硅胶基硝化苯硼酸亲和色谱材料的合成及应用  

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

作　　者：李方楼[1] 赵欣捷[1] 许国旺[1] 

机构地区：[1]中国科学院大连化学物理研究所,大连116023

出　　处：《分析化学》2006年第10期1366-1370,共5页Chinese Journal of Analytical Chemistry

基　　金：国家杰出青年基金(No.20425516);国家科技部"973"项目(No.2003CB716003);国家科技部社会公益性项目;辽宁省科学基金资助

摘　　要：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。A boronic acid-substituted silica was synthesized by using a y-glycidoxypropyhrimethoxysilane as the spacer arm and using a 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 the pressure of 2. 07 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 by 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 mol/L ammonium acetate （ pH 7.95 ） on extraction column, and of 25 mmol/L potassium dihydrogen phosphate （ pH 4.5 ） on analytical column, respectively. Determination of urinary nucleosides was performed on analytical column by using a linear gradient elution comprising 25 mmol/L 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 show good RSD, recovery and excellent linearity （r 〉 0. 995 ）.

关 键 词：亲和色谱 硼酸亲和色谱预柱 多维液相色谱 直接进样 

分 类 号：O657.7[理学—分析化学]