0232(02)00053-3.
Soluble species of phosphorus, sulfur, selenium and eight metals (Mn, Fe, Co, Ni, Cu, Zn, Mo and Cd) in soybean flour and common white bean seeds were investigated by size exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP-MS). Samples were extracted by 0.02 mol l(-1) Tris-HCI buffer solution (pH 7.5). Fractionation of sample extracts by preparative scale SEC was accomplished using a Fractogel EMD BioSEC column (600 x 16 mm) and 0.02 mol l(-1) Tris-HCl buffer solution (pH 7.5) as mobile phase (flow rate: 2 ml min(-1)). A 2-ml sample was injected. Contents of elements in chromatographic fractions were determined by AAS, ICP-AES and ICP-MS. The elution profiles of P, Fe, Co, Ni, Cu, Zn and Mo in both samples were similar. Main species of Co, Ni, Cu, Zn and Mo were found in the low molecular weight region (2-5 kDa), whereas Fe is predominantly bound to high molecular weight compounds (180 kDa). The dominant phosphorus fraction was detected in the medium molecular weight region (10-30 kDa) and the other fraction in the low molecular weight region. Isotachophoretic analysis of chromatographic fractions revealed that the main phosphorus compound in the medium molecular weight region is phytic acid. SEC on Superdex 75 and Superdex Peptide columns (300 x 10 mm) was performed in on-line hyphenation with ICP-MS. The same mobile phase was used with a flow rate of 0.5 ml min(-1); volume of injected sample was 200 microl. Element specific chromatograms were obtained by continuous nebulization of effluent into ICP-mass spectrometer measuring intensities of 47(PO)+ and 48(SO)+ oxide ions and 55Mn, 57Fe, 59Co, 62Ni, 65Cu, 66Zn, 82Se, 95Mo and 114Cd nuclides. Chromatographic profiles of elements are generally analogous to those obtained with a Fractogel column, but better chromatographic resolution of separated species was achieved so that slight differences between samples were revealed. Estimated molecular weights of major phosphorus species in soybean flour and common white bean seed extracts are 6 and 3.6 kDa, respectively, whereas those of minor phosphorus species in both samples are 0.7 kDa. Traces of phosphorus were also detected in the high molecular weight region (130 kDa). Chromatograms of P, Ni, Cu, Zn and Mo compounds in both extracts are similar but not identical. Molecular weights of major Cu and Zn species are approximately 1 and 0.4 kDa for soybean flour and white bean seeds, respectively. In cases of Mn, Fe, Co and Se, the element profiles of soybean flour and white bean seed extracts are significantly different.
采用尺寸排阻色谱法（SEC）和电感耦合等离子体质谱法（ICP-MS）对大豆粉和普通白豆种子中磷、硫、硒以及8种金属（锰、铁、钴、镍、铜、锌、钼和镉）的可溶性物种进行了研究。样品用0.02 mol l⁻¹ Tris-HCl缓冲溶液（pH 7.5）提取。使用Fractogel EMD BioSEC柱（600×16 mm）和0.02 mol l⁻¹ Tris-HCl缓冲溶液（pH 7.5）作为流动相（流速：2 ml min⁻¹），通过制备规模的SEC对样品提取物进行分级分离。进样2 ml。采用原子吸收光谱法（AAS）、电感耦合等离子体发射光谱法（ICP-AES）和电感耦合等离子体质谱法（ICP-MS）测定色谱馏分中的元素含量。两个样品中磷、铁、钴、镍、铜、锌和钼的洗脱曲线相似。钴、镍、铜、锌和钼的主要物种存在于低分子量区域（2 - 5 kDa），而铁主要与高分子量化合物（180 kDa）结合。在中分子量区域（10 - 30 kDa）检测到主要的磷馏分，另一个馏分在低分子量区域。对色谱馏分的等速电泳分析表明，中分子量区域的主要磷化合物是植酸。在Superdex 75和Superdex Peptide柱（300×10 mm）上进行SEC，并与ICP-MS在线联用。使用相同的流动相，流速为0.5 ml min⁻¹；进样体积为200 μl。通过将流出物连续雾化到ICP质谱仪中，测量47(PO)⁺和48(SO)⁺氧化物离子以及55Mn、57Fe、59Co、62Ni、65Cu、66Zn、82Se、95Mo和114Cd核素的强度，获得元素特异性色谱图。元素的色谱图一般与使用Fractogel柱获得的色谱图相似，但分离物种的色谱分辨率更高，从而揭示了样品之间的细微差异。大豆粉和普通白豆种子提取物中主要磷物种的估计分子量分别为6 kDa和3.6 kDa，而两个样品中次要磷物种的估计分子量为0.7 kDa。在高分子量区域（130 kDa）也检测到痕量磷。两种提取物中磷、镍、铜、锌和钼化合物的色谱图相似但不完全相同。大豆粉和白豆种子中主要铜和锌物种的分子量分别约为1 kDa和0.4 kDa。对于锰、铁、钴和硒，大豆粉和白豆种子提取物的元素图谱有显著差异。
