Chromatographic techniques, Lecture notes of Analytical Chemistry

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Basics of chromatographic Techniques

Course 1

Kannan R., Ph. D.

Mass spectrometer: 100 years after J.J. Thomson invented the first mass spectrometer

http://masspec.scripps.edu/mshistory/perspectives/sborman2.php

parabola spectrograph

“ I feel sure that there are many problems in chemistry which could be solved with far greater ease by this than by any other method ”

History

Mikhail Tswett, Russian, 1872-

Botanist

In 1906 Tswett used to 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”

Thin layer chromatography is used to separate the colorful components of a plant extract

Investigator(s) Year Contribution

Way and Thompson

Runge, Schoenbein, and Goeppelsroeder

Lemberg

Reed

Tswett

Karrer, Kuhn, and Strain

Holmes and Adams

Reichstein

Izmailov and Schraiber

Brown

Tiselius

1848 1850-

1876

1892

1903-

1930- 1935 1938

1938

1939 1940-

Recognized the phenomenon of ion exchange in solids. Studied capillary analysis on paper.

Illustrated the reversibility and stoichiometry of ion exchange in aluminum silicate minerals. First recorded column separation: tubes of kaolin used for separation of FeCI 3 from CuSO 4. Invented chromatography with use of pure solvent to develop the chromatogram; devised nomenclature; used mild adsorbents to resolve chloroplast pigments. Used activated lime, alumina and magnesia absorbents. Synthesized synthetic organic ion exchange resins. Introduced the liquid or flowing chromatogram, thus extending application of chromatography to colorless substances. Discussed the use of a thin layer of unbound alumina spread on a glass plate. First use of circular paper chromatography. Devised frontal analysis and method of displacement development.

Historical Developments in Chromatography

Importance

Chromatography has application in every branch of the

physical and biological sciences

12 Nobel prizes were awarded between 1937 and 1972 alone for

work in which chromatography played a vital role

The substances in a mixture are not chemically combined, so

therefore they can be separated through some physical process.

chromatography, technique for separating the components, or

solutes, of a mixture on the basis of the relative amounts of each

solute distributed between a moving fluid stream, called the mobile

phase, and a contiguous stationary phase. The mobile phase may

be either a liquid or a gas, while the stationary phase is either a

solid or a liquid.

Chromatography is the ability to separate molecules using

partitioning characteristics of molecule to remain in a stationary

phase versus a mobile phase. Once a molecule is separated from

the mixture, it can be isolated and quantified.

Chromatography

Thin Layer Chromatography

TLC is a method for identifying substances and testing the purity of compounds.

TLC is a useful technique because it is relatively quick and requires small quantities of material.

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

The stationary phase: is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate.

The mobile phase: is 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

If the spots can be seen, outline them with

a pencil.

If no spots are obvious, the most common

visualization technique is to hold the plate

under a UV lamp.

Many organic compounds can be seen

using this technique, and many

commercially made plates often contain a

substance which aids in the visualization of

compounds.

Identifying the Spots (visualization)

Reagents Compounds

Iodine UV light p-Anisaldehyde Bromocresol green 2,4-dinitrophenylhydrazine Ninhydrin Sulfanilic Acid Reagent (Diazotized), Pauly's Reagent Sulfuric acid Aniline phthalate Antimony trichloride Dragendorff’s reagent

Aromatic compounds Unsaturated compounds Carbohydrate carboxylic acid Mainly for aldehydes and ketones Good for amines phenolic compounds turn orange or yellow with this reagent sprayed on the TLC Sugar Cardiac glycosides Alkaloids

Visualizing Agents

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 past the sample on the

paper (stationary phase).

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 past the sample on the paper (stationary phase).

Partition occurs between the mobile phase and the stationary aqueous phase bound by the cellulose.

The isolation depends on partition coefficient of the solute.

Paper Chromatography

( )

( )

c stationary K c mobile



Factor Effect Decrease of size improves separation (but very small particles need high pressure).

Particle size of solid stationary phase (or of support)

Column dimensions Efficiency increases as ratio length / width increases.

Non uniform packing results in irregular movement of solutes through column & less uniform zone formation, (i.e. band broadning or tailing).

Uniformity of packing

Increase in column temperature results in speed of elution but does not improve separation (tailing). Column temperature

Solvents should be of low viscosity (to give efficient resolution) & high volatility (to get rapid recovery of the substances).

Eluting solvent

Solvent flow rate Uniform & low flow rate gives better resolution.

Continuity of flow Discontinuous flow disturbs resolution

Condition of adsorbent Deactivation of adsorbent decreases separation.

Concentration of solutes Substances of high concentration move slowly.

Factors affecting solutes separation in CC

Mode or type Stationary phase Mobile phase Mechanism

Solutes move at different rates according to the forces of attraction to the stationary phase.

Solid that attracts Liquid or gas the solutes

Adsorption Chromatography

Solutes equilibrate between the 2 phases according to their partition coefficients

Thin film of liquid Liquid or gas formed on the surface of a solid inert support

Partition Chromatography

Solute ions of charge opposite to the fixed ions are attracted to the resin by electrostatic forces & replace the mobile counterions.

Liquid containing electrolytes

Solid resin that carries fixed ions & mobile couterions of opposite charge attached by covalent bonds

Ion Exchange Chromatography

Molecules separate according to their size: 1.Smaller molecules enter the pores of the gel, and need a larger volume of eluent. 2.Larger molecules pass through the column at a faster rate.

Porous gel with no Liquid attractive action on solute molecules

Molecular Exclusion Chromatography

Special kind of solute molecules interact with those immobilized on the stationary phase

Solid on which Liquid or gas specific molecules are immobilized

Affinity Chromatography