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Chromatography

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Last updated date: 19th Apr 2024
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An Introduction to Chromatography

We all know that a mixture is a combination of different elements or compounds. These elements are mixed uniformly and cannot be separated normally. Then how can we separate the elements of a mixture? Chromatography is a physical method of separation of different components of a mixture.


In this article, we are going to understand what is chromatography, types of chromatography, how can chromatography be used to separate mixtures, and how two dimensional chromatography is used for separating mixtures.


What is Chromatography?

Chromatography is a physical method of separation of different components of a mixture. In chromatography, the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction.


Types of Chromatography

On the basis of phases of separation of elements of a mixture, chromatography is divided into the following categories:

  1. Column Chromatography - In column chromatography, mobile phase flows through the packed column.

  2. Thin Layer Chromatography - In thin layer chromatography, the mobile part moves by capillary action.

  3. Partition Chromatography - In partition chromatography, the stationary part is thin film of liquid adsorbable on an essentially inert support.

  4. Paper Chromatography - Paper chromatography is an example of partition chromatography within which liquid present within the pores of paper is stationary phase and a few other liquid is movable part.

  5. Absorption Chromatography - In adsorption chromatography, the stationary part could be a finely divided solid adsorbent and the mobile part is typically a liquid.


How Can Chromatography Be Used to Separate Mixtures?

The most widely used methodology of chromatography is paper chromatography. Chromatography separates components in a mixture according to their solubility within the mobile part and their absorbance to the stationary part. The mobile phase is the part of the chromatography set up that moves; the stationary part is the part of the set up that does not move.

Components that are more soluble within the mobile phase, i.e. adsorb less well to the stationary part, will move through the chromatography set up quicker as a result of there are fewer interactions with the stationary section to slow the component down. These components are going to be observed initially at the end point. Components that adsorb more powerfully to the stationary part have more interactions with the stationary part to block the movement of the component. These parts will be observed last at the end point.


Two Dimensional Chromatography

Two dimensional chromatography is used for separating mixtures. The combination of various separation techniques into one experiment (multi-dimensional chromatography; additionally known as 2D chromatography, orthogonal chromatography and cross-fractionation) permits for tackling the core of restricted chromatographic resolution by vastly improving the peak capability. This is specifically vital for the analysis of advanced polymeric materials.


In 2-dimensional chromatography, the sample is fractionated in a first dimension by one structural parameter and the fractions are collected. These fractions are then afterwards separated in a second chromatographic experiment separating according to a second structural feature.


2D Paper Chromatography


2D Paper Chromatography


Two dimensional chromatography could be an advanced setup that is employed to separate complex mixtures.

  • The solvent is placed at the bottom of the tank and also the filter paper saturated with the stationary part is then kept within the tank. The development occurs upwards but very slowly because it's against gravity, additionally, as the compound could be a complex one.

  • After a few hours, the filter paper is turned 90 degree clockwise and also the tank is filled with a different kind of solvent. If there's no pronounced separation then development proceeds to the “c” stage.

  • Again the filter paper is turned ninety degrees clockwise and another solvent is used. This results in a satisfactory separation.

Although this may take some time, this allows a high degree of separation.


Interesting Facts

  • Chromatography is helpful in determining the antibodies that fight numerous diseases. Scientists used chromatography in the fight against the Ebola virus, responsible for over 11,000 deaths. The process was used to resolve which antibodies are the most effective at neutralising the deadly virus.

  • Chromatography is additionally used to facilitate catching criminals. In line with programs like CSI, gas chromatography (GC) is employed to research blood and fabric samples, helping to identify criminals and bring them to justice.


Conclusion

Chromatography is a physical method of separation of different components of a mixture. In chromatography, the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction. On the basis of phases of separation of elements of a mixture, chromatography is divided into the five different categories. Two dimensional chromatography is used for separating mixtures.

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FAQs on Chromatography

1. What is the basic principle of chromatography?

combination of chemical substances into its individual parts. Chromatography is based on the idea of separating molecules in a mixture added to the ground or solid and liquid stationary state (stable phase) when travelling with the help of a mobile phase. It is defined as the method of separation of the individual components of a mixture based on their relative affinities towards stationary and mobile phases. Principle: The samples are subjected to flow by mobile liquid onto or through the stable stationary part.

2. What is the Rf value in chromatography and how is it useful?

RF stands for retention factor in paper chromatography, or the distance a fluid compound moves up a plate of chromatography. For every specific solvent, all compounds have a standard RF value, and RF values are used to equate unknown samples with known compounds. The distance travelled by sample divided by the distance travelled by the solvent. It's a component characteristic and might be used to classify elements for a given system at a known temperature.

3. What type of chromatography is HPLC and can we use water in chromatography?

High Performance Liquid Chromatography (HPLC) is a type of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). 


Water is the chemical agent utilised in the largest volumes in liquid chromatography and its purity is important, particularly in high sensitivity applications. These will require water for sample pre-treatment, like solid phase extraction, and for the preparation of eluents, chemical agent blanks and standards.