FLASH COLUMN CHROMATOGRAPHY
- 1. GURU GHASIDAS VISHWAVIDYALAYA,BILASPUR SESSION-2023 TOPIC- FLASH COLUMN CHROMATOGRAPHY GUIDED BY PRESENTED BY DR. VINOD D. RANGARI SIR VINAY BISEN M.PHARM 1ST SEM
- 2. INTRODUCTION Flash chromatography is an air pressure driven hybrid of medium pressure and shorter column chromatography which has been optimized for rapid separation of organic compounds. Flash chromatography is also known as medium pressure chromatography(10-15psi) This chromatography was popularized several years ago by Clark still It is a technique used to separate mixtures of molecule into their individual constituents, frequently used in drug discovery process Flash chromatography is a rapid form of preparative column chromatography based on an optimized pre-paked column through which is pumped solvent at a high flow rate It uses plastic columns or cartridges with high flow rates and column packed with silica gel (200-400mesh particle size) is widely used to purify synthetic comounds .
- 3. Principle • The principle is that the eluent which is a liquid, under gas pressure (normally nitrogen or compressed air ) rapidly pushed through a short glass column • Applied pressure to column reservoir to speed up mobile phase • The column is packed with an adsorbent of defined particle size with large inner diameter • The most used stationary phase is silica gel 40-63 µm • Particles smaller than 25µm should only be used with very low viscosity mobile phases, because otherwise the flow rate would be very low • Efficiency of column will be attained by optimum flow rate of mobile phase • Normally gel beds are about 15cm high with working pressures of 1.5-2.0 bars. • The net result is a rapid over in a flash and high resolution chromatography
- 5. THEORY • The basic theory involved in the flash chromatography is similar to column chromatography except the involvement of pressure • In column chromatography the flow of mobile phase is due to the gravitational force • In flash chromatography the flow of mobile phase is under the influence of pressure(10-15psi) • Pressure is applied through a pump to the column by using air/nitrogen. • The mobile phase is pushed rapidly through the stationary phase packed in a short column with large inner diameter • The analytes in the mixture are separated according to their distribution between adsorbent and eluent and reaches the bottom of the column • The solvent polarity decides the separation of the analytes. A mobile phase combination of solvents starting with non polar to higher polar is passed through the column and various fraction are collected
- 6. Phases used in flash chromatography a) Mobile phases This chromatography is usually carried out with a mixture of two solvents, with a polar and a nonpolar component. Occasionally, just one solvent can be used. The only appropriate one- component solvent systems (listed from the least polar to the most polar): Hydrocarbons: pentane, petroleum ether, hexanes Ether and dichloromethane: (very similar polarity) Ethyl acetate b) Stationary phase most commonly used stationary phases are silica gel (sio2) and alumina (Al2 O3 )
- 8. Flash chromatography generally consist of follwing parts:- 1.Pump systems – It consist of a pump controller and vacuum pump/peristalic pump - A pressure range up to either 10 bar or 50bar gives optimum separation results for a broad range of application 2. Sample injection systems – samples are introduced into the column using sample injection valves - the valves are of specific volumes. Around 3-5 ml volume of injection valves are used 3. Column cartridges – Pre packed plastic cartridges • The cartridges are of different sizes , capable of loading from 50 up to 700g of silica • Solvent is pumped through the cartridge , which is much quicker and more reproducible • The introduction of gradient pumps means quicker separation , less solvent usage and greater flexibility.
- 9. 4. Precolumns - pre-column are placed before the main column and used to minimize dead volumes and enhance the life time of the main column by trapping contaminants. 5.Fraction collector – fraction collector consist of a panel that has provision to keep test tubes for collecting fractions 6.Detectors and recorders/software - For most application UV/vis detectors are commonly used - In the absence of adequate UV/Vis absorption, likely for sugar or polymer, a differential refractometer (RI Detector) in combination with a UV/vis detector is used. 7. Computing system – a computer with software is used to monitor the separation process - The peaks are displayed when separation occurs.
- 10. Sample application/sample loading Samples are loaded in two ways- 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. 2. Dry loading method - First dissolve the sample to be analysed in the minimum amount of solvent and add sufficient amount of silica gel. Swirl the mixture until the solvent evaporates and only a dry powder remains. Place the dry powder and transfer it to the top of the prepared column. Elution – apply minimum pressure for continuous flow of the mobile phase Locating the sample – TLC is used
- 11. Method optimization • When optimizing flash purification for component retention ,TLC is the most efficient tool for evaluating solvent ratios. • Initially TLC is carried out to get the desired Rf value • For optimization of flash method just obtaining a separation on TLC is not enough. We need to get the target compound in the proper Rf range • Ratios of the solvent are optimized using the chosen solvent (or solvents) from the selectivity study,to get an Rf value of between 0.1 and 0.4 ,for the target compound . • Compound retention and mass-transfer kinetic are related to separation efficiency and therefore ,loading capacity. • In flash chromatography, retention is expression in column volumes(CV). • A column volume is the space in a column or cartridge not occupied by th staionary phase
- 12. APPLICATION • Flash chromatography used for the purification of synthetic compounds during synthetic process. It is usefull in separation of impurirties present along with product • Non aqueous reversed phase chromatography that can be very effective at separation and purifying very lipophilic compounds and other hydrocarbon with minimal polar functionality • Flash chromatography has been used for the isolation of chemical constituent from crude extract of plants • It has been used for the separation of synthetic peptides to isolate pure peptide . • Puification of fatty acid methyl ester (FAME) ,mixture of glyceroids , mono , di, tristearin and purification of sterols • It is usefull in bile acid purification, impurity isolation during drug purification .
- 13. Natural products/Nutraceuticals application: 1. Separation and Isolation of α-Santalol and β-Santalol from Sandalwood Extraction 2. Isolation and Purification of Chromophoric and Nonchromophoric Compounds in Giant Knotweed Rhizome. 3. Isolation and Purification of Flavonoids from Ginkgo Biloba Leaves Extract. 4. Isolation and Purification of Ginsenosides from Red Panax Ginseng Extract. 5. Isolation and Purification of Catechins from Green Tea Extract .
- 14. Carbohydrate application 1. Purification of Conjugated Quercetin and Rutinose. 2. Impurity Isolation of Valproic Acid from CyclodextrinvDuring Encapsulation. 3. Isolation ofAminosugar and Acarbose . 4. Flavanone Glycoside Purification. 5. Isolation of Aminoglycoside Antibiotics. Lipid application 1. Purification of Fatty Acid Methyl Esters (FAMEs). 2. Purification of a Mixture of Glycerides, Mono-, Di-, and Tristearin. 3. Purification of Sterols.
- 15. Pharmaceutical/small molecule application: 1. Bile Acid Purification During Lead Generation in Drug Discovery. 2. In Impurity Isolation During Drug Purification. 3. Mestranol Purification During Chemical Synthesis. 4 .In Anti-malarial Drug Purification in Drug Discovery.
- 16. Conclusion • Purification of drug is an important step in any branch of research.Preparative chromatography is used to separate the components of a mixture for more advanced use and is thus a form of purification. • Flash Chromatography can be alternative to preparative HPLC as it saves time and solvent. Extrapolation of TLC results on preparative scale can be achieved by Flash chromatography. • Modern Flash chromatography with disposable cartridges and advanced detection techniques is applicable to a wide range of compounds.