Types & Principles of Chromatography

No wonder, being a science student you must be aware of chromatography techniques. We have been studying these techniques since 12th class and are useful in most of the experiments in the lab. Chromatographic techniques are used in Forensic science laboratories for the analysis of samples making it much more necessary for you to learn and understand.

Here, I will be explaining to you different Chromatographic techniques and their principles.

Introduction to Chromatography

It is a separation technique in which a sample is separated based on the principle of adsorption or partition where a sample is separated into different components based on stationary and mobile phases. The stationary phase can be solid or liquid while the mobile phase can be liquid, gas, or supercritical liquid.

The term Chromatography (chroma means color and graphien means to write) is the aggregate term for a lot of research facility methods for the partition of blends. This technique helps to determine the composition, concentration of a sample,

History of Chromatography

The first use of Chromatography dates back to the 19th century by dye chemists. They dipped their clothes or filter papers in a dye vat to analyze their dye mixtures. During that time several German chemists tried to experiment to explore this phenomenon.

A publication was made in 1861 by Friedrich Gopplesroder who described the method and named it “Capillary analysis”.

However, the credit for the discovery of the technique was given to M. Tswett in 1906. He utilized a method to isolate different colors. For example, chlorophylls and xanthophylls bypass the arrangement of these mixes into the glass section which was pressed with finely partitioned calcium carbonate.

Almost following three decades, in 1935 Adams and Holmes watched the Ion Exchange attributes in the squashed phonograph. This perception opened the field for the readiness of Ion Exchanged gums.

The idea of Gas-Liquid Chromatography was first presented by Martin and Synge in 1941. They were likewise answerable for the improvement in Liquid-Liquid chromatography.

In 1944, from Martin’s research facility, the detachment of amino corrosive by paper chromatography was reported. In 1952, its significance was seen when both Synge and Martin were granted with Nobel Prizes.

In 1959, a method known as Gel Filtration chromatography was seen which is utilized to isolate low sub-atomic weight substances from high sub-atomic substances. In 1960, further improvement in fluid chromatography prompted the advancement of HPTLC.

The next decade of the 1970s saw an improvement in the field of adsorption chromatography as Affinity chromatography which was essentially founded on organic associations.

A new field was started which was supercritical liquid chromatography which is a half-breed of gas and fluid chromatography and consolidates favorable elements of the gas and fluid.

Classification of chromatography

Classification on the basis of solute to the stationary phase

On the basis of solute to stationary phase, Chromatographic techniques are classified as:

1. Adsorption Chromatography

It is presumably one of the most established techniques around. It uses a portable fluid or vaporous stage that is adsorbed onto the outside of a strong stationary phase. The equilibration between the portable, what’s more, the fixed stage represents the division of various solutes.

Principle of Adsorption Chromatography

Mikhail Tsvet In 1901 invented this method while working on plant pigments. He separated carotenoid pigments and chlorophyll using this method.

It works on the principle that when the mobile phase moves upwards through the stationary phase, the components of a mixture in the mobile phase (either liquid or gas) are adsorbed in the stationary phase (solid) for the separation of components.

Types of Adsorption Chromatography

This method is further classified into 3 types known as:

• Thin Layer Chromatography
• Column Chromatography
• Gas-Solid Chromatography

2. Partition Chromatography

This technique was invented by the work and research of Archer Martin and Richard Laurence Millington Synge around the 1940s.

This technique is used to separate and identify amino acids, alkaloids, Carbohydrates, etc. using a stationary phase and mobile phase where the stationary phase is a liquid while the mobile phase is either liquid or gaseous.

 Principle of Partition Chromatography

In this technique, the analytes of the sample are separated based on the partition between two phases.

The technique works on the principle that the mobile phase consisting of the components of a mixture moves through the stationary phase thus separating the components.

Types of Partition Chromatography

The two main types of this technique are Liquid-Liquid and Gas-Liquid Chromatography.

3. Ion Exchange Chromatography

Particle Exchange or Ion exchange Chromatography is a procedure that permits the partition of ions and polar atoms depending on their partiality to the particle exchanger.

This technique works on almost all kind of charged molecules such as proteins, amino acids, nucleotides, etc. but the process must be performed under conditions such that they are one unit away from the isoelectric point of the protein.

 Principle of Ion Exchange Chromatography

This technique contains the sample containing charged molecules as a liquid phase and the chromatographic column containing a stationary phase is an oppositely charged site.

The molecules separated on the basis of their charge are eluted using a solution of varying ionic strength. Passing such a solution through the column, highly selective separation of molecules according to their different charges takes place.

Types of Ion Exchange Chromatography

• Cation exchangers

In this method, the stationary phase possesses the negatively charged groups which attract positively charged ions.

• Anion exchangers

In this method, the stationary phase possesses the positively charged groups which attract negatively charged ions.

4. Size Exclusion Chromatography (SEC)

SEC or Molecular sieve Chromatography is a technique in which the components of a mixture are separated on the basis of size and sometimes molecular weight based on their elution from columns filled with porous gels.

It is normally applied to huge particles or macromolecular buildings such as proteins and modern polymers. In this technique, the column is filled with fine porous beads comprising agarose of polyacrylamide or dextran polymers.

Principle of Size Exclusion Chromatography

SEC is one of the HPLC separation mode techniques in which the column is filled with materials containing many pores. When the molecules are dissolved into the column, the smaller molecules flow slowly while the larger ones move fast.

It is because the smaller molecules penetrate deep into the pores while the larger ones go on and thus, are the ones to elute first, effectively separating on the basis of molecular size.

Classification on the Basis of Bed Shape

Chromatographic techniques on the basis of bed shape are classified as Two-dimensional and Three-dimensional.

1. Two-dimensional: Two-dimensional techniques are further classified into Thin Layer and Paper Chromatography.
2. Three-dimensional: The 3-D technique under bed shape classification is Column Chromatography.

Thin layer Chromatography (TLC)

TLC was discovered by M. Tswett in 1906. It is a very common technique used for the purpose of the separation of components of non-volatile mixtures. The experiment is performed on a thin sheet of aluminum foil or glass or similar material coated with absorbent material such as silica gel or cellulose or aluminum oxide.

The absorbent material is known as the stationary phase while the solvent used to run on a TLC plate is known as the mobile phase.

 Paper Chromatography

An inexpensive yet effective technique used for isolating components of a mixture. The technique works on the principle of partition.

It may come as a surprise to hear that this technique is partition-based i.e., based on liquid-liquid separation yet it is true because the paper used for the experiment is made up of cellulose in which polar molecules are absorbed.

In all types of chromatography, a stationary and mobile phase is present. In this method, the stationary phase is the paper while the mobile phase is the solvent.

This method is completely based on TLC and the only difference is the stationary phase.

Classification Based on Physical Condition of Mobile Phase

1. Liquid Chromatography
2. Gas Chromatography
3. Super Critical Fluid Chromatography

1. Liquid Chromatography

Liquid chromatography is a strategy used to isolate a test into its individual parts. This division happens based on the cooperation of the example with the portable and stationary phase. Since there are numerous stationary/mobile phases blends that can be utilized while isolating a blend, there are a few unique sorts of chromatography that are characterized depending on the physical conditions of the phases.

Liquid-solid, the most well-known chromatographic procedure, includes a fluid versatile stage gradually channelling down through the strong fixed stage, carrying the isolated segments with it.

Types of Liquid Chromatography:

• Liquid-Liquid (when stationary phase is liquid)

(i)  Normal phase, (ii) Reverse phase

• Liquid-Solid (when the stationary phase is solid)

(i) Normal Phase, (ii) Reverse Phase

I) Normal phase

In a typical stage, the stationary stage is polar, thus the more polar solutes being isolated will follow more to the fixed adsorbent stage. At the point when the dissolvable or angle of solvents is gone through the section, the less polar parts will be eluted quicker than the more polar ones.

The parts would then be able to be gathered independently, accepting satisfactory detachment was accomplished, arranged by expanding extremity.

II) Reverse phase

In the switch stage, the polarities of the versatile furthermore, fixed stages are inverse to what they were when performing ordinary stage chromatography. Rather than picking a non-polar versatile stage dissolvable, a polar dissolvable and non-polar fixed stage must be picked.

2. Gas Chromatography (GC)

GC is a typical sort of technique utilized in diagnostic science for isolating and breaking down intensities that can be disintegrated without decay. Typical employment of GC incorporates testing the immaculateness of a specific substance or isolating the various parts of a blend (the overall measures of such parts can likewise be decided).

In certain circumstances, GC may help in distinguishing a compound. In preparative chromatography, GC can be utilized to get ready unadulterated mixes from a blend.

Principle of Gas Chromatography

GC is categorized into two based on the type of stationary phase used. The stationary phase used in this technique is either solid or Liquid while the mobile phase is Gas which is used to carry the analyte over the stationary phase for the purpose of isolation. Thus, GC is also known as GLC or GSC because of its stationary phases.

3. Supercritical Fluid Chromatography (SFC)

The discovery of supercritical fluids led to the discovery of a new technique i.e., SFC. A supercritical fluid is any substance at a high temperature or pressure above its critical point, where distinct liquids and gas phases do not exist.

SFC is termed a Column method and is much better and more advantageous than GC and HPLC. SFC has its advantage over HPLC due to its sensitivity and efficiency while it surpasses GC due to its ability to analyze easily decomposable substances.

Conclusion

Chromatographic techniques are very useful in the isolation of amino acids, proteins, drugs, vitamins, carbohydrates, ink, etc. to analyze and distinguish between their properties.

It is useful in different sectors such as pharmaceutical companies, the Food industry, Forensic Science, Chemical industries, molecular biology, etc. These techniques are used based on the characteristics of different mixtures.

With the continuous development in the field of science, new techniques are being established depending on the needs. As more techniques form up, a simple way to isolate different mixtures comes up.

Also Have a Look at:

• Difference Between Paper Chromatography & Thin Layer Chromatography
• Roles and Responsibilities of a Forensic Pathologist
• Difference Between Chromosome, Chromatin and Chromatid