Flash Column Chromatography Methodology

Flash column chromatography was developed in 1978 as an alternative method to long column chromatography.

The method aims to isolate a component from a mixture and thereby purify it. It was built from existing long column chromatography technology, which was time-consuming and often not satisfactory. Flash column chromatography, in short, passes a sample through a column filled with a gel, which separates the sample.

Original Methodology

The creators of flash column chromatography, Still and colleagues, had been using medium pressure chromatography and short column chromatography as alternatives to long column chromatography. They decided to combine these two to overcome the drawbacks of long column chromatography such as being time-consuming and having poor recovery.

The original gel used to line the column was silica gel, which is still widely employed. They use air pressure to drive a solvent through the silica gel column, to compress the column. Then the sample is applied, and the same (can be different, but often the same) solvent is used to pass the sample through the column. The purified components, or fractions, are then collected, with the whole process taking roughly 5-10 minutes. Typically, small fractions are the first to emerge, and larger fractions elute at the end. The original analysis of the collected fractions is done by thin-layer chromatography (TLC) plates.

The General Guidelines

Since its introduction, silica gel flash column chromatography has been widely used in organic chemistry. However, guidelines have often been loose, driven by personal experience, or have not translated well when settings are changed. But, some things still hold true. Increasing sample quantity results in poorer resolution. The resolution of flash column chromatography in comparison to high performance liquid chromatography (HPLC) is already mediocre, but enough for adequate separations, so increasing quantities would only worsen that. Second, the optimal flow rate differs depending on column length and width, as well as the properties of the gel. This is due to the number of plates available, for example, longer and narrower columns would provide more number of theoretical plates, and would thus affect the flow rate. Lastly, resolution is affected by the stationary phase. The stationary phase, or the gel which lines the column, provides better resolution if it is more homogenous, and has smaller particle size. Smaller particle size means it has more surface area, therefore yielding better resolution.

Manipulating all these factors to optimize purity or recovery of components can be quite complex, since they interact with each other, but have different effects when tested independently. For example, mobile phase selectivity has the largest effect on resolution, but it, in turn, depends on the column capacity. This can then, in turn, be affected by the solvent that is chosen.

Therefore, the settings used in flash column chromatography need to be tested or calculated before any real experimentation can begin. Optimal settings can be calculated without the need to test any flash column chromatography settings if there is data from a TLC. Low TLC retardation factor (Rf) provides better separation. Using data like this, the amount of solvent required can be calculated, since they are inversely proportional to each another, and both share a relationship with column void volume (solvent in a column before sample is loaded).

Dry Columns

In the original methodology, preventing the column from getting dry was of big importance. However, since then a more user-friendly alternative has been developed, called dry column flash chromatography. This method has been adapted to be used by first-time students, while still yielding relatively good results rivalling analytical TLC quality. The principles are the same, but the column contains dry silica gel. The powder-like dry gel is packed into the column using suction to end up with an even bed of roughly 1 cm for the solvent and sample. The column is also eluted by suction, and is dried after every fraction.

Further Reading

Last Updated: Sep 18, 2018

Sara Ryding

Written by

Sara Ryding

Sara is a passionate life sciences writer who specializes in zoology and paleontology, which is the study of animals before our time. She has hands-on laboratory experience in genetics, neurobiology, and proteomics.

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