If you’re revising for your upcoming A level organic chemistry exam, you’ll need to have a good grasp of chromatography. Keep reading to learn what it is, how it works, and the different chromatography techniques.
In this post:
What is chromatography?
As a scientific laboratory technique, chromatography is the separation of the components of a mixture, which usually have different colours.
Originally developed by German scientists in the 1930s, chromatography is a combination of two Greek words: the prefix chromato- (colour) and -graphia (writing). Hence, it literally means colour-writing or writing by colour.
The mixture that’s being analysed is first dissolved in a fluid solvent, which can either be gas or liquid. Known as the mobile phase, this stage carries the mixture through a system of either a column, capillary tube, plate or sheet.
During the second stationary phase, a material is fixed onto the system and the various constituents of the test mixture separate. The different physical and chemical affinities of the constituent components cause them to stick to specific surface sites as they travel through the system at different rates of motion. As a result, the constituents of the mixture separate.
Key facts for chromatography and qualitative analysis
A mixture’s constituents are separated through the differential partitioning between the mobile phase and the stationary phase. Each substance in a mixture has a different partition coefficient. This applies to both homogeneous and heterogeneous mixtures (e.g. water and oil).
You can calculate the partition coefficient as the ratio between the concentration of a substance in one medium (phase) and the concentration of the same substance in another medium (phase) when they are in equilibrium.
Coefficient = (C1/C2)equilibrium
Although the differences in the partition coefficients are usually very subtle, they’re sufficient to make it possible for differential retention to occur. This process can either be used for preparative or analytical purposes. If it’s the former, the known components of a mixture may be separated and retained for later use (similar to a purification or distillation process).
Analytical chromatography, however, aims to determine the types and concentrations of the constituents of a mixture. It’s usually done on a small scale with smaller amounts of analytes. In some cases, not all constituents of a mixture have to be identified; the process may simply be used to rule out the presence of a specific component.
The concept of chromatography can be easily demonstrated without using sophisticated laboratory techniques or equipment. All you need is a paper towel and a non-primary food colour, like green.
Place a few drops of green dye on a paper towel and wait for a few minutes until the dye spreads out from the centre. The primary colours (in this case blue and yellow) will then separate, as shown in the photo below.
Regardless of the chromatographic method, a sample material (analyte) is applied onto a stationary material that either absorbs or adsorbs it. The former means the molecules or ions of the sample penetrate the material. In the latter, the molecules or ions simply cling or adhere to the surface of the material (the stationary phase).
The sample is then dissolved and transported by a liquid or gas solvent in one direction (the mobile phase). It’s this motion that causes the components of the sample substance to separate.
Thin layer chromatography (TLC)
Biochemicals are separated and analysed in the laboratory using various techniques. One of the most common methods is thin-layer chromatography or TLC. It works because various types of biochemicals have different attractions or affinity to the stationary and mobile phases to which they’re subjected.
To some extent, the principle behind TLC is the same as paper chromatography. The main difference is the material used for the stationary phase. A thin layer of adsorbent material, like silica gel or cellulose, is used instead of paper.
Laboratories commonly use TLC because of its versatility. For example, more than one sample of the substance can be separated simultaneously using the same layer. This is useful for screening purposes such as drug testing and water supply testing. There’s also a low probability of cross-contamination because each separation process and analysis is carried out on a new layer of stationary phase.
Another advantage of TLC is that it’s faster than traditional paper chromatography, thanks to the adsorbent mineral-based stationary phase. It’s also more accurate and precise, providing better results for quantitative analysis.
One of the many applications of TLC is differentiating chromosomes in a gel medium. The table below shows the different types of materials that are used as the stationary phase, along with their corresponding mechanisms and applications.
As you’ve probably guessed, gas chromatography uses a gaseous solvent instead of liquid. The gas is used as the mobile phase of the system and is pumped into the system via a flow controller. Inert gases like helium (He), nitrogen (N2), hydrogen (H2), and argon (Ar) are used as solvents or carriers of samples.
Meanwhile, the stationary phase is liquid and contained within the columns. Depending on the design of the chromatograph, the columns are usually between 1 and 100 metres long.
The liquid stationary phase is adsorbed onto the surface of an open capillary tube. It may also be adsorbed on a packed solid support inside the tube.
The illustration below demonstrates how a gas chromatography system works.
High performance liquid chromatography (HPLC)
Liquid is used as the mobile phase or carrier of samples in high performance liquid chromatography (HPLC). The liquid chromatographic columns can either consist of liquid-liquid, liquid-solid or ion-exchange.
Stainless steel is commonly used as HPLC columns, but other materials such as thick glass, polymers, and composites can also be used. The liquid is usually a combination of polar and non-polar components.
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