Chromatography is an analytical chemical technique used for the separation, identification, and purification of compounds in a complex mixture. It is an important biophysical technique used for qualitative and quantitative analysis.
All types of chromatography techniques has two phases- a stationary and a mobile phase. The stationary phase contains the mixture while the mobile phase carries the components in the mixture as it passes through. The compounds in the mixture separate at different paces as they pass through the mobile phase.
Chromatography can be used for the separation of any chemical in liquid, solid, or gaseous phases, using specific stationary and mobile phases. The characteristic time for the passage of each component is called retention time.
Depending on the retention time each component travels at different levels which leads to the separation of the compounds.
Types of Chromatography Techniques
Chromatographic techniques are categorized based on several criteria. There are different types based on the chosen stationary and mobile phases and the chosen method. Based on the stationary and mobile phases chosen, there are different types of chromatography techniques.
- Adsorption chromatography uses a mobile phase in liquid or gaseous form which passes through a stationary phase. Here, the mobile phase is adsorbed on the stationary phase.
- Partition chromatography has a stationary phase on which the mobile phase forms a thin liquid film. It is a liquid chromatographic technique that works on how the compounds have an affinity towards the mobile or stationary phases.
- Ion exchange chromatography uses a resin as a stationary phase which attaches covalently with anions or cations. The solute ions from the mobile phase attach themselves to the resin through electrostatic forces.
- Molecular exclusion chromatography does not have any interaction between its two phases. Here, a porous gel separates molecules from the passing mobile phase, based on their molecular size. Since the pores are small and allow only small molecules to enter it, larger molecules will move through the mobile phase faster than the smaller molecules.
- Affinity chromatography depends on specific interactions between the two phases. It is a liquid chromatography technique that uses the binding interaction between the compounds or molecules in the mixture to separate them. An affinity ligand attached to the resin works as the stationary phase. As the mixture passes through the mobile phase, molecules that have more affinity to the ligand stay behind and the others move on.
Different Types of Chromatography Methods
Based on the method used, chromatography can be categorized into,
- Paper chromatography
- Gas chromatography
- Column chromatography
- Flash Column chromatography
- TLC or Thin Layer Chromatography
- HPTLC or High-Performance TLC
- HPLC or High-Performance Liquid chromatography
Paper Chromatography
- Paper chromatography uses filter paper sheets to separate the compounds from all classes of natural materials.
- The component solution is loaded on a single spot on the sheet.
- The sample is repeatedly loaded on the same spot at regular intervals to have a concentrated sample.
- The mobile medium is often a mixture of 3-4 solvents and one of them is usually water.
- The paper is fixed to a system in such a way that it is in contact with the mobile phase.
- The mobile solvent passes through the paper.
- As it passes through the sample, the compounds start separating.
- After the chromatogram, the colored compounds will be visible to the naked eye.
- The colorless compounds are visible only after the paper is subjected to exposure to heat in an oven, spraying with sulfuric acid, UV light, or reagents.
- Heating and spraying of sulfuric acid char the compounds which will appear as black spots.
- UV light shows fluorescence and reagents cause the compounds to be stained in different colors.
- Each method is used for different compounds.
- For radioactive compounds, radioactive detectors or X-rays are used.
Gas Chromatography
Gas chromatography is used for separating volatile compounds. A non-volatile stationary phase such as silicone oils, grease, paraffin, apiezon oils, etc., is used here. An inert gas is used as the mobile phase which will pass through the other one and carries the separated compounds with it.
The column used here could be a capillary or a packed column. For the latter, the columns are packed with fine, solid, inert particles such as diatomaceous earth. This diatomaceous earth is coated with the liquid stationary phase.
The capillary column can be a Wall Coated Open Tubular (WCOT) or Support Coated Open Tubular (SCOT). The former has its wall coated with the liquid stationary phase while the latter has its wall lined with a thin layer of diatomaceous earth where the stationary phase is adsorbed. The capillary columns are more efficient than the packed columns.
Applications of Gas Chromatography
- Gas chromatography is ideal for the analysis of low concentrations of samples.
- Gas chromatography is useful for the identification of a completely new compound as it will require the analysis of its spectral characters.
- A gas chromatography connected to a mass spectrometer can analyze the spectral characters of the compounds which will help in their identification.
- Preparatory gas chromatography can be done using a thermal conductivity detector, FID, and splitting outlet.
Column Chromatography
Column chromatography uses a column to separate the compounds.
- Here, the sample is loaded onto a solid support. The column is filled with the solvent which will flow with gravity.
- As the solvent moves, it separates the compounds in the sample.
- Each compound moves at a different pace or rate of movement.
- The separated mixture is collected separately as they exit the column.
- The solvent is evaporated to collect the compounds.
Flash Column Chromatography
Flash gas chromatography is simple and works faster. It uses a narrow glass tube where the flow of the solvent can be controlled by a pressure valve at the top or a suction at the bottom.
The stationary phase of flash golem chromatography requires smaller particles than what is used in column chromatography. It uses silica, reverse silica, cellulose, etc as the stationary phase depending on the nature of the compounds.
The working mechanism is the same as that in the column chromatography.
TLC or Thin Layer Chromatography
TLC is the common type of chromatographic technique used for the quantitative analysis of compounds. It is simpler and faster than other types of chromatography. The stationary phase uses a thin layer of silica, cellulose, or alumina, coated on a glass plate, aluminum foil, plastic sheet, etc.
Advantages of TLC
- TLC offers quicker fractionation of compounds.
- Works well with smaller quantities of samples.
- It can have separated spots of sample or a single more compact spot.
- Spots are demarcated from each other.
- TLC has better sustenance than filter paper when sprayed with sulfuric acid.
- It is cheaper and more effective than other options.
- It requires less amount of sample, solvents, and adsorbents.
HPTLC or High-Performance TLC
HPTLC stands for High-Performance Thin Layer Chromatography, which is an upgraded version of normal TLC. This is an automated setup used for quantitative and qualitative analysis of compounds. Precoated plates with silica gel coating are used here as the stationary phase. The reverse phase mode uses C18, C8, etc.
HPTLC is more efficient than TLC since the adsorbents have uniform size and are smaller. It needs just a tiny spot of sample but it has to be more concentrated.
A semiautomatic loading apparatus such as the Linomat V apparatus is usually used to load the sample. HPTLC can be done in linear development chambers where the chromatogram can be developed from the sides.
It is usually used for the standardization of herbal formulations and extracts. Moreover, it helps develop the analytical profile of anthracene, lipids, alkaloids, flavonoids, steroids, etc.
HPLC or High-Performance Liquid chromatography
HPLC is similar to Flash column chromatography but uses high pressure to move the mobile phase. Instead of a glass column, HPLC uses a steel column that can withstand high pressure.
It is suitable for all types of liquid chromatography. The HPLC will be connected to a spectrometer that helps study the spectral characters of the compounds that are separated.
HPLC is advantageous over gas chromatography as the sample can be recovered after the analysis and purification.
References
- Coskun, O. (2016). Separation techniques: Chromatography. Northern Clinics of Istanbul, 3(2), 156-160. https://doi.org/10.14744/nci.2016.32757
- Shah, Biren N, Avinash Seth. (2010). Textbook of Pharmacognosy and Phytochemistry. Elsevier.
