This module is designed to provide theory and practical use of size exclusion chromatography (SEC) for separation of proteins.
1. What is an obvious physical difference between an immunoglobulin (antibody), which is a protein, and glucose, which is a monosaccharide?
2. When would it be useful to separate immunoglobulins from glucose in blood?
3. How could you devise an experiment to separate immunoglobulins from glucose utilizing the physical difference between the molecules determined in the first question?
Size exclusion chromatography (SEC), also called gel filtration, gel permeation, molecular sieve, and gel exclusion chromatography, is a separation technique used to separate molecules on the basis of size and shape (hydrodynamic radius). Size exclusion chromatography is called gel filtration chromatography because the gel essentially allows for the filtering of molecules from a sample based upon molecular size. However, unlike other techniques, the larger molecules elute first. This technique is widely applicable to purification or desalting of proteins in complex samples such as blood, which contain molecules of widely different sizes.
4. Depicted below are three different sized molecules. Next to them is shown a pore. What would happen as each molecule interacted with the pore? How would this effect the retention time of each molecule?
Molecules of different sizes can be separated by this technique because of differential time spent inside a solid phase particle which excludes entrance of relatively larger molecules, allows some entrance of medium-sized molecules, and allows free accessibility of the smallest molecules. The particles contain pores with tunnels in which the size can be controlled depending on the size of molecules to be separated. Smaller molecules experience a more complex pathway (like a maze) to exit the particle than do larger molecules. Because molecules that have a large size compared to the pore size of the stationary phase have very little entrance into the pores, these larger sized molecules elute first from the column. Medium sized molecules are relatively large compared to the pore size of the solid phase and therefore may find some pores in which they enter and spend some time. Smaller-sized molecules have more pores that are accessible to them and therefore spend more time inside the pores relative to larger-sized molecules. Therefore, smaller molecules elute last and larger molecules elute first in Size Exclusion Chromatography.
Figure depicting the separation of three molecules by Size Exclusion Chromatography (SEC):
a.The black molecule is much smaller in size compared to the pore size of the solid particle and has total access to the pores (not excluded).
b.The Green molecule is somewhat smaller in size compared to the pore size of the solid particle and has some access to the pores (partially excluded).
c.The purple molecule is larger in size than the pore size of the solid particle and does not have any access to the pores (totally excluded).
The figure below shows a schematic representation of the operation of a size exclusion chromatographic column:
The link below shows an animation of a SEC separation:
The Solid Phase Material:
Solid phase materials used in SEC are usually classified based on their ability to separate different sizes of proteins. Since size is a difficult item to accurately measure for a large molecule, the solid phase materials are identified with a molecular weight range instead and the weight is equated with size. All compounds with a molecular weight less than or equal to the lower number in the range will see the entire internal volume of the beads resulting in no selection and therefore no separation. All compounds with a molecular weight greater than or equal to the higher number in the range are completely excluded from the inside of a bead and therefore no separation is achieved. Molecules with weights or sizes between these two extremes of the range can be separated. This is the numerical pore size range reported for each solid phase material used in SEC. The pore size used for a separation is dependent on the size range of the particular set of molecules to be separated. Smaller pore sizes are used for rapid desalting of proteins or for protein purification. Intermediate pore sizes are used to separate relatively small proteins. Very large pore sizes are used for purification of biological complexes.
5. Other chromatographic methods depend on specific interactions between the molecules being separated and the surface of the solid support. What effect might such specific interactions have on the method you came up with to separate glucose from immunoglobulins? Can you think of how you might limit specific interactions between the molecules and the surface?
Types of Material
Protein separations are generally performed using materials composed of dextrose, agarose, polyacrylamide, or silica which have different physical characteristics. Polymer combinations are also used. Ideally the materials will have no interaction with the proteins or maybe modified so that there is little interaction between the material and protein. The dextran sephadex is most commonly used in protein separations. Typical separation ranges that can be achieved using sephadex are given below. Molecules ranging from 100 to 600,000 Da can be separated depending on the type of sephadex chosen.
G10 (100-800Da), G15 (500-1500 Da), G25 (1000-5,000 Da), G50 (1,500-30,000 Da), G75 (3,000-80,000 Da), G100 (4,000-150,000 Da), G150 (5,000-300,000 Da), and G200 (5,000-600,000 Da).
Therefore, the solid phase packing material is selected based upon some knowledge of the size of the range of molecules to be separated.
Applications of SEC:
SEC is not a high resolution technique; generally the molecules to be separated must differ by at least two-fold in molecular weight. Therefore, SEC is useful for desalting or removal of small molecule contaminants from protein samples, determination of the solution subunit composition of a multimeric protein, and to isolate different multimers from each other.
Size exclusion chromatography is generally used near the end of the purification process for a protein of interest, for example to desalt the protein or separate the correctly folded native protein from the denatured protein.
Other Important Aspects of SEC:
The ability to correctly pack a column is very important in obtaining the desired separation. Overpacking a column can lead to inaccessible pores and poor resolution. Underpacking a column can lead to surface area accessibility problems and cause poor resolution.
6. Would you expect glucose or immunoglobulins to produce a broader peak profile when dissolved in buffer solution at a similar concentration? Which of the peaks below is most likely to belong to glucose? Which peak is most likely to belong to an immunoglobulin? Explain.
Obtaining Size and Molecular Weight Data from SEC:
Calibrations must be performed in order to get information about the size or molecular weight of a protein separated by SEC. Standard protein samples of known molecular size are separated on a SEC column and the retention volumes or elution volumes (i.e. the volume of eluting buffer necessary to remove a particular analyte from a packed column) are recorded. A calibration plot of log molecular mass (Y axis) versus elution volume (X axis) is prepared. The calibration plot can be used to estimate the molecular weight of a protein(s) by separating the protein(s) on the same column as the standards and recording its retention volume. The molecular weight can then be extrapolated from the calibration plot as shown below.
If standard proteins 1, 2, and 3 have molecular masses of 60,000, 30,000, and 15,000 Da, respectively, then an unknown protein which has an elution volume of 9mL will have an estimated log molecular mass of 4.7 and a molecular mass of 45,000 Da.
7. Would it be easier to separate glucose from an immunoglobulin or two immunoglobulins from each other? Explain.