硅胶基硝化苯硼酸亲和色谱材料的合成及应用

合成了硅胶基硝化苯硼酸亲和色谱材料,首先对间氨基苯硼酸进行硝基化,制得3-氨基-4-硝基苯硼酸功能基团,通过γ-缩水甘油醚氧丙基三甲氧基硅烷把功能基团键合到硅胶基体上,在20.7MPa压力下装成亲和色谱预柱(35mm×4.6mmi.d.)。用该预柱通过六通阀后接ODS分析柱(250mm×4.6mmi.d.),构成中心切割二维HPLC。该系统能对含有顺二羟基结构的化合物进行在线富集,实现生物复杂样品直接进样分离分析。使用该系统对尿中多种修饰核苷进行了分离分析,以pH值7.95的0.25mol/LNH4Ac碱性缓冲液把实际尿样(100μL)中核苷保留在预柱上,生物大分子无保留通过,再切换六通阀,以pH4.50的25mmol/LKH2PO4酸性洗脱液把保留在预柱上的核苷洗脱,进样到下一级ODS分析柱柱头上聚焦,然后用梯度洗脱法(pH4.50的25mmol/LKH2PO4缓冲液与体积比60∶40的甲醇-水梯度混合构成洗脱液)完成核苷在ODS分析柱上的分离(紫外检测260nm),11种目标核苷的分离分析取得了良好的定性定量结果。

Synthesis and Application of a Nitroboronic Acid-substituated Silica for Affinity Chromatography

A boronic acid-substituted silica was synthesized using γ-glycidoxypropyltrimethoxysilane as the spacer arm and 3-amino-4-nitrobenzeneboronic acid as the ligand. The 3-amino-4-nitrobenzeneboronic acid was modified from m-aminobenzeneboronic acid. Then, boronic acid-substituted silica was packed in a stainless-steel column (35 mm × 4.6 mm I.D.) under a pressure of 20.7 MPa as an affinity pre-column. A coupled-column liquid chromatographic method for the direct analysis of 11 urinary nucleosides is described. Efficient on-line clean-up and concentration of 11 nucleosides from urine samples were obtained using the affinity pre-column as the first column (extraction column) and an ODS column (250 mm × 4.6 mm I.D.) as the second column (analytical column). The mobile phases applied consisted of 0.25 M ammonium acetate (pH 7.95) on the extraction column, and 25 mM potassium dihydrogen phosphate (pH 4.5) on the analytical column, respectively. Determination of urinary nucleosides was performed on the analytical column using a linear gradient elution comprising 25 mM potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Calibration plots of 11 standard nucleosides showed good RSD, good recovery, and excellent linearity (r > 0.995).