SIZE EXCLUSION CHROMATOGRAPHY AND SAX-HPLC OF TWO SAMPLES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 S-100Sephacryl制备凝胶过滤 将150 mg依诺肝素钠溶于1mL水中，进样于3.2×90 cm Sephacryl S-100色谱柱，用0.25M醋酸铵(pH 6.5)洗脱，流速为1mL/min。洗脱液在254 nm处用紫外检测进行监测，收集4毫升的级分。含有四糖的级分混合在一起，100-ul等分被分析尺寸排除高效液相色谱(见下文)以确定纯度。样品的其余部分被冷冻干燥，然后加水，样品再次冷冻干燥以去除残留的醋酸铵。样品溶于500微升水中，进行SAX-HPLC法。 TSKGel G2000SW分析凝胶过滤 采用TSKGel G2000SW色谱柱(7.8 mm ID×30 cm，5 um)，配备TSKGel防护柱(6.0 mm ID×4.0 cm，7 um)，以0.25M醋酸铵为洗脱液，pH 6.5为洗脱液，流速为0.5mL/min。紫外检测波长232 nm。 分析型SAX-高效液相色谱法 在Agilent系统上，采用4.6×250 mm Waters Spherisorb分析柱，粒径为5微米。分析物通过232 nm处的紫外光吸收进行检测。使用以下系统通过盐度梯度实现分离： 溶剂A：2.5毫米磷酸钠，pH 3.5，含0.2M氯化钠；溶剂B：2.5毫米磷酸钠，pH 3.5，含1.2M氯化钠。在30%B下5分钟后，应用线性梯度以在50分钟后达到60%B。流速为1.4毫升/分钟。进样量为5ul。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Preparative Gel Filtration with Sephacryl S-100 One hundred fifty mg Enoxaparin sodium was dissolved in 1 mL water, injected onto a 3.2 X 90 cm Sephacryl S-100 column, and eluted with 0.25 M ammonium acetate (pH 6.5) at a flow rate of 1 mL/min. The eluate was monitored by UV detection at 254 nm and 4-mL fractions were collected. The fractions containing tetrasaccharide were pooled and a 100-uL aliquot was analyzed by analytical size exclusion HPLC (see below) to determine purity. The remainder of the sample was freeze-dried, then water was added and the samples were again freeze-dried to remove residual ammonium acetate. The sample was then dissolved in 500 uL water for SAX-HPLC. Analytical Gel Filtration with TSKGel G2000SW Separation was carried out on a TSKGel G2000SW column (7.8 mm ID X 30 cm, 5 um), equipped with a TSKGel guard column (6.0 mm ID X 4.0 cm, 7 um), using 0.25 M ammonium acetate, pH 6.5 as eluent at a flow rate of 0.5 mL/min. Detection was by UV at 232 nm. Analytical SAX-HPLC SAX-HPLC was carried out on an Agilent system using a 4.6x250 mm Waters Spherisorb analytical column with 5 um particle size. Analytes were detected by their UV absorbance at 232 nm. Separation was effected by a salt gradient using the following system: Solvent A: 2.5 mM Na-phosphate, pH 3.5, containing 0.2 M sodium chloride; Solvent B: 2.5 mM Na-phosphate, pH 3.5, containing 1.2 M NaCl. After 5 min at 30 % B, a linear gradient was applied to reach 60 % B after 50 min. The flow rate was 1.4 mL/min. Injection volume was 5 uL.