APPLICATIONS OF CHROMATOGRAPHY
APPLICATIONS OF CHROMATOGRAPHY
Chromatography is a process used to separate
Chromatography is a process used to separate mixtures. The word chromatography is derivedmixtures. The word chromatography is derived from the Greek words "khroma" and "graphein" meaning "color" and "to write" or "to
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 represent". The chromatography technique is first discovered by Russian Biologists, Dr Michael Tswett in 1906 for the separation of
Michael Tswett in 1906 for the separation of colored plant pigment on a column of alumina.colored plant pigment on a column of alumina. Now-a
Now-a-days -days variovarious tyus types opes of chrof chromatomatography graphy are usare used to ed to sepaseparate arate almost lmost any gany given iven mixtmixtureure whether colored or colorless into its
whether colored or colorless into its component.component.
Chromatography may be regarded as an analytical technique employed for the
Chromatography may be regarded as an analytical technique employed for the purification andpurification and separation of organic and inorganic substances. It is also found useful for the fractionation of 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
complex mixture, separation of closely related compounds, such as isomers and in the isolationthe isolation of unstable substances.
of unstable substances.
Although there are several different
Although there are several different types of chromatography, in each case a types of chromatography, in each case a substance issubstance is place
placed onto d onto or intor into a meo a medium dium and a and a solvesolvent is pnt is passeassed throd through tugh the tehe test subst substancstance. Ine. In chromatography science, the solvent is called "the mobile phase" or
chromatography science, the solvent is called "the mobile phase" or "the carrier fluid" "the carrier fluid" and theand the medium is called "the stationary phase".
medium is called "the stationary phase". Types of
Types of ChromatogChromatography:raphy: There are three ways to
There are three ways to classify chromatography. They are-classify chromatography. They are-a)
a) ClasClassificasification otion of chromf chromatogatography araphy accordccording ting to mobio mobile phale phase:se:
1.
1. Liquid chromatography: mobile phase is a liquid. (Liquid-LiquidLiquid chromatography: mobile phase is a liquid. (Liquid-Liquid
Chromatography, Liquid-Solid Chromatography). Chromatography, Liquid-Solid Chromatography).
2.
2. Gas chromatography: mobile phase is a gas. (Gas-Solid Chromatography, Gas-Gas chromatography: mobile phase is a gas. (Solid Chromatography,
Gas-Liquid Chromatography) Liquid Chromatography) b)
b) ClasClassificasification tion accoaccording rding to thto the pace packing king of the of the statstationary ionary phasephase:: 1.
1. Thin laThin layer chromyer chromatogatography (TLraphy (TLC): the staC): the stationationary phase is a thiry phase is a thin layen layer supporter supportedd on glass, plastic or aluminum plates.
on glass, plastic or aluminum plates. 2.
2. PapePaper chromar chromatogratography (PCphy (PC): the sta): the stationationary phase ry phase is a thin fiis a thin film of liqlm of liquiduid supported on an inert support.
supported on an inert support. 3.
c)
c) ClasClassificasification tion accoaccording rding to tto the forhe force oce of sepf separatiaration:on: 1.
1. AdsAdsorporptiotion n chrchromaomatogtograpraphy.hy. 2.
2. PaPartrtititioion n chchroromamatotogrgrapaphyhy.. 3.
3. Ion Ion exexchachange nge chrchromomatoatogragraphyphy.. 4.
4. Gel Gel filfiltratratiotion cn chrohromatmatogographraphy.y. 5.
5. AfAffifininity chty chroromamatotogrgrapaphyhy.. Table 01: Different Types of
Table 01: Different Types of chromatographychromatography
Mechanism Mechanism Mobile phase Mobile phase Stationary phase Stationary phase Mode or type Mode or type
Solutes move at different rates Solutes move at different rates according to the forces of according to the forces of
attraction to the stationary phase. attraction to the stationary phase. Liquid or gas
Liquid or gas Solid that attracts the
Solid that attracts the solutes solutes Adsorption Adsorption Chromatography Chromatography
Solutes equilibrate between the 2 Solutes equilibrate between the 2 phase
phases accs according ording to thto their paeir partitirtitionon coefficients
coefficients Liquid or gas
Liquid or gas Thin film of liquid
Thin film of liquid formed on the surface formed on the surface of a solid inert of a solid inert support support Partition Partition Chromatography Chromatography
Solute ions of charge opposite to Solute ions of charge opposite to the fixed ions are attracted to the the fixed ions are attracted to the resin by electrostatic forces & resin by electrostatic forces & replace the mobile counter-ions. replace the mobile counter-ions. Liquid containing
Liquid containing electrolytes
electrolytes Solid resin that
Solid resin that carries fixed ions & carries fixed ions & mobile counter-ions mobile counter-ions of opposite charge of opposite charge attached by covalent attached by covalent bonds bonds Ion Exchange Ion Exchange Chromatography Chromatography
Molecules separate according to Molecules separate according to their size:
their size: 1.
1. SmalSmaller moleler molecules ecules enter thenter the pores
pores of the of the gel, gel, and neand need aed a larger volume of eluent. larger volume of eluent. Liquid
Liquid Porous gel with no
Porous gel with no attractive action on attractive action on solute molecules solute molecules Molecular Molecular Exclusion Exclusion Chromatography Chromatography
2.
2. LargeLarger molecur molecules pass tles pass throughroughh the column at a faster rate. the column at a faster rate.
Special k
Special kind of sind of solute olute moleculesmolecules interact
interact with with those ithose immobilizedmmobilized on the stationary phase
on the stationary phase Liquid or gas Liquid or gas Solid on which Solid on which specific molecules specific molecules are immobilized are immobilized Affinity Affinity Chromatography Chromatography Applications of
Applications of ChromatoChromatography:graphy:
Chromatographic methods will separate ionic species, inorganic or organic, and molecular
Chromatographic methods will separate ionic species, inorganic or organic, and molecular
species ranging in size from
species ranging in size from the lightest and smallest, helium and hydrogen, to particulatethe lightest and smallest, helium and hydrogen, to particulate
matter such as single cells. No
matter such as single cells. No single configuration will accomplish this, however. Little pre-single configuration will accomplish this, however. Little
pre-knowledge of the constituents of a
knowledge of the constituents of a mixture is required. At its mixture is required. At its best, chromatography willbest, chromatography will
separate several hundreds of
separate several hundreds of components of unknown identity and components of unknown identity and unknown concentrations,unknown concentrations,
leaving the components unchanged
leaving the components unchanged. Amounts in the . Amounts in the picogram or parts per picogram or parts per billion ranges can bebillion ranges can be
detected with some detectors.
detected with some detectors.
The
Thesolutessolutes can range from polar to nonpolar— i.e.,can range from polar to nonpolar— i.e., water-soluble to hydrocarbon-soluble.water-soluble to hydrocarbon-soluble.
Substances of low critical temperature or low
Substances of low critical temperature or low molecular weight, such as the gases at laboratorymolecular weight, such as the gases at laboratory
conditions showing dispersive or London intermolecular forces only, are separated with
conditions showing dispersive or London intermolecular forces only, are separated with
molecular sieves or
molecular sieves or gas-solid techniques. Gas-liquid chromatography is applicable to gas-solid techniques. Gas-liquid chromatography is applicable to speciesspecies
with high critical temperatures and normal boiling points as h
with high critical temperatures and normal boiling points as high as 400° C. Substances that areigh as 400° C. Substances that are
solids at normal laboratory conditions with molecular weights below1000 are best separated
solids at normal laboratory conditions with molecular weights below1000 are best separated
with liquid-solid or liquid-liquid systems. Lower members of
with liquid-solid or liquid-liquid systems. Lower members of the molecular weight scale rangethe molecular weight scale range
are amenable to separations. Methods are involved at molecular weights above 1,000.
are amenable to separations. Methods are involved at molecular weights above 1,000.
Field-flow fractionation extends the size range to
flow fractionation extends the size range to colloids and microscopic particles.colloids and microscopic particles.
General uses of chromatography in our real life are: General uses of chromatography in our real life are:
•
• Pharmaceutical CompaPharmaceutical Company – determine amount of ny – determine amount of each chemical found in new product.each chemical found in new product.
•
• Hospital – detect blood or alcohol levels Hospital – detect blood or alcohol levels in a patient’s blood stream.in a patient’s blood stream.
•
• Law Enforcement – to compare a sample found at a crime scene to samples fromLaw Enforcement – to compare a sample found at a crime scene to samples from suspects.
•
• Environmental Agency – determine the level of pollutants in the Environmental Agency – determine the level of pollutants in the water supply.water supply.
•
• Manufacturing Plant – to purify Manufacturing Plant – to purify a chemical needed to make a product.a chemical needed to make a product.
•
• Biotechnology industry – establishing the purity or concentration of compounds inBiotechnology industry – establishing the purity or concentration of compounds in biot
biotechnoechnologilogical recal researchsearch.. •
• Biological applicatioBiological application – n – Chromatography has many applications in biology. It is used toChromatography has many applications in biology. It is used to separate and identify amino acids, carbohydrates, fatty acids, and
separate and identify amino acids, carbohydrates, fatty acids, and other naturalother natural substances. Environmental testing laboratories use chromatography to identify substances. Environmental testing laboratories use chromatography to identify tracetrace quantities of contaminants such as PCBs in waste oil and
quantities of contaminants such as PCBs in waste oil and pesticides such as DDT inpesticides such as DDT in groundwater. It is also used
groundwater. It is also used to test drinking water and to test drinking water and test air quality. Pharmaceuticaltest air quality. Pharmaceutical companies use chromatography to prepare quantities of extremely pure materials. The companies use chromatography to prepare quantities of extremely pure materials. The food industry uses chromatography to detect contaminants such as aflatoxin.
food industry uses chromatography to detect contaminants such as aflatoxin. For example we can consider the use
For example we can consider the use of chromatography in Forensic Toxicology.of chromatography in Forensic Toxicology. Forensic toxicology is the application of toxicology for the
Forensic toxicology is the application of toxicology for the purpose of law.purpose of law. Forensic Chemistry and Toxicology generally concerns
Forensic Chemistry and Toxicology generally concerns the detection and characterizationthe detection and characterization of poisons or
of poisons or toxins exhibiting adverse physiological effects. Laboratory methods used intoxins exhibiting adverse physiological effects. Laboratory methods used in chemical toxicologica
chemical toxicological analysis cover a l analysis cover a wide range and may be broadly classified aswide range and may be broadly classified as follows: (1) physical tests (2) crystal tests (3) chemical spot tests (4) spectrophotometric follows: (1) physical tests (2) crystal tests (3) chemical spot tests (4) spectrophotometric tests (5) chromatographic tests.
tests (5) chromatographic tests. Toxicology is the study of
Toxicology is the study of substancesubstances that are s that are harmful to human beings. Forensicharmful to human beings. Forensic
Toxicologists have the responsibility of detecting and identifying the presence of drugs Toxicologists have the responsibility of detecting and identifying the presence of drugs andand poiso
poisons in ns in fluidfluids, tiss, tissues sues and oand organsrgans. The. Their seir servicervices are nos are not just just reqt required uired in criin crimeme laboratories and medical examiner's offices; they reach in
laboratories and medical examiner's offices; they reach into hospital laboratories where theto hospital laboratories where the identification of an overdose can mean the difference between life and death. The work of a identification of an overdose can mean the difference between life and death. The work of a toxicologist generally falls into three main categories:
toxicologist generally falls into three main categories:
1.
1. Routine testing for alcohol in blood or Routine testing for alcohol in blood or urine samples following a "breathalyzer" test.urine samples following a "breathalyzer" test.
However with the advent of n
However with the advent of new technology much of the testing can be carried ew technology much of the testing can be carried out byout by polic
police wite with feweh fewer caser cases beins being pasg passed osed on to tn to the forehe forensic nsic laborlaboratoryatory. Like . Like GasGas chromatography was, and still is, used to
chromatography was, and still is, used to identify exactly how much alcohol is presentidentify exactly how much alcohol is present in the blood or urine of a suspect. Because it is very accurate, results can be used as in the blood or urine of a suspect. Because it is very accurate, results can be used as evidence in a court of law.
2.
2. Identification of drugs such as heroin, cocaine, cannabis, etc. It is common to performIdentification of drugs such as heroin, cocaine, cannabis, etc. It is common to perform
preli
preliminarminary coly color or mor or microcicrocrystarystalline lline testtests befors before usie using chrng chromatomatograography tphy to ideo identify antify a substance as chromatography yields somewhat inconclusive results. However, both substance as chromatography yields somewhat inconclusive results. However, both thinthin layer and gas chromatography in conjunction with the preliminary tests is
layer and gas chromatography in conjunction with the preliminary tests is well suitedwell suited for drug analysis.
for drug analysis.
3.
3. Detection of drugs and poisons in body fluids, tissues and organs. This area of forensicDetection of drugs and poisons in body fluids, tissues and organs. This area of forensic
toxicology involves the analysis of organs, tissues and body f
toxicology involves the analysis of organs, tissues and body fluids in suchluids in such
circumstances as sudden deaths and suspected poisonings. A post mortem is performed circumstances as sudden deaths and suspected poisonings. A post mortem is performed by a p
by a pathoathologilogist who st who sendsends spes specimecimens of vns of variouarious bods body tisy tissues sues and fland fluids uids to the to the forensforensicic toxicologist for examination. Many technique
toxicologist for examination. Many techniques are s are used in this area of used in this area of work includingwork including chromatography
chromatography
Depend on the mechanisms different types of
Depend on the mechanisms different types of chromatography are specified in uses of chromatography are specified in uses of differentdifferent actions. Applications according to the
actions. Applications according to the mechanism are given bellow:mechanism are given bellow: •
• Applications of Paper Chromatography:Applications of Paper Chromatography:
1.
1. PapePaper chromatr chromatograpography has widhy has widely beely been used for quen used for quantitantitative anative analysalysis of Inorgais of Inorganic,nic, organic and biochemical interest.
organic and biochemical interest. 2.
2. PapePaper chromatr chromatograpography is ideahy is ideally suitlly suited for rapid aed for rapid analysnalysis of reactis of reaction mixtion mixture and so it isure and so it is versatile tool in the hand of
versatile tool in the hand of organic chemists.organic chemists. 3.
3. PapePaper chromatr chromatograpography has beehy has been succesn successfullsfully used for characy used for characterizterizing and isoling and isolating theating the following organic compounds.
following organic compounds.
• • Acids,Acids, • • Alcohols,Alcohols, • • Glycols,Glycols, • • Alkaloids,Alkaloids, • • Amines,Amines, •
• Amino acidsAmino acids
•
• Proteins and peptides,Proteins and peptides,
•
• Antibiotics etc.Antibiotics etc.
4.
4. PapePaper chromar chromatogrtography haaphy has also bs also been useen used in the aed in the analysnalysis of mixtis of mixture of sugure of sugars.ars. 5.
6.
6. It can be useIt can be used for the identd for the identificaification of cotion of compoumpounds in drugnds in drugs, in biochs, in biochemicemical prepaal preparatiorationn and in natural products. It can be used for checking the purity of samples.
and in natural products. It can be used for checking the purity of samples. •
• Applications of Gel-filtration ChromatographyApplications of Gel-filtration Chromatography
1.
1. Analytical Analytical applicationsapplications
Analytical group separationAnalytical group separation
Analytical fractionationAnalytical fractionation
Determination of molecular masses: Determination of M. wt. of pDetermination of molecular masses: Determination of M. wt. of peptides,eptides, prote
proteins & pins & polysolysacchaaccharidesrides 2.
2. Preparative Preparative applicationsapplications
Preparative fractionationPreparative fractionation
Preparative group separationsPreparative group separations
Separation of mixture of Separation of mixture of mono-and polysaccharides.mono-and polysaccharides.
Separation of amino acids from peptides & Separation of amino acids from peptides & proteins.proteins.
Separation of proteins of diffSeparation of proteins of different molecular weights.erent molecular weights.
Separation of mucopolysaccharides & soluble RNA.Separation of mucopolysaccharides & soluble RNA.
Separation of myoglobin & Separation of myoglobin & haemoglobin.haemoglobin.
Separation of alkaloids & purification of enzymes.Separation of alkaloids & purification of enzymes. 3.
3. Biochemical applicationsBiochemical applications: In : In general, Gel-filtration chromatography which is general, Gel-filtration chromatography which is alsoalso known as Size Exclusion chromatography is considered a low resolution
known as Size Exclusion chromatography is considered a low resolution chromatography as it does not discern similar sp
chromatography as it does not discern similar species very well, and is thereforecies very well, and is therefore oftene often reserved for the
reserved for the final "polishing" step of purification. The technique can determine thefinal "polishing" step of purification. The technique can determine the quaternary structure of purified p
quaternary structure of purified proteins that have slow exchange times, since it can beroteins that have slow exchange times, since it can be carried out under
carried out under native solution conditions, preserving macromolecular interactions.native solution conditions, preserving macromolecular interactions. Size Exclusion chromatography can also assay protein tertiary structure, as
Size Exclusion chromatography can also assay protein tertiary structure, as it measuresit measures the hydrodynamic volume (not molecular weight), allowing folded and unfolded
versions of the same protein
versions of the same protein to be distinguished. For example, the apparentto be distinguished. For example, the apparent
hydrodynamic radius of a typical protein domain might be 14 Å and 36 Å for the folded hydrodynamic radius of a typical protein domain might be 14 Å and 36 Å for the folded and unfolded forms,
and unfolded forms, respectivelyrespectively. Size . Size Exclusion chromatography allows the separationExclusion chromatography allows the separation of these two forms, as the folded form will elute much later due to its smaller size. of these two forms, as the folded form will elute much later due to its smaller size. 4.
4. Polymer synthesis: Gel-filtration chromatography or Polymer synthesis: Gel-filtration chromatography or Size Exclusion chromatographySize Exclusion chromatography can be used as a
can be used as a measure of both the size measure of both the size and the polydispersity of a synthesizedand the polydispersity of a synthesized poly
polymermer, that , that is, tis, the abhe ability ility to be to be able table to find o find the dthe distriistributiobution of tn of the sizhe sizes oes of polyf polymer mer molecules. If standards of
molecules. If standards of a known size are run a known size are run previouslypreviously, then a , then a calibration curve cancalibration curve can be cre
be created ated to deto determtermine tine the sizhe sizes oes of polyf polymer mmer molecolecules oules of intef interest irest in the n the solvesolvent chont chosensen for analysis. In
for analysis. In alternative fashion, techniques such as light scattering and/or viscometryalternative fashion, techniques such as light scattering and/or viscometry can be used online with Size Exclusion chromatography to yield absolute molecular can be used online with Size Exclusion chromatography to yield absolute molecular weights that do not rely on
weights that do not rely on calibration with standards of known molecular weight. Duecalibration with standards of known molecular weight. Due to the difference in size of
to the difference in size of two polymers with identical molecular weights, the absolutetwo polymers with identical molecular weights, the absolute determination methods are, in
determination methods are, in general, more desirable. A typical Size Exclusiongeneral, more desirable. A typical Size Exclusion chromatography system can quickly (in about half an hour)
chromatography system can quickly (in about half an hour) give polymer chemistsgive polymer chemists information on the size and polydispersity of
information on the size and polydispersity of the sample. The preparative Sizethe sample. The preparative Size Exclusion chromatography can be used for polymer fr
Exclusion chromatography can be used for polymer fractionation on an analytical scale.actionation on an analytical scale. •
• Applications of Thin Layer Applications of Thin Layer ChromatographyChromatography
1.
1. As a check on process: It has been used for checking of the other separationAs a check on process: It has been used for checking of the other separation
proce
procedures dures and pand purificurificatioation procn processeesses.s. 2.
2. In In OrOrgaganic nic ChChememisistrtry:y:
The main use of Thin The main use of Thin Layer Chromatography is isolation and separation of Layer Chromatography is isolation and separation of
individual components of a
individual components of a mixture.mixture.
The main reasons for popularity of The main reasons for popularity of Thin Layer Chromatography as an analyticalThin Layer Chromatography as an analytical and preparation methods are:
and preparation methods are:
It can be used for most of chemical compounds.It can be used for most of chemical compounds.
It has high speed of separation.It has high speed of separation.
The following are the various applications of The following are the various applications of Thin Layer chromatography inThin Layer chromatography in
organic chemistry: organic chemistry:
For checking the purity samples as a For checking the purity samples as a purification process.purification process.
Examination of reactions.Examination of reactions.
For identifying organic compounds.For identifying organic compounds.
Thin Layer Chromatography has been successfully used for characterizing andThin Layer Chromatography has been successfully used for characterizing and isolating the following organic
isolating the following organic compounds.compounds. i. Acids i. Acids ii. Alcohols ii. Alcohols iii. Glycols iii. Glycols iv. Alkaloids iv. Alkaloids v. Amines v. Amines
vi. Amino acids, proteins and vi. Amino acids, proteins and pept
peptidesides
vii. Antibiotics vii. Antibiotics Besides these, there are
Besides these, there are compounds like carbohydrates, carbonyl compounds, Dyes,compounds like carbohydrates, carbonyl compounds, Dyes, Hydrocarbons, lipids, nucleic acids, p
Hydrocarbons, lipids, nucleic acids, pesticides, natural pigments, pharmaceutical products,esticides, natural pigments, pharmaceutical products, pheno
phenols, stls, steroideroids, terps, terpenesenes, esse, essential ntial oils, oils, vitavitamins, mins, adheadhesivesives, expls, explosiveosives s plastplasticizeicizers etcrs etc.. which have been separated and characterized by Thin Layer Chromatography.
which have been separated and characterized by Thin Layer Chromatography. 3.
3. For For sepseparaaratiotion of n of InoInorgarganic nic IonIons:-
s:-
Recently Thin Layer Chromatography has been used for separatingRecently Thin Layer Chromatography has been used for separating
cationic,
cationic, anionic, anionic, purely purely covalent covalent species species and aand also somlso some organic e organic derivatives derivatives of theof the metals.
metals.
4.
4. Applications of Thin Layer Applications of Thin Layer Chromatography in quantitative analysis:Chromatography in quantitative analysis:
i.
i. SpectrophotomeSpectrophotometrictric Measurement
Measurement
ii. Fluorimetric Method ii. Fluorimetric Method
iii. Visual comparison of spots iii. Visual comparison of spots iv. Spectral reflectance
iv. Spectral reflectance
v. Spot densitometer v. Spot densitometer
vi. Vapour phase chromatography vi. Vapour phase chromatography vii. Radioactive methods
vii. Radioactive methods viii. Volumetric analysis viii. Volumetric analysis
•
• Application of Column Application of Column Chromatography:-
Chromatography:-1.
1. Analytical uses: For analytical purposes, column Analytical uses: For analytical purposes, column chromatography finds limitedchromatography finds limited
applications. V
applications. Vestergaard and Sayegh estergaard and Sayegh could separate seven could separate seven urinary steroids urinary steroids within 5within 5 hours which requires 36 hours
hours which requires 36 hours on a normal column. They have used narrow Teflonon a normal column. They have used narrow Teflon tubing packed with aluminum oxide or silica gel.
tubing packed with aluminum oxide or silica gel.
2.
2. Separation of geometrical isomers: The separation of cis/trans isomer is Separation of geometrical isomers: The separation of cis/trans isomer is based on thebased on the
steric factors. Isomers whose functional groups can approach the surface of the steric factors. Isomers whose functional groups can approach the surface of the adsorbent more easily are more strongly adsorbed.
adsorbent more easily are more strongly adsorbed. 3.
3. SepSeparaaratiotion n of of DiaDiastestereoreomermers.s.
4.
4. Separation of tautomeric mixtures.Separation of tautomeric mixtures.
5.
5. SeSepapararatition oon of raf racecemamatetes.s. •
• Applications of Affinity chromatography:Applications of Affinity chromatography:
1.
1. Purify and concentrate a substance from a Purify and concentrate a substance from a mixture into a buffering solution.mixture into a buffering solution.
2.
2. Reduce the amount of a substance in Reduce the amount of a substance in a mixture.a mixture.
3.
3. Discern what biological compounds bind to a particular substance, such asDiscern what biological compounds bind to a particular substance, such as
drugs. drugs. 4.
4. PPururifify y aand nd coconcnceentntrarate te aan en enznzyyme me ssololututioion.n. •
• Applications of Ion Exchange Chromatograph:Applications of Ion Exchange Chromatograph:
1.
1. WWater softening: Removal of ater softening: Removal of Ca2+, Mg2+ & other Ca2+, Mg2+ & other multivalent ions causing hardness of multivalent ions causing hardness of
water by filtration through a layer of strong cation resin. water by filtration through a layer of strong cation resin.
2.
2. Separation of electrolytes from non-electrolytes.Separation of electrolytes from non-electrolytes.
3.
3. Neutraliz Neutralizationation: Cat: Cationic ionic excexchangehanger in [Hr in [H++] can be used to ] can be used to neutralize alkali hydroxide &neutralize alkali hydroxide &
anionic exchanger in [OH
anionic exchanger in [OH--] form ] form to neutralize the acidity.to neutralize the acidity. 4.
4. Water demineralization:Water demineralization: Removal of cations & Removal of cations & anions dissolved in water. Usuallyanions dissolved in water. Usually
carried by the two step
exchanger in [H
exchanger in [H++] form & strongly basic anion exchanger in [OH] form & strongly basic anion exchanger in [OH--] form are used in] form are used in
sequence sequence.. 5.
5. SepaSeparatioration of n of carbocarbohydrathydrates es & t& their dheir derivaterivativesives::
Uronic acids separated on Uronic acids separated on anion exchangeranion exchanger..
Sugars converted into ionized form by Sugars converted into ionized form by using borate& separated on strong anionusing borate& separated on strong anion
exchanger. exchanger.
HexosaminesHexosamines separated on separated on strong cation exchanger.strong cation exchanger.
•
• Medical ApMedical Applications of plications of High Performance Liquid High Performance Liquid Chromatography:Chromatography: The isolation and purification of compounds is of
The isolation and purification of compounds is of critical importance to medicinalcritical importance to medicinal chemists discovering and developing drugs. In these activities it is
chemists discovering and developing drugs. In these activities it is extremely important toextremely important to be ab
be able tle to prodo produce muce mg qug quantitantities of ies of the tathe target crget compoompoundsunds, from , from a syna synthetthetic reic reactioactionn mixture. Then later on use the
mixture. Then later on use the same separation technologies to provide gram and Kilosame separation technologies to provide gram and Kilo quantities. Similarly, the technique can
quantities. Similarly, the technique can isolate, purify and isolate, purify and concentrate suitable quantitiesconcentrate suitable quantities of low level impurities and metabolites to support compound identification and further of low level impurities and metabolites to support compound identification and further characterization.
characterization.
One of the key advantages of
One of the key advantages of high performance counter current chromatography is that ithigh performance counter current chromatography is that it scales easily and simply. This allows the medicinal chemist to focus on
scales easily and simply. This allows the medicinal chemist to focus on the value-addedthe value-added part
part of the of the devedevelopmelopment procnt process iess i.e. t.e. the chehe chemistmistry, and ry, and allealleviateviates the s the need need to wato waste tste timeime developing scaled-up chromatograp
developing scaled-up chromatography techniques, as demand for hy techniques, as demand for the quantity of the quantity of compound increases.
compound increases.
A further important issue is
A further important issue is sample solubility since this can affect the throughput tosample solubility since this can affect the throughput to produ
produce a ce a specspecified ified quantquantity oity of the f the compocompound ound of intef interest. Trest. Typicaypically, tlly, this cahis can ben become come anan issue when the purification is performed in
issue when the purification is performed in reverse phase (RP) which generates aqueousreverse phase (RP) which generates aqueous fractions. These aqueous fractions are laborious to
fractions. These aqueous fractions are laborious to concentrate by evaporation and thisconcentrate by evaporation and this proce
process cass can lean lead to dd to degraegradatidation of ton of the prohe productduct.. As scale of production increases the volumes of
As scale of production increases the volumes of solvents used and those of productsolvents used and those of product containing fractions also increase. This is particularly an issue
containing fractions also increase. This is particularly an issue where Reverse Phase-where Reverse Phase-High Performance Liquid Chromatography produces large volumes of aqueous fractions. High Performance Liquid Chromatography produces large volumes of aqueous fractions. High performance counter current chromatography can be used in
same separation which means that fractions can be collected in essentially non-aqueous same separation which means that fractions can be collected in essentially non-aqueous solvents which makes their processing simpler, faster and
solvents which makes their processing simpler, faster and less energy consuming.less energy consuming. High Performance Liquid Chromatography has f
High Performance Liquid Chromatography has found many applications in medicinalound many applications in medicinal chemistry, to which the Spectrum or Midi bench top
chemistry, to which the Spectrum or Midi bench top products are normally found to products are normally found to bebe the most suitable for the