MadSci Network: Biophysics

Re: How do we know light not transmitted in a spectrophotometer is absorbed?

Date: Tue Feb 3 02:41:42 1998
Posted By: Dr. Ofer Markman, Post Doc, Physiology, Hebrew U. School of medicine
Area of science: Biophysics
ID: 883826929.Bp

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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