Chromatography: The Most Ancient Separation Technique

Every component in this universe is made up of matter, the basic principle which we all learned in chemistry. We also learned that every compound is a mixture of multiple elements. Starting from water to colored solutions everything is a combination of one or more elements, which is why for ages humans have always been curious to separate these compounds to study what they are made up of.

Scientists succeeded finally in separating components in the year 1855, when Friedlieb Ferdinand Runge, demonstrated a technique to separate dyes and components of chemicals using a filter paper immersed in another chemical. This was the starting of the most colorful and useful technique we today know as Chromatography

But the evolution of chromatography did not stop with Mr. Runge. In 1900 in Russia, Michael (Mikhail) Tsvet, an Italian botanist tried to separate plant pigments adapting and modifying Runge’s technique. The result he obtained was the separation of plant pigments and chlorophyll, carotenes, and xanthophylls which appeared extremely colorful filled with greens and oranges.

The term chromatography was hence coined by Tsvet and is a combination of two Greek words, ‘chroma’ means “color” and ‘graphein’ means “to write or record”. This technique typically separated components into distinct bands of color.

Definition and Principle of Chromatography

Scientifically, chromatography can be defined as the technique used for the separation of mixtures by passing it in solution or suspension through a medium in which components move at different rates.

Even though chromatography was discovered by a botanist, in the early days this technique was used by artists, to study the components of paint, their properties and to create new dyes for them.

The evolution of chromatography since then has broadened into multiple fields of science, agriculture, artistry, manufacturing, and much more with different types and mechanisms of chromatography being introduced. However, in general, chromatography works on the principle of differential adsorption partition and capillary action.

When the complex mixture such as pigments is dissolved in a fluid component (which can be a solvent or gas) called the mobile phase, it is carried upward through a system of a column, capillary tube, plate, or sheet due to capillary action.

On the column is the fixed stationary phase which is usually made up of materials like silica. The mixture travels at different rates concerning the size of its molecules. The large molecules travel slower than the small molecules. The differential partition between the Mobile phase and stationary phase combined with adsorption results in the separation.

Chromatography can be used as two broad categories, as a purification technique to simply purify the components and separate it, and as an analytical tool for establishing and measuring the relative proportions of analysis in a mixture.

Purification Chromatographic Techniques

They can be classified into Column chromatography, Planar chromatography, Paper chromatography, and Thin-layer chromatography(TLC).

1. Column Chromatography

Column chromatography is usually conducted to separate a single compound from a mixture of components into fractions. It works on the principle of differential adsorption, which signifies the process of separation of substituents based on the interaction of the adsorbate with the adsorbent. 

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Column chromatography is performed inside a tube, where the stationary phase is dissolved. The mobile phase along with the mixture that needs to be separated is introduced into the tube from the top. 

There are certain characters that both the stationary and mobile phases possess. Like the mobile phase in which the mixture(solute) is added also acts as a solvent and can generally be ethanol, acetone, and pyridine. It also helps as an eluting and separation agent as it helps in the separation of the mixture into distinct bands.

The stationary phase on the other hand must be inert, to not react with the mixture introduced for separation, should allow free flow of the mobile phase, must contain uniformity in the shape and size of its particles of about 60-200 micrometers and must be colorless and inexpensive. Usually, for most of the chromatographic techniques, silica is suitable as the stationary phase.

Execution

The stationary phase dissolved within the walls of the tube is made wet by the solvent as an initial step. The mixture to be separated is introduced into the column(tube) along with the solvent from the top.

It is done without touching the top level and disturbing the column slowly, through the edges of the tube. It is added throughout the experiment as per the requirement. The tap of the column is then turned on to initiate the movement of the components based upon their molecular size and polarity.

Once it is completed the distinct bands can be viewed which denotes the separated components from the mixture.

Types of Column Chromatography

Based on the type of mobile phase, stationary phase, and its characteristics, column chromatography can be of different types with slight changes in each.

  1. Adsorption column chromatography: In this technique, the components of the mixture are adsorbed on the surface of the adsorbent. 
  2. Partition column chromatography: In this both the stationary and mobile phase will be liquid.
  3. Gel column chromatography: The stationary phase would be made up of any gel component and it will be usually silica gel.
  4. Ion exchange column chromatography: The stationary phase used will always be ion exchange resin.

2. Planar Chromatography

Liquid chromatographic techniques in which the stationary phase is arranged in a form of a flatbed or a plane are referred to as planar chromatography. There are two main chromatographies dealt under this section;

3. Paper chromatography

In this type of Chromatography, the stationary phase is on inkblot paper, cellulose paper, or specialized chromatography paper. The compound to be separated is simply placed on the paper and the system is transferred to a closed setup.

The paper is then dipped in the desired solvent. As a result of the capillary action, the solvent being the mobile phase moves up and separates the components of the mixture based upon their molecular sizes.

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The preferred solvent for paper chromatography can be either methanol or n-propyl acetate. Based on the movement of molecules and the shape of the system, paper chromatography can be further divided into, Ascending, Descending, Ascending-Descending, Horizontal/circular, and two-dimensional paper chromatography.

You can read about the types of paper chromatography in detail.

4. Thin Layer Chromatography (TLC)

Thin Layer Chromatography works on the same principle as that of paper chromatography, but the stationary phase is usually a glass plate coated with silica slurry, alumina, or readymade TLC plates which are pre-coated with Silica or other inert components.

The solvents used could vary from Bromoform to varieties of alcohols. TLC is preferred over paper chromatography in most of the laboratory separation of mixtures, due to its high accuracy, and ease.

The final result of separated bands can be further analyzed by viewing it more distinctively under UV light or by spraying phosphorus over it. TLC is usually preferred to separate non-volatile components.

As TLC is preferred by most scientists, it has been modified into more conventional and high profile versions such as High-Performance Liquid Chromatography(HPLC), Preparative liquid chromatography(PLC), and Thin layer chromatography-mass spectrometry(LC-MS).

Analytical Chromatographic Techniques

Analytical techniques help in both the qualitative and the quantitative analysis of the compound and its components. 

1. Gas Chromatography (GC)

GC is also known as gas-liquid partition chromatography or vapor phase chromatography. It is used for the separation of volatile components, that is those which can be vaporized without decomposition.

Gas chromatography is the process of separating compounds in a mixture by injecting a gaseous or liquid sample into a mobile phase, typically called the carrier gas, and passing the gas through a stationary phase. The mobile phase is usually an inert gas or an unreactive gas such as helium, argon, nitrogen, or hydrogen. 

For this sophisticated technique, the partition occurs inside equipment known as a gas chromatograph.

It is made up of narrow flow through a tube called a column, through which the sample passes in a gas stream at different rates depending on their various chemical and physical properties and their interaction with a specific column lining or filling, chemicals exit the end of the column, which are detected and identified electronically.

The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time. Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, column length, and temperature.

The final output result is concluded using an external detector attached to it. Commonly used detectors are the flame ionization detector (FID) and the thermal conductivity detector (TCD).

While TCDs are beneficial in that they are non-destructive, their low detection limit for most analytes inhibits widespread use. FIDs are sensitive primarily to hydrocarbons and are more sensitive to them than TCD.

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FIDs cannot detect water or carbon dioxide which makes them ideal for environmental organic analyte analysis. FID is two to three times more sensitive to analyte detection than TCD.

2. Gel Permeation Chromatography

Gel permeation chromatography is also called gel filtration or size exclusion chromatography. In size exclusion chromatography, the stationary phase is a porous matrix made up of compounds like cross-linked polystyrene, cross-like dextrans, polyacrylamide gels, agarose gels, etc.

The separation is based on the analyte molecular sizes since the gel behaves like a molecular sieve. This technique is used for the separation of proteins, polysaccharides, enzymes, and synthetic polymers.

As a technique, size exclusion chromatography was first developed in 1955 by Lathe and Ruthven. It works on the principle that it is a technique in which the separation of components is based on the difference in molecular weight or size.

The stationary phase used is a porous polymer matrix whose pores are filled with the solvent to be used as the mobile phase. The molecules in the sample are pumped through specialized columns containing such microporous packing material (gel).

The basis of the separation is that molecules above a certain size are excluded from the pores, while smaller molecules access the interior of the pores partly or wholly.

The flow of the mobile phase hence will cause larger molecules to pass through the column unhindered, without penetrating the gel matrix, whereas smaller molecules will be retarded according to their penetration of the gel.

The detectors used may be concentration-sensitive detectors, bulk property detectors, refractive index (RI) detectors, etc.

Few other chromatographic techniques include Dye ligand, pseudo affinity, hydrophobic interactions, and affinity chromatographies.

Applications of Chromatography

Going back to the definition of chromatography quoted, as it says a technique for the separation of components, thus it serves the basic purpose of separation of the constituents of a mixture.

  1. Chromatography is known to separate any form of a mixture, due to this property it is highly useful in drug analysis. Most of the chemistry divisions under forensic labs make use of these chromatographic techniques for the separation of sample evidence of drug obtained and to study its components.
  2. Chromatography is highly used in the analysis of poisons in forensic toxicology. Separation of desired components from the obtained visceral organs could be done using the techniques discussed above. 
  3. Chromatography is still the most favorite technique for botanists in studying plant varieties, their components, composition of pigments and so much more. They are also used widely in the industrial area and chemical manufacturing companies to determine the composition of distinct chemicals. 
  4. Chromatography is a boon for researchers. It is the most convenient and less expensive technique that could be used.
  5. Recent studies have tried to even analyze soil and stone particles from different planets to learn the possibility of life. Hence there goes a wide application of chromatography in astronomy!

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