Molecular exclusion chromatography

In molecular exclusion chromatography (also called size exclusion or gel filtration or gel 

Large molecules pass through the column faster than small molecules do. 

In pure molecular exclusion, all molecules are eluted between Km = 0 and = 1. 

The total volume of mobile phase in a chromatography column is Vm, which includes solvent inside and outside the gel particles. The volume of mobile phase outside the gel particles is called the void volume, V0. The volume of solvent inside the gel is therefore Vm — V(). The quantity Km (read K average ) is defined as 

Void volume is measured by passing a large, inert molecule through the column. Its elution volume is defined as F0. Blue Dextran 2000, a blue dye of molecular mass 2 X 106, is commonly used for this purpose. The volume Vm can be calculated from the measured column bed volume per gram of dry gel. For example, 1 g of dry Sephadex G-100 produces 15 to 20 mL of bed volume when swollen with aqueous solution. The solid phase occupies only 1 mL of the bed volume, so Vm is 14 to 19 mL, or 93-95% of the total column volume. Different solid phases produce widely varying column bed volumes when swollen with solvent. 

Small molecule Large molecule ( StaUonaiy phase 0 Frit retains statkxiaiy phase 

In molecular exclusion chromatography, the volume of mobile phase (the solvent) in the column outside the stationary phase is called the void volume, Vq. Large molecules that are excluded from the stationary phase are eluted in the void volume. Void volume is measured by passing through the column a molecule that is too large to enter the pores. The dye Blue Dextran (2 X 10 Da) is commonly used. 

Retention volume is the volume of mobile phase required to elute a particular solute from the column. Each stationary phase has a range over which there is a logarithmic relation between molecular mass and retention volume. We can estimate the molecular mass of an unknown by comparing its retention volume with those of standards. For proteins, it is important to use eluent with an ionic strength high enough (such as 0.05 M NaCl) to eliminate electrostatic adsorption of solute by occasional charged sites on the gel. 

Proteins were passed through a gel filtration column and retention volumes (Vr) were measured. Estimate the molecular mass (MM) of the unknown. 

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Glucose oxidase (from Aspergillus niger) [9001-37-0] Mf 186,000, [EC 1.1.3.4]. Purified by dialysis against deionized water at 6° for 48hours, and by molecular exclusion chromatography with Sephadex G-25 at room temperature. [Holt and Cotton J Am Chem Soc 109 1841 1987.]... 

Molecular exclusion chromatography. The stationary phase in molecular exclusion chromatography is a material containing pores, the dimensions of which are chosen to separate the solutes present in the sample based on their molecular size. This can be perceived as a molecular sieve allowing selective permeation. This technique is known as gel filtration or gel permeation, depending on the nature of the mobile phase, which is either aqueous or organic. The distribution coefficient in this technique is called the coefficient of diffusion. 

If the molecular masses of solutes are >2 000 and if they are soluble in organic solvents and their molecular diameter is >30 nm. Figure 25-14 tells us to try molecular exclusion chromatography.. Stationary phases for this type of separation are described in Chapter 26. If the molecular masses of solutes are >2 000, and they are soluble in water, but not ionic, and have diameters <30 nm, the decision tree says to use reversed-phase chromatography, or hydrophobic interaction chromatography. 

Figure 26-13 Separation of proteins by molecular exclusion chromatography with TSK 3000SW column. [Courtesy Varlan Associates. Palo Alto, CA.]...

Nanoparticles can be separated by molecular exclusion chromatography just as proteins are separated. Figure 26-15 shows the relation between measured size and retention time of CdSe quantum dots. These are particles containing 2 000 CdSe units in a dense, crystalline core capped by alkyl thiol (RS) groups on Cd and trialkylphosphine (R3P) groups on Se. 

Molecular exclusion chromatography is based on the inability of large molecules to enter small pores in the stationary phase. Small molecules enter these pores and therefore exhibit longer elution times than large molecules. Molecular exclusion is used for separations based on size and for molecular mass determinations of macromolecules. In affinity chromatography, the stationary phase retains one particular solute in a complex mixture. After all other components have been eluted, the desired species is liberated by a change in conditions. 

Ferritin (molecular mass 450 000), transferrin (molecular mass 80 000), and ferric citrate were separated by molecular exclusion chromatography on Bio-Gel P-300. The column had a length of 37 cm and a 1.5-cm diameter. Eluate fractions of 0.65 mL were collected. The maximum of each peak came at the following fractions ferritin, 22 transferrin, 32 and ferric citrate, 84. (That is, the ferritin peak came at an elution volume of 22 X 0.65 = 14.3 mL.) Assuming that ferritin is eluted at the void volume and that ferric citrate is eluted at Vm, find Kay for transferrin. 

Gel filtration chromatography (also known as size or molecular exclusion chromatography) separates molecules based on their ability to penetrate into the pores or channels in agarose or dextran beads. As a mixture of molecules in a fluid permeate through the beads of gel the volume available for diffusion is determined by their diameter and the size of the channels in the gel beads. The... 

Molecular exclusion chromatography Medium-low High Medium-low Medium ... 

In industry, dialysis is not widely used because it is slow and labor-intensive. Here, desalting and/or buffer exchange is usually performed by diafiltration or molecular exclusion chromatography, which are discussed later. 

Molecular exclusion chromatography, also known as gel filtration, size exclusion chromatography, gel permeation chromatography, or simply gel chromatography, is another separation method based on differences in molecular size. 

Schematic illustration of the separation principle involved in molecular exclusion chromatography.

The most commonly used matrices for molecular exclusion chromatography are cross-linked polymer gels, such as agarose, dextran, and polyacrylamide. Since dextran and agarose are biodegradable, they should be stored in the presence of antimicrobial agents. 

In molecular exclusion chromatography, resolution is directly related to the sample loading volume, since the higher the sample volume, the higher the volume in which the protein is eluted. Usually, sample volume should be 0.5-5% of the total bed volume. For volumes below 0.5% the sample becomes too diluted, while volumes above 5% work well only for molecules with large differences in size. For desalting, due to the pronounced difference in the molecular size of the components, sample volumes can reach up to 30% of the column capacity without a significant decrease in resolution. 

Therefore, to achieve high resolutions in molecular exclusion chromatography, it is common to use columns with large height-to-diameter ratios (in the range of 20-40), although at an industrial scale this can become difficult. 

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