2 Spec-20 Instrument Wavelength control knob Light control knob Amplifier control knob Sample holder cover 3 Spec-20 Absorbance Insert Photograph Spec-20 100 Absorbance 0 Transmittance Absorbance 0 Absorbance 100 Transmittance l (wavelength) 4 Schematic representation of a spectrophotometer I Io Detector Monochromator Meter Light Source (1) Sample cell (3) (2) (4) (5) A spectrophotometer is an instrument that measures the fraction (I/Io) of an incident beam of light (Io) which is transmitted (I) by a sample at a particular wavelength. For a given substance the amount of light absorbed depends on a) the concentration b) the cell or path length c) the wavelength of light and d) the solvent. 5 Absorbance of Chlorophyll 1.5 1.2 0.9 Absorbance 0.6 0.3 0.0 300 400 500 600 700 800 Wavelength (nm) 6 Amount absorbed 663 nm 400 500 600 700 Wavelength (nm) Kelter Carr Scott Chemistry A World of Choices 1999 page 480 7 Calibration Curve (fixed wavelength) 100 Absorbance 0 Transmittance out of linear range (too concentrated)
Absorbance 0 Absorbance 100 Transmittance x 2 Concentration Dilute sample with water 5050. Run sample read concentration. 8 Dilution of Solutions Dilution of Solutions Dilution of Solutions Dilution of Solutions Dilution of Solutions Keys http//www.unit5.org/chemistry/Solutions.html
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