What is Chromatography?
Chromatography is derived from the Greek terms “chromo= color & gram=bands.”
Hence as the name indicates, in chromatography, there is the formation of colored bands.
These bands are indicative of different components in the sample.
In the initial stages, it was developed as column chromatography to separate mixtures.
Here into a long vertical column, sample along with a mobile phase was allowed to percolate through a solid stationary phase like sand.
So there were bands of separated compounds at different heights or lengths of the column appearing as colored bands.
Further passage of the mobile phase through the column leads to leakage of the components from the column bottom into different beakers kept for each band.
With the rise in further requirements in analysis, the mixture molecule separated is identified by using detectors.
“Chromatography is an analytical technique wherein a sample mixture under test is separated into different components.”
This is both a qualitative and quantitative method. The sample gets separated under the influence of a mobile phase (moving phase) over a stationary phase.
These separated components are later identified and also quantified.
Principles of chromatography
Chromatography is based on the principle of separation of compounds into different bands (color graphs) and the identification of those bands.
The preferential separation is done due to differential affinities of compounds towards stationary and mobile phase. After separation of the compounds, they are identified by suitable detection methods.
The differences in affinities arise due to relative adsorption or partition coefficient between components towards both phases.
Adsorption varies due to the polarity of components towards the stationary phase. If both the stationary phase and components in the sample are polar, the rate of travel for the polar component is slow and hence get separated from the rest of sample is passed out of the column last. Similarly, if both the stationary phase and component in the sample are non-polar, then the non-polar component comes out last due to the slow rate of travel in the column under the influence of the mobile phase. So the stationary phase and mobile phase are always opposite. I.e., if the stationary phase is polar, the mobile phase used is non-polar and vice-versa.
Ex: In a sample of a mixture of lipids and proteins. Lipids are non-polar and proteins are polar in nature. Suppose here Sand, which is polar is used as stationary phase and non-aqueous solvents like Hexane or Acetone which are non-polar in nature are used as mobile phase. During separation, lipids come out of the column first while proteins come out later due to their higher adsorption to sand stationary phase.
Partition varies due to the solubility of components into different liquids. So here both mobile phase and stationary phase must be liquids. The stationary phase liquid is in the form of a thin layer of film on a hard background in the column. So when a sample mixture having components of differences in solubility in different liquids, of same nature, i.e., either polar or non-polar, they get separated into two liquid phases, i.e., mobile phase and stationary phase based on their partition coefficient in between two liquids.
Ex: Iodine in between water and trichloromethane. Though iodine is soluble in both, it is non-polar in nature and hence is more soluble in trichloromethane than that in water. So most of the iodine separates into it, and a tiny amount stays in the water.
There are different techniques in chromatography. For details go through Types of Chromatography, But common technical requirements to all the types include
1. Stationary phase: The stationary phase is one which stays motionless and allows the sample to move over it. This phase used can be solid or liquid. If it is a solid stationary phase, it should have particles of uniform size and shape. Moreover, their shape should be preferably spherical. If a liquid is used as the stationary phase, the liquid is spread as a uniform layer on the solid background. The chromatography columns are house the stationary phases in all the types of chromatography except on paper and thin layer chromatography as they do not have a column.
2. The mobile phase: This is the chromatography liquid and it helps the sample move over the stationary phase. The mobile phase used is a liquid or gas and it should be free of particle matter and other impurities. Technically, the mobile phase should have opposite polarity to that of stationary phase material. I.e., if the stationary phase is of polar in nature, then the mobile phase has to be non-polar and vice-versa. In normal phase chromatography, the mobile phase is non-polar in nature while in reverse phase, the mobile phase is polar in nature.
3. Flow rate: the rate of flow of the mobile phase over the stationary phase is always kept constant. It should be uniform over the entire period of the experiment for reliable results. The high rate will help on faster separation, but the band can be very closure. If the rate of flow is slow, it will be time-consuming and also requires more mobile phase.
4. Temperature: The temperature of the experimental chamber or chromatography lab is kept uniform. Alteration in temperature can alter the flow rate, the state of mobile phase and also the detector efficiency.
5. Treatment of sample: Samples sometimes need to be treated for better separation or detection by the detector. In such cases, the sample is chemically altered before or after the process of separation. This is termed as pre-column or post-column derivation. This is especially followed in gas chromatography for some solvents.
6. Other techniques like ascending, descending and radial mode of development of chromatograms are followed.
7. The test sample or standard sample used in the analysis is moistened with the solvent preferably the mobile phase and it should be soft, powder form or homogeneous or pulpy but not hard.
8. The preparative or analytic technique is employed. For preparative HPLC, the sample quantity is more and is aimed to get the pure compound. While in analytic technique samples just analyzed for identification of sample and also the determination of its quantity.
Also, see the Difference between Chromatography and Electrophoresis
Chromatography Uses :
Chromatography is one of the widely used analytic technique in many industries.
- It is used in the food industry to check for permissible limits of ingredients.
- It is used in clinical diagnosis to see the levels of the drug in the blood at different time intervals (Kinetics & bio-availability study)
- In scientific research to check the biological substances and their quantity in the extracts.
- In quality control of drugs, in pharmaceutical industries. Once the drug formulation is ready, it is evaluated for quantities of different ingredients. This is done to comply with USP and other pharmacopeial standards.
- It is specifically used to identify and measure the molecules having similar chemistry, optical absorbance, etc.