The column
- 2. The column Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling.
- 3. The classical preparative chromatography column is a glass tube with a diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter (a glass frit or glass wool plug – to prevent the loss of the stationary phase) at the bottom. Two methods are generally used to prepare a column: the dry method and the wet method. Column chromatography proceeds by a series of steps
- 4. What is column chromatography? Column chromatography is one of the most useful methods for purification & separation (Isolation) of individual desire compound from mixture of unwanted compounds. It is often used for preparative applications on scales from micrograms up to kilograms. It is a solid - liquid technique in which the stationary phase is a solid & mobile phase is a liquid. The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for column chromatography is Silica Gel, followed by Alumina Oxide. The mobile phase or eluent is a liquid. It is either a pure solvent or a mixture of different solvents. It can be used for molecules whose molecular weight is < 2000 g/mol.
- 5. Types of Chromatography Paper Chromatography Gas Chromatography Thin Layer Chromatography Solid - Liquid Chromatography (Column Chromatography
- 6. Methods of Column Packing i. Dry Method ii. Wet Method Dry Method: Add dry silica / Alumina to the column and apply to the bottom of the column. This will compress the silica gel and keep it compressed for the next steps. Packing can be improved by tapping the column. While applying vacuum; pour solvent in it. Allow the solvent to move though the column until reaches to the bottom. At this stage vacuum is not require. Allow 5–6 columns value of solvent to flow through the column to make sure it is complete packed. Drain the solvent till the solvent level is just even with the surface of the stationary phase
- 8. Wet Method: Fill the column about one third with solvent In a beaker, measure out the required amount of silica / alumina. In another beaker, take solvent approximately one and a half times the amount of silica / alumina. Add silica/alumina to the solvent while swirling in small quantity at a time. Use a glass rod to mix the slurry. Pour some of the slurry into column & allow solvent to drain to avoid overflowing. Tap the column carefully to encourage bubbles to rise and the silica to settle. Continue to move the slurry to the column until all the silica or alumina is added. Wash the inside of the column by pouring solvent down the inside edge. Drain the solvent till the solvent level is just even with the surface of the stationary phase
- 10. Adsorbents used in chromatography method Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used for column chromatography. These adsorbents are sold in different mesh sizes such as 60-230 mesh, 100- 200 mesh, 200-400 mesh & tailor made. Adsorbent particle size affects how the solvent runs through the column. Smaller particles (higher mesh size i.e. 230-400 mesh) are used for flash chromatography & larger particles (lower mesh size i.e. 60120/60-200) are used for gravity chromatography.
- 11. Difference between Normal Phase & Reverse Phase Chromatography Normal Phase Chromatography It uses a polar stationary phase and a non-polar (low Polarity Solvents) mobile phase. Non-polar compounds elute faster than polar compounds. When we increase polarity of mobile phase elution time will increase. It can not be reused / reproducible. Mobile phase are non polor i.e. IPA, hexane, dichloromethane, chloroform, ethyl ether, and isopropyl alcohol (IPA). Reverse Phase Chromatography It uses a non polar stationary phase and a polar mobile phase. Polar compounds elute faster than non polor compounds. When we increase polarity of mobile phase elution time will decrease. It Can reused / reproducible. Mobile phase are polor compounds such as water, acetonitrile, methanol
- 12. Application Separation of mixture of compounds Purification process Purification of Phytochemical Isolation of metabolites i.e. Small molecules Estimation of drugs Process Development Purify Natural compounds To separate active component from Plant material Herbal Extraction
- 13. Advantages: Any type of mixture can be separated by column chromatography. Any quantity of the mixture can also be separated. In preparative type, the sample can be separated and reused. Automation is possible. It can be used in both analytical and preparative applications. It is used to identify the number of components of a mixture. It is also used to separate and purify important quantities of those components for subsequent evaluation. There is wider choice of Mobile Phase (Solvents). It is low cost process and disposability of the stationary phase once it is used in the process. Process can be scale up form lab scale to commercial scale.
- 14. Disadvantages: Time consuming Process. More amounts of Mobile Phase (Solvents) required . Scale up process will take a long time to properly prepare & use. Automation makes the techniques more complicated & expensive.
- 15. Types of Company we need to focus Pharmaceutical Industries – Bulk Drugs & API Nutraceuticals Herbal Extracts products manufacturers Research Laboratories Laboratories Chemical Repacks Contract Research Laboratories Types of Department we need to contact • R & D – Research & Development • Organic Synthesis Lab, • Medicinal Chemistry lab, • Novel Drug Discovery, • Clinical Research, • Pilot Scale lab, • Preparative Lab, • Semi Preparative Lab