Scanning Ultraviolet/Visible Spectroscopy (Spectronic 20
and Automated Instruments)
When measuring the absorbances
of several samples that have different concentrations, it is important
to identify an appropriate wavelength at which to take measurements.
This wavelength is the one at which the samples absorb the most
light and is called lambda max, since the Greek
letter is used to symbolize wavelength.
The Spectronic 20 or a scanning spectrophotometer can
be used to help generate a plot of absorbance versus wavelength,
which can then be used to find lambda max.
Spectronic 20 at Variable Wavelength
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. These steps are described in
the Spectronic 20 at Fixed 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.
A plot of absorbance versus wavelength shows lambda max.
Video: Animation of graphing
process ( 4.53 M )
Use this wavelength (lambda max) to measure the absorbances of
other samples of this solution that have different concentrations.
Below is a graph of absorbance versus wavelength for a solution.
What is lambda max? Can you predict the color of this solution?
Below is an absorption spectrum of a solution as measured
with a spectroscope.
Sketch a graph of absorbance versus wavelength. What is lambda max
for this solution? Answer
A scanning ultraviolet/visible (UV/Vis) spectrophotometer
operates on the same principles as a Spectronic 20.
A scanning spectrophotometer's main advantage over the Spectronic
20 is that the scanning spectrophotometer can rapidly scan
a range of wavelengths and record absorbances at each wavelength.
In this module, the Perkin Elmer Lambda 11, a single beam
spectrophotometer, is described but similar principles apply to
all UV/Vis spectrophotometers. For specific operating instructions,
refer to the operator's manual or other instructions.
The Perkin Elmer Lambda 11
Single Beam UV/Vis Spectrophotometer
Preparing the Cuvettes
Use a clean rectangular cuvette that is free of cracks. Plastic
cuvettes are acceptable when absorbance measurements will be recorded
in the visible range of the spectrum (about 400 to 700 nm wavelengths),
but quartz cuvettes are necessary if measurements are made at
lower wavelengths or if the sample liquid will destroy a plastic
To aid in proper placement of the cuvette within the sample
compartment, first cover one face of the cuvette with tape.
Video: Marking with tape ( 2.06 M )
Next, prepare the reference liquid. The reference liquid should
be the same as the solution to be analyzed except that it must
have zero concentration of the absorbing species. Rinse the cuvette
three times with a small volume of the liquid that the sample
is dissolved in. Then fill the cuvette about three-fourths full
with the liquid. Gently tap the cuvette until any air bubbles
are dislodged. Wipe the outside of the cuvette with a laboratory
Zeroing the Spectrophotometer
Place the cuvette containing the reference liquid into the
cell compartment in the spectrophotometer. Make sure that the
cuvette's clear sides are perpendicular to the light source when
the cuvette is placed into the sample compartment.
When the cuvette containing the reference sample
is placed into the compartment, its taped side
should be perpendicular to the light source.
Next, set up the wavelength range on the spectrophotometer.
Using the computer, set the spectrophotometer to scan the desired
wavelengths (usually 400 to 700 nm to scan the visible region
of wavelengths). In addition, the step size must also be set.
The step size indicates how often the spectrophotometer will record
an absorbance measurement. A step size of one nanometer is common.
With the sample compartment lid closed, zero the spectrophotometer.
After the spectrophotometer has been zeroed, remove the reference
sample, pour out the reference solution, rinse the cuvette three
times with the sample, and place the cuvette into the sample compartment.
Why use the same cuvette? Again, be
sure that the clear faces of the cuvette are perpendicular to
the light source. Close the lid, and use the computer to begin
scanning the wavelengths and recording absorbance measurements.
Video: Using the sample ( 5.17 M )
The computer should display a plot of absorbance versus wavelength.
If the spectrum goes off the scale, dilute the sample solution
and re-measure the spectrum.
The wavelength at which the absorbance is greatest is lambda max.
Copyright © 1995-1996 NT Curriculum Project, UW-Madison