Question

In an experiment, why would the concentration of a sample decrease when changing the wavelength for a spectrophotometer from 235 nm to 280 nm?

This is just a general question, I would like to know why the absorbance peak would be higher at a wavelength of 235nm rather than at 280nm. Is this normal? or could it an experimetnal error?

Answer 1

If I understand your question correctly, then you want to ask that why the absorbance (not the concentration) of a sample decreases when we change the wavelength from 235 nm to 280 nm.

This change in absorbance is attributed to several factors which are given below:

1. The absorbance of a particular solution depends upon the identity of compound present in it. It means substance A has different absorbance pattern than substance B.

2. The absorbance of a substance covers a range of wavelength known as absorption envelope that gradually increases from starting wavelength reaches its maximum value at a certain wavelength ( known as _max ) of that substance and then decreases again. It means if the absorbance of compound is measured at _max it will be maximum and lesser on other wavelengths.

3. Since the concentration of a solution is related to absorbance by the formula ; where A is absorbance, is molar extinction coefficient and l is pathlength of cell, so we can calculate the concentration by recording absorbance at a particular wavelength provided at that wavelength is known.

Now coming to your question;

If you are observing a decreased absorbance at 280 nm then it is because of the fact that wavelength 280 nm is far from the _max of that compound. The higher absorbance at 235 nm is obtained because it should be near to _max. But this entirely depends on the range of absorption envelope of that substance and not merely true for absorbance of all compounds at 235 nm and 280 nm. If a substance has _max at 280 nm then you will gate higher absorbance at 280 nm.

Lastly the concentration of the solution doesn't change on changing the wavelength. It is the absorption property of that substance which changes with change in wavelength and results in different absorption value.