Size exclusion chromatography total volume

The total stationary-phase volume required to process a given feed stream is proportional to the inlet concentration and volume of the feed. For example, for a typical inlet concentration of protein of 10 g/L, in a 100 L volume of feed, a column volume of at least 100 L is needed for size-exclusion chromatography. In comparison, an ion-exchange column having an adsorption capacity of 50 g/L would only require 20 L of column volume for the same feed. 

Polymerization conditions temperature 35 °C time 18.5 h solvent CH2C12, 100 mL total volume b TON turnover number, E ethylene, P propylene, H 1-hexene c Determined by size exclusion chromatography with polystyrene standard "Temperature 25 °C... 

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),... 

The separation range in size-exclusion chromatography for a particular column is relatively narrow, and it lies between the total volume of the liquid phase in the column (void volume) and the exclusion volume, Ve. The difference between these two volumes is the total pore volume of the packing material in the column. Indeed, if some molecules of studied polymers are small enough to penetrate inside all pores of the packing material, they will elute with the column void volume. On the other hand, polymers with significant molecular size that cannot penetrate inside the particles will all travel together around the particles and elute early with exclusion volume. 

Separation in size-exclusion chromatography requires careful matching of the pore size of the stationary phase material with the size of the molecules to be separated. Small molecules in a sample will be able to penetrate all the pores of the stationary phase and will elute with an elution volume, Fe, which is equal to the void volume of the column, Fm- Very large molecules will be excluded from all the pores of the stationary phase and will elute with an elution volume, which is equal to the interstitial volume of the Uquid between the particles, Fj. Molecules of intermediate size will be able to penetrate some but not all of the pores and will elute with an elution volume that is between the interstitial volume and the void volume of the column. The void volume is the total volume of the liquid in the column and is related to the interstitial volume and the pore volume, Fp by equation (3.38) ... 

Size Exclusion Chromatography A mode of chromatography using porous particles having pores of a size comparable to that of the molecules to be separated. Large molecules do not access the pores and elute at the external porous volume. Small molecules access all pores and elute at the total pore volume. Intermediate size molecules access part of the pores. This method applies to polymers, and especially to proteins. The solvent and the porous soUd must be selected to avoid adsorption of the feed components. 

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. 

Differentiation and separation only occurs over a certain range of molecular sizes, typically between molecular weights of 2 kDa and 200 kDa, although this can be increased up to 1,000 kDa by the use of more specialised gels. This size range is dependent on the sizes of the pores and pore size distribution in the gel matrix. Retention volumes Vr are often used in size exclusion chromatography instead of retention times tR. The total volume V, of the separation column is the sum of the volume of the gel particles Vg, the volume of the solvent inside the pores, also called the intrinsical volume V and the volume of the free solvent outside the pores, the inter particle volume Vq ... 

The largest elution volume in size-exclusion chromatography is the total mobile-phase volume in the column the sum of the pore volume and the interstitial volume, thus the separation space is constrained. Therefore, how good a separation can be obtained depends strongly on the width of the individual band, namely, on the plate count. System plate count is very important in SEC. 

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... 

Size exclusion chromatography is usually carried out by applying a sample in a volume that is small relative to the total volume of the column, as illustrated in figure la. This results in a large dilution of the sample on the column, in some cases ranging from 10 to 100-fold. In many situations this dilution on the column causes no problems with the analysis. However, for protein samples in which a rapid equilibrium between monomer and oligomers exists, dilution of this magnitude can seriously affect the results. 

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