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Lecture5-AAA - BIOC*2580Lecture5. 1 Synopsis Amino acid...

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BIOC*2580 Lecture 5. Analysis of amino acids 1 Page 1 of 7 Synopsis: Amino acid analysis usually involves two distinct processes, first separation of the individual amino acids from each other and from other contaminants, and then detection of the separated components. Separation is based on the different properties of the side chains, such as polarity or charge. Separation is generally achieved by some form of chromatography . Detection is based on chemical reactions that generate coloured or fluorescent amino acid derivatives that can be seen and measured. Reading: Lehninger, p. 85‐92 (4 th ed p.89‐95) Amino acid analysis Amino acid analysis is a necessary aspect of experiments to determine protein structure Analysis involves two processes: 1. The mixture must be separated into individual components 2. The components of interest must be detected Detection can be qualitative and determine what is present Detection can be quantitative and measure how much is present. Chromatography is an important method for separating components of a mixture Particles of solid are chosen with a given property. For example, silica gel contains HO‐Si‐OH groups that are effective in forming hydrogen bonds with polar amino acids. This makes up the stationary phase Liquid solvent or buffer flows past the particles and is nonpolar. This makes up the mobile phase. Amino acids rapidly exchange between phases. Polar amino acids ( P ) spend more of their time hydrogen bonded to the stationary silica ‐ they move more slowly Nonpolar amino acids ( N ) spend more time in the moving solvent, and move almost as fast as solvent.
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BIOC*2580 Lecture 5. Analysis of amino acids 2 Page 2 of 7 Thin layer chromatography The silica gel is spread as a thin layer on a plastic or glass sheet. Samples are applied to the silica gel in a small drop of solvent. Each sample forms a spot, and different sample spots are arranged in a row near the bottom edge of the sheet. The lower edge of the sheet is dipped in solvent. As solvent soaks up the sheet, the sample spots shift as the solvent moves past. Different substances move at different rates, so the components of an initial mixture are separated. Pure samples of substances suspected to be in the mixture are also applied. Spots in the mixture can be identified if they move the same distance as one of the pure samples. Polarity is the basis for separation of substances by thin layer chromatography. The rate at which a given sample, e.g. an amino acid, moves up the sheet depends on its relative preference for stationary phase (silica gel) or mobile phase (nonpolar solvent) . A very polar amino acid such as aspartate will spend most of its time stuck to the silica gel and will barely move. A very non‐polar amino acid such as leucine will spend most of its time in the solvent, and will move up the sheet almost as fast as the solvent.
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