氨基酸和多肽在碱性硅胶载体上的固相衍生研究

为进行复杂体系中痕量生理活性物质 (如氨基酸和多肽等 )的高灵敏度分析 ,往往需要对其进行柱前或柱后的荧光衍生 -高效液相色谱或毛细管电泳分析 [1] .在一个存在着竞争反应的体系中 ,为保证样品有足够的反应产率 ,往往需使衍生试剂过量很多 ,这就使得衍生后的样品中必然含有高浓度的衍生试剂及其水解后形成的副产物 ,从而大大地干扰了分离与分析 .为解决这一问题 ,通常可采取溶剂萃取[2～ 5] 或加入 1 -金刚烷胺 [6 ,7] 或羟胺 [8,9] 等方法除去过量试剂 .但这些额外的处理使衍生方法更加烦琐 ,有时还导致收率的降低 .也有使用固相化的衍…

Solid Phase Derivatization of Amino Acids and Peptides on Alkaline Silica Gel Support

A solid phase derivatization method of amino acids and peptides was described. Samples were added on alkaline silica gel cartridge and dried. The derivatization was carried out by passing the derivatization regent dissolved in anhydrous solvent through the cartridge slowly, then the cartridge was washed with a selected solvent, finally, the derivatives were eluted. The eluates were separated and detected by means of HPLC with fluorescence detector. Three derivatization reagents, 9|fluorenylmethyl chloroformate(FMOC|Cl), {7|chloro|4|nitrobenzo|2|oxa|1,3|diazole}(NBD|Cl) and fluorescein isothiocyanate(FITC) were investigated with this method. The results showed that the method was suitable for derivatization of amino acids and some of peptides with FMOC|Cl because of its highly reacting activity and low polarity, the side|products of the FMOC|Cl with water were greatly decreased, the excess FMOC|Cl was eliminated completely, and the silica gel support did not significantly affect the reaction rate and yield for amino acids. NBD|Cl was not suitable for this method because of the low yield of derivatization. FITC was also not suitable for this method because of its strong polarity.