Chromatography: Working Principle and Applications

Chromatography is an analytical technique used for the separation, quantification, purification, and identification of components of the mixture. This separation takes place between two phases i.e, the stationary phase and the mobile phase. The word chromatography is derived from two words chroma and graphy. Chroma means color and graphy means writing.

Chromatography was named and invented by Russian botanist Mikhail Tswett in the 19th Century. He was separating plant pigments i.e, chlorophyll, and xanthophylls, by using a glass column packed with finely divided calcium carbonate. These pigments are separated as color bands on the glass column. Because of these color bands, this method was named chromatography or color writing.

Initially, chromatography was used by artists for color writings. It was also used for dying textile materials.

Definitions of chromatography

The term chromatography is very difficult to define properly because it is a combination of systems and techniques. Chromatography is differently defined by scientists in different eras.

“An analytical technique used for resolution of solute by differential migration through porous medium caused by the flow of solvent” Strain

“Chromatography is the separation of components of the mixture in the concentration zones irrespective of the nature of force and phases where they were present originally”

William and Weil

“It is a separation of mixtures between two phases i.e thin phase and bulk phase. These phases are contacted through differential counter current manners” Classid

“Separation of substances by filtering them through glass column packed with suitable adsorbent and then washing the column with solvents” Tswett

Principle

The basic principle of all types of chromatography is

“Relative affinities of components of the mixture toward mobile phase and stationary phase”

In general, it is the distribution of analytes between the mobile phase and stationary phase or the interaction of the sample with the mobile phase and stationary phase. Greater the interaction of the analyte with the mobile phase faster would be separation and greater the interaction of the analyte with the stationary phase slower would be separation.

All chromatographic separations are based on the difference in the extent to which solutes are partitioned between the mobile phase and the stationary phase. The equilibria involved can be described quantitatively with the temperature dependent constant

Where K= partition coefficient

Cs =solute concentration in stationary phase

CM =solute concentration in mobile phase

K depends upon the following factors

  • Temperature
  • Type of compounds
  • Stationary phase
  • Mobile phase

K with larger has strong interaction between solute and stationary phase and vice versa.

Types of chromatography

Chromatography can be classified into different types on the basis of:

  1. Stationary phase
  2. Mobile phase
  3. Force / Method of separation

On the basis of the mobile phase

  • Liquid chromatography (solution chromatography)
  • Gas chromatography

On the basis of the stationary phase

  • Solid chromatography
  • Liquid chromatography
  • Gas chromatography

On the basis of the method of separation

  • Adsorption chromatography
  • Partition chromatography
  • Exclusion chromatography
  • Ion exchange chromatography

Name of different types of chromatography

  • Thin layer chromatography (TLC)
  • Paper chromatography
  • Column chromatography (CC)
  • Adsorption chromatography
  • Partition chromatography
  • Ion exchange chromatography (IEC)
  • Affinity chromatography
  • Gel filtration
  • Gas chromatography (GC)
  • Electrochromatography
  • High Performance Liquid Chromatography (HPLC)

Types of chromatography

Working

Although, all types of chromatography are based on the same working principle which is relative affinities of an analyte with mobile phase and stationary phase. But the method of separation is different in different types of chromatography.

Working principle of all types of chromatography
Type of chromatography Working principle
Paper chromatographyPartition on a sheet of paper. (filter paper)
Adsorption column chromatographyDistribution of solute between solid and liquid phase on a column
Thin layer chromatographyAdsorption or partition on a thin layer of sheets/plates.
Molecular sieve chromatographySeparation on the basis of the size of solutes.
Ion exchange chromatographyexchange of ions.
Gel permeation chromatographySeparation on the basis of the size of solute.
Gas chromatographyDistribution of solutes between gas and liquid or solid phase.
Partition column chromatographyDistribution of analyte between two liquid phases.

(i) Partitioned chromatography

In partitioned chromatography, an analyte is differently distributed between the mobile phase and stationary phase. In this type, stationary phases may be liquid or solid. Partitioned chromatography can be carried out as

  • Paper chromatography
  • Column chromatography
  • Gas chromatography
  • Thin layer chromatography

(ii) Adsorption Chromatography

In adsorption chromatography, a suitable adsorbent is used. The adsorbent acts as a stationary phase. The working principle is the differential adsorption by the adsorbent. In the adsorption chromatography, the analyte is adsorbed on the stationary phase. It bonds directly to the stationary phase. The adsorbent may also produce a partitioned effect by retaining some of the analytes. In this type, the stationary phase is always solid.

(iii) Exclusion chromatography

In exclusion chromatography, sample components are separated according to their molecular size. It can be carried out as

  • Gel permeation
  • Sieving separations

In gel permeation, there is a three dimensional network of cross linked polymer chains. In the presence of a suitable solvent, gel undergoes swelling and the space between the polymer chains is increased. Molecules of smaller size are retarded whereas large molecules pass unhinderedly.

In sieving separations, natural and synthetic zeolites are used as molecular sieves. They have cavities and channels of different sizes in their structures. The compounds of smaller size fall into the cavities while passing whereas larger particles pass unhindered.

(iv) Ion exchange chromatography

Ion exchange chromatography is the form of solid liquid chromatography. Bead shaped ion-exchange resins act as a stationary phase. These resins are highly polymerized, cross-linked, organic materials containing a large number of acidic and basic functional groups. These groups are known as active groups. These groups are of hydrophilic nature. Cation exchangers contain cation whereas, anion exchangers contain anions.

Ion exchange chromatography is mostly performed on columns with adsorbents and cationic or anionic exchangers. The columns are designed in such a way that there should be no hindrance in the flow of liquid. This form of chromatography relies on the attraction between the oppositely charged ion exchanger, and the mobile phase containing the analyte.

Quantitative analysis

Results are calculated in different ways. Because chromatography may be instrumental or noninstrumental.

Instrumental chromatography
In the case of instrumental chromatography, different types of detectors i.e

  • UV detectors
  • Refractive index
  • Conductometric
  • Mass Spectrometric
  • Fluorescence
  • Thermal conductivity detectors
  • Atomic emission detectors
  • Flame ionization detectors
  • Differential viscosity detectors,

are used. Some of these converts the obtained signals into the area and provide us with a chromatogram. Similarly different other techniques are used for result calculations i.e

  • Spectrophotometry
  • Polarography
  • Conductometry
  • Radiochemical methods

Non Instrumental chromatography

In the case of instrumental substances colored substances are easily detected. But the colorless substances are detected through physical and chemical methods i.e.

  • Organic compounds are detected by spraying a solution of iodine or sulfuric acid.
  • A UV lamp is used for the detection of fluorescent substances.
  • Ninhydrin is selective for the detection of amino acids.
  • Potassium chromate is used for the detection of inorganic ions i.e Pb, Hg, etc.
  • Rubeanic acid is used for the detection of ions of Co, Fe, Ni, Cu, etc.
  • Protein can be located by the staining process.

Advantages of chromatography

Advantages of different types of chromatography are given as:

  • It is a very precise technique of analysis
  • It requires less amount of samples for analysis
  • Similarly, chromatography can be used for the separation, quantification, and identification of analytes.
  • Moreover, it can be used for the purification of hydrocarbons, antibiotics, and plant extracts.
  • Chromatography can separate chemicals having almost the same properties.
  • Several compounds can be analyzed in a single run.

limitations of chromatography

Limitations of different types of chromatography are given below:

  • It is a time-consuming technique. (HPLC)
  • Separation of complex mixtures is difficult
  • It can not differentiate between the enantiomers
  • Volatile compounds are difficult to analyze by using chromatography

Applications

Chromatography is the most widely used technique for various types of analysis. It has many applications in different industries. A few of these are given below:

In pharmaceutical industry

  • Chromatography is used for the purification, separation, and identification of drug materials.
  • It is used to study the stabilities of different drug products.
  • It is used for the quantification of API in pharmaceutical products.
  • Moreover, it can be used in research and developmental processes in the pharmaceutical industry.
  • Chromatography can also be used to determine impurities in the products.
  • It is used to study the formulation of different medicines.

Food and beverages

  • Chromatography is used to determine the nutritional quality of food.
  • It is used for the detection of additives, pesticides, and other contamination in food.
  • It is used for the analysis of proteins, vitamins, and other preservatives in food.
  • Moreover, it is used for the determination of organic acid in food which helps in spoilage detection.

Hospitals

  • It is used for the detection of drugs in body fluids.
  • Chromatography is used for the identification of species.
  • HPLC is used to analyze vitamins, proteins, and amino acids in body fluids.
  • Similarly, it can be used to find out glucose levels in the bloodstream.
  • HPLC is also used to find catecholamine in urine.

Chemical analysis

  • It is used to purify materials and analyze chemical compounds for trace contaminants
  • Chromatography is used to separate chiral compounds

Environmental analysis

  • Chromatography is used to monitor air quality.
  • It is used to test the quality of drinking water.
  • Similarly, it is used to find pollutants in soil.

Concepts Berg

Define chromatography?

Chromatography is an analytical technique used for the separation, quantification, purification, and identification of a mixture of compounds.

What is meant by the term elution?

The term elution means “wash out”. In chromatography, Elution is the process through which an analyte is passed through the stationary phase with the help of the mobile phase.

What is meant by retention time?

The retention time is the time taken by the solute (analyte) to pass through a column (stationary phase). It depends upon relative affinities of solute with mobile phase and stationary phase. It is denoted by “RT”

What is the Rf value in chromatography?

Rf is known as the retardation/retention factor. It is the ratio of the distance moved by the solute from the base to the distance moved by the solvent from the baseline.

How we can reduce the elution time in chromatography?

We can reduce elution time in different ways:

  • By increasing temperature.
  • By increasing flow rate.
  • Similarly by changing the concentration of the mobile phase.
  • By using smaller columns.

Reference Books:

Vogel’s Textbook of Quantitative Chemical Analysis sixth edition by J Mendham, RC Denney, JD Barnes, M Thomas.

First edition of Analytical Chemistry Instrumental Techniques by Mahinder Singh

Reference links:

Chromatography Overview(news medical.net)

Chromatography Definition Types and Facts(britannica.com)

 

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