Size Exclusion Chromatography

Introduction

Chromatography was first developed in the first decade of the 20th century, mainly for the separation of plant pigments such as chlorophyll, carotenes and xanthophylls (green, orange, and yellow, respectively). The technique got its name from the property of colourful separation of the pigments in the process. Since the separation of chemical constituents is essential in any type of chemical analysis, chromatography became a vital and irreplaceable tool. Although later on, chromatography took many forms and applications, the same principles of chromatography cover all of them. However, its definition did change to a more generalised version.

Chromatography is a lab technique used to separate the constituents of a mixture dissolved in a mobile phase, using another phase. This process is based on the principle of differential speeds of the constituents in different phases, causing them to separate. Let us move to SEC.

The size exclusion chromatography or the molecular sieve chromatography is a method to separate macromolecules (10-5 to 10-3mm) based on their size and shape. Since a correlation between the size and weight can be drawn the method is also used to determine molecular weight.

The Process

The chromatography column tube is tightly filled with porous beads which are mainly made of agarose, polyacrylamide or dextran polymers. Then the buffer is added in the column and degassed so that there are no air bubbles.

After degassing the column, the sample is loaded at the top of the column tube. Followed by this, the buffer is gradually added from the top. As the buffer flows through the column, it forces the solute out of the pores. Now, as the mixture moves down, components of the sample start moving at different rates based on their size.

The final step is to collect equal-sized fractions of the buffer (known as the eluate) eluting out of the column. These collected fractions are then tested by spectroscopic techniques to determine the concentration of the macromolecules in it. A plot of the eluted buffer fractions (volume) versus the absorbance, known as a chromatogram, is used to determine the success of the experiment performed.

Some Notes

The buffer used should have the pH and salt concentrations optimised for the target molecules to remain active. The working range of pore size is a decisive parameter for the separation of macromolecules. Therefore, beads are selected based on the estimated size of the macromolecule so that the molecule is not too large to get into the pore (known as the exclusion limit) and elute with all the large macromolecules or too small to reach all the pores (known as the permeation limit) and end up with small molecules like peptides and nucleotides.

How to optimise the results?

Size Exclusion Chromatography Optimisation

One may achieve a better resolution by increasing peak-to-peak distance known as selectivity. Therefore, using a longer column or slower buffer flow rate may increase selectivity and thus the resolution.

If a column is not packed correctly, it will increase the width of the elution curve known as zone broadening. Increasing the column efficiency will reduce the zone broadening and also increase the resolution.

Large sample volume can also reduce the resolution due to zone broadening known as extra-column effects. Increasing the column diameter will increase the column capacity and thus reduce the extra-column effects.

The Working

The diffusing ability of a molecule through the beads (stationary phase) is based on the size. Same happens here, the larger molecules hardly diffuse into the beads and comes out faster than smaller ones with better diffusing ability. The large molecules which do not fit into the pores, flow down the interstitial space (remain in the mobile phase) and comes out of column first. This volume is known as void volume (Vo). Molecules with the best diffusion ability come out last at total accessible volume (Vt). Therefore, for an optimum result, one should select a pore size that results in the target molecule to elute between the Vo and the Vt.

Total accessible volume (Vt) = Void volume (Vo) + Pore volume (Vp)
Total column volume (Vc) = Void volume (Vo) + Bead volume (Vi) + Pore volume (Vp)
Note: Bead volume (Vi) = Total bead volume (Vb) – Pore volume (Vp)
Therefore, Vi is the bead volume inaccessible to buffer.

The shape and size of the target molecule is described by partition coefficient (Kp). Partition Coefficient is defined as the relative concentration of the molecules in the stationary to the mobile phases.

So, the molecules eluting at Vo have Kp = 0 because they cannot access the stationary phase. The molecules eluting at Vt will have Kp = 1 and if a particular molecule interacts with the column or beads, which is not ideal, will have Kp > 1.

It is not easy to determine the actual concentration in the stationary and mobile phases, and this is not even required because Kp can be expressed in other parameters, the volumes. Let us say the target molecule M elute at Ve then

Size Exclusion Chromatography Partition Coefficient Determination

How to determine the molecular weight?

To determine the molecular mass, one has to run the standard proteins markers (markers have a known molecular mass) through a similar column and then make a plot of Kav vs log of molecular weight. This graph gives a unique linear function f(x) which can be used to determine the molecular mass of the target molecule.

Size Exclusion Chromatography Molecular Weight Determination

The Drawbacks

The standard proteins markers are globular; thus, if the target molecule is not globular, we get incorrect molecular mass. Sometimes the molecular mass of a monomer increases with the amount of sample loaded due to concentration-dependent oligomerisation. Hence, other methods should be used to determine precise molecular mass like PAGE or AGE and ultracentrifugation.

A limited number of samples can be separated, which falls in the right range and even within the range, a limited number of samples can be purified. For the separation of two molecules with a proper resolution, there should be at least a 10% difference in molecular mass.

The Advantages

One can easily separate the target molecules within a minimum volume, much less than the parent solution. This is possible because the eluted fractions used are much smaller in volume than the total solution.

SEC is the best approach for macromolecules sensitive to changes in pH, salt concentration, or other harsh conditions. One can smoothly perform separation in the presence of essential components like – other proteins, co-factors, urea, or high or low salt concentration.

The buffer composition used in any other chromatography will generally be the same for SEC, and this gives an option to perform SEC directly after other types of chromatography.

Major Applications

One can easily separate the folded and unfolded protein structures, thus separate functional and nonfunctional proteins. This is possible because of different hydrodynamic volumes of the two structures. Some studies have shown a relation between the hydrodynamic volumes and the elution volume.

Desalting: a process used to remove salts from larger molecules such as protein and nucleic acids. This can be done quickly under high flow rates and by choosing beads such that the macromolecules are near the exclusion limit and elute foremost near Vo.

References:

  1. Burgess RR. A brief practical review of size exclusion chromatography: Rules of thumb, limitations, and troubleshooting. Protein Expr Purif. 2018;150:81-85. doi:10.1016/j.pep.2018.05.007
  2. Protein Purification: Principles, High Resolution Methods, and Applications, 3rd Edition. Edited by Jan-Christer Janson. doi:10.1002/9780470939932
  3. Size Exclusion Chromatography: Principles and Methods. GE Healthcare Handbook. PDF link
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