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 too 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 = a color and graphien = 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 the 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 bypassing 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 Prize.
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 HPLC.
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:
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.
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 stationary phase (solid) for separation of components.
This method is further classified into 3 types known as:
- Thin Layer Chromatography
- Column Chromatography
- Gas-Solid 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.
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.
The two main types of this technique are Liquid-Liquid and Gas-Liquid Chromatography.
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.
This technique contains the sample containing charged molecules as a liquid phase and the chromatographic column containing a stationary phase is 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.
- 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.
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 building such as proteins and modern polymers. In this technique, the column is filled with fine porous beads comprising of agarose of polyacrylamide or dextran polymers.
SEC is one of the HPLC separation mode technique 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.
Two-dimensional techniques are further classified into Thin Layer and Paper Chromatography.
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 separation of components of non-volatile mixtures. The experiment is performed on a thin sheet of aluminium foil or glass or similar material coated with absorbent material such as silica gel or cellulose or aluminium 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.
TLC is based on the principle of adsorption. The detachment relies upon the relative partiality of mixes towards the stationary and mobile phases.
In this technique, the sample is placed on the stationary phase. When the mobile phase runs upwards along with the stationary phase due to the capillary action, the components of a mixture also moves along with it based on their affinity towards the stationary phase.
Components having higher affinity moves slowly while the components with low affinity move fast thus, separating the components of the given sample.
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.
The principle of this method is based on the principle of partition. In this technique, the sample is placed on the paper and the solvent moves through the stationary phase.
As the mobile phase runs on the paper, the components of the mixture also run along with it and separate on the basis of their migration rate on the paper.
This method is divided into 4 types:
- Ascending Paper Chromatograph
- Ascending-Descending Paper Chromatograph
- Radial or Circular Chromatograph
- Two-dimensional Paper Chromatograph
Classification on the basis of physical condition of mobile phase
- Liquid Chromatography
- Gas Chromatography
- Super Critical Fluid 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 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.
High-performance liquid chromatography (HPLC)
HPLC It It is a type of Liquid chromatography also is known as High-pressure chromatographic technique due to the high pressure being applied to the column. The pressure works by forcing the mobile phase through at much higher rate thus, increasing the resolution power.
It is based on the principle that the analyte (sample) is separated based on the stationary and mobile phase. The mobile phase in HPLC is the solvent and the stationary phase is the column.
Gas Chromatography (GC)
GC is a typical sort of technique utilized in diagnostic science for isolating and breaking down intensifies that can be disintegrated without decay. Typical employments of GC incorporate 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.
GC is categorised 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 used to carry the analyte over stationary phase for the purpose of isolation. Thus, GC is also known as GLC or GSC because of their stationary phases.
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 as a Column method and is much better and 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 analyse easily decomposable substances.
Chromatographic techniques are very useful in the isolation of amino acids, proteins, drugs, vitamins, carbohydrates, ink, etc. to analyse and distinguish between their properties.
It is useful in different sectors such as pharmaceutical companies, 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 this particular field, new techniques are being established depending on the needs. As more techniques form up, a simple way to isolate different mixtures come up.