|MadSci Network: Biophysics|
Message ID Number: 883826929.Bp How do we know light not transmitted in a spectrophotometer is absorbed? Your question is well placed! While we usualy would check absorbance only for materials that are not reflecting, a wrong choice of the tube, an unclean tube or very dirty one may lead to errors in readings due to reflectance or turbidity. The problem of turbidity (light interference) is even more serious. Turbidity would lead to loss of light reaching the detector due to light diffraction by particles with size around (and more then) half of the light wave length. In microbiology it is actually common to use specrophotometers to determine the density of bacteria by turbidity. We choose light wavelength, in which absorbance is negligable and determine the density of bacterial "particles" by turbidity. In this case the light not reaching the detector is actually diffracted. One way to make sure is to take apart from the reading in a certain wavelength also the spectrum (Absorbance vs. Wavelength) since the dependance of absorbance in wavelength is stronger then the dependence of turbidity on wavelength. Thus a sharp "peak" should arise in the right wavelength. Thus remember, the reading in your spectrophotometer depends on the state of your tube (reflectance or turbidity), on the amount of dust and visible particles in it (turbidity) and on the absorbance of your sample. We can usualy correct for reflectance and tube dependant inaccuracies by calibrating the spectrophotometer with the same tube with water or buffer only, but we can not correct for turbidity unless we check also in other wavelengths.
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