Size exclusion chromatography total permeation

The effect of limited penetration of the pores by the largest molecules may also be applied beneficially for the separation of very large molecules. Depending on the size of the molecules (in solution), they will be more ore less excluded from the pores, and hence the retention times will be affected. This effect is used in size exclusion chromatography (SEC) or gel permeation chromatography (GPC). In this technique, any interactions between the solute molecules and the stationary phase are purposefully avoided. The solute molecules remain exclusively in the mobile phase, but the accessible mobile phase volume, and hence the retention volume, may vary between the total volume of the mobile phase and the so-called exclusion volume, which is the total volume of mobile phase outside the pores. The latter elution volume applies to very large solute molecules (excluded solutes),... 

Gel permeation chromatography (GPC), sometimes called size-exclusion chromatography (SEC), is based on a very simple principle a gel is constructed with a narrow range of pore sizes and packed into a chromatography column. If a polymer of infinite molecular weight is applied at the top of the column, it cannot fit into any of the pores and is eluted in volume Vq, the excluded volume. A small molecule, on the other hand, can fit into all the pores and is this eluted in volume Ft, the total volume of the column. Fractionation according to molecular size thus occurs between elution volumes Fq and Ft with molecules eluted in order of decreasing size. 

Size exclusion chromatography separates molecules according to their size in solution. As a sample passes through the column, molecules which are too large to penetrate the pores of the packing are excluded and remain in the interparticle volume, Vq (this is also called the void or interstitial volume and is not to be confused with the solvent elution time Iq normally encountered in other forms of liquid chromatography). These molecules are eluted first from the column at the point of total exclusion. Small molecules which can permeate all the pores elute at the solvent front or total solvent volume F. This is the point of total permeation. Molecules of intermediate size will penetrate... 

The degrees of alteration caused by frying foods are commonly estimated by determining total polar materials by simple column chromatography, polymers by gel-permeation chromatography, or size-exclusion chromatography, and... 

In exclusion chromatography, the total volume of mobile phase in the column is the sum of the volume external to the stationary phase particles (the void volume, V0) and the volume within the pores of the particles (the interstitial volume, Vj). Large molecules that are excluded from the pores must have a retention volume VQ, small molecules that can completely permeate the porous network will have a retention volume of (Vo + Fj). Molecules of intermediate size that can enter some, but not all of the pore space will have a retention volume between VQ and (V0 + Fj). Provided that exclusion is the only separation mechanism (ie no adsorption, partition or ion-exchange), the entire sample must elute between these two volume limits. 

To understand how steric exclusion differs from the other forms of chromatography, refer to Equation 21.4. In this context, V a and Va are referred to as the void volume and the total pore volume, respectively. The distribution coefficient depends on the molecular weight of the sample and on the pore size of the packing. The equilibrium established in exclusion chromatography is described by Equation 21.1 ATx is defined by Equation 21.2. In a true permeation process, assuming all pores to be accessible to a small solute molecule, and = 1- If none of the pores... 

Size exclusion (SEC) or gel permeation chromatography (GPC) is the most widely used chromatographic method in biochemistry. The biomolecules are separated by size during elution through a neutral and hydrophilic packed bed of porous particles. Large molecules are excluded from the pores and elute first, whereas small molecules totally invade the pores and elute last. 

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