One of the most convenient, accurate and sensitive methods for measuring the concentrations
dilute solutions is by colorimetry or absorption spectrophotometry. The technique is based upon
the measurement of the amount of light energy a solution absorbs from a beam of light of a
wavelength. The wavelength chosen, is usually that one at which the absorbance, of the species
to be analyzed, is at a maximum.
The picture below is a representation of a Spectronic 20-D colorimeter which is the instrument you will be using in both the manganese and iron determinations. Use of the instrument is easy and results are excellent provided you follow all directions carefully.
Before using the instrument, record the number of the blue tag on the front of the machine in your laboratory notebook. This will make it possible for you to identify the instrument that you used when beginning an analysis. Since you are going to calibrate this instrument it is important that you always use the same instrument during subsequent lab periods.
For your purposes in Chemistry 230 the following controls are going to be the most
1. Power Switch / Zero Control
2. Transmittance / Absorbance Control
3. Wavelength Control
4. Mode Control
5. Sample Compartment
6. Status Indicator
1. Turn the instrument ON by turning the Power Switch knob clockwise (toward the right).
Allow the colorimeter to warm up for 15 minutes in order to stabilize the light source and
2. After the warm-up period, set the desired wavelength with the Wavelength Control
For instance, the wavelength for the manganese determination is 525 nm.
3. Set the display mode to transmittance by pressing the MODE control key until the light
beside the "Transmittance" is lit.
4. Adjust the display to 0.0% T with the Zero Control knob. Make certain the sample
compartment is empty and the cover is closed tightly while make this adjustment.
5. Fill a cuvette with the blank solution to the top of the triangle on the side of the cuvette.
Wipe the cuvette with a Kimwipe to remove any liquid and fingerprints on the outside of
the cuvette. Both of these will interfere with light transmission and will cause erroneous
6. Position the cuvette with the vertical guide line (fiducial line) facing toward your right
insert the tube gently but completely into the cuvette compartment. Next rotate the
cuvette by 90 in a clockwise direction in order to align the guide line on the cuvette with
the guideline on the sample compartment. This technique prevents scratching of the
cuvette in those areas through which the light will pass. Scratches on the cuvette can lead
to erroneous measurements. Close the cover of the compartment.
7. Using the Transmittance / Absorbance control knob adjust the display to read 100.0%.
8. Press the MODE control key and switch the Status Indicator light to read Absorbance. If the Transmittance calibration was done correctly the display should read 0.0. No further adjustment is required. If the display does not indicated 0.0 use the Transmittance / Absorbance control knob to adjust it to that value. Using the MODE key switch the display back to Transmittance.
9. Remove the cuvette from the compartment by reversing the previous procedure: rotate
cuvette 90 in a counterclockwise direction then remove it. Fill another cuvette with the
solution whose absorbance you wish to measure. Insert it into the compartment in the
same manner as before.
a. Read the %T value directly from the digital display.
b. Using the MODE key select Absorbance and read the A value directly from the
digital display. Again select Transmittance
10. Remove the cuvette from the sample compartment by reversing the procedure used
insert the it. Close the lid to the compartment.
A. Make sure the cuvettes are clean.
B. Fill cuvettes two-thirds full or to the top of the triangular mark.
C. Always use the same cuvette for the blank solution and the other one for the sample.
D. Make certain that the index mark on the cuvette is lined up with the index mark on the
sample compartment before taking a reading. Doing so insures that the cuvette is in the
same position for each measurement.
E. If the wavelength control is moved in the slightest it is necessary to reset zero and 100
F. If you suspect that your cuvettes are showing differences greater than 3% T, you should search for a pair which has closer agreement. To find such a pair, fill two clean cuvettes with distilled water. Calibrate the instrument using one as a blank. Then read %T on the other. If your reading is less than 97%T or greater than 103%T, fill a third with distilled water and take a new reading. Any pair showing differences greater than 3%T may adversely affect your grade on an experiment which uses the Spectronic 20-D spectrophotometer.