Gel Filtration Chromatography: Separation And Purification Of Biomolecules

Mechanism of Gel Filtration Chromatography

Discuss about the Gel filtration chromatograph technique.

The principal method that is used for the separation and purification of the biomolecules in biochemical laboratories is chromatography. In chromatographic separation, partition occurs in between two phases one is mobile phase and another one is stationary phase. Gel filtration chromatography or size exclusion chromatography is a type of chromatography (Murray, Granner, Mayes and Rodwell 2009). It is used to separate protein molecules based on stokes radius. Stoke radius is the measured diameter of the sphere occupied by the protein when they tumble in the solution. Stoke radius can also be defined as the function of molecular mass and shape (Murray, Granner, Mayes and Rodwell 2009).  As protein with large stroke radius are too large to enter inside the porous bed (stationary phase) and are thus eroded away (mobile phase) where as protein with smaller stoke radius enter the indentation of the porous bed and remain there until they are drifted back by the mobile phase. The protein thus separated emerge from the gel filtration column in the descending order of their measured stroke radii (Murray, Granner, Mayes and Rodwell 2009; Nelson, Lehninger and Cox 2008).

The following report gives a detailed analysis of concept behind the gel filtration chromatography along with the potential advantages and disadvantages associated with it. The report further illustrates the mechanism of gel filtration chromatography via diagrammatic representations and two video links. After illustrating the concept, the report tends to shed light on the practical application of this biochemical procedure in research along with different porous beds with their size limits, which are commonly employed as stationary phase. At the end, the report will provide a detailed breakup of the formula used to calculate the protein size in gel filtration chromatography.

Gel filtration chromatography is the simplest technique used for the separation of the complex component by according  to their molecule size only, measuring by how dynamic they penetrate the pore of the stationary phase, this  chromatography is also know a size exclusion chromatography or molecular exclusion chromatography. This type of method  permits to purified polypeptide from other polypeptides based to their size by moving through the gel porous into the column. Gel filtration is application use to analyze the separating of molecules size such as protein, polysaccharides -and nucleic acids by separating and removing large proteins and complexes.

This is used for the separation of the molecules according to their molecular weight, the proteins are aqueous in solution (mobile phase), the protein solution will be transfer to the top of chromatography column, these columns composed of a gel bed of porous beads, this is known as (stationary phase).

Practical Applications of Gel Filtration Chromatography

During the analyzing gel filtration the smaller molecules able to penetrate the bed of porous of beads, and will take time and elute last, while the larger molecules will not be able to enter bed of the porous of beads and pass through the sides of the gap between the porous and elute first.

The substance has been chosen for the fraction and the recognition of the molecular size depending on the size rage of the molecules within the sample. The molecules pass through the column in the pores including their aqueous solution to complete the elution. Subsequently, the molecule is measured. The volume is known as the total value TV.

During the separation process of the protein large molecules will elute first by passing through the sides of the gel of the stationary phase. However, the small molecules will take some time to elute as they enter the pore of the beads. There are many other technique or ways that can be purified the protein. Nevertheless, this process is of the ways to carry out.  

Gel chromatography is one of the methods that is used to analyze in the attempt to determine the size of the protein. This process separates the protein based on their size, solution and their shape as they pass through the stationary phase or gel pores. Usually protein has three-dimensional polymers of dextran, agarose or silica which is composed pores of controlled size ranges to give free movement to the liquid phase and limited diffusion of the protein to be separated.

Desalt and buffer exchange of gel filtration is used for the separation of the macromolecules from the smaller size molecules. Desalting and the buffer exchange are one of those applications used gel filtration chromatography. Both desalt and buffer exchange uses the same substance. The process to extract salt from the sample is known as “desalt” The buffer exchange occurs when extracting the salt from the test. There are different types of salt exchanges. In chromatography desalt is balanced with water while the buffer exchange is balanced with salt. Buffer exchange is a method used to exchange a set of buffer salts in substance to another. The applications used for desalting is the salt extraction from protein solution extracting phenol or unincorporated nucleotides from nucleic acid preparation.

Buffer exchange is generally used to place a protein solution into a more appropriate buffer before the subsequent applications such as:

1. Electrophoresis
2. Ion exchange
3. Affinity chromatography

Advantages and Disadvantages of Gel Filtration Chromatography

Advantages

1. It does not depend on any temperature, pH, and buffer composition. which means separation can be carried out under any conditions
2. There is very little adsorption present
3. There is zonal spreading than in other techniques
4. The elution volume is related to the molecular weight

Disadvantages

1. Limited number of peaks that can be resolved within short time scale of GPC run.
2. Filtration must be performed before using instrument to prevent dust and other practical from ruining the column and interfering with the detectors.
3. Most of the chains in the molecular masses will be too close for the GPC separation to show anything more than broad peaks. ‘‘(GPC) By Asabuwa N. F. Published on Jul 19, 2015’’

Figure: 1

From the lab, which took place during the year, the following picture (Figure 1) was taken for observation. This clearly shows that the blue dextran protein eluted first as it was the larger molecule.

This also shows that the potassium chromate protein eluted last as it was the smaller molecule. This stayed on top of the blue dextran and eluted at a slower rate. This was due to its size and molecular weight as stated in applications/uses in research below.

Figure 2

The picture in (figure 2) shows three different columns, each with porous beads and solution. In the first column the two solutions are added into the porous beads.The second column shows the two proteins eluting through the porous beads, clearing showing that the smaller proteins on top of the larger proteins as they are eluting through the mobile phase at a faster rate. The third column shows the smaller proteins eluting at a slower speed allowing the larger molecules to elute at a quicker rate.

Video Link Section

https://www.youtube.com/watch?v=ZA6rgm8wnXE

This silent video shows notes on the basics of gel filtration chromatography. Each slide shows clear diagrams and definitions explaining the process. In the first slide it explains what gel filtration chromatography is and some of its examples in the stationary phase, also what gels can be used this was most associated with dextran and polyacrylamide. (Figure 2) is shown in this clip,  as this is an important diagram and can be followed quite easily. In the last few slides a clear labelled diagram is presented explaining the process of what molecules are eluted first. This video was chosen as it helped with the understanding of the basics of gel filtration chromatography and really gave a clear explanation. I would recommend this video to anyone who wants to know more about this topic.

https://www.youtube.com/watch?v=KqF6KuXaY0w

This is another silent video, which shows how the experiment of gel filtration chromatography was carried out. In the first few slides of the clip, it tells some information about the gel in the mobile and stationary phase and other properties associated with the product. Following all that information, the experiment is carried out beginning with what equipment is used. Throughout the clip and the experiment each step is clearly explained and outlined. This clip was chosen as it shows how to carry out the experiment without looking up information in a book. In each step it was clearly explained how to carry out this experiment and made it more understandable and easy to learn in the future.

Porous Beds Used as Stationary Phase

The main application of gel filtration chromatography is in the purification of the macromolecules. the separation of molecules based on the molecular size and shape is referred as the gel filtration. It is a type of size exclusion and be used to fractionate molecules and compounds in a sample into fractions with a particular size and separate compounds like small and large proteins (Bio-rad laboratories 2017). The common material used are polymeric organic compounds that process three dimensional network of pores. Large molecules that are excluded from the pores will pass through the interstitial spaces around the gel particles first as they are quite big molecules, an example of large molecule is blue dextran with a size of 2000 kdaltons. The smaller molecules can freely enter the pores as the larger molecules move with the mobile phase very quickly, making them elute first. Molecules that are small enough to enter the pores take a more longer route through the gel particles, therefore they move slowly and are retained on the column for longer periods. An example of a small molecule is potassium chromate with a size of 194 kdaltons. Sephadex G-100 and Sephadex G-200 are most frequency used.

Table 1: Gel Filtration Media

The technique that was chosen was estimating a Protein's Molecular Weight by Gel Filtration chromatography. In this technique the protein sample is suspended in solution, this is normally the mobile phase and applied to the top of the chromatography column which is filled with a matrix of porous beads.This known as the stationary phase. The smaller molecules will enter into the pores of the beads

First of all the column is calibrated, The volume of the liquid it takes to elute the molecule is measured, this volume is referred as the total volume, and is represented as V_{t  .}This is also done with the larger molecules. The volume of the liquid it takes to elute the molecule is measured, this volume is referred to as the void volume V₀

The total volume usually results in 90% of the bed column. This is calculated as πr²h where r is the inner radius of the column and h is the height of the packed column material.)  The void volume is usually 40% of the bed column. To calculate the molecular size of a molecule, the gel filtration column is calibrated again with a set of known molecular size standards. (Elsevier. 2017). The amount of volume taken to elute each molecule of the standard is recorded. This volume is referred to as V_e  To get a accurate result a distribution coefficient (K_D) for each molecule is determined. This is calculated with the following formula.

Formula Used to Calculate the Protein Size in Gel Filtration Chromatography

K_d=(V_e−V_o)(V_t−V_o)

When plotting the log₁₀ of the molecular weights of the standards and the K_D values this results in a straight line standard curve. From this  unknown protein can be run through the column and the molecular weight can be identified from the standard curve.

The following is an example of a problem that could take place in the lab. A column having a fractionation range of 5000 to 250,000 is calibrated and gives a void volume, V₀, of 40 mL and a total  volume, V_t, of 90 mL. The molecular weight standards are applied to the column and their elution volumes (V_e values) are measured. A purified protein has elution volume of 60 mL. What is its approximate molecular weight?

This is the solution to the problem. First calculate the distribution coefficient, K_D, for each of the standard. Since the V₀ for this column has been measured to be 40mL and the V_t has been measured to be 90 mL, the K_D is calculated as

K_d= (V_e−V_o) (V_t−V_o)

K_d= (V_e−40) (90−40) = (V_e−40) x 50

For standard sample A, we have

K_d=(82.5−40) x 50 = 42.550 = 0.85

There are two types of applications in chromatography these include

1. Gas liquid chromatography (GLC)
2. High performance liquid chromatography (HPLC)

Gas liquid chromatography is composed of an injection which is heated to 100-300 degrees, this also has a sample size as low as a tenth of a microlitre. the capillary column has to components these include a support coated open tubular system and a wall coated open tubular system. the oven is the most important part of the equipment. This can operate from 30-400 degrees and can heat up or cool down at 0.5-50 degrees per minute, last is the detector this detects a change in the sample or material being used.

High performance liquid chromatography (HPLC) is composed of a a number of equipment such as a pump which delivers the mobile phase between 0.1 and 10 ml/min. The guard column is similar to the analytical column it protects the original column. The detector has the same job as the gas liquid chromatography, it detects a change eg the UV detector.

Conclusion

Thus from the above discussion, it can be concluded that gel filtration chromatography is an important biochemical procedure that has a huge range of application in the laborites. It is one of simplest procedure that is used for the separation of protein bio-molecules based on their molecular size. Here porous bed is used as stationary phase and desalt buffer is used a mobile phase. Few of the widely used porus bed are sephadex, superdes and sephacryl. The porous bed differs from each other based on their size. Porous bed with large exclusion size is used for the separation of dextran sulphate (2000-kilo Daltons). Some of the notable advantages of this biochemical procedure are: the process is independent to external temperature, pH and buffer condition and thus can be carried out under any condition. Moreover, the formula that is employed for the calculation of the protein’s molecular mass is also easy to execute. However, the procedure has sudden disadvantages too like only a very limited number of picks can be resolved on the basis of the molecular mass in gel filtration chromatography. In order to negate the disadvantages of gel filtration or size exclusion chromatography, High-Pressure Liquid Chromatography (HPLC) is employed. Here incompressible silica or alumina microbeds are used as stationary phase.

Thus, overall chromatography, with a special mention to gel filtration chromatography is an important pillar in biochemical research procedures as it help in the separation of protein or other small macromolecules or micro molecules of proteins like amino acids and sugars.

References

Bio-rad laboratories. 2017. Gel filtration Chromatography. [ONLINE] Available at:https://www.bio-rad.com/featured/en/gel-filtration-chromatography.html. [Accessed 11 December 2017

Creative Biostructure. 2005. Gel Filtration Chromatography. [ONLINE] Available at: https://www.creative-biostructure.com. [Accessed 9 December 2017]

Elsevier. 2017. Science Direct. [ONLINE] Available at: https://www.sciencedirect.com/science/article. [Accessed 9 December 2017]

https://www.ncbi.nlm.nih.gov/books/NBK22410/ .  access 19/01/2018 ‘‘(GPC) By Asabuwa N. F. Published on Jul 19, 2015’’

Murray, K., Granner, D.K., Mayes, P. and Rodwell, V., 2009. Harper's Illustrated Biochemistry. 28 (p. 588). New York: McGraw-Hill.

Nelson, D.L., Lehninger, A.L. and Cox, M.M., 2008. Lehninger principles of biochemistry. Macmillan