Chromatography: Working Principles and Applications

Chromatography is a precise analytical technique, used to separate components of a mixture on the basis of relative affinity. This separation takes place between two phases i.e, the stationary phase and the mobile phase. In brief, quantification, purification, and identification of components of the mixture can be done using various chromatographic techniques.

The word chromatography is derived from two words chroma and graphy. Chroma means color and graphy means writing. It was 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.

Background

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

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” Classic

“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 chromatography is given below:

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

In general, it is the Distribution of analyte 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 

K=Cs /Cm

Where

  • K= partitioned coefficient 
  • Cs =solute concentration in stationary phase
  • Cm =solute concentration in mobile phase
  • K is Partition coefficient

K depends upon the following factors 

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

Note that “K” with larger values 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 the following categories

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

 

On the basis of the mobile phase

  • Liquid chromatography (solution chromatography) 
  • Gass 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 (PC)
  • Column chromatography (CC) 
  • Adsorption chromatography
  • Partition chromatography
  • Ion exchange chromatography (IEC)
  • Affinity chromatography
  • Gel filtration
  • Gas chromatography  (GC)
  • Electrochromatography
  • High Performance Liquid Chromatography (HPLC)

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 

(i) Partitioned chromatography

In partitioned chromatography, an analyte is differently distributed between the mobile phase and stationary phase. In partition chromatography, 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 adsorption chromatography, the stationary phase is always solid.

(iii) Exclusion chromatography 

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

  • Gel permeation 
  • Sieving separations 

In gel permeation, there is 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 unhinderedly.   

(iv) Ion exchange chromatography 

Ion exchange chromatography is the form of solid liquid chromatography. Bead shaped ion-exchange resins act as stationary phase. These resins are highly polymerized, crossed 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 

etc are used. Some of these convert 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 

Noninstrumental chromatography 

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

Advantages of chromatography 

limitations of chromatography

Applications 

The important applications of chromatography in various industries are listed below:

  • To monitor air quality and to test drinking water
  • In the detection of drugs in urine and other body fluids
  • Used in chemical fingerprinting and species identification
  • In the pharmaceutical industry:
    • to purify materials and analyze chemical compounds for trace contaminants
    • to separate chiral compounds
  • For quality control in the food industry:
    • separation and analysis of additives, preservatives, vitamins, and proteins
    • to detect toxins and contaminants in food

Key takeaways

 

Concepts Berg

Define chromatography?

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

What are the different uses of chromatography?

What is meant by the term elution?

The term elution means “wash out”. In chromatography, Elution is the process through which analyte is passed through stationary phase by the help of 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 statioanryu phase. It is denoted by “RT”

What is 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 base line.

Rf=distance moved by a compound form base line distance moved by a solvent form base line

 

 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:

Was this article helpful?