SIZE EXCLUSION CHROMATOGRAPHY AND SAX-HPLC OF TWO SAMPLES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Sephacryl S-100凝胶过滤 将150 mg依诺肝素钠溶于1 mL水中，进样至3.2 X 90 cm Sephacryl S-100柱上，用0.25 M乙酸铵（pH 6.5）以1 mL/min的流速洗脱。通过254 nm处的UV检测监测洗脱液，收集4 mL馏分。 合并含有四糖的级分，并通过分析型尺寸排阻HPLC（见下文）分析100 μ L等分试样以测定纯度。 将剩余的样品冷冻干燥，然后加入水，并将样品再次冷冻干燥以除去残留的乙酸铵。 然后将样品溶于500 μ L水中用于SAX-HPLC。 使用TSKGel G2000 SW进行分析凝胶过滤 在配备有TSKGel保护柱（6.0 mm ID X 4.0 cm，7 um）的TSKGel G2000 SW柱（7.8 mm ID X 30 cm，5 um）上进行分离，使用0.25 M乙酸铵（pH 6.5）作为洗脱液，流速为0.5 mL/min。 分析型SAX-HPLC SAX-HPLC在Agilent系统上使用具有5 μ m粒度的4.6 × 250 mm沃茨Spherisorb分析柱进行。通过232 nm处的UV吸光度检测分析物。 使用以下系统通过盐梯度进行分离： 溶剂A：2.5 mM磷酸钠，pH 3.5，含0.2 M氯化钠;溶剂B：2.5 mM磷酸钠，pH 3.5，含1.2 M NaCl。在30% B下5分钟后，施加线性梯度以在50分钟后达到60% B。流速为1.4 mL/min。注射体积为5 μ L。

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.