The key difference between gel filtration and affinity chromatography is that gel filtration chromatography depends on the differences in molecular weight or size of the analyte sample, whereas affinity chromatography depends on the affinity of an analyte to an immobilized ligand.
The term chromatography in analytical chemistry refers to the process of the separation of components in a mixture using different techniques. Chromatography is a collective name that represents a various number of different methods for separation.
1. Overview and Key Difference
2. What is Gel Filtration Chromatography
3. What is Affinity Chromatography
4. Gel Filtration vs Affinity Chromatography in Tabular Form
5. Summary – Gel Filtration vs Affinity Chromatography
What is Gel Filtration Chromatography?
Gel filtration chromatography is an analytical technique in which the separation of components depends on the difference in molecular weight or size. This technique is also known as size-exclusion chromatography or molecular-sieve chromatography. The separation technique in this process depends on the differing ability of molecules in the sample to enter into the pores of the gel-filtration medium.
In the gel filtration technique, the stationary phase contains beads of hydrated, sponge-like material having pores of molecular dimensions containing a narrow range of sizes. If we pass an aqueous solution consisting of molecules of various sizes through a column containing these “molecular sieves,” the molecules that are larger than the pores of the filtration medium move quickly through the column. In contrast, the smaller molecules enter the pores of the gel and tend to move slowly through the column. Molecules elute from the column in order of decreasing molecular size. Here, the exclusion limit of the gel is the molecular mass of the smallest molecules unable to penetrate the pores of a given gel.
There are different types of gel filtration techniques, such as indirect gel filtration method, steady-state gel filtration, gel bead dialysis, etc. Indirect gel filtration chromatography is useful in the determination of free thyroid hormones. Steady-state gel filtration chromatography is an indirect technique useful mostly for the measurement of free steroids like cortisol, testosterone, etc. Gel bead dialysis, on the other hand, is a modification of steady-state gel filtration that is comparatively simpler.
What is Affinity Chromatography?
Affinity chromatography is an analytical technique and a separation method that depends on a specific binding interaction between an immobilized ligand and its binding partner. Some examples include antibody-antigen binding, enzyme-substrate binding, and enzyme-inhibitor binding. Therefore, this technique has many important applications in nucleic acid purification, protein purification from cell extracts, and purification from blood.
In this technique, the most important property is ligand immobilization. We can use various materials such as acrylates and silica gel for this. It is important to prevent steric interference of the target molecule to the ligand. Moreover, an inhibitor is attached to the solid phase. We call this inhibitor a spacer. Classically, a spacer consists of a hydrocarbon chain.
The stationary phase of the affinity chromatography contains the core, spacer, and ligand. Sometimes, it also has a metal ion that is coupled to the ligand. The most preferred solid phase for the stationary phase in this technique is agarose gel because it is easily dispensed to fill and pack the column with resin beds of any size, and it is large enough for biomolecules to flow freely into and through the beads. The ligands can covalently attach to the bead polymer in various ways. The most common spacer compounds are cyanogen bromide, epoxide, epoxide with C6 acid, and diamin. On the other hand, the ligand we can use differs according to the target, e.g., antibody-antigen, iron or aluminum ions-phosphoproteins, avidin-biotin, glutathione-GST, chelator-His-tagged proteins, etc.
What is the Difference Between Gel Filtration and Affinity Chromatography?
Chromatography is an important analytical technique. Gel filtration chromatography and affinity chromatography are two important variations of chromatography. The key difference between gel filtration and affinity chromatography is that gel filtration chromatography depends on the differences in molecular weight or size of the analyte sample, whereas affinity chromatography depends on the affinity of an analyte to an immobilized ligand.
Summary – Gel Filtration vs Affinity Chromatography
There are different types of chromatographic techniques according to their application and the nature of the analyte sample. Gel filtration and affinity chromatography are two such types of techniques. The key difference between gel filtration and affinity chromatography is that gel filtration chromatography depends on the differences in molecular weight or size of the analyte sample, whereas affinity chromatography depends on the affinity of an analyte to an immobilized ligand.