Medical brain notes 14

  INTRODUCTION The  size-exclusion chromatography  (or  gel-chromatography ) is a means of separation which is exclusively dependent on the exchange of solute molecules between the solvent of the mobile-phase and the same solvent within the pores of the column-packing material. In reality, it is the pore-size-range of the packing material that solely determines the molecular-size-range within which a particular separation can take place effectively.   The timely adoption of the  cross-linked dextran gels  ( i.e.,   Sephadex ) in late-fifties as a packing mate-rial for column chromatography opened an altogether new horizon of chromatographic separation whereby substances, in general, undergo separation more or less as per their molecular size.   In actual practice, the inert gels of dextran (I)-a polyglucose or other types of polymers, for instance : agarose and polyacrylamides, wherein the macromolecules invariably are cross-linked to afford ...

  THEORY The efficiency and ability of a gel to slow down the movement of various substances downwards in a packed column with the respective gel entirely depends on the molecular size of the substance  vis-a-vis  to the pore sizes prevailing within the gel matrix. Evidently, a substance with high molecular weight is unable to diffuse into the pores of the gel and thereby moves down the column more rapidly through the channels between the grains of the gel. On the contrary, a substance having molecular size distinctly smaller than the largest pores of the gel shall naturally penetrate the pores and move with a slower pace down the column. In this manner the substances having molecular size greater than the pores shall undergo exclusion thereby affecting their elution from the column into the space immediately ahead of the relatively small molecular weight compo-nents. In other words, the substances are found to be eluted from the column strictly in order of the decreasing...

  APPARATUS The apparatus for ‘ size-exclusion chromatography ’ essentially comprises of a chromatographic column generally made up of glass having a diameter to height ratio of between 1 : 10 and 1 : 20, packed with an appropriate separation material ( e.g.,  different grades of  Sephadex ) which is capable of fractionation in the suitable range of molecular size and may be adequately temperature controlled. It is an usual practice to allow the mobile phase to pass through the column at a constant rate either by the aid of a suitably pump or simply by gravity.   1. APPLICATION OF SAMPLE   The sample is normally applied to the column by adopting one of the  five  following methods, namely :   ( i ) Directly to the drained-bed-surface with permitting the bed to dry,   ( ii ) Layered beneath the mobile-phase, provided the sample is denser than the mobile-phase,   ( iii ) Using a flow adaptor,   ( iv ) Using a syringe through a septum,...

APPLICATIONS OF SIZE EXCLUSION CHROMATOGRAPHY IN PHARMA-CEUTICAL ANALYSIS The size-exclusion-chromatography may be used for  two  specific purposes in the analysis of pharma-ceutical substances, such as :   ( i ) Determination of relative component composition, and   ( ii ) Determination of molecular weight.   1. DETERMINATION OF RELATIVE COMPONENT COMPOSITION   The assay method along with specific experimental parameters are duly stated in the  official mono-graph . Here,  two  situations arise, namely :   ( a )  Equivalent Responses :  In case, all of the components of the sample exhibit equivalent responses to the detector, then the relative quantity of each component may be determined conveniently by dividing each peak area by the sum of the peak areas of the components of interest, and   ( b )  Non-equivalent Responses :  In case, the responses achieved are not equivalent, calculate the relative component co...