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 scale.
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 logarithmic scale.
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 to test.
5.Wipe the outside of the cuvette with a lint-free, soft tissue (a Chemwipe) to remove any moisture or fingerprints from the outside surface.
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 the knob.
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 the cover.
4.Read either the absorbance or percent transmittance as needed.
A schematic diagram of a Spectronic 20 is shown below.
