Flash chromatography is also known as medium pressure chromatography

Flash chromatography is also known as medium pressure chromatography

1. Noida Institute of Engineering and Technology (Pharmacy Institute) Greater Noida FLASH CHROMATOGRAPHY Submitted to: Chandana Majee (Assistance Professor ) 1 Unit: 4 (MPC201T )Advanced Spectral Analysis Course Details M.PHARM 2 nd Semester Submitted by: Rachit Sharma ( 0231PCH006)

2. CONTENTS INTRODUCTION PRINCIPLE INSTRUMENTATION Modern Flash Chromatography Applications REFERENCES

3. INTRODUCTION Flash chromatography is also known as medium pressure chromatography It is widely used for laboratory scale fractionation of mixtures from organic synthesis or for analysis. Flash Chromatography is a rapid form of preparative column chromatography based on an optimized pre-packed column through which is pumped solvent at a high flow rate. It uses glass or plastic columns with high flow rates and column packed with silica gel is widely used to purify synthetic compounds in many labs. It is simple and easy to use, but it is time consuming, irreproducible and difficult to pack a good column.

4. PRINCIPLE The principle is that the eluent is, under gas pressure (normally nitrogen or compressed air) rapidly pushed through a short glass column with large inner diameter. The glass column is packed with an adsorbent of defined particle size. The most used stationary phase is silica gel 40-63 µm, but obviously packing with other particle sizes can be used as well. Particles smaller than 25 um should only be used with very low viscosity mobile phases, because otherwise the flow rate would be very low.

5. INSTRUMENTATION

6. INSTRUMENTATION The conventional flash chromatography apparatus consists of a glass column of a suitable length containing a small glass-wool plug and a layer of acid- washed sand or glass frit at its base. This column is partially filled with sorbent using either dry-packing or slurry- packing technique. Sample is placed on the top of the sorbent layer. Above the sample a layer of glass wool sand is placed to prevent disturbance of the column bed by solvent added for elution. Above this, a solvent reservoir may be placed or there will be sufficient space for solvent of volume equal to the volume of fraction to be collected. The level of the solvent is maintained throughout the chromatographic procedure. On the top of the column an air inlet adaptor is provided and which in turn connected to air or gas supplier through rubber tubing. Continue...

7. Continued... Pressure of the gas or air is regulated by regulation valves. The typical pressures employed are less than 1-2 atm , with the various parts of the apparatus held in place by springs, clamps or screw thread connectors. In this type of apparatus the apparatus should be disassembled and reassembled while changing the solvent. In further development of the apparatus, a solvent reservoir with tap at thereservoir -to-column inlet is attached to the side of the column. The inlet for the reservoir is situated above the height of the sorbent bed. A second tap at the top of the column allows air pressures to be equalized for rapid solvent addition to the column.

8. 1. Packing Technique a. Dry Packing The sorbent is added into the column in small increment followed by tapping of the column with a hard object. The addition of sorbent in small increments is better for dry packing than filling of the column in bulk with the sorbent. After packing, a weak solvent is forced through the column to remove any air present between the sorbent the column. Several column volumes are passed through the sorbent thus the sorbent bed is made stable. b. Slurry Packing The column is initially partially filled with a weak solvent such as hexane. The sorbent is powdered well in a mortar to remove any clumps and then mixed with the same weak solvent to prepare slurry. This slurry is poured into the column in small increments. The excess solvent in the column is intermittently drained away. Generally tapping the column in this method is not followed. Periodic pressurization of the sorbent bed is used to aid consolidation.

9. 2. Phases used in Flash Chromatography a) Mobile Phases Mobile phase in flash chromatography is organic solvents. A fixed combination of organic solvents is used for isocratic elution whereas a different composition of solvents usually starting from non polar to polar range is used for method optimization. b) Stationary Phases Generally silica or alumina is the choice of stationary phases. However there are many sorbents are used as stationary phases. Hence depending upon the nature of the samples to be separated choice of columns can be done.

10. 3. Columns a) 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. b) Plastic and Glass Column Plastic and Glass-coated Glass Columns are available for larger sample amounts and higher pressure applications on a high safety level. The columns are designed for sample amounts from 1-100 g and pressures up to 50 bar during preparative separations. An integrated cooling jacket allows separations under constant conditions. c) Precolumns Precolumn are minimizing dead volumes and enhance the life time of the main column by trapping contaminants. The small Precolumn, fits to Glass Columns.

11. Modern Flash Chromatography In the modern flash chromatography system the glass columns are replaced with pre-packed plastic cartridges which are much safer and also more reproducible. Solvent is pumped through the cartridge, which is much quicker and more reproducible. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps means quicker separations, less solvent usage and greater flexibility. Modern Flash chromatography generally consist of following parts Pump Systems Sample Injection Systems Columns-cartridges, Filling Sets and Column Valves Precolumns Fraction Collector Detectors and Chart Recorders Computing system

12. A. Pump Systems It consists of a pump Controller and vacuum Pump/peristaltic Pump. 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 various units. These units are specially designed for isocratic or gradient or for both. B. Sample Injection Systems Unlike the conventional flash chromatography, in modern instruments 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. Continued...

13. C. Columns Cartridges i. Pre-packed plastic cartridges The cartridges are of different sizes, capable of loading from 50 up to 700 g of silica. They may be disposable, bidirectional and stackable. Solvent is pumped through the cartridge, which is much quicker and more reproducible. The introduction of gradient pumps means quicker separations, less solvent usage and greater flexibility. Column Characteristics Plastic cartridges are disposable with different sizes. This allow easy scale-up and reproducible results. Solid sample module and injection valve helps in easy sample loading. The pressure can be applied up to 100 psi thus leads to rapid separation. Low back pressure and higher efficiency can be achieved through narrow particle distribution provides Continued...

14. ii. Filling Sets for Glass Columns a) 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 25-200 µm can be packed with this method. b) Slurry Filling Set The Slurry Filling Set is used for wet filling and conditioning of glass columns with silica gel particles smaller than 25 µm. Continued...

15. D. 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. The small Pre-columns are usually made up of glass columns with inner diameter of 15, 26, 36 and 49mm whereas the large Pre-column, have inner diameter of about 70 and 100 mm. E. Fraction Collector Fraction collector consists of a panel that has provision to keep test tubes for collecting fractions. A tube connects the outlet of detector and the fraction collector. This setup is automated and can be controlled by software so that the required fractions will be collected in the collection tubes automatically. Continued...

16. F. Detectors and Recorders/Software For most applications UV/Vis detectors are commonly used. These detectors detect the analytes and generate signals as peaks. Compounds that have absorption in the UV-Vis region can be conveniently analysed using these detectors. In the absence of adequate UV/Vis absorption, likely for sugars or polymers, a Differential Refractometer (RI Detector) in combination with a UV/Vis detector is used. G. Computing system A computer with software is used to monitor the separation process. The peaks are displayed while separation occurs. The desired peaks can be collected using fraction collector. Continued...

17. Applications Flash chromatography use for the purification of synthetic compounds during synthetic process. It is useful in separation of impurities present along with the product. Non-aqueous reversed-phase chromatography that can be very effective at separation and purifying very lipophilic compounds and other hydrocarbons with minimal polar functionality. Flash chromatography has been used for the isolation of chemical constituents from crude extracts of plants. It has been used for the separation of synthetic peptides, to isolate pure peptide. Purification of Fatty Acid Methyl Esters (FAME), Mixture of Glycerides, Mono-, Di Tristearin and purification of sterols. It is useful in bile acid purification, in impurity isolation during drug purification.

18. REFERENCES http://www.labhut.com/education-centre/flash-chromatography/what-is-flash- chromatography.html http://www.pharmatutor.org/articles/flash- chromatography-area-applications http://www.ijprr.com/File_Folder/45-49.pdf www. Buchi Preparative Chromatography www.biotage.com AB Roge , SN Firke RM Kawade , SK, Sarje , SM Vadvalkar . Brief Review On: Flash Chromatography. IJPSR, 2(8), 2011, 5-11. www.pretech.nu/products Still WC. Kahn M, Mitra A. Flash chromatography. J. Org. Chem., 43(14), 1978, 2923- 2925. 6. www.Flash Column Chromatography Guide