TECHNIQUES OF ANALYSIS

1) 2) 3)

Chromatography Electrophoresis

X-ray diffraction

Candidates should be able to: (a) describe the basic principles of paper chromatography in pigment separation, electrophoresis for protein and nucleic acid separation.

1) CHROMATOGRAPHY

Chromatography is the collective term for a set of laboratory techniques 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 is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase.

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.

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)

TWO DIMENSIONAL PAPER CHROMATOGRAPHY

VIDEO

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 at one time. 2) when the solutes are too similar, they are not separable using this technique.

2) ELECTROPHORESIS

Technique that used to separate substances with different charges as protein in an electric field. In other words, electrophoresis is a separations technique that is 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 enzymes 2) diagnose disease as blood plasma proteins are separated. 3) DNA fingerprinting.

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3) X-RAY DIFFRACTION

Technique that used to analyze three dimensional structure or internal structure of crystals and solid macromolecules such as nucleic acids and proteins. Principle involved is like that of a spectroscope in which the substance is irradiated with X-ray. The dispersion of electron is caught in a film that can be developed and many angle for the same substances are taken. This technique can be used to: a) determine 3D structure of proteins such as haemoglobin and myoglobin. b) determine the double helix structure ( Watson and Cricks model) c) determine the structure of other vitamins and membrane.