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Separation by Chromatography

  • Last Updated : 14 Jul, 2021

Chromatography is a technique used to simulate the separation of two or more dissolved solids contained within a solution in very small quantities. It is a physical process in which the solutes, that is the components of a sample mixture are segregated as a result of their differential distribution between the stationary and mobile phases. In Greek, the word ‘chroma’ means colour and ‘graphein’ is used to indicate writing. Initially, the technique was used for the separation of colours. 

The substance that has to be segregated during the process of chromatography is known as an analyte. It basically refers to the component needed from the mixture. 

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Principle behind chromatography

The separation technique of chromatography is dependent on the principle of partition of the constituent components between two phases – Mobile Phase and a Stationary Phase. The mobile phase is used to indicate the mixture of the substances to be separated dissolved in a liquid or a gas. The stationary phase is used to describe a porous solid matrix through which the sample present in the mobile phase percolates.

The method of chromatography is based on the fact that the dissolved substances in the same solvent may have differing solubility. The solute with a greater solubility rises faster and segregates from the mixture. 

Types of chromatography

The chromatography can be classified into different types based on the following categories based on the physical state of mobile and stationary phases:

  • Liquid chromatography:
    The principle behind liquid chromatography is the varying distribution of the solutes in a liquid mobile phase as well as a stationary phase. HPLC is an extensive chromatography technique, which is used to refer to a term when the particles of small diameter are used as stationary phase.
  • Gas chromatography:
    • In the technique of gas chromatography, the gas mobile phase is taken in order to transfer a solution containing volatile solutes through a column that contains the stationary phase. The gas mobile phase is termed as the carrier gas. In the majority of the cases, the gas chosen for the gas mobile phase is an inert gas, for instance, nitrogen or helium.
    • In gas chromatography, the separation of the solute is dependent on the relative differences in the solutes vapour pressures and their corresponding interactions with the stationary phase in the procedure. As a result of this process application, the solute with greater volatile property is eluted from the column.
    • It is visibly noticeable that the solutes are categorized based on the time of retention.
    • This process illustrates the following observations : 
      • The solutes are separated using the column effluent in order of their elution.
      • The amount of solute detected is directly proportional to the peak size. 


  • Paper chromatography to separate colours of a dye
    A thin strip of filter paper is taken and a line using a pencil is drawn at a noticeably visible length from the lower corner of the paper.
    A drop of ink using a fountain pen is projected at the centre of the drawn line. It is dried further.
    The filter paper is dipped in a beaker containing water as the solvent, in such a way that the ink drop is right above the water level. 
    When the water rises up on the filter strip, it takes along with it the dye particles. 
    Since the dye is soluble in water, the component in the dye with a greater solubility rises higher. As a result of this, the components of the dye get separated out.

  • Food department
    • Chromatography can be used to estimate the shelf life of food items by providing an insight into when the food item gets spoiled.
    • It can be used to understand the nutritional value of the food sample.
    • It can be also used to analyse the presence of chemical additives in food.
    • Can be used for the clarification and skimming of milk.
    • Extraction of cream and production of cheese.
  • Pigments from natural colours
  • Chemical domain
    • The purity detection of water samples.
    • The purity detection of air samples.
    • Detection of the presence of toxic contaminants in pesticides and oils. The most commonly used technique is GC and HPLC.
    • Check DDT in groundwater.
    • PCBs in waste oils.
  • Pharmaceutical industry
    • Chromatography can be used to detect the presence of chemicals and elements in a given sample, present in traces. These chemical compounds can also be segregated and extracted on the basis of their molecular masses and constituent elements.
    • Separation of drugs from the bloodstream- The removal of impurities and unknown compounds from the drugs as well as the estimation of drug sample purity can be analysed using the chromatography technique.
  • Molecular biology
    • Nucleic acid research can be simulated by the chromatography technique.
    • EC-LC-MS, an extensive chromatography technique can be used to perform the study of proteomics and metabolomics.
    • HPLC chromatography technique can be used to carry out protein separation operations.
    • Several procedures like plasma fractionation and enzyme purification are based on chromatography.
    • DNA fingerprinting and informatics.

Sample Questions

Question 1: Describe the nature of solvents used in chromatography. 


The used solvents should have the following characteristics: 

Solvents with low viscosity since the viscosity of a solvent is inversely proportional to the rate of solvent flow.

Question 2: Elaborate the criteria used for solvents used for paper chromatography technique?


The following criteria is used for selection of solvents for paper chromatography : 

  • Non-toxic or carcinogenic solvents
  • The solvent should not react with any of the sample constituent particles.
  • Low volatility
  • Solubility of sample components.
  • The chosen solvent should not hinder the detection of separated spots.

Question 3: Explain developing technique in chromatography?


In case the substances to be segregated are colourless, then during the process of chromatography, the components may not be visible on the chromatogram. Therefore, developing technique is the process of spraying a suitable reagent on the chromatogram such that the different components become visible on the chromatogram. The reagent is known as the developing reagent. 

Question 4: Elaborate the shortcomings of paper chromatography.


Paper chromatography has some limitations such as:

  • Greater components’ concentration. This may lead to streaking.
  • It is semi-quantitative in nature.
  • Irregular sample spotting
  • Overlap in the spots with close Rf value components.
  • Uneven flow of solvent may lead to error in the calculations of Rf values.

Question 5: Explain any one detector used in HPLC. 


A detector named UV-VIS detector, is used in HPLC, since it has a property of giving a specific response to a particular compound. Detector has variable wavelength range which is used for absorption by many organic compounds.

Question 6: Define analytical chromatography 


Analytics chromatography is used to determine the existence and the concentration of the substance to be separated during the process, that is analyte. 

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