Applications Of Chromatography 545l16

Applications of Chromatography 1

APPLICATIONS OF CHROMATOGRAPHY Chromatography is a process used to separate mixtures. The word chromatography is derived from the Greek words "khroma" and "graphein" meaning "color" and "to write" or "to represent". The chromatography technique is first discovered by Russian Biologists, Dr Michael Tswett in 1906 for the separation of colored plant pigment on a column of alumina. Now-a-days various types of chromatography are used to separate almost any given mixture whether colored or colorless into its component. Chromatography may be regarded as an analytical technique employed for the purification and separation of organic and inorganic substances. It is also found useful for the fractionation of complex mixture, separation of closely related compounds, such as isomers and in the isolation of unstable substances. Although there are several different types of chromatography, in each case a substance is placed onto or into a medium and a solvent is ed through the test substance. In chromatography science, the solvent is called "the mobile phase" or "the carrier fluid" and the medium is called "the stationary phase". Types of Chromatography: There are three ways to classify chromatography. They area) Classification of chromatography according to mobile phase: 1. Liquid chromatography: mobile phase is a liquid. (Liquid-Liquid

Chromatography, Liquid-Solid Chromatography). 2. Gas chromatography: mobile phase is a gas. (Gas-Solid Chromatography, Gas-

Liquid Chromatography) b) Classification according to the packing of the stationary phase: 1. Thin layer chromatography (TLC): the stationary phase is a thin layer ed on glass, plastic or aluminum plates. 2. Paper chromatography (PC): the stationary phase is a thin film of liquid ed on an inert . 3. Column chromatography (CC): stationary phase is packed in a glass column.

Applications of Chromatography 2

c) Classification according to the force of separation: 1. Adsorption chromatography. 2. Partition chromatography. 3. Ion exchange chromatography. 4. Gel filtration chromatography. 5. Affinity chromatography. Table 01: Different Types of chromatography Mode or type

Stationary phase

Mobile phase

Mechanism

Adsorption

Solid that attracts the Liquid or gas

Solutes move at different rates

Chromatography

solutes

according to the forces of attraction to the stationary phase.

Partition

Thin film of liquid

Liquid or gas

Solutes equilibrate between the 2

Chromatography

formed on the surface

phases according to their partition

of a solid inert

coefficients

Ion Exchange

Solid resin that

Liquid containing Solute ions of charge opposite to

Chromatography

carries fixed ions & electrolytes

the fixed ions are attracted to the

mobile counter-ions

resin by electrostatic forces &

of opposite charge

replace the mobile counter-ions.

attached by covalent bonds Molecular

Porous gel with no

Exclusion

attractive action on

Chromatography

solute molecules

Liquid

Molecules separate according to their size: 1. Smaller molecules enter the pores of the gel, and need a larger volume of eluent.

Applications of Chromatography 3

2. Larger molecules through the column at a faster rate.

Affinity

Solid on which

Liquid or gas

Special kind of solute molecules

Chromatography

specific molecules

interact with those immobilized

are immobilized

on the stationary phase

Applications of Chromatography: Chromatographic methods will separate ionic species, inorganic or organic, and molecular species ranging in size from the lightest and smallest, helium and hydrogen, to particulate matter such as single cells. No single configuration will accomplish this, however. Little preknowledge of the constituents of a mixture is required. At its best, chromatography will separate several hundreds of components of unknown identity and unknown concentrations, leaving the components unchanged. Amounts in the picogram or parts per billion ranges can be detected with some detectors. The solutes can range from polar to nonpolar— i.e., water-soluble to hydrocarbon-soluble. Substances of low critical temperature or low molecular weight, such as the gases at laboratory conditions showing dispersive or London intermolecular forces only, are separated with molecular sieves or gas-solid techniques. Gas-liquid chromatography is applicable to species with high critical temperatures and normal boiling points as high as 400° C. Substances that are solids at normal laboratory conditions with molecular weights below1000 are best separated with liquid-solid or liquid-liquid systems. Lower of the molecular weight scale range are amenable to separations. Methods are involved at molecular weights above 1,000. Fieldflow fractionation extends the size range to colloids and microscopic particles. General uses of chromatography in our real life are: • Pharmaceutical Company – determine amount of each chemical found in new product. • Hospital – detect blood or alcohol levels in a patient’s blood stream. • Law Enforcement – to compare a sample found at a crime scene to samples from

suspects.

Applications of Chromatography 4

• Environmental Agency – determine the level of pollutants in the water supply. • Manufacturing Plant – to purify a chemical needed to make a product. • Biotechnology industry – establishing the purity or concentration of compounds in

biotechnological research. • Biological application – Chromatography has many applications in biology. It is used to

separate and identify amino acids, carbohydrates, fatty acids, and other natural substances. Environmental testing laboratories use chromatography to identify trace quantities of contaminants such as PCBs in waste oil and pesticides such as DDT in groundwater. It is also used to test drinking water and test air quality. Pharmaceutical companies use chromatography to prepare quantities of extremely pure materials. The food industry uses chromatography to detect contaminants such as aflatoxin. For example we can consider the use of chromatography in Forensic Toxicology. Forensic toxicology is the application of toxicology for the purpose of law. Forensic Chemistry and Toxicology generally concerns the detection and characterization of poisons or toxins exhibiting adverse physiological effects. Laboratory methods used in chemical toxicological analysis cover a wide range and may be broadly classified as follows: (1) physical tests (2) crystal tests (3) chemical spot tests (4) spectrophotometric tests (5) chromatographic tests. Toxicology is the study of substances that are harmful to human beings. Forensic Toxicologists have the responsibility of detecting and identifying the presence of drugs and poisons in fluids, tissues and organs. Their services are not just required in crime laboratories and medical examiner's offices; they reach into hospital laboratories where the identification of an overdose can mean the difference between life and death. The work of a toxicologist generally falls into three main categories: 1. Routine testing for alcohol in blood or urine samples following a "breathalyzer" test.

However with the advent of new technology much of the testing can be carried out by police with fewer cases being ed on to the forensic laboratory. Like Gas chromatography was, and still is, used to identify exactly how much alcohol is present in the blood or urine of a suspect. Because it is very accurate, results can be used as evidence in a court of law.

Applications of Chromatography 5

2. Identification of drugs such as heroin, cocaine, cannabis, etc. It is common to perform

preliminary color or microcrystalline tests before using chromatography to identify a substance as chromatography yields somewhat inconclusive results. However, both thin layer and gas chromatography in conjunction with the preliminary tests is well suited for drug analysis. 3. Detection of drugs and poisons in body fluids, tissues and organs. This area of forensic

toxicology involves the analysis of organs, tissues and body fluids in such circumstances as sudden deaths and suspected poisonings. A post mortem is performed by a pathologist who sends specimens of various body tissues and fluids to the forensic toxicologist for examination. Many techniques are used in this area of work including chromatography Depend on the mechanisms different types of chromatography are specified in uses of different actions. Applications according to the mechanism are given bellow: • Applications of Paper Chromatography:

1. Paper chromatography has widely been used for quantitative analysis of Inorganic, organic and biochemical interest. 2. Paper chromatography is ideally suited for rapid analysis of reaction mixture and so it is versatile tool in the hand of organic chemists. 3. Paper chromatography has been successfully used for characterizing and isolating the following organic compounds. •

Acids,

•

Amines,

•

Alcohols,

•

Amino acids

•

Glycols,

•

Proteins and peptides,

•

Alkaloids,

•

Antibiotics etc.

4. Paper chromatography has also been used in the analysis of mixture of sugars. 5. It can be used to detect traces of pollutants in water food or in soil.

Applications of Chromatography 6

6. It can be used for the identification of compounds in drugs, in biochemical preparation and in natural products. It can be used for checking the purity of samples. • Applications of Gel-filtration Chromatography

1. Analytical applications 

Analytical group separation



Analytical fractionation



Determination of molecular masses: Determination of M. wt. of peptides,

proteins & polysaccharides 2. Preparative applications 

Preparative fractionation



Preparative group separations 

Separation of mixture of mono-and polysaccharides.



Separation of amino acids from peptides & proteins.



Separation of proteins of different molecular weights.



Separation of mucopolysaccharides & soluble RNA.



Separation of myoglobin & haemoglobin.



Separation of alkaloids & purification of enzymes.

3. Biochemical applications: In general, Gel-filtration chromatography which is also

known as Size Exclusion chromatography is considered a low resolution chromatography as it does not discern similar species very well, and is therefore often reserved for the final "polishing" step of purification. The technique can determine the quaternary structure of purified proteins that have slow exchange times, since it can be carried out under native solution conditions, preserving macromolecular interactions. Size Exclusion chromatography can also assay protein tertiary structure, as it measures the hydrodynamic volume (not molecular weight), allowing folded and unfolded

Applications of Chromatography 7

versions of the same protein to be distinguished. For example, the apparent hydrodynamic radius of a typical protein domain might be 14 Å and 36 Å for the folded and unfolded forms, respectively. Size Exclusion chromatography allows the separation of these two forms, as the folded form will elute much later due to its smaller size. 4. Polymer synthesis: Gel-filtration chromatography or Size Exclusion chromatography

can be used as a measure of both the size and the polydispersity of a synthesized polymer, that is, the ability to be able to find the distribution of the sizes of polymer molecules. If standards of a known size are run previously, then a calibration curve can be created to determine the sizes of polymer molecules of interest in the solvent chosen for analysis. In alternative fashion, techniques such as light scattering and/or viscometry can be used online with Size Exclusion chromatography to yield absolute molecular weights that do not rely on calibration with standards of known molecular weight. Due to the difference in size of two polymers with identical molecular weights, the absolute determination methods are, in general, more desirable. A typical Size Exclusion chromatography system can quickly (in about half an hour) give polymer chemists information on the size and polydispersity of the sample. The preparative Size Exclusion chromatography can be used for polymer fractionation on an analytical scale. • Applications of Thin Layer Chromatography 1. As a check on process: It has been used for checking of the other separation

procedures and purification processes. 2. In Organic Chemistry: 

The main use of Thin Layer Chromatography is isolation and separation of

individual components of a mixture. 

The main reasons for popularity of Thin Layer Chromatography as an analytical

and preparation methods are: 

It can be used for most of chemical compounds.



It has high speed of separation.



Its selectivity is high.

Applications of Chromatography 8



The following are the various applications of Thin Layer chromatography in

organic chemistry:





For checking the purity samples as a purification process.



Examination of reactions.



For identifying organic compounds.

Thin Layer Chromatography has been successfully used for characterizing and

isolating the following organic compounds. i. Acids

v. Amines

ii. Alcohols

vi. Amino acids, proteins and

iii. Glycols iv. Alkaloids

peptides vii. Antibiotics

Besides these, there are compounds like carbohydrates, carbonyl compounds, Dyes, Hydrocarbons, lipids, nucleic acids, pesticides, natural pigments, pharmaceutical products, phenols, steroids, terpenes, essential oils, vitamins, adhesives, explosives plasticizers etc. which have been separated and characterized by Thin Layer Chromatography. 3. For separation of Inorganic Ions:

Recently Thin Layer Chromatography has been used for separating

cationic, anionic, purely covalent species and also some organic derivatives of the metals. 4. Applications of Thin Layer Chromatography in quantitative analysis:

i. Spectrophotometric Measurement ii. Fluorimetric Method iii. Visual comparison of spots iv. Spectral reflectance

v. Spot densitometer vi. Vapour phase chromatography vii. Radioactive methods viii. Volumetric analysis

Applications of Chromatography 9

• Application of Column Chromatography:1. Analytical uses: For analytical purposes, column chromatography finds limited

applications. Vestergaard and Sayegh could separate seven urinary steroids within 5 hours which requires 36 hours on a normal column. They have used narrow Teflon tubing packed with aluminum oxide or silica gel. 2. Separation of geometrical isomers: The separation of cis/trans isomer is based on the

steric factors. Isomers whose functional groups can approach the surface of the adsorbent more easily are more strongly adsorbed. 3. Separation of Diastereomers. 4. Separation of tautomeric mixtures.

5. Separation of racemates. • Applications of Affinity chromatography: 1.

Purify and concentrate a substance from a mixture into a buffering solution.

2.

Reduce the amount of a substance in a mixture.

3.

Discern what biological compounds bind to a particular substance, such as

drugs. 4.

Purify and concentrate an enzyme solution.

• Applications of Ion Exchange Chromatograph: 1. Water softening: Removal of Ca2+, Mg2+ & other multivalent ions causing hardness of

water by filtration through a layer of strong cation resin. 2. Separation of electrolytes from non-electrolytes. 3. Neutralization: Cationic exchanger in [H+] can be used to neutralize alkali hydroxide &

anionic exchanger in [OH-] form to neutralize the acidity. 4. Water demineralization: Removal of cations & anions dissolved in water. Usually

carried by the two step technique in which two columns of strongly acid cation

Applications of Chromatography 10

exchanger in [H+] form & strongly basic anion exchanger in [OH-] form are used in sequence. 5. Separation of carbohydrates & their derivatives: 

Uronic acids separated on anion exchanger.



Sugars converted into ionized form by using borate& separated on strong anion

exchanger. 

Hexosamines separated on strong cation exchanger.

• Medical Applications of High Performance Liquid Chromatography:

The isolation and purification of compounds is of critical importance to medicinal chemists discovering and developing drugs. In these activities it is extremely important to be able to produce mg quantities of the target compounds, from a synthetic reaction mixture. Then later on use the same separation technologies to provide gram and Kilo quantities. Similarly, the technique can isolate, purify and concentrate suitable quantities of low level impurities and metabolites to compound identification and further characterization. One of the key advantages of high performance counter current chromatography is that it scales easily and simply. This allows the medicinal chemist to focus on the value-added part of the development process i.e. the chemistry, and alleviates the need to waste time developing scaled-up chromatography techniques, as demand for the quantity of compound increases. A further important issue is sample solubility since this can affect the throughput to produce a specified quantity of the compound of interest. Typically, this can become an issue when the purification is performed in reverse phase (RP) which generates aqueous fractions. These aqueous fractions are laborious to concentrate by evaporation and this process can lead to degradation of the product. As scale of production increases the volumes of solvents used and those of product containing fractions also increase. This is particularly an issue where Reverse PhaseHigh Performance Liquid Chromatography produces large volumes of aqueous fractions. High performance counter current chromatography can be used in normal phase for the

Applications of Chromatography 11

same separation which means that fractions can be collected in essentially non-aqueous solvents which makes their processing simpler, faster and less energy consuming. High Performance Liquid Chromatography has found many applications in medicinal chemistry, to which the Spectrum or Midi bench top products are normally found to be the most suitable for the quantities of compound required.