Chromatography: Definition, Principal and Types

Did you know that chromatography is utilized in over 60% of chemical analyses globally?

Chromatography is a critical analytical method in all branches of biological sciences, from chemistry to forensics and research. It is an effective technique to separate the components of a mixture and analyze them. 

Chromatography was first used in the early 1900s and continues to serve as an efficient analytical tool in laboratories. It is typically used to separate organic and inorganic components from homogenous mixtures. 

Let us tell you about the principle and types of chromatography in detail.

What is Chromatography?

The word chromatography is a combination of “chroma” and “graphy.” Chroma means color, and graphy translates to writing. 

Thus, chromatography is defined as a separation technique in which the components give different colors on separation when passed through a solution.

The process comprises a stationary phase containing the mixture to be analyzed. The mobile phase (solvent) moves slowly over the stationary phase to carry the components according to their solubility. Each of the components moves to a particular distance per their affinity and solubility.

Different types of microscopes like fluorescence combined with HPLC and other chromatography techniques prove helpful in finding solutions to complicated analytical problems. 

Principle of Chromatography

Chromatography makes use of a stationary and mobile phase. The analyte is incorporated in a gaseous or liquid mobile phase and pumped through the stationary phase. It allows the components of the analyte to separate without dissolving in the mobile phase completely. One of the phases is usually hydrophilic, while the other is lipophilic. 

The polarity of the analyte components interacts with the mobile phase in their specific way. It enables the separation of the sample. The time taken by each constituent is known as the retention time which helps identify the elements.

Types of Chromatography

Analytical studies around the world make use of different chromatography techniques for the needed results. All chromatography techniques might not be suitable for all substances and types of analysis. Over fourteen types of chromatography techniques are used including gas chromatography, thin-layer chromatography, HPLC, paper chromatography, liquid chromatography, column chromatography, etc.

Here are some of the widely utilized chromatography types for better understanding.

Paper Chromatography

Paper chromatography is a technique that uses specialized paper for analysis. It is of two kinds; paper adsorption chromatography and paper partition chromatography. 

Paper adsorption chromatography works on the varying degree of interaction between the stationary phase and molecules. Molecules with a higher affinity have a lesser speed, whereas lower affinity molecules move faster.

On the contrary, paper partition chromatography works on the principle that the moisture on the paper is the stationary phase. The molecules separate depending on their adsorption onto the stationary phase. The ratio of distance traveled by the molecule to the distance traveled by the mobile phase gives the retention value. The unique retention value of each molecule makes it easy to identify them.

It is typically used to separate colored drinks, ink, and reaction mixtures. It also helps detect the purity of pharmaceutical products.

Thin-layer Chromatography

Thin-layer chromatography involves applying the stationary phase as a thin layer on a solid plate with a liquid mobile phase. It is widely utilized in small-scale labs. The mobile phase elutes the components. This technique also works on the affinity principle that binds the constituents with a higher affinity to the stationary phase.

The mixture passes through the mobile phase, and the substances with a high affinity bind to the substrate in the stationary phase. The other components are removed or eluted with the mobile phase. The molecules appear as spots in the stationary phase after separation. You can detect the components using different techniques.

Thin-layer chromatography is adapted in forensics to analyze fiber, and in medical plant identification. It also gives assay of pharmaceutical products for quality-check.

Liquid Chromatography

Liquid chromatography is a popular type of chromatography that uses a liquid as a mobile phase, and separation occurs on a plain surface or column. 

When the components have a higher affinity to the mobile phase, they come out of the column faster. Alternatively, constituents with a lower affinity elute out of the column later. Two molecules with different polarities will move at different speeds, making it easy to distinguish.

Liquid chromatography is often used for the separation of solids insoluble in water. You can also separate colored solution components due to two different bands.

Gas Chromatography

Gas chromatography converts the sample into vapors to analyze it. The specimen is introduced as a gas or liquid which is vaporized. 

The mobile phase carries the sample through the gas column. The constituents are collected when they come out of the column. The sample passes through the detector to find the retention time. The components with a higher affinity to the stationary phase take more time to exit the columns and have a higher retention time.

Gas chromatography is used to quantify drugs in sports, water samples, and soil. It also helps analyze different chemicals, oil spills, etc.

HPLC

High-performance liquid chromatography, also known as HPLC, is a reformed form of column chromatography. It uses 400 atm instead of relying on gravity, like column chromatography.

HPLC uses the stationary phase affinity principle to separate the sample’s constituents. It works on differential adsorption. The molecules separate on the basis of their degree of interaction with the absorbent in the stationary phase.

Molecules with a higher affinity have a lesser speed as they are adsorbed for longer. On the other hand, lower affinity molecules move fast. This difference in speed due to different adsorption lets the molecules separate in different fractions. 

HPLC chromatography technique is used in various industries such as pharmaceuticals to maintain product quality. It is utilized in purity analysis and used to separate biological molecules.

The Bottom Line

Chromatography is one of the widely used analytical methods. Around 60% of chemical analysis is done through chromatography worldwide. It typically consists of two phases; stationary and mobile phase. However, different types of chromatography are used for varying purposes. Gas chromatography, paper chromatography, liquid chromatography, HPLC, etc., work on similar principles using different equipment. They are utilized in labs and industries for various purposes like separating and analyzing components.

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