Use of the Spectronic 20
Spectrophotometer
The Science Learning Center
The University of Michigan-Dearborn
Revised by: Bette Kreuz, Ruth Dusenbery and
Dawn Wisniewski
Slide 1a-Introduction
• Spectrophotometers are
instruments that measure the
amount of light absorbed by
solutions.
• The absorption of light is
directly proportional to the
concentration of the absorbing
species in the solution.
Overview Spectronic 20 Controls
Meter
Absorbance and % Transmittance Scales
Turn POWER
ON with
Power/Zero
control (left)
knob.
Wavelength
Scale
Wavelength
Control
Red light will
go on.
Allow 15 min
warmup time.
Sample Power/Zero 100% Trans/Light
Holder
Control
Control
Slide 2a-Power Control
The instrument controls are shown in the diagram.
The instrument is turned on by using the Power/Zero
Control (left) knob.
The red signal light on the front will go on.
Allow 15 minutes warm up time before making
measurements.
Turn Light Control (right) knob Counterclockwise
one half turn to protect the phototube.
Set Wavelength
Use the wavelength control knob
to select the desired wavelength
on the wavelength scale.
Read the scale directly
perpendicular to avoid
“parallax” errors.
Slide 3-Set Wavelength
• Use the wavelength selector knob on the top
right side of the instrument to select the
desired wavelength.
• Stand up and view the wavelength scale
from directly above.
• Your line of sight must be perpendicular to
the scale to read the dial without a
“parallax” error.
Reading the Meter
Slide 4-Reading the Meter
The meter simultaneously reads:
Percent Transmittance--the portion of incident light
passing through the sample--on the top scale, and
Absorbance--the portion of incident light absorbed
by the sample--on the bottom scale.
Reading the Meter
Top scale is read to the right.
Bottom scale is read to the left.
Slide 5a-Reading the Meter
• The top scale (%T) is divided into increments of
constant size and must be read from left to
right.
This linear scale is easy to read.
• The bottom scale (Absorbance) has increments
between tick marks that vary across the scale
and must be read from right to left.
This nonlinear scale is more difficult to read
accurately.
Reading the Meter
Avoid Parallax Error
Position your
head directly
in front of the
meter.
Reading the Meter
Avoid Parallax Error
Adjust
viewing angle
until the
needle and its
shadow on
the mirror
are aligned.
Calibrating the Instrument
Slide 8a-Calibrating the Instrument
In order to make a measurement using the
Spectronic 20, you must first set
the 0% and the 100% transmittance
readings.
Setting 0% Transmittance
With an EMPTY
sample chamber adjust
the LEFT knob
until the meter
reads 0% transmittance.
Setting 100% Transmittance
With a cuvette containing
the BLANK solution
(water) adjust the
RIGHT knob
until the meter reads
100% transmittance.
Sample Handling
11a--Sample Handling
• All readings are made using cuvettes, which
resemble small glass test tubes, but are made
from higher quality glass.
• For qualitative work two cuvettes are used, one
for the sample solutions and one for the blank
solution.
• A blank solution consists of all of the components
in the sample solution except the substance that
you wish to measure.
Rinse Cuvettes
If the cuvettes are not
clean and dry, rinse
them thoroughly with
the solution that you
will be measuring.
Rinsing several times
with small volumes of
the solution is
preferable to rinsing
once with a large
volume of the solution.
Use Correct Volume of Solution
Filled properly.
Not filled enough.
13a--Use Correct Volume of Solution
• Fill the cuvette with the solution to a
sufficient height so that the internal light
beam passes through the solution in the
cuvette, and not through air.
• The Spectronic 20 cuvettes need to be
filled about ¾ full.
Remove Trapped Air Bubbles
Tapping to remove bubbles
14a-Remove Trapped Air Bubbles
Trapped air bubbles can be removed by
tapping the bottom of the cuvette to
dislodge the them.
Filling the cuvette with a Pasteur
pipette reduces the chance of trapping
bubbles in the sample solution.
Clean Cuvettes with Kimwipes
15a--Clean Cuvettes with Kimwipes
• It is important to clean the outside, lower portion
of a cuvette before taking any readings.
• Fingerprints, liquid droplets, and smudges on the
cuvette surface can give false absorbance readings.
• Wipe the cuvette first with a damp Kimwipe and
then with a dry one.
• After cleaning handle cuvettes only by their tops.
Sample Holder
Sample
Holder
16a--Sample Holder
Once the sample or blank is free from
bubbles and in a clean cuvette, it can be
inserted into the sample holder.
The sample holder is located on the left, top
surface of the Spec 20.
It is fitted with a cover which must be
closed before taking readings.
Inserting and Aligning the Cuvette
in the Sample Holder
Align the
Vertical
Index
mark on
the cuvette
with the
raised nub
on the
front of the
sample
holder.
Vertical Index Mark
Nub
17a--Inserting and Aligning the Cuvette
in the Sample Holder
Insert the cuvette into the sample chamber by
gently pushing it into position.
Hard pushing could damage the instrument.
To ensure reproducible positioning in the sample
holder align the Vertical Index mark on the
cuvette with the raised nub on the front of the
sample holder.
Final Step--Take the Reading
Carefully close the cover to the sample chamber.
Stray light can enter and give false readings.
Record the % Transmittance or Absorbance
value.
Remove the cuvette from the sample holder as
soon reading has been completed.
Summary--Making a Measurement
• Calibrate the Spec 20 with an empty chamber
by turning the left dial to 0% T.
• Place a cuvette ¾ filled with distilled water into
the chamber. Using the right dial, adjust the
Spec 20 to 100%T.
• Remove the cuvette, rinse with sample and fill
to ¾ with sample solution.
• Read % Transmittance and/or Absorbance of
the sample carefully.