Proteomics/Protein Separations - Chromatography/Molecular Exclusion

Chapter written by: Laura Grell and Alexander Butarbutar
Contact llg3875@rit.edu or nbb3924@rit.edu for contributions
Chapter modified by Kai Burnett and Dalia Ghoneim
Contact kab9783@rit.edu or dxg6098@rit.edu

Molecular Exclusion

Figure 1. Molecular exclusion column - Larger molecule move faster as they are excluded from the beads. Smaller molecules have to travel through the beads causing them to move slower.

   Molecular Exclusion / Gel Filtration / Gel Permeation / Size Exclusion is one of the simplest mechanism for separating molecules. This method separates proteins based on their size. The stationary phase is packed with porous bead with a range of pore sizes. When a mixture of protein is introduced into the column, a range of protein from small, intermediate and large will be filtered through the column

• Small proteins can fit inside any pores and enter many pores before reaching the end of the column. Thus the movement of small proteins are greatly impeded which cause them to elute last in SEC.
• Intermediate size protein may fit in some pores but not in all. These protein will generally elute somewhere between the small size and large size protein.
• Large size proteins cannot enter the beads. Their movements in the column are not impeded by beads which make them the first protein to be eluded from the column.

   In practice, gel filtration can be used to separate proteins by molecular weight at any point in a purification of a protein. It can also be used for buffer exchange - a protein dissolved in a sodium acetate buffer, pH 4.8, can be applied to a gel filtration column that has been equilibrated with tris buffer, pH 8.0. Using the tris buffer, pH 8.0, as the mobile phase, the protein moves into the tris mobile phase as it travels down the column, while the much smaller sodium acetate buffer molecules are totally included in the porous beads and travels much more slowly than the protein.

Resolution in Molecular Exclusion Chromatography

Achieving high resolution protein separation depends mainly on two factors:

1. Sufficient selectivity: creating conditions that create enough space between sample zones
2. Sufficient efficiency: counteracting "zone broadening effects"

The resolution can be expressed in the following formula: $R={\frac {Vr1-Vr2}{(W1+W2)/2}}$

Vr1 and Vr2: the elution volumes of two adjacent peaks. Vr2-Vr1 therefore represents the distance between two peaks.

W1 and W2: the widths of the peaks

Achieving Sufficient Selectivity

The numerator of the Resolution formula represents the selectivity or the distance between two different curves in a chromatogram. When the distance is too short, two adjacent curves may start to merge resulting in an adequate separation of the two proteins. The selectivity is controlled by two factors: the selectivity of the actual medium, and the length of the column.

Different mobile phase medium have different selectivity curves. Some mediums provide high selectivity while others provide lower selectivity. The medium chosen usually reflects the type and size of the proteins being separated.

The length of the column also has an effect on the selectivity. Molecular Exclusion does not involve any gradients; therefore, the molecules will move down the column at a fairly constant rate. If two molecules are traveling at different rates, increasing the travel distance will subsequently increase the distance between the two molecules. Increasing the column length is one method that can improve resolution. One caveat to this, however, is that as the column lengthens, the sample zones broaden. This zone broadening effect will counteract some of the gained resolution.

Achieving Sufficient Efficiency

Increasing the efficiency involves counteracting zone broadening effects. Ideally all of the sample of a specific protein should reach the end of a column at the same time; however, several factors have an effect on the level of zone broadening that occurs. The two most significant ones are: uniformity in beads, and bead size and flow rate.

It is essential to have uniformity in the beads in a column. Irregularities will can different parts of a sample to travel down the column at different speed. The range of time over which the sample reaches the eng of the column becomes large which is not ideal.

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Resources

1. Craig, P., Rochester Institute Of Technology Gel Filtration Chromatography
2. GE HealthCare Molecular Exclusion Animation || Introduction to Gel Filtration & Products
3. Bio-Rad Size Exclusion Chromatography Kit || Size Exclusion Chromatography & Products
4. Molecular Exclusion Image

References

1. Archer J.P. Martin – Nobel Lecture The development of partition chromatography *
2. Richard L.M. Synge – Nobel Lecture Applications of partition chromatography *

.* Denotes Free Article