Flash Chromatography.pdf
- 1. Flash Chromatography Presented by Dhanashree R. Kavhale M. Pharm. (Pharmaceutical Chemistry) Sem- II Department of Pharmaceutical Sciences Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440033
- 2. Contents Introduction Principle of flash chromatography Instrumentation Applications References 2
- 3. What is Flash chromatography? It was popularized several years ago by Clark Still of Columbia University. Flash chromatography, also known as medium pressure chromatography. An air pressure driven hybrid of medium and short column chromatography optimized for rapid separation. An alternative to slow and often inefficient gravity-fed chromatography. Differs from the conventional technique in 2 ways: Slightly smaller silica gel particles (250-400 mesh) are used. 3
- 4. Continue.. Due to restricted flow of solvent caused by the small gel particles, pressurized gas (10-15 psi) used to drive the solvent through the column of stationary phase The net result is a rapid "over in a flash" and high resolution chromatography. 4
- 5. Principle The principle is that the eluent is, under gas pressure rapidly pushed through a short glass column. The glass column is packed with an adsorbent of defined particle size with large inner diameter. The most used stationary phase is silica gel 40 -63 um, but obviously packing with other particle sizes can be used as well. Particles smaller than 25 µm should only be used with very low viscosity mobile phases, because otherwise the flow rate would be very low. 5
- 6. Continue.. Normally gel beds are about 15 cm high with working pressures of 1.5- 2.0 bars. In the meantime, however, and parallel to HPLC, reversed phase materials are used more frequently in flash chromatography. The computerized system control the Working of Flash chromatography. 6
- 7. Instrumentation of Flash chromatography 7 Fig: Components of flash chromatography
- 8. Adsorbents Silica: Slightly acidic medium. Best for ordinary compounds, good separation is achieved. Alumina: Basic or neutral medium. Can be effective for easy separations, and purification of amines. Reverse phase silica: The most polar compounds elute fastest, the most non-polars lowest. 8
- 9. Selection of stationary phase The most important stationary phase in column chromatography is silica. Silica gel (SiO2) and alumina (Al2O3) Adsorbent particle size affects how the solvent flows through the column. Smaller particles (higher mesh values {70-230} ) are used for flash chromatography; larger particles (lower mesh values {230-400}) are used for gravity chromatography. The amount of silica gel depends on the Rf difference of the compounds to be separated, and on the amount of sample. 9
- 10. Solvent Systems Flash column chromatography is usually carried out with a mixture of two solvents, with a polar and a nonpolar component. 1. Hydrocarbons: pentane, petroleum ether, hexanes. 2. Ether and dichloromethane (very similar polarity) 3. Ethyl acetate The most common two-component solvent system: 1. Ether/Petroleum Ether 2. Ether/Hexane 3. Ether/Pentane Ethyl 4. Acetate/Hexane 5. Methanol/Dichloromethane 10
- 11. Pump Systems A pressure range up to either 10 bar or 50 bar gives optimum separation results for a broad range of applications. The pump modules can be controlled by three different units. Pump Controller C610 Pump Manager C615 Control Unit are designed for isocratic separations. The flow rate can be easily adjusted by turning a knob and is indicated by a large illuminated LCD-display. Delivered with a overpressure sensor for maximum safety. 11
- 12. Sample Injection Systems Way of injecting sample Injection systems are designed to facilitate column loading with liquids and low solubility oils and solids. 1. Columns Glass Columns : A wide range of columns offer maximum flexibility for every situation. Depending on the nature and the quantity of the sample offers a series of column types which vary in form, size and performance. 12
- 13. 2.Column Plastic +Glass Column : Plastic + Glass-coated Glass Columns are available for larger amounts of samples and higher pressure applications on a high safety level. Precolumns : Pre column are minimizing dead volumes and enhance the life time of the main column by trapping contaminants. The small Pre column, fits to Glass Columns of inner diameter of ID 15, 26, 36 and 49 mm. 13
- 14. Continue.. The large Pre column, fits to Glass Columns of ID 70 and 100 mm inner diameter. Filling Sets for Glass Columns Dry Filling Set : The Dry Filling Set is employed for filling glass columns with silica gel using compressed gas. Silica gel in the size range of 25 -200micro meters can be packed with this method. 14
- 15. Sample loading Two different methods are used to load the column. Wet Method Dry Method 15
- 16. 1. Wet Loading Method In the wet method, the sample to be purified(or separated into components) is dissolved in a small amount of solvent, such as hexanes, acetone, or other solvent. This solution is loaded onto the column. Sometimes the solvent choice to load the sample onto the column is more polar than the eluting solvents. In this case, if we use the wet method of column loading, it is critical that we only use a few drops of solvent to load the sample. If we use too much solvent, the loading solvent will interfere with the elution and hence the purification or separation of the mixture. In such cases, the dry method of column loading is recommended. 16
- 17. 2. Dry Loading Method First dissolve the sample to be analyzed in the minimum amount of solvent and add about 100 mg of silica gel. The mixture until the solvent evaporates and only a dry powder remains. Place the dry powder on a folded piece of weighing paper and transfer it to the top of the prepared column. Add fresh eluting solvent to the top now we are ready to begin the elution process. 17
- 18. Advantages & Disadvantages Advantages Disadvantages 1. Low cost Low resolution 2. It is possible to employ stepped gradient and isocratic solvent elution. It is difficult and time-consuming to separate complicated crude reaction mixtures. 3. The chemist totally regulates the introduction of stationary phase and packing techniques. 18
- 19. Applications It is now one of the most widely used methods for purifying pharmaceutical intermediates as well as final products. Moreover, it is commonly used in the study of natural products. For examples; • Isolation and separation of catechin from green tea extracts. • Purification of fatty acids • Purification of steroids 19
- 20. Thank you.. 20