For Impurities of Higher Molecular Weights

Materials Required Corticotrophin 1 mg acetic acid (1 M) [prepared by dissolving 57 ml of glacial acetic acid in 1000 ml of DW] 100 ml sodium dodecyl sulphate (1% w/v) 10 ml  

Procedure Dissolve accurately weighed 1 mg of corticotophin in 1 ml of 1 M acetic acid containing 1% w/v of sodium dodecyl sulphate. Heat the solution at 100 °C for 10 minutes and allow to cool. 

The chromatographic procedure may be performed using (a) a column (about 85 cm x 10 mm) packed with polyacrylamide or cross-linked dextran for chromatography having a fractionation range for peptides with 

Equilibrate the column with 1 M acetic acid. Apply the cold solution to the top of the column using 0.4 ml per cm2 of column cross-sectional area. The sum of the areas of any peaks eluted before the principal peak is not greater than 5.0% of the sum of the areas of all the peaks in the chromatogram. 

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Size Exclusion Chromatography (Chapter 31) has also been included as a means of analysis for substances that undergo separation more or less as per their molecular size, viz., insulin and human insulin—for proteins of higher molecular weight corticotrophin—for impurities of higher molecular weights and plasma-protein solution—for polymers and aggregates. 

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