Chromatography is a physical separation process in which the different compounds are distributed between two phases. Learn the principle, types, and applications of chromatography.
What is chromatography?
Chromatography is one of the powerful and versatile methods that is used in all branches of science particularly in analytical chemistry. It is a separation technique that is obtained by distributing the analytes of a sample mixture between two phases, a stationary and a mobile phase. Each analyte has its properties, so they have a different affinity towards the stationary phase.
The analytes of the sample mixture move at different speeds, causing them to separate from each other. It depends on the nature of the stationary phase and the mobile phase, which analytes travel more rapidly or more gradually. These different travel times of the separated analytes are called retention time.
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The principle of chromatography (How does chromatography works?):
Chromatography is based on the principle of applying the mixture of the compounds to the surface or the stationary phase, they travel with the aid of a mobile phase and get separated. The factors that are involved in this process of isolation include molecular characteristics of a partition, affinity, and adsorption.
Due to these variations, certain compounds remain in the stationary phase longer, and travel gradually throughout the chromatography process, whereas others are quickly eluted through the mobile phase.
Chromatography uses differences in the polarity of the molecules of the sample mixture. In this separation process, a liquid or solvent acts as a mobile phase and it runs through a layer of particles which is known as the stationary phase. A sample consisting of a mixture of compounds is introduced or injected into the mobile phase and passes through the stationary phase.
Based on the relative affinity for mobile and stationary phases the compounds are isolated. The compounds having more affinity they are traveling slowly as compared with the compounds with a lower affinity. The results of isolated molecules are then used as qualitative and quantitative analysis by comparing them with standards.
Different types of chromatography:
The chromatographic separation can be executed with several different setups. Based on the molecular interaction, there is ion-exchange chromatography for ionic molecules, partition chromatography based on solubility, affinity chromatography with specific interactions, size exclusion chromatography based on molecular size, and adsorption chromatography for non-ionic polar molecules.
1. Planar chromatography:
The planar chromatography includes paper chromatography and thin-layer chromatography, which relies on the adhesion and cohesion forces of a solution that passes through the media. In paper chromatography, the cellulose paper acts as a stationary phase and in TLC a sheet which is coated with a thin layer of an adsorbent such as silica or alumina is used as a stationary phase.
Analytes are separated according to their attraction to the adsorbent and appear after chromatographic separation as individual spots on the surface. Finally, the retention factor (Rf value) is used to identify an unknown substance.
The principle of paper chromatography and thin-layer chromatography (TLC) depends on the relative affinity of solutes towards both the phases. Paper chromatography has five different types such as ascending paper chromatography, descending paper chromatography, ascending-descending paper chromatography, circular paper chromatography, and two-dimensional paper chromatography, etc.
2. Column chromatography:
Column chromatography is a widely used technique for separating and purifying of different chemical compounds. It consists of a glass tube placed vertically, in which the mixture of samples along the mobile phase passes through a solid stationary phase and compounds gets separates according to their affinity.
The column is packed with a solid adsorbent material which is called a stationary phase. Generally, silica gel or alumina used to pack the column using the dry method or wet method according to application.
Most commonly used column chromatography works on the principle of "partition" using the force of gravity. There are four different types of column chromatography are used in chemistry, such as adsorption chromatography, ion-exchange chromatography, partition chromatography, and gel chromatography, etc.
3.High-performance liquid chromatography (HPLC):
HPLC is the most popular and widely used analytical technique for the separation of the solutes. It is an advanced version of column chromatography. In HPLC, the sample is injected into the flow of the mobile phase, and then it is pumped under high pressure into an HPLC column.
The isolation depends on the affinity of the solutes for particles of the analytical column and mobile phase. The mobile phase consists of a solvent/mixture of solvents or solvent with a buffer solution.
Different types of HPLC chromatography are used for various applications that are dependent on the stationary phase such as reversed-phase, normal phase, size-exclusion, and ion-exchange. For HPLC the different types of columns, different types of HPLC detectors (UV/PDA), different types of injectors (Rheodyne, septum/stop flow), and different types of HPLC pumps. (Reciprocating/syringe/pneumatic) are available.
4. Gas chromatography (GC):
In GC, an inert gas such as helium or nitrogen or argon is used as the mobile phase and is usually a solid or a liquid stationary phase composed of silicon polymers. The gas chromatography works on the mechanism of “partition”. The volatile mixture of sample is injected into the column with the stationary phase and is selectively adsorbed. The isolated molecules are recognized using a detector as they eluted from the column.
There are different types of columns (packed or capillary) and different types of detectors (flame ionization, thermal, and GC-MS) can be used for different applications of GC, usually, it depends on the type of sample used in GC. Results obtained from GC and HPLC have peak areas used as qualitative and quantitative analysis of the molecule.
The shape of the peak needs to be symmetrical and it should not be beyond the limit of the tailing, fronting, and resolution of the peak.
5. Ion-exchange chromatography:
The ion-exchange chromatography is used to isolate ion and polar molecules, the isolation depends on the affinity toward the ion-exchanger. The basic principle involved is the exchange of ions, which can be used for almost any kind of charged molecule, such as amino acids, proteins, and nucleotides. Cationic and anionic exchangers are the types of ion-exchange chromatography.
Applications of chromatography:
Chromatography has many essential applications in several fields such as the pharmaceutical industry, food industry, chemical industry, environmental, beverage, and it's also used in forensics. Some of the important applications of chromatography are discussed below.
- Chromatography is used to detect unknown components and purity of sample mixtures.
- The HPLC, GC, and MS are used in forensic detection and crime scene investigation.
- Chromatography is used in the separation of organic as well as inorganic components.
- It is used for the determination of toxins, contaminants, nutrition, and food quality.
- Chromatography used to monitor the drug synthesis reaction.
- In the clinical diagnostics, it is used to determine the amount of substance found in blood and urine.
- The applications of column chromatography include the separation of several classes of drugs such as plant extracts, aggregates, alkaloids glycosides, and amino acids.
- Chromatography is used in the food manufacturing process for quality control.
- The applications of TLC chromatography include the isolation and identification of preservatives, sweetening agents, dyes, and many other cosmetic products.
- For testing the quality of water and air, it can be used.
- Chromatography is used to isolate and analyze of vitamins, proteins, additives, and preservatives.
- It is used to detect various contaminants in pesticides and oils.
- The applications of paper chromatography consist to detect and separate color mixtures including pigments, it is used in analytical chemistry as a qualitative technique.
Advantages of chromatography:
- The major advantage of chromatography is that a wide range of samples can be separated using different separation mechanisms.
- It is a simple method for separating a complex sample mixture using the chromatography method.
- It does not require a large amount of sample. To detect the sample is the use of a sample in just grams, ppm, and ng.
- One person can perform or handle the experimental process of chromatography.
- Chromatography results are rapid and precise.
The commonly asked questions on chromatography are as follows.
What is the major advantage of HPTLC over TLC?
The major advantage of HPTLC over TLC is that the HPTLC has more resolving power with faster development times and the consumption of solvent is less than TLC.
What are the factors that affecting resolution in HPLC chromatography?
It is an elution is a quantitative determination of how the two peaks can be distinguished in chromatographic separation. Three factors mainly affect the resolution i.e. separation, retention, and efficiency.
What is the principle of column chromatography?
Column chromatography is a type of chromatography in which the solutes to be isolated are distributed between a mobile phase and a stationary phase. In this method, different distributions depend on selective adsorption.
What type of chromatography is paper chromatography?
Paper chromatography is classified in planar chromatography in which processes are carried out on a specialized cellulose paper.
What is the importance of pKa value in HPLC method development?
The pKa is a way used to indicate the strength of an acid. It is a negative log of the acid dissociation constant or the value of. A low PKA value of a substance indicates a strong acid.
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