The spectrophotometer is an instrument which measures the amount of light of a specified wavelength which passes through a medium. According to Beer’s law, the amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present. Thus the concentration of a colored solute in a solution may be determined in the lab by measuring the absorbency of light at a given wavelength.
Wavelength (often abbreviated as lambda) is measured in nm. The spectrophotometer allows selection of a wavelength pass through the solution. Usually, the wavelength chosen which corresponds to the absorption maximum of the solute. Absorbency is indicated with a capital A.
To familiarize yourself with the spectrophotometer, illustrate and label the following features which are important to its proper use. You should know the function and/or significance of each of these features before you use the instrument.
At the spectrophotometer, you should have two cuvettes in a plastic rack. Solutions which are to be read are poured into cuvettes which are inserted into the machine. One should be marked “B”for the blank and one “S” for your sample. A wipette should be available to polish them before insertion into the cuvette chamber. Cuvettes are carefully manufactured for their optical uniformity and are quite expensive. They should be handled with care so that they do not get scratched, and stored separate from standard test tubes. Try not to touch them except at the top of the tube to prevent finger smudges which alter the reading. For experiments in which minor imprecision is acceptable, clean, unscratched 13 x 100 mm test tubes may be used.
WARM-UP: 1. Plug in and turn on (left hand front dial, labeled ZERO in the illustration). Allow about 30 minutes for warm up.
ZERO ADJUST: 2. With no cuvette in the chamber, a shutter cuts off all light from passing though the cuvette chamber
Under this condition therefore, the machine may be adjusted to read infinite absorbance (zero% transmittance) by rotating zero adjust knob (front left on Spectronic 20). Do not touch this knob again during the rest of the following procedure.
SELECT WAVELENGTH: 3. Select the desired wavelength of light at which absorbance will be determined by rotating wavelength selection knob (top right knob) until the desired wavelength in nanometers appears in the window. A nanometer (nm), formerly millimicron, equals 10-9 meter.
BLANK ADJUST: 4. Fill the B (blank) cuvette with the solvent used to dissolve specimen (often distilled water).
Polish to clean.
Insert into the cuvette chamber.
Close chamber cover.
5. Rotate blank adjust knob (front right knob) to adjust absorbency to read zero . 6. Remove blank cuvette, place in plastic test tube rack.
7. Pour the sample into the S (specimen) cuvette.
Polish and insert into the chamber, aligning mark to the front.
8. Note that the scale for absorbency is the lower scale on the dial, and should be read from R to L.
For for all readings of the dial, line up the reflection of the needle in the mirror behind the dial with the needle itself. Otherwise, parallax error will occur, giving an erroneous reading. The illustration shows the correctly aligned dial with a reading of 0.116.
PARALLAX ERROR: Here, the picture was taken of the identical solution as in the previous image, but with the point of view too far to the right. Note that the needle reflection is to the right of the needle. The apparent reading is 0.120.
PARALLAX ERROR: Here, the picture was taken of the identical solution as in the previous image but with the point of view too far to the left. Note that the needle reflection is to the left of the needle. The apparent reading is 0.113.
9. If you read additional specimens, you should confirm that the machine is still zeroed and blanked out, as in steps 2, 4 and 5 for all readings. Here are a few practice dials to read: What is the reading of dial A?
What is the reading of dial B?
What is the reading of dial C?
Answers to the spectrophotometer dial readings:
A: 0.182
B: 0.063
C: 0.116
Clean Up:
10. Remove cuvette from machine, carefully wash and store spectrophotometer cuvettes keeping them separate from regular test tubes.
