Chromatography: Principles, Types, Applications


Chromatography is a technique for separating a mixture of components into their constituents by distributing them in equilibrium between two phases, the stationary phase, and the mobile phase.

The basis for the chromatography method is the variation in the rate at which various mixture components pass through the stationary phase when a suitable mobile phase is present.

M. S. Tswett discovered chromatography at the beginning of the 20th century. Tswett provided an in-depth explanation of the separation of complicated mixtures based on adsorption in 1903. Later he referred to it as “chromatography”. This name, which means “color writing” in Greek, refers to the method of separating plant pigments by showing a colorful illustration of the process.

It is a process for separating the constituents, or solutes, of a mixture based on the relative amounts of each solute distributed between a moving fluid stream, known as the mobile phase, and a contiguous stationary phase.

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What is chromatography?

Chromatography is a biophysical technique that separates, identifies, and purifies a mixture to analyze its constituents. It relies on two distinct phases, the mobile and stationary phases, to accomplish this.

The stationary phase is either a solid phase or a liquid phase on the surface of a solid phase. A gaseous or liquid phase is the mobile phase. Along with the mobile phase, stationary phase molecules also move. When the mobile phase is liquid, the technique is known as liquid chromatography (LC), and when a gas is utilized as the mobile phase, the technique is known as gas chromatography (GC).

Chromatographic methods are most beneficial for separating mixture components, purifying chemicals, and separating natural products. They are the most effective, quick, and adaptable instrumental approach for the chemical analysis of complicated mixtures.

Principe of chromatography

According to the underlying principle of chromatography, molecules in a mixture applied to a surface or into a solid and a fluid stationary phase (stable phase) separate from one another while moving with the help of a mobile phase. It works by measuring how quickly various components of a mixture move through a stationary phase when a solvent is present. The mixture’s various components travel at varying speeds in the stationary phase, which causes them to separate into their pure states.

The molecular features related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights are the factors that have an impact on this separation process.

Due to these variations, some mixture components flow quickly into the mobile phase and exit the chromatographic system more quickly, while others pass slowly into the stationary phase and travel slowly through the system.

Types of chromatography

Based on the mobile phase, stationary phase, forces of separation, or mechanism of separation, chromatographic techniques can be classified into different types. There are several types of chromatographic techniques, some of them are:

Chromatographic techniques

Paper chromatography

Paper is employed as the stationary phase in this form of chromatographic technique. The solvent flows through a piece of cellulose-based specialized paper. The flow of the solvent carries the chemical along with it, separating the compound. The rate of migration over the paper determines how quickly the chemical components separate. The material is divided or disseminated among the liquid phases. Water is the stationary phase that resides in the filter paper’s pores, and the other is the solvent that flows or runs over the paper. The capillary action of the paper’s pores causes the solvent to migrate. Due to variations in the compounds’ affinities for water, the compounds present in the sample segregate.

Column chromatography

It is a method where the substances to be separated are added to the top of a column packed with an adsorbent, passed through the column at various rates depending on the affinities of the substances for the adsorbent and the solvent or mixture, and typically collected in solution as they exit the column at various times.

It is a solid—liquid technology with a solid stationary phase and a liquid or gas mobile phase.

Adsorption chromatography

The stationary phase in this chromatographic technique is solid. It’s called liquid-solid chromatography. An appropriate adsorbent is utilized as a stationary phase in this method. Alumina, Silica gel, activated charcoal, starch, etc. are some examples of different adsorbents. These adsorbents provide a partitioning effect by retaining some of the samples in the mobile phase.

It functions similarly to partition chromatography. The principle is based on the adsorbent’s differential absorption and is commonly used in column chromatography.

Affinity chromatography

It is a liquid chromatography technique that employs biospecific interactions to separate compounds. The molecule that needs to be purified is selectively and irreversibly adsorbed to a certain ligand.

Proteins’ biological functions frequently entail binding to or interacting with certain ligands, substrates, co-factors, inhibitors, other proteins, and so on. These unique interactions between proteins and ligands are exploited in this technique. A protein combination with a suitable moiety covalently bonded to a solid support is run through the column. Only the protein that recognizes the ligand of interest will bind to the column, while the other proteins are washed away.It can be exceedingly selective, allowing for protein isolation in a single step.

Gel filtration chromatography

Gel filtration, also known as size exclusion chromatography, is a technique for separating proteins based on their molecular size. A protein solution is run through a column of tiny cross-linked polymer beads. The degree of cross-linking determines pore size. Solutes with pore sizes bigger than this are omitted from the matrix and flow freely through the column.

Smaller solutes will enter the gel matrix and remain on the column for a longer period of time. The retention duration of a solute is inversely proportionate to its size.

Ion exchange chromatography

Ion exchange chromatography, also known as affinity chromatography, is based on the ionic attachment of molecules (such as proteins) to an inert matrix material. The column is packed with resin/inert material, and the solution is allowed to percolate through it. Ion exchangers are made up of charged groups that are covalently bonded to the surface of an insoluble matrix. Attraction occurs between the sample and the oppositely charged stationary phase (ion exchanger).

The primary basis of separation is the interchange of ions in the sample solution with those on ion exchangers. Ions and polar compounds are separated according to their affinity for ion exchangers.

Partition chromatography

Partition chromatography is frequently referred to as liquid-liquid chromatography. The stationary phase in this chromatographic technique is liquid. In most cases, the liquid stationary phase is a water molecule found in inert porous materials such as cellulose. The mobile phase might be a mixture of liquid solvents based on the requirements.

Gas chromatography

Gas chromatography (GC) is a separation technique in which volatile, thermally stable solutes migrate at rates specified by their distribution ratios across a column containing a stationary phase. GC allows separation and quantitative analysis for volatile, thermally stable compounds in a wide range of combinations, from the simplest (such as purity tests of individual compounds) to the most complex (such as petrochemical assays of samples consisting of hundreds of distinct components).

High-performance liquid chromatography

The high-performance liquid chromatography (HPLC)  technique involves the separation of analytes dissolved in the mobile phase using a particular interaction with a stationary phase.

In general, it is a greatly enhanced form of column chromatography. A solvent is forced through a column at high pressures rather than being permitted to flow through it under gravity. As a result, it is considerably quicker.


The migration of individual or colloidal particles through a solution under the influence of an electrical field is known as electrochromatography. It combines size exclusion chromatography with gel electrophoresis. Charged particles travel towards oppositely charged electrodes when a potential difference is applied between two electrodes.

It can be done with or without a supporting medium. Strips of paper serve as the supporting medium. These are simple to manipulate and produce excellent effects. Electrochromatography can be performed using graphite rods, stainless steel, silver chloride, and platinum electrodes. A rectifier with a voltage range of 100-300 volts serves as the direct current source. The nature of the sample affects the voltage used and the amount of time required for fractionation.

Supercritical fluid chromatography

Supercritical fluid chromatography (SFC) utilizes a mobile phase consisting of fluid in the supercritical state. As a result of this phenomenon, there are enhancements observed in the isolation of thermolabile compounds, as well as in the separation of compounds with high molecular weight, among other applications.

Applications of chromatography

  • It can be used to identify which antibodies are most effective at fighting and neutralizing specific diseases.
  • It is used in the food business to separate and analyze additives, vitamins, proteins, amino acids, and other nutritious components. It can also be used to establish expiration dates by distinguishing the number of organic acids present, as well as to detect any dangerous contaminants that may have been added to the food item.
  • We can detect Perfluoroalkyl compounds in the environment and our drinking water using solid-phase extraction, LC, and mass spectrometry, even at extremely low levels.
  • It is an essential part of practically every protein purification approach. Protein purification and analysis are carried out using a variety of chromatographic techniques.
  • It is primarily employed in bioanalytical chemistry for the separation, isolation, and purification of proteins from complex sample matrices.
  • HPLC and GC are widely used for detecting pollutants in pesticides and oils, such as polychlorinated biphenyls (PCBs).
  • Various hyphenated techniques, such as EC-LC-MS, are used in the study of metabolomics and proteomics, as well as nucleic acid research.
  • HPLC is utilized in protein separation procedures such as insulin purification, plasma fractionation, and enzyme purification, as well as in other industries such as the fuel industry, biotechnology, and biochemical processes.
  • It is used in forensic pathology and crime scene investigation, such as analyzing blood and hair samples from crime scenes.
  • It analyses a urine sample to identify the substances that have been consumed, determining whether any dangerous or illegal drugs have been used, whether conducting a clinical toxicology report, drug testing a new hire, or testing a professional athlete for performance-enhancing drugs.
  • GC is used to analyze finished gas products and refining operations. It is most commonly used to determine BTU amount and hydrocarbon composition in natural and industrial gas.


  • E. Heftmann 2004. Chromatography, Sixth Edition: Fundamentals and applications of chromatography and related differential migration methods – Part B: Applications

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Kabita Sharma

Kabita Sharma, a Central Department of Chemistry graduate, is a young enthusiast interested in exploring nature's intricate chemistry. Her focus areas include organic chemistry, drug design, chemical biology, computational chemistry, and natural products. Her goal is to improve the comprehension of chemistry among a diverse audience through writing.

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