Scientific Equipment Source
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Oshawa, ON, L1H 3V8
The Spectronic 21 Colorimeter (spectrophotometer)
The Spectronic 21 Colorimeter (spectrophotometer) is an update of the
Specronic 20 Colorimeter (spectrophotometer) The operation is very
simple and similar to that of the Spec 20. At the top of the
instrument is a meter that displays both absorbance and percent
transmittance of the sample being measured. On the left side is the
compartment for holding the sample to be measured. On the top right
are the wavelength selector and indicator. On the lower front left is
the knob that controls the 100% position on the %T scale. On the lower
front right is the switch that turns on the power.
Here are the steps to follow when using a Spec 21.
- Turn the instrument on using the switch at the lower right front of
the instrument. Allow about 5 minutes for warm up when first turned
- Select the appropriate wavelength for the sample to be measured.
- With the Spec 21 no adjustment is necessary to set 0%T, this is
- Fill a tube half full with water. This is called a blank. Place it
in the sample holder and close the cover.
- With the blank in the sample holder and the cover closed, adjust
the meter needle all the way to 100%T using the light control knob on
the lower left front of the instrument.
- Remove the blank and place the sample to be measured in the sample
holder and close the cover.
- Read absorbance value (or %T) from meter. In this case the readings
are 20%T and 0.70 absorbance units.
- Repeat this step with additional known samples if making a
calibration curve or verifying proportionality (Beer's Law).
- Repeat this step with a solution of unknown concentration, so that
its absorbance can be compared to the absorbance of a known solution.
Additional details on operation of the spectronic 21
- SET WAVELENGTH
The wavelength selector is on the top right side of the instrument. To
set the wavelength correctly, you must view the dial from directly
above. Otherwise, you may read the dial wrong. Such 'parallax' errors
occur if your line of sight is NOT PERPENDICULAR to the face of the
All readings are done in cuvettes, which resemble small glass test
tubes, but are made from higher quality glass.
Usually you will need two cuvettes to take the readings -- one to
hold the water blank and one to hold blue sample solutions.
- RINSE CUVETTES
In case your cuvettes are not clean and dry before using, you should
rinse them thoroughly with the solution, which you will be reading in
them. Several small rinses are preferred to using one big rinse in
order to coat the inside of the cuvette.
- FILLING VOLUME
When pouring a liquid into the cuvette, the solution must fill the
cuvette to a sufficient height so that the internal light beam passes
through the solution in the cuvette, and not through air.
The Spec 21 cuvettes have a horizontal index mark to show the
minimum required filling volume.
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 light absorbance readings.
The proper procedure for cleaning the surface of a cuvette is to
use laboratory tissues, called Kimwipes.
- CLEAN WITH KIMWIPES
Wipe the cuvette first with a damp Kimwipe and then with a dry
After cleaning the cuvettes, you should handle them by their tops.
Don't touch the lower portion of the glass.
- AIR BUBBLES
Even after cleaning the cuvette errors may still occur in a reading
if air bubbles are present in the solution.
Before reading a sample of even a blank, you must REMOVING AIR BUBBLES
Removing air bubbles can be done by tapping the bottom of the cuvette
to dislodge the bubbles.
Remove all air bubbles.
- REMOVING AIR BUBBLES
If tapping does not work, then cover the top of the cuvette with
Parafilm (a stretchy plastic covering) and slowly invert the cuvette
several times until all the bubbles are removed.
- 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
21. It is fitted with a cover, which must be closed before taking
- INSERTING A CUVETTE
When inserting a cuvette into the sample chamber, GENTLY push the
cuvette into its position. Hard pushing could damage the instrument.
- VERTICAL INDEX MARK
To assure reproducible positioning in the sample chamber, the
cuvette has a vertical index mark near its top.
When inserted properly, the vertical index mark on the cuvette must
be exactly aligned with the small nub on the top of the sample holder
as seen here.
- FINAL STEP
Close the cover to the sample chamber. Stray light can enter and give
- Rinse the inside several times with solution.
- Fill the cuvette above the horizontal index mark.
- Remove any air bubbles by tapping or inversion.
- Clean the outside surface with Kimwipes.
- Insert gently into sample chamber.
- Align the vertical index mark on the cuvette in holder.
- Close the chamber cover before taking a reading.
- ADJUSTING THE BLANK
The blank solution is used to calibrate the instrument so that the
internal light beam passes through the cuvette to the light-sensing
device. This is indicated by a dial reading of 100% Transmittance or
With the water blank properly in the sample holder with the chamber
cover closed, you may now adjust the light control knob to the right
until the readout meter on the face of the Spec 21 reads exactly
100%T. The light control switch is on the lower left of the
- AVOID ERROR
To avoid parallax error when reading the meter, your head must be
directly in front of the needle as shown here. If positioned
correctly, you will not see a reflection of the needle in the mirror
- REMOVE CUVETTE AFTER READING
Always remove the cuvette from the sample holder as soon as the
necessary adjustment or reading has been completed. Leaving the
cuvette in the sample holder for an extended period can damage the
- MATCHING CUVETTES
To reduce reading errors due to imperfections or scratches on the
cuvettes, the sample and blank cuvettes should be matched to one
This entails finding two cuvettes, which give exactly the same
reading when containing the same solution.
An alternative to using matched cuvettes is to read all solutions,
both blanks and samples, in a single cuvette. This is cumbersome, but
it assures accuracy.
If the second cuvette reads within 1% of the blank cuvette, then
they may be considered as matching.
- LEARNING TO READ THE METER
Before you begin to work with the blue sample solutions, it is wise
to learn how to read the Spec 21's scale for light absorption.
The meter simultaneously indicates Absorbance (the amount of light
absorbed by the sample) on the lower scale and Percent Transmittance
(the portion of light passing through the sample) on the top scale.
- HOW TO READ THE METER
The top scale (%T), which is divided into increments of constant
size, must be read from left to right. This is an easy scale to read.
The bottom absorbance scale has increments between tick marks,
which vary across the scale, making for some difficult readings. Also,
the scale is read from right to left, the opposite of the %T scale.
- PRACTICE TAKING ABSORBANCE READINGS
- Now that you have two matching cuvettes and have learned to
properly read the Spec 21 absorbance scale, you can begin taking
absorbance readings of the copper sulfate solutions.
- Select one of the three concentrations of CuSO4 to work with.
Remember to handle the chemicals carefully, avoiding skin contact.
- The Spec 21 must first be adjusted to 100% T using the blank
cuvette. Verify that the Spec 21 is set to 100% T when the blank
cuvette is inserted properly into the chamber.
- Now prepare the sample cuvette. Pour the water out of the second,
matched cuvette, rinse it with the selected blue solution several
times, fill it to above the mark, wipe the outside clean, remove any
bubbles, gently insert it into the sample chamber, and close the
- First 'blank' the Spec 21 to 100% T (zero A)
- Rinse the inside of the cuvette with sample solution
- Fill cuvette to above horizontal index mark
- Remove all air bubbles by tapping or inversion
- Clean outside of cuvette with when, then dry, tissue
- Insert GENTLY into sample chamber
- Align vertical index mar on cuvette in holder
- Close cover on sample chamber before reading
- Read the %T and/or the absorbance on meter
- Remove cuvette immediately after taking reading
Read both the absorbance and %T for the sample and check its accuracy
against the sign hanging behind the Spec 21. Are your readings with
the limits of acceptable error? Take your time to be certain.
- CHANGE THE WAVELENGTH
Now that you have determined the absorbance and %T at a particular
wavelength, you should change the wavelength and read the values for
Absorbance and %T again for the same sample of copper sulfate. You
should use the same cuvette to take the reading.
However -- and this is important to remember -- every time the
wavelength is changed, you must readjust the Spec 21 using the blank
Insert the blank and adjust the light control so that the meter reads
100% T (also zero absorbance) before reading the sample at the new
wavelength. You may not see much change in the 'blanking' adjustment
for this wavelength change; but in may parts of the spectrum, the
change is dramatic. Forgetting to 'blank' the instrument can give very
- CHECK ABSORBANCE AT ALL WAVELENGTHS
You should determine the absorbance and %T for your sample at all
three suggested wavelengths, checking the accuracy of all the
readings. Then if you want more practice using the Spec 21, try a
different concentration of copper sulfate sample.
When you feel confident on your ability to use the Spec 21, ask the
Science Learning Center staff for a posttest. The posttest consists of
determining the absorbance and percent transmittance of an unknown
concentration of copper sulfate.