Pharmaceutica Analytica Acta

The Significance of Chromatography-Principles and Working of Its Various Techniques

Wenin Geng^* Department of Chemistry and Biochemistry, University of Texas at Arlington, USA
^*Correspondence: Wenin Geng, Department of Chemistry and Biochemistry, University of Texas at Arlington, USA, Email:

Received: 04-Apr-2022, Manuscript No. PAA-22-16687; Editor assigned: 07-Apr-2022, Pre QC No. PAA-22-16687 (PQ); Reviewed: 22-Apr-2022, QC No. PAA-22-16687 ; Revised: 29-Apr-2022, Manuscript No. PAA-22-16687 (R); Published: 06-May-2022, DOI: 10.35248/2153-2435.22.13.668

Description

Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on properties such as size and shape, overall charge, surface hydrophobic groups, and ability to bind to stationary phases. Four separation techniques based on molecular properties and interaction types employ mechanisms of ion exchange, surface adsorption, partitioning, and size exclusion. Other chromatographic techniques are based on fixed beds such as columns, thin layers, and paper chromatography. Column chromatography is one of the most common methods of protein purification [1].

Chromatography is based on the principle that molecules in a mixture applied to a surface or a solid and liquid stationary phase (stable phase) separate while moving with the help of a mobile phase. Factors that work with this separation method include adsorption (liquid solid), distribution (liquid solid), and molecular properties related to affinity or difference between molecular weights. Due to these differences, some components of the mixture stay in the stationary phase longer and move slowly through the chromatography system, while others move quickly to the mobile phase and exit the system faster [2].

The nature of the interactions between the substances contained in the stationary phase, mobile phase, and mixture is an effective basic component for separating molecules from each other. Partition-based chromatography is very effective in separating and identifying small molecules such as amino acids, carbohydrates and fatty acids. However, affinity chromatography (ie, ion exchange chromatography) is more effective at separating macromolecules such as nucleic acids and proteins. Paper chromatography is used to study protein separation and protein synthesis. Gas-liquid chromatography is used to separate alcohols, esters, lipids and amino groups and observe enzyme interactions, while molecular sieving chromatography is used specifically to determine the molecular weight of proteins. Agarose gel chromatography is used to purify RNA, DNA particles and viruses [3].

Column chromatography, Proteins have a variety of characteristics such as size, shape, net charge, stationary phase used, and binding capacity, so each of these characteristic components can be purified using chromatographic methods. Of these methods, column chromatography is the most commonly used. This technique is used to purify biomolecules. First apply the sample to be separated to the column (stationary phase) and then to the wash buffer (mobile phase). Their flow is guaranteed by the column material inside the fiberglass carrier. Depending on the time and amount, the sample will accumulate at the bottom of the device [4].

Ion exchange chromatography, Ion exchange chromatography is based on electrostatic interactions between charged protein groups and solid supports (matrix). The matrix has an ionic charge opposite to that of the protein to be separated, and the protein's affinity for the column is achieved through ionic bonding. Proteins are separated from the column by changing the pH, ionic salt concentration, or ionic strength of the buffer. The positively charged ion exchange matrix is called the anion exchange matrix and adsorbs negatively charged proteins. Templates bound to negatively charged groups are known as cation exchange templates, and positively charged proteins adsorb [5].

Paper chromatography, in paper chromatography, the carrier material consists of a highly water-saturated cellulose layer. In this method, thick filter paper formed a support and water droplets settled in its pores formed a stationary "liquid phase". The mobile phase consists of the appropriate liquid in the processing tank. Paper chromatography is "liquid" chromatography.

Conclusion

Initially chromatographic techniques were used to separate substances based on their color as was the case with herbal considerably. Nowadays, chromatography is accepted as an extremely sensitive, and effective separation method. Column chromatography is one of the useful separation, and determination methods. Column chromatography is a protein purification method realized especially based on one of the characteristic features of proteins.

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