Chromatography

In chemistry, chromatography is a process for separating different components from a mixture. This is achieved by passing a sample mixture in a stream of neutral solvent through some form of material that will provide resistance by virtue of chemical interactions (NOT reactions) between the components of the sample and the material. Usually, each component has a characteristic separation rate that can be used to identify it and thus the composition of the original mixture.

Paper chromatography

A small spot of the solution containing the sample is applied to a strip of chromatography paper about one centimetre from the base. This sample is adsorbed onto the paper. This means that the sample will contact the paper and may form interactions with it. Any substance that will react with (and thus bond to) the paper cannot be measured using this technique. The paper is then dipped in to a suitable solvent? (such as ethanol or water) and placed in a sealed container. As the solvent rises through the paper it meets the sample mixture which starts to travel up the paper with the solvent. Different compounds in the sample mixture travel different distances according to how strongly they interact with the paper. Paper chromatography takes some time and the experiment is usually left to complete for some hours.

The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes. Whereas compounds can be identified by calculating R_f values which can be compared with values in a data book. These can be calculated by:

  Rf = (distance moved by spot) / (distance moved by solvent)

Two-way paper chromatography involves using two solvents and rotating the paper 90^o inbetween. This is useful for separating complex mixtures of similar compounds.

Thin layer chromatography

This involves the adsorbent (the solid which the sample is adsorbed to) being in a thin layer on the surface of glass plate. The adsorbent (eg silica gel or calcium sulphate) is pasted on to the glass and baked. The process if the same for paper chromatography. The advantage is that wider seperations can be achieved in less distance, and different adsorbents can be used.

column chromatography

gas-liquid chromatography

HPLC
High performance or high pressure liquid chromatography

Gel electrophoresis

Gas chromatography

In gas chromatography (GC) a carrier gas, usually hydrogen, continuously flows through a column. At a given time, a known volume of a gaseous or liquid sample is injected into the entrance of the column. The carrier gas carries the sample molecules through the column, but this motion is inhibited by adsorption of the molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the column materials. Since each type of molecule has a different rate of progression, the various components of the sample mixture are separated as they progress along the column and reach the end of the column at different times. A detector is used to monitor the outlet stream from the column, and thus the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified by the order in which they emerge from the column.

Two types of columns are used in gas chromatography. Packed columns contain a finely divided, inert, solid support material (eg. diatomaceous earth) coated with a liquid or solid stationary phase. The nature of the coating material determines what types of materials will be most strongly adsorbed. Thus numerous columns are available that are designed to separate specific types of compounds. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm. The outer tubing is usually made of stainless steel. Capillary columns have a very small internal diameter, on the order of a few tenths of millimeters. The column walls are coated with the active materials. Most capillary columns are made of fused-silica with a polyimide outer coating. These columns are flexible, so a very long column can be wound into a small coil.

Molecular adsorption and thus the rate of progression along the column depends on the temperature, thus for precise work the column temeprature is carefully controlled to within a few tenths of a degree. Reducing the temperature produces the greates level of separation, but can result in very long elution times. For some cases temperature is ramped either continuously or in steps to provide the desired separation.

A number of detetectors are used in gas chromatography. The most common is the thermal conductivity detector (TCD), which monitors changes in the thermal conductivity of the effluent. The main advantage of the TCD is that it can detect any substance (except the carrier gas). Some of the other detectors are only sensitive to specific types of substances. Other detectors include the Flame ionization detector (FID), Electron capture detector (ECD), Flame photometric detector (FPD), Photo-ionization detector (PID) and Hall electrolytic conductivity detector.

An example of the use of gas chromatography is in the study of the selectivity of Fischer-Tropsch synthesis catalysts. The outlet from this process contains a number of light gases including N₂, H₂, CO, CO₂, H₂, CH₄, and Ar, as well as heavier parafinic and olefinic hydrocarbons (C2-C40). In a typical experiment, a packed column is used to separate the light gases, which are then detected with a TCD. The hydrocarbons are separated using a capillary column and detected with an FID.