Chromatography, HPLC

Dr.ANSHUL

HistoryMikhail Tswett, Russian,

Botanist

In 1906 used chromatography to separate plant pigments.

He called the new technique chromatography because the result of the analysis was 'written in color' along the length of the adsorbent column.

Chroma means “color” and graphein means to “write”

Importance

Chromatography has application in every branch of the physical and biological sciences12 Nobel prizes were awarded between 1937 and

1972 alone for work in which chromatography played a vital role

Chromatography 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 (mobile phase) moves in a definite direction.

The chromatographic process occurs due to differences in the distribution constant of the individual sample components.

Chromatography Technique used to separate and identify the components of a mixture.

Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium.

Molecules that spend most of their time in the mobile phase are carried faster.

Classification of chromatography according to mobile phase:

1- Liquid chromatography: mobile phase is a liquid. (LLC, LSC).

2- Gas chromatography : mobile phase is a gas. (GSC, GLC).

Classification according to the packing of the stationary phase:

1- Thin layer chromatography (TLC): stationary phase is a thin layer supported on glass, plastic or aluminium plates.

2- Paper chromatography (PC): stationary phase is a thin film of liquid supported on an inert support.

3- Column chromatography (CC): stationary phase is packed in a glass column.

Classification according to the force of separation:

1- Adsorption chromatography.2- Partition chromatography.3- Ion exchange chromatography.4- Gel filtration chromatography.5- Affinity chromatography.

Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase.

The stationary phase: a thin layer of adsorbent (usually silica gel or

alumina) coated on a plate. The mobile phase: a developing liquid which travels up the stationary

phase, carrying the samples with it. Components of the samples will separate on the

stationary phase according to- how much they adsorb on the stationary phase

versus how much they dissolve in the mobile phase.

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

ProcedureA TLC plate is a sheet of glass, metal, or plastic which is

coated with a thin layer of a solid adsorbent (usually silica or alumina).

A small amount of the mixture to be analyzed is spotted near the bottom of this plate.

The TLC plate placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid.

This liquid, mobile phase, slowly rises up the TLC plate by capillary action.

As the solvent moves, an equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and the molecules which are in solution.

components differ in solubility and in the strength of their adsorption to the adsorbent so components will be carried farther up on the plate than others.

When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized.

Identification of the Spots If the spots can be seen, outline them

with a pencil.

If no spots are obvious after staining- hold the plate under a UV lampMany organic compounds can be

seen using this technique.many commercially made plates also

contain a substance which aids in the visualization of compounds.

Visualizing Agents

Ninhydrin- Amino acids: Rhodamine B- Lipids.Antimony trichloride- SteroidsAniline phthalate- Sugar.

Interpreting the Data

The Rf (retention factor) value for each spot should be calculated.

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

Hence, known Rf values can be compared to those of unknown substances to aid in their identifications.

Application

• Identification of drugs in urine.• Analysis of amino acids & carbohydrates in urine.• Analysis of lipids in amniotic fluid.(L/S ratio in RDS.)

Limitation-

Two different substance may exhibit same Rf value.

Paper Chromatography

A method of partition chromatography using filter paper strips as carrier or inert support.

The factor governing separation of mixtures of solutes on filter paper is the partition between two immiscible phases.

One is usually water adsorbed on cellulose fibres in the paper (stationary phase).

The second is the organic solvent flows and migrate the sample on the paper (mobile phase).

Paper Chromatography

Paper Chromatography

Application-• Detects amino acid, sugar and pigments.• Identification of type of aminoaciduria-

Cystinuria, PKU.

Gas Liquid Chromatography• Type of partition chromatography used for

qualitative and quantitative analysis of large no. of compounds.

• It provides high speed of resolution very good reproducibility and high sensitivity.

• It is based on partition of a compound between liquid and gas phase.

Gas Liquid Chromatography

• Principle :–Volatile molecule (lowest boiling point)

swept rapidly along with carrier gas.–Molecules soluble in liquid phase – retained

in liquid . Volatility and solubility affect – rate of flow of sample molecule in gas column.

Gas - Liquid Chromatography

Sample introduced by syringe.

Column separates components.(Heated in oven)

Detector monitors compounds emerging from outlet.

Recorder plots signals as a chromatogram.

Uses Of GLC

• Volatile organic compound separation.• Non volatile substance separation (converting

into volatile by oxidation, acylation, alkylation)• Alcohol, ester, fatty acid and amines present in

biological sample can be separated.• Concentration of individual element such as

carbon and hydrogen - determined accurately.• Functional group can be identified.• Position of double bond can be determined by

cleaving the bond by oxidation.

Adsorption ChromatographyPrinciple :• It is differential adsorption of solute on the

surface of stationary phase.• Certain solid material have ability to hold

molecule at their surface.• This adsorption forces involves weak & non

ionic forces such as hydrogen, Vanderwal’s forces.

• This is used for separation of organic compound.

Adsorption Chromatography

Adsorbent :• Should be inert (should not react with eluting solvent).• Should be stable for long peroid.• Should be cheap.Most commonly used adsorbent are :• Silica gel- AA and steroids.• Alumina -small organic molecule .• Activated carbon –carbohydrate and proteinMost strongly adsorb component forms top most band while least adsorb component forms lower most band on adsorbent media

Adsorption Chromatography

Factors Affecting :• Nature of adsorbent.• Nature of solvent.• Rate of flow of solvent through column.• Dimension of column.• Temperature of column during separation.

Adsorption Chromatography

Applications : Separation of• Aromatic compound phenol, amine etc.• Aliphatic hydrocarbon from aromatic

hydrocarbon• Nucleic acid

Ion Exchange ChromatographyPrinciple :Based on exchange of ions b/w charges stationary phase and ions of opposite charge in mobile phase.Soluble mixture to be separated, allowed to pass through a column of ion exchange resins which are insoluble polymer containing ionic group.Ion exchange resin made of-cellulose, dextran, acrylamide polymer etc.

Ion Exchange Chromatography

Ion exchange resin : two type1: Cation exchange resin- contain negatively charge ions (-carboxyl, -phosphate ions and bind covalently to positively charge ion in mobile phase.2: Anion exchange resin-contain positively charge ion & binds to negatively charged ions in mobile phase

Ion Exchange Chromatography

Application: Separation of – Amino acid – Glycated hemoglobin– Oligonucleotides – Removal of inorganic ions from aqueous mixture.

Disadvantage :– Different substance with same ionic charge – can

not be separated

Gel Filtration Chromatography

Syn.(size exclusion, gel permeation, molecular exclusion chromatography)Principle :Separation based on –molecular size, shape, weight.A mixture of molecules of different size placed on top of gel filtration column large molecule pass through interstitial space b/w beads without any resistanceSmall molecule enter in pores of beads and effectively removed from stream of eluting solvent.Movement of small molecule is retarted- eluting out slowly

Gel Filtration ChromatographyAdvantage• Separation of substance - independent of ph,

temperature, ionic strength and buffer composition.

• No adsorption- labile substance (enzyme) not affected

• Elution volume- directly related to molecular weight.

Gel Filtration Chromatography

Application :• Use to purifying enzymes and other

proteins and fractionating nucleic acid.• To determine molecular weight of

protein.• Agarose gel – various type of RNA and

viruses are fractionated and purified.

Affinity Chromatography

Principle :Based on specific and non covalent binding of substance like protein & enzyme to specific ligand which is attached to gel matrix.Ex. Separation of LDH from RBC- using NAD+ cofactor as ligand which is linked to affinity gel.

Affinity Chromatography

Application :• Isolation of albumin, DNA polymerase,

coagulation factors.• Isolation of glycoproteins.• Purifying human immunoglobulins.• Purification of bone collagen, HBsAg,

ribosome.

High Pressure Liquid Chromatography (HPLC) Method in which the solution to be analyzed is passed, under high pressure, through a long, thin column packed with tiny beads such that analyses are completed in minutes rather than hours and with improved resolution.

(HPLC)• Columns are packed in strong stainless steel

cylinder - withstand high pressure.• Non compressible resin - used as supporting

media.• UV absorption, fluorescence, regenerative

index detectors are used.• All physiochemical mechanism(adsorption,

partition, affinity, ion exchange) are possible in HPLC.

• Entire HPLC process can be completed in a few minutes of effective separation and detection.

HPLC

HPLC

Uses :• Spectrophotometer detector- identification of

drugs in urine and serum.• Flurometer- amino acids & other primary amine

can be detected.• Electrochemical detector- urinary catecholamine

& compounds(unsaturated FAs & PGs) can analyzed.

• Urinary VMA(vanillylmandelic acid), metanephrins, homovanillic acid can be analyzed.

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