Chromatography – Definition, Principles, Types, & FAQs

Chromatography

We may have come across these questions while studying analytical chemistry. Chromatography is a chemical term used for separating, testing and purification of compounds. 

What is Chromatography?

Chromatography is considered the method utilised for chemical compounds’ purification, separation and testing. The term ‘chromatography’ comes from a Greek word. In that chroma indicates ‘colour’, whereas graphein represents ‘to write’. 

In chromatography, the mixture will be separated in a sanitary phase, maybe solid or liquid. A pure solvent, namely any gas or water, is permitted to move at a slower rate. In the pure solvent, it will move over the stationary phase and carry the components separately according to their solubility.

Principles of Chromatography

Chromatography is a separation technique where we combine the analyte within a gaseous or liquid mobile phase. It has to be pumped through a stationary phase. In general, one phase is lipophilic while the other one is hydrophilic. The components contained in the analyte are capable of interacting in a varied manner using these two phases.

Moreover, the components spend less or more time interacting with the stationary phase depending on the polarity. It will make them pass through to a greater or lesser extent. It further leads to the separation of various components contained in the sample.

Furthermore, all the sample components are removed from the stationary phase at a particular time known as the retention time. The signal of the components will be recorded as they pass through the detector. It ultimately gets plotted in a chromatogram form.

Types of Chromatography

Chromatography consists of four major types. They are:

1. Column Chromatography

Column chromatography is widely utilised to separate the components present in the mixtures. It uses a suitable adsorbent column packed with a glass tube. We can place the mixture on the column’s top, and a much-needed removal is made. It is then enabled to flow gradually down the column.

The following is the figure that explains the same:

The components will experience separation based on the degree of adsorption. These components are located on the wall adsorbent column. The component with the highest absorptivity can remain at the top. At the same time, the other will flow down to multiple heights accordingly.

2. Thin Layer Chromatography

Thin Layer Chromatography is also known as TLC. This process is regarded as the mixture of substances separated into their components using the assistance of a glass plate. This plate is coated with a very thin adsorbent layer, alumina and silica gel.

The following is the diagrammatic representation of the same:

In this process, a chrome plate is used. The solution contained in this mixture is separated. It is separated using the application of a small spot at a 2 cm distance above the plate’s end. After doing so, the plate is fixed in a closed jar that consists of a fluid called eluent. It rises on top of the plate by carrying various mixture components to multiple heights.

3. Adsorption Chromatography

The process of adsorption chromatography includes the adsorption of various compounds on the adsorbent. The degree of adsorption differs depending on the component’s absorptivity. In adsorption chromatography, moving over a stationary phase would require the involvement of the mobile phase. Therefore, carrying components that contain higher absorptivity to a lower distance is much better than carrying a lower absorptivity. The below-mentioned figure includes the main chromatographic methods which are utilised in industries:

4. Partition Chromatography

In partition chromatography, the components tend to perform continuous differential partitioning of a mixture. There will be occurrences of the mobile phase and stationary phase. An ideal example of partition chromatography would include paper chromatography. Here, we are using chromatography paper as a stationary phase. Moreover, it acts as a mobile phase because it is suspended in the solvents’ mixture.

We place a spot at the chromatographic paper’s base during this process. It has to be kept with the mixture in order to be separated. Depending on their retention, the components will be carried to varying degrees as soon as the solvent tends to rise to the paper. Therefore, the components are separated at different heights.

The following is the figure that indicates partition chromatography:

Differential Extraction

Differential extraction is a technique that involves the separation of any organic component contained in an aqueous solution. In differential extraction, organic solvents are used. The solvent’s solubility will be more than the desired component. Moreover, an organic solvent is selected so that it is immiscible in an aqueous solution. It can further form layers that can easily be separated with the help of a separating funnel.

Later on, the organic compound is recovered by evaporation or distillation process. The continuous extraction process is utilised in such cases where the compound’s solubility is less in the organic solvent.

Provided below are the before and after pictures of extraction:

Applications of Chromatography

Let us see some of the real-time applications of chromatography below:

• Security: Chromatography is used in security practices. Gas chromatography is utilised in order to identify volatile gases. In addition, safety precautions at various locations, including large gatherings, airports, sporting events, concerts, etc., are some places where we can use chromatography.
• Drug Testing: Chromatography is highly useful in clinical toxicology reports and testing of drugs. The main advantage of chromatography is that it can separate and analyse substances contained in urine samples. Running a toxicology report, testing for drugs on a new employee, drug testing a professional athlete, etc., are some drug testing areas where chromatography is used. The main objective of chromatography is to determine the drug content present in the urine.
• Petroleum: One of the major uses of gas chromatography can be found in refining processes and finished gas products. We can utilise chromatography for analysing natural and refinery gas for hydrocarbon composition and BTU content. Nowadays, we can use chromatography in developmental, analytical and quality control labs because of its wide variety of applications.
• Molecular Biology Studies: A very complex usage of chromatography is seen in molecular biology studies. Hybrid methods between mass spectrometry and electrochemistry are frequently applied to various studies resulting in peptides, proteins and nucleic acids. This combination can be widely used for biotransformation reactions, namely proteomics and oxidative reactions. In addition, it is used in the purification of antibodies, plasma proteins and hormones.
• Forensics: Forensics is quite similar to security measures. Chromatography is used for various in-depth forensic procedures. For instance, police use crime scene analysis in order to test the evidence, namely hair, fabric samples, blood, etc. It will help them understand the case further and eventually solve them. It will also provide them with an idea of what might have happened and who did the crime while matching the evidence with the government database. 
• Chemical and Environmental Industry: It is essential for the chemical and other such industries to adhere to the environmental conditions set by the high commission. PFAS substances are considered to be harmful to our bodies and the environment. It is usually found in fabrics, shoes, clothes, firefighting foams and electronics. It is understandable that it makes the product very durable. However, it is also very dangerous and should be taken care of.

Conclusion

To conclude, chromatography is a chemical term commonly used for purifying, separating and testing various compounds. Therefore, from this blog, we have understood the chromatography definition and how we can use it on different occasions. 

Furthermore, it would have given you much-needed knowledge regarding chromatography and other related concepts.

Frequently Asked Questions

1. What is the purpose of chromatography?

The main purpose of chromatography is to separate the different substances that make up a mixture. Chromatography uses might range differently based on various factors, starting from purity verification of a provided compound to the quantitative representation of the components contained in a mixture.

2. What is the chromatography technique?

It is a method generally utilised for separating the solutes or the components of a mixture based on each solute’s relative amounts. It is distributed among a moving stream of fluid. It is called the mobile phase. On the other hand, if it is contiguous, it is then termed the stationary phase.

3. What type of mixtures are separated by chromatography?

One of the standard practices for the separation of mixtures includes paper chromatography. It helps in separating peptides, complex amino acid mixtures, steroids, carbohydrates, purines and a few simple organic compounds.

4. Mention the four major types of chromatography.

 The method of chromatography involves four main types. They are:

• Paper chromatography
• Gas chromatography
• Thin-layer chromatography
• High-performance liquid chromatography

5. Mention the phases present in chromatography.

In chromatography, there are two major phases. They are:

• Stationary phase
• Mobile phase

That stationary phase indicates that the components are fixed in one place, whereas the mobile phase denotes that the components are in movement.