Plasma Protein Solution For Polymers and Aggregates

Materials Required Plasma protein solution 2.0 ml mixed phosphate buffer pH 7.0 with azide [To 1000 ml of a solution containing 1.8% w/v of disodium hydrogen orthophosphate and 2.3% w/v of sodium chloride and sufficient of a solution containing 0.78% w/v of sodium dihydrogen orthophosphate and 2.3% w/v of sodium chloride (about 280 ml) to produce a pH of 7.0 Dissolve sufficient sodium azide in the resulting solution to give a 0.02% w/v solution] 1000 ml  

Procedure The chromatographic procedure may be carried out at room temperature using (a) a column (1 M x 25 mm) packed with a cross-linked dextran suitable for fractionation of globular proteins in the range of molecular weights from 5,000 to 350,000 (Sephadex G-150 is suitable), (b) mixed phosphate buffer pH 7.0 with azide as the mobile-phase with a flow rate of about 20 ml (4 ml per square centimetre) of column cross-sectional area) per hour, and (c) a detection wavelength of 280 nm. 

Collect the eluate in fractions of about 4 ml and combine the fractions corresponding to each peak. For each combined fraction carry out the determination of nitrogen as per BP (1993). Not more than 10% of the total nitrogen is present in the combined fraction associated with non-retained proteins. 

What is the importance of Size-exclusion chromotography (SEC) in the assay of drugs Explain. 

Expatiate the theoretical aspects of SEC with specific reference to the following  

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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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