APPLICATION OF COLUMN CHROMATOGRAPHY IN PHARMACY
Analytical Separation and isolation of chemical compound from a mixture is called as Chromatography and purification of such compounds in larger quantities is achieved prominently using Column Chromatography. Column Chromatography is a separation Technique which is applied in various fields among which Pharmacy is the one, where preparation and dispensing of drugs is performed based on this technique, to ensure the effectiveness of the drug.In Column Chromatography, there are different types of columns such as Gravity Columns, Flash Columns, Low and Medium pressure Columns, Vacuum Columns and High-Pressure Columns. But the similar point in all these columns is that all require an Absorbent, which acts as a Stationary Phase through which sample containing different compounds flow down at differing rates. On the Other hand, the Adsorbent is applied to the Column in 2 ways namely-Slurry packing method and Dry packing Method. Slurry packing method is often used for macro scale separations and Dry packing method for microscale separations.
Silica gel for Column Chromatography
Silica gel is often used for macro scale separations using Slurry pack method where the adsorbent is mixed with a small amount of Non-polar solvent until a consistent paste is formed and then poured in the column. In dry pack method, a steady stream of the absorbent is poured through a funnel into the column by tapping the sides of the column so that packing remains even in the column. Silica gel for Column Chromatography is considered as the best absorbent in column packing for both dry pack and Slurry pack method, as it gets eventually distributed and forms a packed Stationary phase. The sample is then mixed with a polar solvent and added to the column where the component molecules which are to be separated are either adsorbed on the particle surface or adsorbed into the particle pores. Thus, the different components of a sample get split into separate bands in the column and get eluted at different rates.
The elution is at different rates because of the particle size and pores of Silica gel absorbent which are available in different sizes called as Silica Gel mesh with small size and more pores which act as a Sieve. These Silica gel mesh sizes chromatography is typically used in Column as it retains the compounds for a longer time thereby enhancing the purification of the product. Silica gel for Column chromatography mesh size refers to the number of holes present in the mesh, per unit area of the absorbent to be used in the Column. Silica Gel 100, 200 mesh Merck are employed in Gravity Columns, whereas higher mesh Merck is used in Flash Columns.
Gravity Columns are mainly preferred in Gel Permeation Chromatography and are followed by Adsorption chromatography technique containing Silica gel 100-200 mesh adsorbent gives the higher efficiency of separations, improved reproducibility and require low solvent consumption. Whereas, Flash Columns containing Silica gel (250-400 mesh) with small particle size, restricts the flow of solvent, therefore pressurized gas is applied to drive the solvent resulting in high resolution.The amount of Silica gel to be used for these Columns mainly depends on the amount of the sample. It means nearly 30 to 100 grams of Silica gel is required for easy separations but for difficult separations more than 30:1 ratio of silica gel is required. With the increase in the quantity of Silica gel, the time consumption for the separation also increases.
Column chromatography applications
Purification of reaction mixture in chemical synthesis such as –
- Through Flash column chromatography using Silica gel 60(220-240 mesh), β-Ketoester and Desired alcohol were purified,
- Purification of Polychlorinated biphenyls (PCB) from traces of sulphur using Silica gel 60(70-230 mesh) and Silver nitrate impregnated silica gel.
- Silica gel (100-200 mesh) for Organ chlorine Pesticides, Phenols and Polynuclear aromatic compounds
Purification of biomolecules such as proteins for pharmaceutical research
- Synthesis of Pramlintide which is an analogue of Amylin, a peptide hormone, for treating type 1 and type 2 Diabetics is also purified using Silica gel.
- Purification of bioactive glycolipids, showing antiviral activity towards HSV-1(Herpes Virus)is also performed by Column chromatography using Silica gel as absorbent.
- Purification of Nucleic acids in vivo and vitro cultures is also done using Silica gel as it absorbs the Nucleic acid.
Analysis of environmental samples
- Purification of target chemical from co-extracted, non-chemical samples from the environment is done using Gas chromatography, in which the environmental samples are passed through an inert gas where the target chemical gets desorbed from aqueous phase to gas phase and gets separated from a stream of gas by absorption on the Silica gel mesh. On heating it the trapped chemical get released and can be analyzed
- Extraction of pesticides from solid food samples of animal origin containing lipids, waxes and pigments as contaminants are performed using Silica gel adsorption chromatography in atmospheric condition.