When measuring the absorbances
of several samples that have different concentrations, it is important
to identify an appropriate wavelength. This wavelength is the
one at which the samples absorb the most light and is called lambda
max.
To find lambda max, set the wavelength on the Spectronic 20™
to 400 nm and calibrate the maximum and zero absorbance using
the reference solution. Insert the sample solution and record
its absorbance at this wavelength.
Increase the wavelength in 20 nm increments and record the
absorbance at each wavelength. Remember to recalibrate the maximum
and zero absorbance each time you change the wavelength.
When the absorbance begins to rise rapidly, decrease the wavelength
increment from 20 nm to 2-5 nm. This smaller increment allows
a more accurate determination of the wavelength of maximum absorbance
(lambda max). After the absorbance has peaked and again begins
to decrease, change the wavelength increment back to 20 nm. Take
measurements from 400 nm up to 600 nm.
Lambda max is determined by plotting absorbance versus wavelength and noting where the sample absorbs the most light.
Video: Animation of graphing process ( 4.53 M )
