Gel Filtration Chromatography Principle, Components, Steps, Types, Application

Gel-filtration chromatography can be described as a type of partition chromatography which is used to distinguish molecules with different molecular dimensions. The technique has also been called by a variety of different names, such as gel-permeation or gel-exclusion size-exclusion and molecular-sieve chromatography.

Principle of Gel Filtration Chromatography

Gelfiltration is also referred to as molecular-sieve chromatography. This method of separation is determined by the different capacity (due to different molecular size) of the molecules present in the sample to penetrate to the pores within the medium. The stationary phase of this method is comprised of beads made of a sponge-like, hydrated material with pores that are of molecular dimension and a small size range. If an aqueous solution with molecules of different dimensions, is passed through a column that contains “molecular sieves,” molecules that are bigger than those of the filtration medium are able to move swiftly throughout the column. Smaller molecules get into the gel’s pores and are able to move more slow across the column. They are then eluted in an sequence of decreasing molecular size. A molecular weight of the most tiny molecular that is in a position to penetrate the pores the gel is thought to be the “exclusion limit” of the gel.

A column is composed of swollen gel particles as well as the solvent used to expand the gel inside a tubular container. The following equation can be found:

Vt = V0 + Vi + Vm 

where, 

• Vt = the total volume of the column (which can be measured), 
• V0 = the volume of liquid outside the gel matrix (known also void or dead volume), 
• Vi = the volume of liquid inside the matrix, 
• Vm = the volume of the gel matrix

Components of Gel Filtration Chromatography

1. Stationary Phase 
2. The Mobile Phase 
3. The Columns 
4. The Pump
5. Detectors

1. Stationary phase

Comprised of semi-permeable and polymer gel beads that are porous and have a an equivocal range of pores.

Properties of gel beads

• Chemically inert
• Mechanically Stable 
• It has a homogeneous and ideal porous structure (wide pores with a small size, which gives the lowest resolution).
• A uniform particle size and pore.
• The size of the pore of the gel needs to be to be controlled.

Types of Gel

a. Dextran gel

• natural linear polysaccharide that contains one link
• insoluble when in the aqueous media
• made by the cross-linking of OH groups of dextran, and an epichlorohydrin dispersion in an organic medium
• Chemical stability is high

b. Agarose gel

• large porous size gel
• formed from the neutral fraction of agar and the agarose units cross-linked with alternating 1,3 linked-b-D-galactose and 1,4-linked 3,6-anhydro-a-L-galactose units
• created below 30 degrees C
• Useful for large molecule separation

c. Polyamide gel

• Low solubility
• Used to separate organic acids, phenols etc.
• ability to create strong H-bonds between its amides and phenolic groups of the -OH group.

2. The Mobile Phase: Made of a liquid which is used to dissolve biomolecules and create the mobile phase, which allows high detection responses and soaking the surface of the packing. 

3. Columns: Columns Commercially Available include: 

• Analytical column- 7.5–8mm diameters, 
• Preparative columns-22–25mm 
• Usual column lengths-25, 30, 50, and 60 cm.  
• Narrow bore columns- 2–3mm diameter have been introduced

4. The pump: Are an syringe pump or a reciprocating pump that have a very steady flow

5. Detectors: 

Concentration sensitive detectors

• Bulk Property Detectors- Refractive Index (RI) Detector
• Solute Property Detectors- Ultraviolet (UV) Absorption Detector
• Evaporative Detectors- Evaporative Light Scattering Detector (ELSD)

Molar mass sensitive detectors

• Light Scattering Detectors
• Low Angle Light Scattering (LALS) Detectors
• Multiangle Light Scattering (MALS) detectors

Types of Gel Filtration Chromatography

A. Group Separations

Gel filtering is employed as a group separation method to separate small molecules from the group of larger molecules. It is also an efficient, quick solution to exchange buffers. Small molecules like excessive salt (desalting) or unlabeled labels can be easily separated. The samples can be prepared for storage, or used for other chromatography methods and tests. Gel filtration using group separation is commonly utilized in purification strategies for proteins to remove salts and for buffer exchange.

B. High resolution fractionation

Gel filtration can be used in fractionation mode to distinguish different components of an experiment on the basis of the size differences. The objective could be to separate one or more components, or to determine molecular weight, or determine the molecular weight distribution in the sample . The greatest results from high-resolution fractionation are obtained using samples that initially contain only a few components, or samples that are partially purified using other techniques of chromatography (in to get rid of the proteins that are similar in size and aren’t of interest).

Steps in Gel Filtration Chromatography

1. Spherical particles from gel filtering medium are put into the form of a column.
2. The sample is then applied onto the column.
3. Buffer (mobile phase) and sample moves across the column.
4. Molecules are able to diffuse out and in from the pores within the matrix (also described as the division in the matrix between the mobile and stationary phase).
5. Smaller molecules are able to move deeper into the matrix, and remain longer on the column.
6. Since buffer is continuously passing across the column, the molecules larger than the matrix’s pores are not able to be absorbed into the pores, and move into the columns.
7. Smaller molecules get absorbed by the pores, and delay their travel through the column.
8. Separation takes place at various intervals, which are followed by the detection of the components.

Factors affection on Resolution of gel filtration

Many factors influence the final resolution (the degree of separation between peaks of a gel

filtration separation): These include: (1) matrix choice, (2) sample size and concentration, (3) column parameters, (4) choice of eluent, (5) effect of flow rate, and (6) column cleaningand storage.

1. Matrix Choice

The primary consideration when creating an extensive chromatographic purification procedure concerns the properties of the matrix. The solid particles contained in the columns used for chromatographic recovery are called matrix. The matrix must be macroporous, hydrophilic and hydrophilic. It should also be rigid, sphericaland that is chemically solid, stable durable, and reusable. It should also be easily accessible and affordable.

Example of the commonly used matrix;

• Superdex is the best option to get high-resolution, quick time to run and high recovery.
• Sephacryl is a good choice for quick high recovery separations in industrial and laboratory scales.
• Superose has a broad separation range, however it does not work well for large and industrial-scale separations.
• Sephadex is ideal for fast group separations, such as the desalting process and exchange of buffers. Sephadex is utilized in laboratories and in production, prior to and after purification processes for chromatography.
  • Sephadex G-25 is suggested in the vast majority of separations which involve globular proteins. This medium is ideal for eliminating salt and other minor pollutants from molecules with a size greater than Mr 5 000.
  • Sephadex G-10 is ideal for segregating biomolecules, such as Peptides (Mr greater than 700) and lesser compounds (Mr 100+).
  • Sephadex G-50 is a good choice to separate molecules with a value of more than 30 000 from molecules with a value of less than 500, such as protein that is labeled or DNA from unlabeled label.

2. Sample Size and Concentration

The resolution of gel-filtration chromatography is dependent on the use to the specimen in a very small amount, usually 1-5 % of the bed volume. Because of this, gel-filtration chromatography is characterized by a lower capacity for handling of samples, and consequently, should be conducted later in the purification process in cases where the number of molecules present in the sample are small. 

The amount of the sample put into the column is limited because of its viscosity (which increases with the amount of sample) in relation to the fluid being used to make it. High viscosity results in an irregular flow of the sample through the column (with consequent reduction in clarity) as well, and in certain situations, may reduce the flow rate of the column. If you are separating proteins using gel filtering, the sample must not contain a concentration of protein greater than 20 mg/mL.

3. Column Parameters

The best resolution in gel-filtration chromatography is attained with long columns. The ratio of the column’s length to diameter can vary between 1:20 and 1:100.

4. Choice of Eluent

Because gel-filtration chromatography distinguishes molecules solely by their size this technique is independent of the kind of eluent employed. Elution conditions (pH, essential ions, cofactors, protease inhibitors etc.) must therefore be chosen to meet the needs of the molecule in question. But, the Ionic strength of the liquid must be sufficient to reduce protein-matrix and protein interactions via electrical or van der Waals interactions. In addition, adding 0.1 M NaCl or KCl to the eluent in order to prevent these interactions is popular.

5. Effect of Flow Rate

Low flow rates provide the highest resolution during gel-filtration chromatography because resolution and flow rate are inextricably related. The optimal flow rate to resolve proteins is 2 mL/cm2/h. However, greater flow rates may be employed, particularly when using rigid matrices, such as that of the Sephacryl HR range of GE Healthcare (30 mL/cm2/h). Unfortunately, the lower flow rates result in longer intervals of separation. So, a compromise in resolution and speed should be made.

6. Column Cleaning and Storage

The majority of gel-filtration matrices can be cleaned using 0.2 M sodium hydroxide or other nonionic detergents. If left unattended for long durations, the they should be stored at 4°C in darkness in the presence of an antimicrobial (e.g., 0.02-0.05 % sodium azide w/v or 20 % v/v of ethanol).

Applications of Gel-Filtration Chromatography

Gel filter chromatography is utilized to:

• Fractionation of complexes and molecules within a certain size range

• Analysis and determination of size
• Removal of complexes and proteins that are large
• Buffer exchange
• Desalting
• Elimination of tiny molecules like primers, nucleotides and dyes, and other contaminants.
• Test of purity of the sample
• Separation of bound from nonbound radioisotopes

Advantages of Gel Filtration Chromatography

• Quick analysis time.
• Well defined separation
• Good sensitivity and narrow bands
• There isn’t any loss sample
• Very little mobile phase needed.
• The flow rate is determined.

Limitations of Gel Filtration Chromatography

• There are a limited number of peak that can be resolved within the small time frame within GPC. GPC run.
• The filtering process must be completed prior to using the device to avoid dust and other particles from destroying the columns and disrupting the detectors.
• The molecular mass of the majority of the chains are too close to GPC separation. GPC separation to reveal anything other than broad highs.

Gel Filtration Chromatography Mechanism In detail (What happened during Gel filtration chromatography?)

In a column of gel filtration chromatography it is made up by a porous matrix which is what forms the buffer which flows between matrix beads. The beads are characterized by a the size of their pores, also known by the term “fractionation. Complexes and molecules too large to fit into the pores remain inside the liquid phase, and then move through the column according to the fluid flow. Smaller molecules and complexes that are able to move into the pores enter the stationary phase and move through the gel filtration column by a longer path through pores of the beads.Medium-pressure gel filtration chromatography system

Any complex or molecule that is outside the threshold of fractionation for a specific column of gel filtration will pass through the column much faster than any other molecule that could move into in the stationary. So, any component in the specimen that falls in the fractionation range will be eluted first (in the volume of voids) prior to anything else that is within the range of fractionation. The minimum size that can stay on the surface of the liquid phase but not be able to enter to the stationary stage is referred to by the term “exclusion. Bio-Rad provides gel filtration chromatography chromatography media and columns with exclusion limits that vary up to 3 orders between 100 daltons and 100,000 daltons (100 kDa).

Complexes and molecules that be introduced into the stationary phase are divided according to the size of their molecules. Smaller molecules will be able to move into the pores and be slowed more than larger molecules which are not as easily able to enter the pores and thus are eluted from the column faster. This variation in pore mobility causes the fragmentation of the components according to size, with those that are most massive eluting first.

In gel filtration chromatography columns that are designed to remove buffer exchange, desalting and removal of small molecules, such as nucleotides. The salts and smaller compounds easily penetrate the pores, and are impeded, and move more slowly than larger nucleic acids or proteins. Thus, the elements that are important to the sample are eliminated ahead of salts, nucleotides and so on. DNA cleanup kits employing this method typically include spin columns for gel filtration.

Resolution, which is defined here as the degree of sharpness of the lines between sizes is determined by bead diameter and a host of other variables. Beads with smaller sizes generally provide more resolution in a filter column for chromatography. Small molecules move throughout the stationary phase more quickly that linear ones. Size exclusion, the range of fractionation, and elution rates are affected by the composition of the buffer, the Ionic strength, and pH. When it comes to the fractionation of complex mixtures made up of proteins, the elution time and the size exclusion limits might require a more empirical determination.

What are the criteria for Gel?

The standard for gels includes:

• The gel should have a chemical composition that is inert.
• It has to be mechanically solid.
• It should be carefully crafted with a reproducible, porous form.
• It should have a homogeneous size of particles.

References

• Prapulla, S.G. (2014). Encyclopedia of Food Microbiology || FERMENTATION (INDUSTRIAL) | Recovery of Metabolites. , (), 822–833. doi:10.1016/B978-0-12-384730-0.00109-9 
• Stellwagen, Earle (2009). [Methods in Enzymology] Guide to Protein Purification, 2nd Edition Volume 463 || Chapter 23 Gel Filtration. , (), 373–385. doi:10.1016/S0076-6879(09)63023-8 
• Evans, David R.H. (2009). [Methods in Enzymology] Guide to Protein Purification, 2nd Edition Volume 463 || Chapter 9 Concentration of Proteins and Removal of Solutes. , (), 97–120. doi:10.1016/S0076-6879(09)63009-3 
• Ó’Fágáin, C., Cummins, P. M., & O’Connor, B. F. (2017). Gel-Filtration Chromatography. Methods in molecular biology (Clifton, N.J.), 1485, 15–25. https://doi.org/10.1007/978-1-4939-6412-3_2
• https://www.slideshare.net/asabuwangwa/gel-permeation-chromatography-gpc
• https://library.um.edu.mo/ebooks/b28050630.pdf
• https://www.slideshare.net/shovameghbalika/gel-chromatography
• https://kirschner.med.harvard.edu/files/protocols/GE_gelfiltration.pdf