Chromatography Principle, Types, Applications

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Definition chromatography

Chromatography is a crucial biophysical process that permits separate, recognition and purification of a mix for an analysis that is both qualitative as well as quantitative. Chromatography was invented by the Russian botanist Mikhail Tswett coined the term Chromatography in the year 1906.

The first use for analytical purposes of chromatography was first described in 1952 by James as well as Martin in the year 1952, to describe gas chromatography as a method to study fat acid mixtures. The vast array of chromatographic methods make use of variations in binding affinities, size charges, size, and other characteristics to distinguish different substances. It is a highly effective separation instrument used in every field of science. It is usually the sole method of segregating elements of complex mix-ups.

Chromatography Overview

“Chromatography” is an analytical method that is used to separate the chemical mixture into its constituents in order that the distinct elements can then be fully examined. There are a variety of chromatography e.g. gas chromatography, liquid Ion Exchange Chromatography as well as affinity chromatography. However, all of them employ the same fundamental principles.


Chromatography is an separation method which every organic chemist as well as biochemist is aware of. Imyself, as an organic chemist, have regularly performed chromatographic separations of various mixtures of chemicals in the lab. In actuality I was looking through some of the research slide when I discovered a picture of a real separation chromatographic that I performed within the laboratory. It seems like that image could make a great start base for this post!

Let me first describe what I was trying accomplish here. I had two reactionants ‘A” and “B”. I allowed them to react with each with respect to certain conditions of reaction, to create a product called known as ‘C’. Once the reaction was complete I had the reaction mixture which contained unreacted A, unreacted C and my desired C. The next step was to isolate A C, B and A in order to identify and analyze pure C.

In the beginning, as illustrated in the left section, I used the thin layer analysis (TLC) plate. It is essentially an rectangular glass, which is which is coated in a fine layer silica. I placed a tiny amount of the reaction mix just above the surface of the dish (denoted by an elongated line) and then placed the plate into a jar that was filled with an organic solvent that was suitable (in this instance, a the 1:1 volume-by-volume mixture of hexane and ethyl acetate was utilized) and only enough volume to cover into the bottom of the plate. Then, by capillary force, the solution began ascending up the silica plate and as you can see, the reaction mixture was separated into three distinct spots. shades by the time it been able to reach the mark on the front of the solvent.

Chromatography Overview
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Then, to perform separation I put together the glass column (as as shown on the right side of the image). I made a glass-based column with an attached stopcock at the bottom, then inserted an elastomer plug at the base of the column and filled it with a mixture made of silica gel (prepared with the presence of an organic solvent). After the column was filled and the volume of solvent above the bed was reduced by less than five millimeters at a time, I carefully poured reaction mixture onto the silica-filled bed on the upper part of the column using the glass pipette. I shut the stopcock then let the liquid flow slow through the column. I continuously added solution from top to bottom of column. As you can see, the reaction mix began to split into 3 distinct band: orange, pink and yellow which correspond to unreacted B unreacted A, and C, which is the product I wanted in turn. I separated the individual bands into separate flasks and was capable of obtaining pure C!

Principle of Chromatography

The basis of Chromatography lies in the concept where the mixture of molecules is applied to the surface or into the solid the stationary phase (stable phase) is separated from one by moving using the mobile phase. The elements that are effective in the separation process are molecular properties related to the adsorption (liquid-solid) as well as partition (liquid-solid) and affinity, or the differences between the molecular masses of their constituents.

Principle of Chromatography

Due to these distinctions because of these differences, some elements of the mix remain more time in the stationary stage and move slower in the chromatography system. However, others move quickly to the moving phase and then leave the system more quickly. Three components form the foundation of the technique of chromatography.

  • The stationary phase is composed of the “solid” phase or “a layer of a liquid adsorbed on the surface solid support”.
  • Mobile phase It is comprised by “liquid” or a “gaseous component.”
  • Separated molecules

The kind of interactions between the stationary mobile phase, and the substances that are in the mix is the most important element in separating molecules from one another.

Principle of Chromatography (how does chromatography work)
Image Source: Khan Academy

Types of Chromatography

  • Substances can be classified by different techniques and particular characteristics like shape and size and total charge, as well as hydrophobic groups on the surface and binding capability with that of the stationary phase.
  • This results in different types of chromatography methods that each have their specific instrumentation and operating principles.
  • For example the four separation techniques built on molecular features and interaction types employ mechanisms for ion exchange the surface adsorption process, partition as well as size-exclusion.
  • Other chromatography techniques are built upon the bed stationary such as thin layer, column as well as paper chromatography.
  • Chromatographic techniques can be divided into two main categories: Partition Chromatography, Adsorption chromatography.

Adsorption chromatography

It is a form of chromatography that is the separation of the components in a mix is based on the different levels of the adsorption of these components on the stationary phase in the column for chromatography. The components of the mixture move at different rates because of differences in their non-covalent interactions with the stationary phase. Adsorption chromatography is a stationary solid phase (known as an adsorbent). The mobile phase can be fluid or gas. Adsorbents may be non-polar or polar molecules.

Adsorption chromatography
Adsorption chromatography

Partition chromatography

Partition chromatography
Partition chromatography

In this kind of chromatography, the constituents in the mix are separated in two phases because of variations in the distribution coefficient (Kd) that is the ratio of the concentration of solutes between two distinct phases. The distribution of solutes in both phases are dependent on the differences in solubility. In partition chromatography, there is a stationary while the mobile phase can be gas or liquid.

Partition chromatography

Liquid-liquid partition chromatography

In this form of chromatography that is partitioned the separation process is based on the partitioning of solutes between two liquid phases: stationary phase. Substances that are more easily soluble within the mobile phase will move swiftly through the system while those that are more soluble in more stationary phases will get removed.

It comes in two varieties:

  • Normal-phase partition chromatography: Elution chromatography where the stationary phase is much more far from that of the mobile. In elution, the most polar analyte, the least polar is eluted first, and the most polar is eluted last.
  • Reversed-phase partition chromatography: Reversed-phase partitioning chromatography (RPPC) is an elution method that uses the mobile phase which is more in polarity that the stationary. In this scenario, most non-polar substances elute first while the those that are least polar will elute last.

Size exclusion chromatography

They’re also referred to as molecular sieve Chromatography. It is the process of separating molecules based on dimensions and shapes. In this case, the column matrix is packed with highly porous, granular gel comprised of an insoluble and hydrated polymer , such as dextran, polyacrylamide or agarose that acts as a stationary phase. Gel permeation chromatography usually employs organic mobile solvents, whereas gel filtration chromatography employs the mobile solvent of aqueous to separate and analyze molecules.

Size exclusion chromatography
Size exclusion chromatography

Size exclusion chromatography can be described as two kinds:

  • Gel permeation chromatography.
  • Gel Filtration analysis

Affinity chromatography

Affinity chromatography involves purifying a biomolecule according to its their biological function or structure. The material to be removed is specifically and reversibly adsorbs to a chemical ligand that is then bound by a covalent bond to a bed material for chromatographic.

Affinity chromatography
Affinity chromatography

The process of affinity chromatography consists of these steps:

  1. Selection of the appropriate ligand.
  2. Incorporation of the ligand to an underlying matrix.
  3. The binding of molecules that are of interest to the ligand.
  4. Removal of molecules that are not bound specifically.
  5. Elimination of the molecules of interest in a purified version.

Choices of ligand

  • Specificity
  • Reversibility
  • Stability
  • Size
  • Affinity

High performance liquid chromatography

HPLC is a form of column chromatography. It is a technique for liquid chromatography i.e. the liquid phase is mobile. In this method, stationary phase could be liquid or solid. This method of chromatography could be used in a variety of types of chromatographic processes: size exclusion. Adsorption, ion exchange and partition. Instead of being let to drip through column due to gravity and then being forced through with high pressure. This results in speed and performance that is superior in comparison to conventional column chromatography techniques because the the mobile phase gets pumped by high pressure.

High performance liquid chromatography
High performance liquid chromatography

Ion exchange chromatography

It is used to separate charged molecules. In this form of chromatography, ionic substances have reversible electrostatic interaction with an charged stationary phase. A stationary solid phase typically is composed of covalently attached anions or cations, which are insoluble matrix known as Ion Exchangers.

Ion exchangers

It is composed of two components: an insoluble matrix as well as charged chemically bonded groups and on top part of it.

  • Cation exchanger: Cation exchanger, also known as acidic ion exchanger is used to separate of Cation.
  • Anion exchanger: Cation exchanger, also known as acidic ion exchanger is used to separate of Cation.
Ion exchange chromatography
Ion exchange chromatography

Gas chromatography

In this kind of chromatography, a gas serves as the mobile phase. the stationary phase could be an adsorbent solid that is known as gas-solid or a liquid placed on an inert substrate, which is referred to by the term gas liquid chromatography. This method is typically employed to study volatile substances that are in gas phases. Gas chromatography is the only method that doesn’t use the mobile phase to interact in conjunction with an analyte.

Gas chromatography
Gas chromatography

Commonly employed chromatography techniques include

TechniqueStationary phaseMobile phaseThe basis of separationNotes
*Paper Chromatographysolid (cellulose)liquidPolarity of moleculescompound directly on cellulose paper
*Thin Layer Chromatography (TLC)Solid (silica or Alumina)liquidPolarity of moleculesglass is coated with a thin layer of silica. On it is seen the chemical
*Liquid column chromatographySolid (silica or Alumina)liquidPolarity of moleculesGlass column is filled with silica-slurry
Size exclusion chromatographySolid (microporous pieces of silica)liquidthe size of moleculessmall molecules get stuck in tiny pores within the stationary phase while larger molecules move through the spaces between the beads and have very short retention time. Thus, larger molecules emerge first. In this form of chromatography there isn’t any interplay whether chemical or physical, between the analyte and stationary phase.
Ion-exchange chromatography(cationic or anionic resin) (cationic or anionic resin)liquidThe charge of the ions in moleculesmolecules with the same charge to resins will be able to bind strongly to the resin. molecules with similar charges to the resin will move through the column, and then elute out first.
Affinity ChromatographySolid (agarose as well as porous glass beads which immobilized molecules are like antibodies and enzymes)liquidthe binding affinity of the chemical analyte to the molecule that is immobilized on the stationary phaseIf the molecule acts as an enzyme’s substrate that it binds to the enzyme, and the non-bound analytes will move through the mobile phase and then elute out of the column leaving the substrate attached to the enzyme. It is then removed of the stationary part and then eluted from the column using the appropriate solvent.
Gas chromatographySolid or liquid supportgas (inert gas such as Helium or argon)the boiling point of moleculesThe samples are dissolved and the sample with the lowest boiling point is taken out first. The molecule that has the highest boiling point exits the column last.

Applications of Chromatography

1. Pharmaceutical sector

  • To determine and analyze samples to determine the presence of trace elements or chemical compounds.
  • Separation of substances based on their molecular weight as well as element composition.
  • It detects compounds that are not known and the the purity of mixture.
  • In drug development.

2. Chemical industry

  • Testing water samples is a way to test water quality and also to check the air quality.
  • HPLC along with GC are extensively employed to detect diverse contaminants, such as PCBs, also known as polychlorinated Biphenyl (PCBs) found in pesticides as well as oils.
  • In numerous life science applications

3. Food Industry

  • In food spoilage as well as detection of additives
  • The determination of the nutritional value of food items

4. Forensic Science

  • Forensic pathology is a part of crime scene analysis, such as analysing hair and blood samples from crime scenes.

5. Molecular Biology Studies

  • Numerous hyphenated techniques used in the field of chromatography like EC-LCMS are used in the research of proteomics and metabolomics, as well as research on nucleic acids.
  • HPLC is utilized in Protein Separation such as Insulin Purification, Plasma Fractionation as well as Enzyme Purification and in many departments, such as Fuel Industry, biotechnology, and biochemical processes.
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