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.
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