光学Dec 21st.pptVIP

Absorption Absorption Light passing through a material medium will a) travel at a slower v = c/n where n(f) = dispersion b) have some absorption – depending on the electric dipole moment and orientation of the incident light E c) the absorbed energy become the heat or light. Absorption Gross description: where c=concentration, ? = extinction coeff. Integrates to (Beer-Lambert Law) Lambert law Beer law Optical Density In terms of A = εcl = absorbance, we have ln(Io/I) = A or log10(Io/I) = A/2.303 = O.D. OD = 1 means 1/10 is transmitted OD = 2 means 1/100 is transmitted OD 1, followed by OD 2 results in OD 3 Transmittance T = I/Io = e-A % Transmittance = (I/Io) x 100 Absorption In simpler molecules there will be a number of discrete absorption lines In macromolecules there are so many lines that a broad absorption band forms The type of incident light used (uv, IR, etc.) determines the type of absorption that is probed – uv probes electronic; ir probes vibrational, etc. How are the measurements made? Spectrophotometer Used to measure OD vs l Simplest: l varies with dial setting (rotation of prism or grating; photocell = detector – single beam; compare sample to blank of buffer If e known you can determine concentration Fancier double-beam instruments with monochromator and recorder coupled to grating ; read OD vs l directly with a constant comparison to blank Details I Animation Light source = incandescent tungsten for near IR to vis; Hg arc lamp for 250 – 350 nm; Xe lamp for 250 nm DNA hypochromism More order means less absorption – perfect crystals are clear (diamonds) So, ss DNA absorbs more than ds DNA at same concentration; more base stacking means less absorption- so can monitor DNA melting by absorption Applications II Monitor denaturation/renaturation – e.g. helix-coil in ds transition of DNA Maxwell 理论 吸收与色散 吸 收 吸 收 吸 收 吸 收 吸 收 吸 收 色 散 色 散 色 散 色 散 色 散 色 散 色 散 Scattering Scattering of L