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Biology 2290F/G
Robert Dean

1 Spectronic 20 optical system LAMP light 340660nm goes to DIFFRACTION GRATING splits light like a glass prism and then goes to EXIT SLIT through SAMPLE then DETECTOR diffraction grating is also connected to lever and wavelength CAMspec 20 gives information on both thetransmittance and absorbance Spectrophotometer DOES NOT measure absorbance measures the amount of light transmitted by a sample 100 T when blankwhen the wavelength is changed you MUST adjust the spectrophotometer for the blank before reading each sample 2 Transmittance fraction of incident light that passes through is transmitted by the sample TIIo where Ioradiant power of the incident light Iradiant power of light that passes through the sampleAbsorbance amount of light absorbed by the sample Alog 1Tlog IoI3 Lamberts Law for a thin layer of sample the same proportion of light is absorbed regardless of the radiant power of incident light Io Layer one absorbs 12 layer two ALSO absorbs 12 First two layers transmit 14122 Light quantity decreases exponentially as it passes through the sampleBeers Law absorbance of a solution is proportional to theof absorbing particles conc in solution AElc if l1cm4 Appropiate blank contains all the substances EXCEPT the material whose absorbance we wish to measure 5 principal uses of spectrophotometer1to produce absorption spectra that can be used to characterize or identify compounds in solution2 to make quantitative determinations of the concentration of materials in solution by reference to your own standard curves3to study the rate at which biochemical reactions proceed6 Absorption spectra An absorption spectrum is a graph of the absorbance values for a substance measured at a series of different wavelengths Note the wavelength at which absorption is maximal the max for each solutiontake T readings of DCPIP at different wavelengths starting from 340 to 600 nm Zero the blank whenever the wavelength is changed7 Standard curve Use the stock 1 x 104 M solution of DCPIP to make a dilution series as indicated in the following tableRead the absorbance A of each solution at the wavelength of maximum absorbance max Draw a graph of absorbance vs dye concentration The line should pass through the origin because at zero dye concentration the absorbance will be zero This graph will exemplify Beers Law which states the absorption of light is proportional to the number of absorbing particles in solution Spectrophotometry can therefore be used to meas
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