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Transcript
Introduction to Analytical Techniques I. Analytical Techniques A. Def.- Any procedure that allows one to determine the molecular composition of an unknown substance. 1. May be qualitative or quantitative 2. Must be accompanied by control substances that allow the experimenter to assess the proper function of the procedure. 3. Frequently requires the separation of substances from a mixture. (Cell extracts, tissue extracts, and biological fluids represent a complex mixture of biological molecules.) B. Types of techniques 1. Separation based on molecular size * a.. Agarose electrophoresis i. frequently used to separate and identify DNA fragments from a mixture of fragments extracted from a biological sample ii. Required for DNA fingerprinting, DNA sequencing, DNA cloning, gene mapping, etc. iii. Involves “pulling” components of a mixed sample through a mesh network of agarose gel. An electrical field generates the “pull”. The “mesh network” provides an “obstical course” or series of pores through which the molecules in the sample must travel. Small molecules move quickly, large molecules move slowly. b. Gel filtration chromatography i. Frequently used to separate and purify large proteins from biological samples. ii. Involves separation of molecules within a mixture by “pulling” them through a gel matrix of pores that has been packed into a vertical column. The “pull” is provided by gravity. A column of beads that contain specified pore sizes provides the matrix of pores. c. Differential sedimentation- centrifugation i. Relies on the concept that heavier substances will sediment at a faster rate when spun at high speeds in a centrifuge. ii. Frequently used to prepare cell fractions as a preparative step prior to one of the other analytical techniques discussed. 2. Separation based on specific characteristics a. differential solubility. * i. Thin layer chromatography ii. Paper chromatography iii. Requires a mobile phase and an immobile phase. iv. Solubility in the mobile phase allows for characteristic mobility across the immobile phase. The mobile phase is the solvent, the immobile phase is the solid support (either a thin layer of silica or paper) v. Solubility is based on the “Like dissolves like” rule. Polar substances will dissolve in polar solvents, nonpolar substances will dissolve in nonpolar solvents. b. Electrical charge i. Some forms of electrophoresis (Isoelectric focusing) - Used to separate proteins based on the nature of the R groups found in their specific amino acid sequence. - Can be used to separate isotypes of the same protein. ii. Ion exchange chromatography - Used to separate proteins or nucleic acids based on their electrical charge. - Frequently used as a preparative technique. c. Antigen/antibody reaction * i. Enzyme linked immunosorbent assay (ELISA) - Antibodies are used to “catch” an specific substance from an unknown sample. - The presence of the substance is then detected by a visible enzymatic reaction that is attached to the specific antibody. ii. Affinity chromatography - Antibodies or other specific “sticky” chemical groups are used to “catch” a substance from an unknown sample. - The “catching” substance is attached to beads that are placed into a vertical column. - Sample is poured over the column allowing the specific “target” molecule to be “caught” - The “caught” molecules are then eluted by disrupting the “sticky” reaction that allowed them to become “caught” 3. Analysis of light wave absorption or scatter * i. Visible spectrophotometry - Qualitative- what wavelengths of light are absorbed by the sample? - Quantitative- How much light of a specific wavelength is absorbed by a sample containing an unknown quantity of a known substance? ii. Ultraviolet spectrophotometry - Same as above but with ultraviolet wavelengths of light - Most common mechanism for quantitating DNA and RNA in research and clinical settings. iii. Infrared spectroscopy - Same as above but using infrared wavelengths of light - Commonly used to determine the identity of substances in an unknown mixture. C. Web addresses for additional information 1. Chromatography http://www.chem.ubc.ca/courseware/154/tutorials/exp3A/columnchrom/ 2. Thin layer chromatography. http://www.chem.ucla.edu/~bacher/General/30BL/tips/TLC1.html 3. Gel electrophoresis. http://www.life.uiuc.edu/molbio/geldigest/electro.html 4. ELISA http://www.chemicon.com/resource/ANT101/a2C.asp 5. Spectroscopy http://chemistry.about.com/library/weekly/aa021302a.htm