1. Connect the Spectronic 20
to an electrical outlet. Turn on the instrument with the zero
adjust/on-off control and allow about five minutes warm-up time.
Turn the wavelength control knob to select the desired wavelength.
2. With no sample tube in place and the cover closed, turn
the zero adjust knob to bring the meter to 0% transmittance.
3. Fill the spectrophotometer tube to about 2/3 with distilled
water. Make sure that the outside of the tube is clean and dry
(do not handle the tube on the sides so as to avoid fingerprints)
and insert it in the cuvette holder, pushing it all the way down
and aligning it so that the reference line on the cuvette holder
matches a line (or other mark) on the tube. Close the cover of
the cuvette holder.
4. Adjust the light control knob on the spectrophotometer so
that the meter reads 100% transmittance. Steps (2) - (4) should
be repeated before each absorbance measurement.
5. Fill (to 2/3) a tube with the solution to be determined.
Wipe the outside of the tube to remove water and fingerprints
and insert into the cuvette holder, aligning as above. Close the
cover and read the % transmittance (or absorbance) from the meter
The same sample tube should be used for all absorbance measurements.
Care must be taken to align the tube in exactly the same way each
time. A separate tube may be used for the distilled water blank,
but again always align it in the same way.
Because the sample may be heated in the light beam or photochemical
decomposition may occur, the
absorbance reading should be made quickly after the tube is inserted.
It is good practice to read and record the % transmittance
meter reading and to convert to absorbance later. The transmittance
scale is linear and thus less subject to reading error than the
Cuvette -- The cuvettes for use in the
Spec 20 resemble small test tubes. Each cuvette is marked so that
it can be positioned properly in the sample holder. The mark is
at the top of the cuvette and must be positioned toward the front
of the spectrophotometer when taking measurements. Handle these
tubes with extreme care to keep both the inside and outside surfaces
clean and free of scratches.
Cleaning the Cuvette:
1.NEVER use a brush to clean the inside of the cuvette.
2.Rinse the tube with distilled water a few times.
3.Add about 1 mL of the solution to be measured. Tilt and turn
the cuvette so that the solution has contact with all the surfaces.
Discard this solution and repeat this rinse once more.
4.Fill the cuvette about 3/4 full of the solution you wish
5.Wipe the outside of the cuvette with a lint-free, soft tissue
(a Chemwipe) to remove any moisture or fingerprints from the outside
Calibrating the Spectronic 20
1.Plug in and turn on the Spec
20. It must warm up for 30 minutes before use. A schematic of
an older instrument is shown at the left.
2.Set the instrument to the proper wavelength by turning the
knob located on the right hand surface of the spectrophotometer.
The wavelength setting can be seen through the window next to
3.Obtain a properly cleaned cuvette and fill it about 3/4 full
of the reference solution (usually water).
4.With no cuvette in the sample holder, close the cover and
rotate the zero light control knob (left front knob) to display
a reading of 0.0% transmittance. Provided that the instrument
is not turned off and this knob is not moved, no other adjustments
to this control are needed.
5.Place the reference solution cuvette in the sample holder,
close the cover, and rotate the light control knob (front right
knob) to display a reading of 100.0% transmittance. This procedure
must be repeated every time measurements are taken at a new wavelength
or if several measurements are made at the same wavelength.
Taking measurements with the Spectronic 20
1.Calibrate the instrument (see above instructions) at the
wavelength you wish to measure. You will use the solvent of your
sample solution as your reference (this will usually be water).
2.Fill a properly cleaned cuvette 3/4 full of you sample solution.
3.Place your sample cuvette in the sample holder and close
4.Read either the absorbance or percent transmittance as needed.
A schematic diagram of a Spectronic 20 is shown below.