Column chromatography is a technique used in chemistry to isolate individual chemical compounds from mixtures in a laboratory. This type of chromatography can be used to purify compounds on a scale ranging from micrograms (ug) to kilograms (kg). Chromatography columns used in this technique are disposable, which prevents cross-contamination and degradation of the chromatography columns due to reuse. Although this requires many columns to be used, they are rather inexpensive, so overall column chromatography is still cost-effective.
As with all types of chromatography, column chromatography has a stationary phase and a mobile phase (also called an eluent). The stationary phase in column chromatography is a solid powder – usually silica gel – but can also be alumina or cellulose powder. The mobile phase or eluent is either a pure solvent or a mixture of different solvents.
There are two types of column chromatography: the dry method and the wet method. In the dry method, the stationary column is filled with a powder, and the mobile phase is flushed through the column until it is completely saturated. In this method, the chromatography column is kept saturated throughout the chromatography separation process.
In the wet method, a slurry of the eluent and stationary phase powder is prepared and poured carefully into the chromatography column. The organic material to be separated is pipetted onto the stationary phase and topped with a small layer of sand and cotton or glass wool to avoid disturbing the shape of the organic layer from the addition of new eluent (solvent). Eluent is then added to the column slowly to advance the organic material through the column.
Separation of the sample compound occurs when each unique analyte partitions between the packing material (silica or alumina) and the mobile phase eluent. The difference in each analytes’ absorption affinity for the packing material determines the rate of separation, which also then determines the resulting color pigmentation when the analytes are filtered through the absorbent. As the analytes elute off the column, they leave pigmented bands that are subsequently flushed out to produce a pigmented chromatograph.
Column chromatography relies on gravity to pull the organic material through the column. There is no pressurization to force the material through the column and to the detector. Each unique analyte in the sample mixture elutes off the column at different times and are referred to as fractions. The various fractions are collected at different time points and can be analyzed separately. Additional types of chromatography, such as thin-layer chromatography, can be conducted on the fractions to determine what components each fraction contains and to determine if the separation of components was successful.
The column used in column chromatography is tubular with two narrowed openings and is usually made of glass, metal, or plastic. The bottom opening is stuffed with enough cotton to hold the sand or silica gel in the tube, but not enough to prevent the eluent from filtering through at an acceptable rate. The column is then clamped to a ring stand, the attached tubing is clamped, and then packed with sand.
The silica slurry is then prepared by adding silica to the selected eluent. The amount of eluent should be approximately double the volume of the silica. Stir into a slurry and immediately pour into the open end of the column. The tubing clamp is then removed and tubing placed into an empty flask. Eluent is used to rinse the slurry down the column, with remaining eluent dripping into the flask. Sand is packed on top of the silica. All remaining eluent should be allowed to drip out prior to introducing the sample.
A pipette is used to add the liquid sample to the column. The pipette is then rinsed with eluent multiple times to pass through the column. The liquid is allowed to drain out, and then more eluent is added to rinse all sample separations out. The column should be rinsed 2-3 times. The resulting column will have layers of solution with different color pigments, depending on the sample. Each layer is then collected in a test tube, flask, or beaker. The layer solution is then used in thin-layer chromatography in order to determine separation of the column chromatography analysis.
Although it is not commonly used in industrial laboratories in practice, column chromatography theory is the basis for the chromatography systems and instrumentation that are used world-wide in a variety of applications. The benefit of column chromatography is that it can be used as a preparative step in order to purify a sample solution prior to thin-layer chromatography or other chromatography analyses. In the instance of failed instrumentation or other obstacles, column chromatography utilizes common laboratory tools to purify and separate sample mixtures and can be set up easily. The fractions generated by the column chromatography analysis can also be used for determining sample separation.