1.6 techniques of analysis

7/31/2019 1.6 Techniques of Analysis

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TECHNIQUESOF ANALYSIS

1) Chromatography

2) Electrophoresis

3) X-ray diffraction


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Candidates should be able to:

(a) describe the basic principles of paper

chromatography in pigment separation,electrophoresis for protein and nucleic acidseparation.


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1) CHROMATOGRAPHY

Chromatography is the collective term for a set of laboratorytechniques for the separation of mixtures.

It involves passing a mixture dissolved in a "mobile phase"through a stationary phase, which separates the analyte to be

measured from other molecules in the mixture. The phases are chosen such that components of the sample

have differing solubilities in each phase. A component which isquite soluble in the stationary phase will take longer to travelthrough it than a component which is not very soluble in thestationary phase but very soluble in the mobile phase.

As a result of these differences in mobilities, samplecomponents will become separated from each other as theytravel through the stationary phase.


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Chromatography can separate other mixtures, which

include proteins, amino acids, nucleic acids, nucleotides,

fatty acids, monosaccharide and disaccharides.

The solid media are; paper, gel layer or column of

cellulose, and achrimide polymer.


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Types of chromatography:

1) Paper Chromatography (for leaf extraction)

2) Two dimensional paper chromatography (for

mixture of amino acids)3) Thin-layered chromatography (extraction of

green leaf)

4) Column Chromatography (isolation of plant

pigments)


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TWO DIMENSIONAL

PAPER

CHROMATOGRAPHY


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VIDEO


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Factors that influence the rate of adsorption are:

1) solubilities

2) molecular size

3) charges

This technique is useful because:

1) simple and can be easily carried out.

2) it takes short time to carry out.

3) require only simple apparatus.

Limitation in using this technique:1) only small amount of substances can be separated atone time.

2) when the solutes are too similar, they are notseparable using this technique.


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2) ELECTROPHORESIS

Technique that used to separate substances with differentcharges as protein in an electric field.

In other words, electrophoresis is a separations technique thatis based on the mobility of ions in an electric field.

Ions have different migration rates depending on their total

charge, size, and shape, and can therefore be separated. It also can separate other mixtures include amino acids and

nucleic acids fragments especially DNA.

The medium used can be paper, gel layer, or in a column.

This technique used to:

1) separate proteins. i.e separation of enzymes2) diagnose disease as blood plasma proteins are separated.

3) DNA fingerprinting.
http://elchem.kaist.ac.kr/vt/chem-ed/sep/sepintro.htmhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/sepintro.htm


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VIDEO


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3) X-RAYDIFFRACTION

Technique that used to analyze three dimensionalstructure or internal structure of crystals and solidmacromolecules such as nucleic acids and proteins.

Principle involved is like that of a spectroscope in whichthe substance is irradiated with X-ray. The dispersion of

electron is caught in a film that can be developed andmany angle for the same substances are taken.

This technique can be used to:

a) determine 3D structure of proteins such ashaemoglobin and myoglobin.

b) determine the double helix structure ( Watson andCricks model)

c) determine the structure of other vitamins andmembrane.

1.6 techniques of analysis

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