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Saturday, May 2, 2020

Principle and applications of column chromatography

Column chromatography is a basic and simple type of chromatography technique used for separating and purifying the compounds. It is a kind of adsorption chromatography in which the compounds are separated based on their different affinity to the stationary phase. In column chromatography, generally, silica gel is used as a stationary phase (adsorbent), and solvent or a mixture of solvent used as a mobile phase. Column chromatography has a polar stationary phase. The separation of the compounds based on its polarity since the polar compounds elute slowly and nonpolar compounds travel rapidly through the column.
The principle of column chromatography:
The adsorption is the basic principle involved in the separation process of column chromatography. The liquid mobile phase is allowed to travel through a solid stationary phase with the sample mixture under the force of gravity. The analytes of the sample become adsorbed in the stationary phase throughout their travel and they get separated due to differences in affinity of individual analytes. The analytes have low affinity to the stationary phase they rapidly eluted, while those analytes have greater affinity they elute slowly from the column. The solute reversibly adsorbs to the stationary phase. Therefore, with a constant input of the solvent from above all the samples flow out.
Various essential components involved in the column chromatography process.
Tubular glass column, stationary phase, solvent, stand with clamp and funnel, etc.
Major steps involved in the process of column chromatography.
1. Preparing the column
2. Selecting the suitable solvent to run the column
3. Loading the column
4. Fractions Collection

The applications of column chromatography are as follows.

One of the major applications of column chromatography is that it can be used for isolation and purification of many classes of components.
Essential components can be separated from plant extracts, formulations, active components, from plant extracts.
It is very useful in the removal of impurities from the sample.
It can be used to isolate amino acids, glycosides, and alkaloids.
This method is used in the purification of reaction mixtures in chemical synthesis.
Metabolites from biological fluids such as blood, serum, etc. are isolated using column chromatography.
The impurities of the sample can be separated by using the appropriate mobile and stationary phases.
This is used for the determination of drugs from formulations.
Pharmaceutical manufacturers typically used to purify compounds.
It is used for the purification of proteins (biomolecules) for pharmaceutical research.
The column chromatography is used to isolate any type of sample mixture.
The column chromatography is used to analysis of environmental samples.

Commonly asked questions on column chromatography are as follows.

What are the types of column chromatography?
Based on the stationary phase there are four types of column chromatography are adsorption chromatography, ion-exchange chromatography, partition chromatography and, gel chromatography

What is the most common absorbent in column chromatography?
The silica gel (SiO2) and alumina (Al2O3) are the most widely used adsorbents in column chromatography. The calcium phosphate, calcium carbonate, starch, and magnesia, are also used as stationary phase in column chromatography.

What are the factors affecting column chromatography?
The dimension of the column, the particle size of the adsorbent, the temperature of the column, flow rate of solvent, nature of the solvent used, and pressure on the system, etc.

How do you pack columns for column chromatography?
Usually, two methods are used to prepare a column i.e. dry method and wet method.

Principle and applications of TLC

TLC is one of the economical, simple, and widely used chromatographic methods in qualitative and quantitative analysis, and is used for separation and analyzes the purity of organic compounds and test the purity of compounds. Thin-layer chromatography is a type of liquid chromatography in which a stationary phase (a thin plate coated with a silica gel or alumina) and a mobile phase (a mixture of solvents) are used to analyze the molecule on a flat surface, and this process works under the capillary action, ambient temperature, and atmospheric pressure.

The principle of thin-layer chromatography (TLC):

Thin-layer chromatography also relies on the separation principle, like other chromatographic methods. The separation depends on the relative affinity of the analytes against the stationary phase and mobile phase. The analytes in the solvent mixture move over the surface of the stationary phase. The movement occurs in such a way that analytes that have a high affinity towards the stationary phase move slowly while other analytes move quickly that have less affinity. Consequently, separation of the complex mixture is achieved. When the separation process is complete, the individual analytes from the sample mixture visualized as spots at particular levels on the TLC plate.

Various essential components involved in the TLC process.

TLC plates, mobile phase, TLC chamber, filter paper, and TLC pipettes, etc.

Major steps involved in TLC procedure:

Saturation: It consists of equilibration the TLC chamber with solvent vapors.

Spotting: It consists of transferring a small volume of sample to one end of the plate.

Development: It is a method of putting the bottom of the TLC plate in a solvent of development.

Visualization: It is a technique for viewing the distance traveled by molecules along with the stationary phase.

The various applications of TLC are as follows.

To check the sample purity: The purity of the compound can be determined by comparing it with the standard. When the impurity is observed, it shows additional spots so these can be easily identified.

Used in chemistry: Thin layer chromatography is widely used in chemistry to determine the retention factor (Rf value) by the separation and identification of molecules and also used in the determination of cations and anions in inorganic chemistry.

To identification of analytes: Another major application of TLC is it can use to separate, purification, and identification of natural substances such as glycosides, essential oils, alkaloids, pure oils, steroids, volatile oil, waxes, etc.

Used in food and cosmetic industries: The thin layer chromatography technique is used to isolate and identify the sweetening agents, colors, preservatives, foodstuffs, and some cosmetic products.

To separation of biochemical metabolites: To isolate and evaluate the biochemical constituent from blood plasma, urine, and body fluids the thin layer chromatography is useful.

Used in pharmaceuticals: Many pharmaceutical manufacturers use the TLC technique to identify impurities in a chemical substance as well as pharmaceutical formulations.

Used to determine the mobile phase: It is quick, easy, requires a minimum amount of solvent to operate, hence it is used to determine a suitable solvent system for column chromatography.

To analyze the reactions: TLC can be employed to analyze the reaction or rate of reaction and also used to check whether the reaction is completed or not.

Used in forensic science: TLC is useful in forensics to identify chemicals, including adulteration, poisoning, illegal drugs, and drug abuse.

Commonly asked questions on TLC chromatography are as follows.

What is the basic principle of TLC?

The TLC, paper chromatography, and high-performance thin-layer chromatography (HPTLC) are works on the same basic principle that is adsorption.

What are the three major applications of thin-layer chromatography?

The pharmaceutical, clinical, and food testing applications are 3 major application of TLC.

Why is TLC better than paper chromatography?

TLC provides better separations of analytes as compared to paper chromatography because as the stationary the TLC uses silica or alumina and paper chromatography uses cellulose paper.

What are the uses of chromatography?

Chromatography is the method used to isolate compounds or a sample mixture according to the relative affinity of each compound distributed amongst a stationary phase and a mobile phase. The compounds can be separated based on the different processes and the presence of characteristics, for example, hydrophobic groups present on the surface, size and shape, binding capacity with the stationary phase, and total charge. There are four main types of chromatography techniques are available such as partition chromatography, ion-exchange chromatography, adsorption chromatography, and size exclusion chromatography. Other chromatography methods are also available based on the stationary bed, such as high-performance liquid chromatography (HPLC), column chromatography, thin-layer chromatography (TLC), gas chromatography (GC), and paper chromatography, etc.
The chromatography has many applications in various fields which are mentioned below.

The chromatography is used in food processing for quality control.

Chromatography is used to check air quality and also the analysis of water samples.

It can use to determine the number of organic acids in foods that give significant information about food quality.

Chromatography is used by beverage manufacturers for its testing.

Chromatography is also used to determine the amount of drug present in blood samples, or urine.

It is used for the detection of contaminants (pesticides) or heavy metal in foods and food water.

It is mainly used in pharmaceuticals to verify the quality of the product or drug substance.

Chromatography is used in qualitative and quantitative analysis of molecules.

It is used to isolate solutions of dyes and pigments.

It is widely used in the forensic department to determine the alcohol or drug abuse.

Chromatography is also used in environmental applications, clinical diagnosis of diseases, petroleum industries, cosmetics, and herbal products, etc.

Classification of chromatography based on the mechanism of separation

Chromatography is one of the widely used methods of separation in which the analytes to be separated are distributed between the stationary phase and mobile phase which travel on the stationary phase in a certain direction. The analytes of a sample mixture redistribute themselves between two phases by a process that may be partition, adsorption, ion exchange, or size exclusion. The stationary phase is either a solid or liquid and the mobile phase is either a liquid, gas, or a supercritical solution. The molecules are separated at different retention times as per affinity towards the stationary phase.
Below is the classification of chromatography based on the mechanism of separation.

Adsorption Chromatography:

Adsorption chromatography is the widely used chromatography technique for separation of components, it is working on the principle of adsorption in which the separation relies on the interaction of adsorbate with the adsorbent. The separation generally depends on the difference amongst the adsorption affinities of the molecules to the surface of an active solid. The three major types of adsorption chromatography are column chromatography, thin-layer chromatography (TLC), and gas-solid chromatography.

Partition Chromatography:

In this type of chromatographic technique, the compounds of the sample mixture distribute more probability in the two liquid phases caused by the difference in the partition coefficient. This relies on the molecules' distribution coefficient (Kd) and retention factor (K) using the mobile phase or liquid for stationary phases. The types of partition chromatography are the bonded-phase liquid chromatography and liquid-liquid chromatography.

Ion-Exchange Chromatography:

Ion-exchange chromatography is a kind of chromatography that separates components on the basis of charge. A column is used in the separation process that filled with a charged stationary phase on solid support, called an ion-exchange resin. The two types of ion chromatography are anion-exchange and cation-exchange

Exclusion chromatography:

Exclusion chromatography is also known as gel filtration or gel permeation chromatography, in this type of chromatography the separation of the analytes mainly depends on exclusion effects, that is the differences in molecular shape or size or charge. The mobile phase moves through a porous gel that separates the molecules according to their size.

Affinity Chromatography:

Affinity chromatography is a process in which the absorption separation based on the particular relationship between the compounds and the desired component in the ligand. It utilizes a particular interaction between a type of solute and another molecule, which is stable in the stationary phase.

Commonly asked questions on chromatography are as follows.

What is chromatography and its types?

Chromatography is a widely used separation technique to obtain pure compounds from sample mixtures. The 5 major types of chromatography include high-performance liquid chromatography, thin-layer chromatography, size exclusion chromatography, gas chromatography, and affinity chromatography.

What are the factors affecting separation in chromatography?
The flow rate of the mobile phase, the composition of the mobile phase, solvents used in the mobile phase, length of the column, nature, and composition of the stationary phase, the temperature of the column and working condition, pressure on the system, and structure, polarity, and size of the component is the factors that affecting the separation in chromatography

What is the factor responsible for the separation in column chromatography?
The differences of polarity between the solutes are responsible for the separation in column chromatography.

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What is the principle of chromatography technique?

Chromatography is a technique applicable to a wide variety of separation methods based on a solute distribution or partitioning between a mobile phase system (liquid or gas) and stationary phase (solid or liquid). The relative interaction of a molecule with the stationary phase and mobile phase is illustrated by the partition coefficient (k) which is the ratio of the molecule concentration in the stationary phase, to the concentration of the molecule in the mobile phase. The field of chromatography may be subdivided as per the specific methods used or the physicochemical principles used in the process of separation. The chromatographic processes developed using various combinations of the mobile phases and stationary phases. As different as the nature of interactions amongst the analytes and stationary or mobile phases, these processes can separate different types of solutes. The sample of different compounds is separated through the techniques at dissimilar rates. Such a difference in migration rates provides a separation as the mixture passes on the adsorptive materials. When there is more adsorption in the stationary phase, the component will gradually move and when there is less adsorption in the stationary phase, the component will move rapidly. Consequently, the disparity between the described factors determines the differential rates at which different components can travel through a stationary phase. The adsorption and solubility of an analyte can be changes by choosing the appropriate mobile phase and stationary phase.

All the techniques of chromatographic separations work according to the same basic principle. Each analyte of sample mixture interacts with other stationary phases in an attribute manner; hence they separate at different retention times. Since each analyte has a different affinity to the stationary phase and mobile phase, the analytes that have more interaction they will stay longer in contact with the stationary phase and thus will slowly elute. The high-performance liquid chromatography (HPLC), column chromatography, thin-layer chromatography (TLC), paper chromatography, and gas chromatography (GC) are the widely used techniques in chemistry.

Commonly asked questions on chromatography are as follows.

What is the definition of chromatography?
It is known as the technique of separating the individual mixture components based on their relative affinity to stationary and mobile phases. There are several types of chromatography; each has some kinds of mobile phase and stationary phase although the basic principle is always identical.

What are the 4 types of chromatography?
The partition chromatography, ion-exchange chromatography, molecular exclusion chromatography, and affinity chromatography are the major types of chromatography based on the mechanism of separation.

What are the 4 applications of chromatography?
Pharmaceutical applications, environmental applications, forensic applications, clinical diagnosis of diseases and disorder, petrochemical, analytical chemistry, biochemistry, quantitative & qualitative analysis are the major applications of chromatography.

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