FLASH CHROMATOGRAPHY Also called as “medium pressure chromatography” “An air pressure driven hybrid of medium and short column chromatography optimized for rapid separation" Popularized by Clark Still of Columbia University An alternative to slow and often inefficient gravity-fed chromatography
Introduction.. Differs from the conventional technique in 2 ways: Slightly smaller silica gel particles (250-400 mesh) are used, and 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.
Column vs Flash Chromatography Traditional Column Flash chromatography chromatography • Pre-packed plastic• Glass columns with silica cartridges gel • Solvent is pumped• Separation is very slow through the cartridge (typically many hours) • Much quicker and more• End of the run, silica gel reproducible must be removed, cleaned, dried • Remaining solvent and re-packed flushed out of the column using pressurized gas• Both time consuming and hazardous
Modern flash chromatography systems are sold as pre-packedplastic cartridges, and the solvent is pumped through thecartridge.Systems may also be linked with detectors and fractioncollectors providing automation.The introduction of gradient pumps resulted in quickerseparations and less solvent usage.Flash chromatography is not expected to provide the resolutionor reproducibility of HPLC; it is a technique that can quicklyimprove the purity of samples to an acceptable level.
Selection of solvent systemSolvent system Compound should have TLC Rf of 0.15 to 0.20 in the solvent system Binary solvent system – Polarity can be adjusted Rate of elution can be determined Common solvents used : dichloromethane/hexane, ether/hexane, hexane/ethyl acetate, and dichloromethane/methanol High polarity of solvents increase the rate of elution of all compounds.
Quantity of silica gel required.. 40-63 µm silica gel particles are used Amount depends on 2 factors : – Rf difference of the compounds to be separated – Amount of sample ↑ silica gel -↑ the length of time for chromatography For, – Easier separations, ratios closer to 30 : 1 are effective – Difficult separations, more silica gel is often required
Packing the column Glass column / plastic cartridges Has either a glass frit or a plug of cotton wool directly above the stopcock (To prevent the silica gel from escaping from the column through the stopcock) ~1/2 inches layer of clean sand above the plug of glass wool Make sure that surface is flat Pour in the silica gel using a funnel.(Do this step in hood)
Method• A solvent is chosen which gives good separation and moves the desired component to Rf = 0.20 on analytical TLC• A column of the appropriate diameter is selected and filled with 5-6 in. of dry 40-63 µ silica gel• Column is solvated – The column is filled with solvent and pressure is used to rapidly push all the air from the silica gel• The sample is applied and the column is refilled with solvent and eluted at a flow rate of 2 in./min• Top of the column should never run dry
Procedure for Microscale Flash Column Chromatography In microscale flash chromatography, the column need neither a pinchclamp or a stopcock at the bottom of the column to control the flow, nor does it need air-pressure connections at the top of the column. Instead, the solvent flows very slowly through the column by gravity until you apply air pressure at the top of the column with an ordinary Pasteur pipet bulb.
(1) Prepare the column.1. Plug a Pasteur pipet with a small Add dry silica gel adsorbent, 230-400amount of cotton; use a wood applicator mesh -- usually the jar is labeled "forstick to tamp it down lightly. flash chromatography." One way to fill the column is to invert it into the jar of2. Take care that you do not use either silica gel and scoop it out . . .too much cotton or pack it too tightly. Youjust need enough to prevent theadsorbent from leaking out.
. . . then tamp it down before scooping Another way to fill the column is to pour themore out gel into the column using a 10 mL beaker.
(3) Load the sample onto the silica gel columnTwo different methods are used to load the column: the wetmethod and the dry method:Wet loading methodThe sample to be purified (or separated into components) is dissolved in a small amountof solvent, such as hexanes, acetone, or other solvent. This solution is loaded onto thecolumn.
5) Elute the column with the second elution solvent.If separating a mixture of one or more compounds, at this pointchange the eluting solvent to a more polar system, Elution wouldproceed as in step (4).(6) Analyze the fractions.If the fractions are colored, simply combine like-coloredfractions, although TLC before combination is usually advisable.If the fractions are not colored, they are analyzed by TLC(usually). Once the composition of each fraction is known, thefractions containing the desired compound(s) are combined.
Advantages• Large quantities of the sample can be separated (0.5-2g)• Fast ( 1o to 15 minutes)• Cost efficient• Elaborate equipment and the purchase of expensive equipment is not necessary• If high resolution is required, flash chromatography is carried out before HPLC to avoid contamination of the expensive plates
Applications• Purification of various peptides, antibiotics• Separation of closely related organic compounds• Purification of closely related drug intermediates• High speed fractionation of natural products – tocopherols, alkaloids, lignans, xanthones, stilbenes, flavonoids• Drug discovery• Agrochemistry• Petrochemistry