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Transcript of CHROMATOGRAPHY
High-performance liquid chromatography
Chromatography is basically a set of laboratory methods for separation of mixtures.
This is mainly used either to purify by separation or analyze mixtures.
The techniques of chromatography serves a wide variety of applications in the fields of ,
diagnostics, food development and testing
and also surprisingly in F1 racing!!
The mixtures are separated based on their solubility on a fluid solvent which is known as the
, which is held by another material, namely, the
This separation is caused by the differences in the compounds'
History: Paper chromatography
Richard Laurence Millington Synge
Archer John Porter Martin
Paper chromatography was first introduced by two scientists,
A British biochemist, Richard Laurence Millington Synge (1914-1994)
A British chemist, Archer John Porter Martin (1910-2002)
In 1952, they shared the Nobel Prize for Chemistry for this invention of partition chromatography which has done a massive contribution to science.
History: Thin-layer chromatography
The technique of TLC was first used in 1937 to 1938
at the Institute of Experimental Pharmacy of the
State University of Kharkov, Ukraine, by Nikolai A.
Izmailov (1907–1961), the young head of this institute, and Maria S. Shraiber (1904–1992), his graduate student.
Apparatus: Paper chromatography
Example: paper chromatogram
Higher the relative solubility of the compound in the mobile phase, higher its relative Rf value and its relative rate of movement.
How do the compounds separate?
Apparatus: Thin-layer chromatography
Separation Process and Principle
High-performance liquid chromatography
History : High-performance liquid chromatography
As we have discussed before,Liquid chromatography was initially discovered as an analytical technique in the early twentieth century and was first used as a method of separating colored compounds. A Russian botanist named Mikhail S. Tswett used a rudimentary form of chromatographic separation to purify mixtures of plant pigments into the pure constituents.
• Beginning of the 60’s: start of HPLC as High Pressure Liquid Chromatography
• End of the 70’s improvements of column material and instrumentation – High Performance Liquid Chromatography
Apparatus: High-performance liquid chromatography
Flow diagram: HPLC
Retention time, separation process and chromatogram
History : Gas-liquid chromatography
Flow diagram : GLC
Analysis of a gas chromatogram
It is commonly used technique for the separation of molecules. For example, it is used to remove pesticides and insecticides like DDT in the water and polychlorinated biphenyls.
It is widely used for testing the purity of drinking water.
In pharmaceutical companies, it is used for producing pure materials for medicines and also for checking the contamination presence in medicines.
It is used in pharmacy for detecting the chiral compounds (Enantiomers and optical isomers).
In the food industry, this technique is very useful for analysing and the separation of additives, proteins and amino acids etc.
It used in forensic science for detecting the presence of drugs.
In forensic studies paper chromatography is used in crime scene investigation and DNA and RNA sequencing along with other studies.
Paper chromatography is used as an analytical chemistry technique for identifying and separating colored mixtures like pigments.
Sugars, amino acids, lipids and nucleic acids and other biomolecules can be easily identified by spraying with appropriate reagents to detect these specific compounds.
This technique is also used for drug testing.
Paper chromatography is also primarily a forensic method to determine the ink and therefore pen used in a given crime such as a bank fraud.
Purity of any sample : Purity of sample can be carried out with TLC. Direct comparison is done between the sample and the standard or authentic sample; if any impurity is detected, then it shows extra spots and this can be detected easily.
Identification of compounds: Thin layer chromatography can be employed in purification, isolation and identification of natural products.
TLC is used in forensic science where it can be used to detect drugs and explosive residues.
Examination of reactions: Reaction mixture can be examined by thin-layer chromatography to assess whether the reaction is complete or not.
This method is also used in checking other separation processes and purification processes like distillation, molecular distillation etc.
High-performance liquid chromatography
Chemical Separations: It is based on the fact that certain compounds have different migration rates given a particular column and mobile phase.
Purification: Purification is defined as the process of separating or extracting the target compound from a mixture of compounds or contaminants.
Identification: Generally assay of compounds are carried using HPLC. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph.
This is used to prepare pure samples for further analysis.
The technique is used to separate mixtures of gases.
GLC also facilitates analyze the air quality.
It can be used to analyze the fuel quality.
Gas-liquid chromatography is also used in industrial scale to test for quality of chemicals.
Specific examples of these applications include:
analyzing ceramides and fatty acids,
detection of pesticides or insecticides in food and water,
composition of fibers in forensics,
assaying the radio chemical purity of radio pharmaceuticals,
or identification of medicinal plants and their constituents.
The separation process in TLC is known as adsorption.
Adsorption is the process by which separation takes place when,
molecules with higher polarity in the mixture is more attracted to the polar stationary phase, thus slowing down their motion through the chromatogram,
meanwhile, the molecules with a lower polarity stays in the mobile phase, thus moving up with the solvent.
The separated compounds can be spotted and thus the calculation and the comparison of their Rfs can take place.
Retention time is the time taken by the compound to travel through the column.
Archer John Porter Martin, in collaboration with Anthony T. James, went on to develop gas chromatography beginning in 1949. In 1952, during his lecture for the Nobel Prize in Chemistry.
Martin announced the successful separation of a wide variety of natural compounds by gas chromatography.
German chemist Fritz Prior had achieved limited success with gas chromatography, separating oxygen and carbon dioxide, in 1947 during his Ph.D. research. The method of Martin and James, however, became the basis for subsequent developments in gas chromatography.
Chromatography was first initiated by the Russian botanist, Mikhail Tswett in 1903, during his research on plant pigments and he discovered
A British chemist, Archer John Porter Martin and a British biochemist, named Richard Synge further developed Mikhail's theory of adsorption and discovered liquid-liquid chromatography in 1941.
Later, in 1945, Archer Martin and Richard Synge in collaboration with Frederick Sanger, a British biochemist, discovered paper chromatography.
By 1950, Archer Martin, laid the foundations for gas chromatography and then later he discovered gas-liquid chromatography.
Archer John Porter Martin and Richard Synge were rewarded with a Nobel Prize for Chemistry in 1952.
Meanwhile, Erika Cremer, a German physical chemist, first came up with a proper gas chromatography method, and developed the technique with Fritz Prior, a graduate student.
• Use of low skilled operators
• Addition of sophisticated detectors
• Micro and nano flow rates
• Standards and support for software and hardware are likely to increase support
• Better ways to collect, store and analyze data
• Improve productivity
• Improve quality and reliability
• Guard bed technologies
• Distillation and purification methods
• Pre treatment
• Down stream chemistry