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 chromatography

• Glass columns with silica gel

• Separation is very slow (typically many hours)

• End of the run, silica gel must be removed, cleaned, dried and re-packed

• Both time consuming and hazardous

Flash chromatography

• Pre-packed plastic cartridges

• Solvent is pumped through the cartridge

• Much quicker and more reproducible

• Remaining solvent flushed out of the column using pressurized gas

Column chromatography Flash chromatography

Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge.

Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage.

Flash chromatography is not expected to provide the resolutionor reproducibility of HPLC; it is a technique that can quickly improve the purity of samples to an acceptable level.

Selection of solvent system

Solvent 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 determinedCommon 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 amount of cotton; use a wood applicator stick to tamp it down lightly. 2. Take care that you do not use either too much cotton or pack it too tightly. You just need enough to prevent the adsorbent from leaking out.

Add dry silica gel adsorbent, 230-400 mesh -- usually the jar is labeled "for flash chromatography." One way to fill the column is to invert it into the jar of silica gel and scoop it out . . .

. . . then tamp it down before scooping more out

Another way to fill the column is to pour the gel into the column using a 10 mL beaker.

(2) Pre-elute the column.

(3) Load the sample onto the silica gel column

Two different methods are used to load the column: the wet method and the dry method:

Wet loading methodThe 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.

Dry loading method

(4) Elute the column.

5) Elute the column with the second elution solvent.

If separating a mixture of one or more compounds, at this point change the eluting solvent to a more polar system, Elution would proceed as in step (4).

(6) Analyze the fractions.

If the fractions are colored, simply combine like-colored fractions, 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, the fractions 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

Conclusion