Chromatography

Dr Shivraj Shetty

Chromatography
What is chromatography ? Chromatography is the physical separation of a mixture into its individual components It can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatography
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Why is chromatography called chromatography?

First application by M. S. Tswett in 1906 For the separation of plant pigments. Since the components had different colors, the Greek chromatos; for color, was used to describe the process.

Chromatography

Principle
Method for separating the components of a mixture by Differential Distribution of the components of the mixture between a stationary phase and a mobile phase.

Chromatography
Principle Different affinity of the different components to stationary phase causes the separation. Interactions- hydrogen bond, London force

Classification

Major components
Mobile phase flows through column, carries analyte. Stationary phase stays in a place, does not move. The SEPARATION is based on the partitioning between the mobile and stationary phase.

Based on the principle

Partition Ion exchange Size exclusion Adsorption Affinity

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Retardation factor ( Rf )

Rf

Distance travelled by the substance/solute Distance travelled by the solvent front

Constant for a particular substance in a particular solvent system

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Histidine -0.24 Valine 0.66 Proline -0.49 Phenyl alanine-0.69

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Thin layer chromatography

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Advantages of chromatography
can separate very complex mixtures
drugs, plastics, flavorings, foods, pesticides, tissue extracts, fuels, air samples, water samples, ...

very small sample sizes separated components can be collected individually analyses can be highly accurate and precise

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Aplications
Detection of inborn errors of metabolism To differentiate between overflow and renal amino acidurias

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In born errors of metabolism

Homocystinuria- Cystathionine beta synthase deficiency Alkaptonuria- homogentisate 1,2 dioxxygenase Tyrosinemiafumarylacetoacetate hydrolase tyrosine aminotransferase 4-hydroxyphenylpyruvate dioxygenase Maple syrup urine disease-branched-chain alpha-keto acid dehydrogenase complex deficiency Galactosemia- Galactokinase, G1PUT, UG epimerase Fructosuria- Fructokinase
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Thank you

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Chromatogram

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Video of column chromatography

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Advantages of HPLC
It is the method of choice for analytical purposes Speed of analysis, resolution Sample requirement is also very low (few femto gram ) Used in separation of proteins, nucleic acids, amino acids, polysaccharides, plant pigments, pesticides, steroids, drugs and their metabolites, animal and plant hormones and complex lipids
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Disadvantages of HPLC
Expensive Complexity Low sensitivity to certain compounds, because of speed

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Queries ?
What is use of the mixture of acetic acid, butanol & distilled water. Partition coefficient Paper chromatography ascending descending Thin layer chromatography The one we are doing is circular or ascending chromatography. The amino acids which moves fastest ? What carbohydrates r we using ?
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