CHROMATOGRAPHYCHROMATOGRAPHY Dr. Tasnim AraDr. Tasnim Ara

MD(part-I)MD(part-I) Phase -A StudentPhase -A Student

Department of BiochemistryDepartment of Biochemistry Sir Salimullah Medical CollegeSir Salimullah Medical College

ChromatographyChromatographyA group of separation techniques that separate

analytes by differential distribution between a stationary phase and a mobile phase.– mobile phase = solvent– stationary phase = column packing material

HistoryHistory •1906 Tsvett – • A scientist named tsvett separated a mixture of plant pigments by adsorbing on finely divided chalk & components of the mixture are separated as coloured band . The process is denoted as chromatography.•1931 Lederer & Kuhn - LC of carotenoids•1938 TLC and ion exchange•1950 Reverse phase LC•1954 Martin & Synge (Nobel Prize)•1959 Gel permeation•1965 instrumental LC (Waters)

MeaningMeaning.….…• Chromatography is derived

from Greek terms chromo-color & gram-bands.

• Hence as the name indicates, in chromatography there is the formation of colored bands.

• These bands are indicative of different components in the sample.

PurposePurpose AnalyticalAnalytical - determine chemical composition of a

samplePreparativePreparative - purify and collect one or more components of a sample Importance Chromatography has application in every Chromatography has application in every branch of the physical and biological sciencesbranch of the physical and biological sciences

1212 Nobel prizes were awarded between 1937 and Nobel prizes were awarded between 1937 and 1972 alone for work in which chromatography 1972 alone for work in which chromatography played a vital roleplayed a vital role

Real UseReal UseReal-life examples of uses for chromatography:

• 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• Environmental Agency – determine the level of pollutants in the water supply• Manufacturing Plant – to purify a chemical needed to make a product

DefinitionDefinitionChromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other moves one definite direction (mobile phase). Chromatography is a qualitative and quantitative analytical technique where in a sample mixture under test is subjected to preferential separation into different components under the influence of a moving phase (mobile phase) over a stationary phase. These separated components are later identified for their quantity.

PhasesPhases• Stationary phase- a bed or column of supportive

material of solid, liquid or gel.• Mobile phase- flowing stream of gas or liquid in

a definite direction

Chromatographic principleChromatographic principle

1. Sample is introduced into the mobile flowing phase that passes through the appropriate stationary phase

2. A varieties of attractive forces between the stationary phase and the components of the sample causes selective and differential movements of the analytes along the mobile phase

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3. The solutes that do not interact with the stationary phase, remain in the mobile phase and comes out first, a solute with greater affinity will move slowly than a solute with lesser affinity.4. The mobile phase with separated solute zones emerges from the column and allowed to pass through a detector or a series of detectors. 5. The detector detects the solute zones and forward an electronic signal to a processor. Eluted solutes are displayed graphically as a series of peaks.

ClassificationClassification.According to the patterns of stationary

phase 1. planar chromatography

2. column chromatography•Technique by separation mechanisms

1. adsorption chromatography2. affinity chromatography3. ion exchange chromatography4. partition chromatography5.size exclusion chromatography.

•Depending on the patterns of mobile phase column chromatography is of two types-

1. gas chromatography 2. liquid chromatography

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1. Planar Chromatography- stationary phase is contained or spread on a flat surface such as a sheet of paper or a glass plate.

2. Column Chromatography- support particles on which the stationary phase is coated or chemically bonded, are packed into a tube or the tube has a coating of the stationary phase onto its inner surface, having an open path in the middle of the tube.

Planar chromatographyPlanar chromatography 2 types1. Paper chromatography - stationary phase

is a layer of water or a polar solvent coated onto the paper fibers.

2. Thin layer chromatography- a thin layer of large particles of a material, such as silica gel (0.2 mm thick) is spread uniformly on a glass plate or a plastic sheet.

Paper chromatographyPaper chromatography In paper chromatography , the stationary

phase is a layer of water or a polar solvent coated into the paper fibers.This paper is made of cellulose,a polar substance,and the compounds within the mixture travel further.

Use of paper chromatographyUse of paper chromatography

1. Identification of unknown organic and inorganic compounds from mixture.2. In forensic study it is used in crime secne investigation.3. Sugar,AA,lipid, NA can easily identified.

Thin layer chromatographyThin layer chromatography Stationary phage is constructed as a thin layer

of sorbent, such as silica gel is spread uniformly ( 0.2 mm thick) on a glass plate/ plastic sheet/ aluminum sheet.

Sorbent may be- silica gel- microcellulose

An appropriate organic solvent is detected as mobile phase.

The specific Retension factor Rf of each chemical can be used to aid in the identification of an unknown sub.

TLCTLC....

RRff value valueThe RThe Rff value for each component is worked  value for each component is worked out using the formulaout using the formula::

  

For example, if the red component travelled For example, if the red component travelled 1.7 cm from the base line while the solvent 1.7 cm from the base line while the solvent had travelled 5.0 cm, then the Rhad travelled 5.0 cm, then the Rffvalue for value for the red dye isthe red dye is::

TLC UseTLC Use Analysis of drugs and metabolites in

serum,urine,and body fluids. Separation and quantification of

phospholipid,VMA etc.

Advantage of TLCAdvantage of TLC

1. Ease with which compounds are separated and visualized

2. Potential to chromatograph the sample directly without clean up step

3. Capacity to analyze multiple samples in a single run

4. Relatively low cost

Gas chromatographyGas chromatography• One type of planar chromatography.Here mobile phase is

gas.it always carried out in a column ,which is typically packed or capillary.

• Liquid chromatography• Here mobile phase is liquid.it can be carried out either in

a column or plane• HPLC(high performance liquid chromatography)

High performance liquid High performance liquid chromatography(HPLC)chromatography(HPLC)

• It is a highly improved form of column chromatography.Here a tightly packed column of stationary phase is constructed by very small particle.Then a liquid mobile phase is allowed to pass through the stationary phase under high pressure .One detector is used outside the column for individual detection and is reabsorbed quantitatively in computer software in the form of peaks.The peaks are then compared with standard.

HPLCHPLC.….…

Use of HPLCUse of HPLC Used for chemical and biochemical

research. Purification of chemical compound . Used in quality control to ensure the purity

of raw materials Used to analysis the air and water

pollutants. Monitoring of pesticide level in

environment

Adsorption ChromatographyAdsorption Chromatography•Active adsorption occurs at the surface of the solid stationary phase column in a glass tube and the mobile phase is gas or liquid (GC or LC)•.Least adsorbed solutes will come first and strongly adsorbed solutes will come last

Types of adsorbents used in LC- charcoal, polystyrene divinyl benzene (non polar), silica gel (acidic polar), alumina (basic polar)

Types of adsorbents used in GC- polystyrene divinyl benzene (non polar), alumina (basic polar)

Partition ChromatographyPartition Chromatography •Differential distribution of solutes between two immiscible liquids is the basis for separation.one immiscible lipid serves as stationary phase.separation is based on differences in the relative solublity of solute bet.two phase.Types•Gas liquid chromatography•Liquid liquid chromatography

Ion exchange chromatographyIon exchange chromatography• Involves separation of the molecules on the

basis of their electrical charges.• Principle: Surface of the particles of plastic

resin or silica serve as stationary phase. Stationary phase has functional groups with fixed anionic or cationic charges. To maintain electrical neutrality, an exchangeable ion termed counter ion is found in close proximity to the fixed charge. Solute ion in the mobile phase exchange with the counter ions. The solute ion are then eluted selectively by the variation of the mobile phase pH, or ionic strength or both.

Ion exchange resignIon exchange resign Types –1. cation exchange resin .Contain covalently bound negatively

charged functional groups.• 2. anion exchange resin . .Contain covalently bound positively

charged functional groups.

example of ion exchange resin• Poly styrene resins- Dowex 1, Dowex 50• Diethyl aminoethyl cellulose.

Ion exchange chro.useIon exchange chro.use

1. Separation of amino acids, proteins, oligonucleotides, nucleotides, nucleic acids

2. Deionization of water3. Separation of Hb variants4. Separation of isoenzymes of CK, LDH5. Removal of interfering ions from mixtures

e.g. urinary porphobilinogens

Size exclusion chromatographySize exclusion chromatography Also known as steric exclusion, gel filtration, gel permeation, molecular exclusion, molecular sieve chromatography

• This type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.

Affinity chromatographyAffinity chromatography Principle is based on the property of

specific and non covalent binding of proteins to other molecules, referred to as ligands.

Specificity resulting from enzyme- substrate, hormone- receptor, antigen- antibody has been used Uses-

1. LDL and VLDL separated with heparin columns

2. glycated Hbs separated by phelyl boronate columns.

Identifying the Spots (visualization)Identifying the Spots (visualization)If the spots can be seen, If the spots can be seen, outline them with a penciloutline them with a pencil..

If no spots are obvious, the If no spots are obvious, the most common visualization most common visualization technique is to hold the technique is to hold the plate under a UV lampplate under a UV lamp..Many organic compounds can Many organic compounds can be seen using this be seen using this technique, and many technique, and many commercially made plates often contain a substance often contain a substance which aids in the which aids in the visualization of compoundsvisualization of compounds..

Visualizing AgentsVisualizing Agents

Alkaloids: DragendorffAlkaloids: Dragendorff’’s reagents reagent

Cardiac glycosides: Antimony trichlorideCardiac glycosides: Antimony trichloride

Sugar: Aniline phthalateSugar: Aniline phthalate

Amino acids: NinhydrinAmino acids: Ninhydrin

Interpreting the DataInterpreting the Data

The RThe Rff (retention factor) value for each spot should be (retention factor) value for each spot should be calculatedcalculated . .

It is characteristic for any given compound on the same It is characteristic for any given compound on the same stationary phase using the same mobile phase for stationary phase using the same mobile phase for

development of the platesdevelopment of the plates . .Hence, known RHence, known Rff values can be compared to those of values can be compared to those of

unknown substances to aid in their identificationsunknown substances to aid in their identifications . .

ChromatogramChromatogram• A chromatogram is a representation of

the separation that has chemically [chromatographically] occurred in the HPLC system.

• A series of peaks rising from a baseline is drawn on a time axis.

• Each peak represents the detector response for a different compound.

• The chromatogram is plotted by the computer data station

Common detectorsCommon detectors

1. Flame ionization detector (FID)2. Photo-ionization detector (PID)3. Thermal conductivity (TCD)4. Electron capture detector (ECD)5. Mass spectrometry

Use of ChromatographyUse of Chromatography..1. Separation and measurement of plasma

proteins.2. Separation of LDL and VLDL3. Separation of aa,peptides,and protein.4. Separation of neucleiotides5. Separation of haemoglobin variants6. Separation of isoengymes7. Analysis of drugs,urine,blood sample.8. Use in forensic study.

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