Fiehn BIS103 lecture 17_fall 2010

Quantitative analysis seeks the amount of a given

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Quantitative analysis seeks the amount of a given compound in a sample. detection (w/ identification) separation types: (a) physicochemical or (b) biochemical properties (a) electrical charge, lipophilicity, hydrophilicity, volatility, size… (b) binding, metabolism, precipitation… detection types: (a) physicochemical or (b) biochemical properties (a) mass, vibration, rotation, magnetic resonance, radiation absorption/fluorescence/scattering, redox potential, thermal conductivity, refractive index… (b) antibody, protein folding/binding barb2right FRET
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4 Separation by liquid chromatography Most typical is high performance liquid chromatography (HPLC), gas chromatography (GC) and capillary electrophoresis (CE) LC based on biochemical property (binding) LC based on physicochemical property (lipophilicity=‘reversed phase’) A Separation by gas chromatography two major separation forces: volatility and interaction with film inside the column 250ºC ramping from 80-330 º C capillary column (30 m) 0.25 mm i.d. column diameter 0.25 μ m stationary phase film
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5 Mass Spectrometry ‘The separation of ions based on their mass / charge ratio’ Compound (after chromatography) Ionization (w/ fragmentation) atmospheric pressure vacuum MS
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