Applications of Chromatography

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  Now-a-days various various types types of of chromato chromatography graphy are used used to separate separate almost almost any given given mixture 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  placed onto or into into a medium medium and a solvent solvent is passe passedd through through the the test test substanc substance. e. 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: Chromatography:

There are three ways to classify chromatography. They area) Classifica Classification tion of of chromatog chromatography raphy accord according ing to to mobile mobile phase: 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) Classifica Classification tion according according to the the packing packing of the stationary stationary phase: phase: 1. Thin layer layer chromatog chromatography raphy (TLC): (TLC): the stationa stationary ry phase is a thin thin layer layer supported supported on glass, plastic or aluminum plates. 2. Paper Paper chromatogra chromatography phy (PC): (PC): the stationa stationary ry phase is a thin film film of liquid liquid supported on an inert support. 3. Column Column chromato chromatograph graphyy (CC): statio stationary nary phase phase is packed packed in a glass glass column. column.

Applications of Chromatography 2

c) Classifica Classification tion according according to the the force force of of separati separation: on: 1. Adsorp Adsorptio tionn chroma chromatog tograp raphy. hy. 2. Part Partit itio ionn chro chroma mato togr grap aphy hy.. 3. Ion excha exchange nge chrom chromato atogra graphy phy.. 4. Gel filtra filtratio tionn chro chromat matog ograph raphy. y. 5. Affi Affini nity ty chro chroma mato togr grap aphy hy.. 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  phases according according to their their partiti partition on

of a solid inert

coefficients

support 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 Smaller molecules molecules enter enter the  pores of the gel, and need need a larger volume of eluent.

Applications of Chromatography 3

2. Larger Larger molecules molecules pass throug throughh the column at a faster rate.

Affinity

Solid on which

Liquid or gas

Special kind kind of solute solute molecules

Chromatography

specific molecules

interact with those immobilized immobilized

are immobilized

on the stationary phase

Applications of Chromatography: 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. 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 h igh 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 members 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 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

 biotechno  biotechnologi logical cal research research.. •

Biological application 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 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 substances that are harmful to human beings. Forensic Toxicologists have the responsibility of detecting and identifying the presence of drugs and  poisons  poisons in fluids, fluids, tissues tissues and organs organs.. Their Their service servicess are not not just just required required in crime crime laboratories and medical examiner's offices; they reach into in to 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 n ew technology much of the testing can be carried out by  police  police with with fewer fewer cases cases being being passed passed on on to the the forensic forensic laboratory 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  preliminar  preliminaryy color color or microc microcrysta rystalline lline tests tests before before using using chromat chromatogra ography phy to to identify 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 f luids in such circumstances as sudden deaths and suspected poisonings. A post mortem is performed  by a patho pathologi logist st who sends sends specime specimens ns of variou variouss body body tissues tissues and fluids fluids to the forensic forensic toxicologist for examination. Many techniques 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 Paper chromatograp chromatography hy has widely widely been been used for quantit quantitative ative analys analysis is of Inorganic, Inorganic, organic and biochemical interest. 2. Paper Paper chromatograp chromatography hy is ideally ideally suited suited for rapid analys analysis is of reaction reaction mixture mixture and so it is versatile tool in the hand of organic chemists. 3. Paper Paper chromatograp chromatography hy has been been successfull successfullyy used for characteriz characterizing ing and isolating isolating the following organic compounds. •

Acids,

•

Amines,

•

Alcohols,

•

Amino acids

•

Glycols,

•

Proteins and peptides,

•

Alkaloids,

•

Antibiotics etc.

4. Paper Paper chromatogr chromatography aphy has has also been been used used in the analys analysis is of mixture mixture of sugars. sugars. 5. It can be be used used to detect detect traces traces of of pollutant pollutantss in water water food food or in soil. soil.

Applications of Chromatography 6

6. It can be used used for the identifica identification tion of compou compounds nds in drugs, drugs, in biochemic biochemical al preparatio preparationn 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, p eptides,

 proteins  proteins & polys polysaccha accharides rides 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 diff erent molecular weights.



Separation of mucopolysaccharides & soluble RNA.



Separation of myoglobin & haemoglobin.



Separation of alkaloids & purification of enzymes.

applications: In general, Gel-filtration chromatography which is also 3. Biochemical applications: known as Size Exclusion chromatography is considered a low resolution chromatography as it does not discern similar species sp ecies very well, and is therefore therefor e often reserved for the final "polishing" step of purification. The technique can determine the quaternary structure of purified proteins p roteins 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. 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  polymer,, that is, the the ability ability to be able to to find the distri distributio butionn of the the sizes sizes of of polymer  polymer  molecules. If standards of a known size are run previously, previously, then a calibration curve can  be created created to determ determine ine the the sizes sizes of of polymer polymer molec molecules ules of of interest interest in in the solvent solvent chosen 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 fr actionation on an analytical scale. •

1.

Applications of Thin Layer Chromatography As a check on process: It has been used for checking of the other separation  procedures  procedures and purific purificatio ationn processe processes. s.

2. In Orga Organic nic Chem Chemis istr try: y: 

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  peptides vii. Antibiotics

Besides these, there are compounds like carbohydrates, carbonyl compounds, Dyes, Hydrocarbons, lipids, nucleic acids, pesticides, p esticides, natural pigments, pharmaceutical products,  phenols,  phenols, steroid steroids, s, terpenes terpenes,, essential essential oils, vitamins, vitamins, adhesive adhesives, s, explosive explosivess plasticize plasticizers rs etc. etc. which have been separated and characterized by Thin Layer Chromatography. 3. For separa separatio tionn of Inorga Inorganic nic Ions:Ions:

Recently Thin Layer Chromatography has been used for separating

cationic, anionic, purely covalent species and also also some some organic derivatives of the metals. 4.

Applications of Thin Layer Chromatography in quantitative analysis: i. Spectrophotometric 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

•

1.

Application of Column Chromatography:Analytical uses: For analytical purposes, column chromatography finds limited applications. Vestergaard 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. Separa Separatio tionn of Diaste Diastereo reomer mers. s. 4.

Separation of tautomeric mixtures.

5. Sepa Separa rati tion on of of race racema mate tes. s. •

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. •

1.

Purif urifyy and conc conceentra ntrate te an enz enzyyme solut olutio ion. n.

Applications of Ion Exchange Chromatograph: Water softening: Removal of Ca2+, Mg2+ & other multivalent ions causing hardness of  water by filtration through a layer of strong cation resin.

2.

3.

Separation of electrolytes from non-electrolytes.  Neutraliz  Neutralization ation:: Cationic Cationic exchange exchangerr 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. Separatio Separationn of carbohydrat carbohydrates es & their their derivat derivatives ives:: 

Uronic acids separated on anion exchanger. exchanger.



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

exchanger. 

•

Hexosamines separated on strong cation exchanger.

Medical Applications 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 able to to produce produce mg mg quantit quantities ies of the target target compo compounds unds,, from a synthet synthetic ic reactio reactionn 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 support 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 developme development nt process process i.e. i.e. the the chemist chemistry, ry, and alleviate alleviatess the need to waste waste time time developing scaled-up chromatography 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  produce a specified specified quantity quantity of of the compound compound of of interest. interest. Typica Typically, lly, this this can can become 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  process can can lead lead to degra degradati dation on of the the product 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 f ound 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.