Use of the Spectronic 20 Spectrophotometer The Science Learning Center The University of Michigan-Dearborn Revised by: Bette Kreuz, Ruth Dusenbery and Dawn Wisniewski

Slide 1a-Introduction Spectrophotometers are instruments that measure the amount of light absorbed by solutions. The absorption of light is directly proportional to the concentration of the absorbing species in the solution.

Overview Spectronic 20 Controls Meter Absorbance and % Transmittance Scales Turn POWER ON with Power/Zero control (left) knob. Red light will go on. Allow 15 min warmup time. Wavelength Scale Wavelength Control Sample Holder Power/Zero Control 100% Trans/Light Control

Slide 2a-Power Control The instrument controls are shown in the diagram. The instrument is turned on by using the Power/Zero Control (left) knob. The red signal light on the front will go on. Allow 15 minutes warm up time before making measurements. Turn Light Control (right) knob Counterclockwise one half turn to protect the phototube.

Set Wavelength Use the wavelength control knob to select the desired wavelength on the wavelength scale. Read the scale directly perpendicular to avoid “parallax” errors.

Slide 3-Set Wavelength Use the wavelength selector knob on the top right side of the instrument to select the desired wavelength. Stand up and view the wavelength scale from directly above. Your line of sight must be perpendicular to the scale to read the dial without a “parallax” error.

Reading the Meter

Slide 4-Reading the Meter The meter simultaneously reads: Percent Transmittance--the portion of incident light passing through the sample--on the top scale, and Absorbance--the portion of incident light absorbed by the sample--on the bottom scale.

Reading the Meter Top scale is read to the right. Bottom scale is read to the left.

Slide 5a-Reading the Meter The top scale (%T) is divided into increments of constant size and must be read from left to right. This linear scale is easy to read. The bottom scale (Absorbance) has increments between tick marks that vary across the scale and must be read from right to left. This nonlinear scale is more difficult to read accurately.

Reading the Meter Avoid Parallax Error Position your head directly in front of the meter.

Reading the Meter Avoid Parallax Error Adjust viewing angle until the needle and its shadow on the mirror are aligned.

Calibrating the Instrument

Slide 8a-Calibrating the Instrument In order to make a measurement using the Spectronic 20, you must first set the 0% and the 100% transmittance readings.

Setting 0% Transmittance With an EMPTY sample chamber adjust the LEFT knob until the meter reads 0% transmittance.

Setting 100% Transmittance With a cuvette containing the BLANK solution (water) adjust the RIGHT knob until the meter reads 100% transmittance.

Sample Handling

11a--Sample Handling All readings are made using cuvettes, which resemble small glass test tubes, but are made from higher quality glass. For qualitative work two cuvettes are used, one for the sample solutions and one for the blank solution. A blank solution consists of all of the components in the sample solution except the substance that you wish to measure.

Rinse Cuvettes If the cuvettes are not clean and dry, rinse them thoroughly with the solution that you will be measuring. Rinsing several times with small volumes of the solution is preferable to rinsing once with a large volume of the solution.

Use Correct Volume of Solution Not filled enough. Filled properly.

13a--Use Correct Volume of Solution Fill the cuvette with the solution to a sufficient height so that the internal light beam passes through the solution in the cuvette, and not through air. The Spectronic 20 cuvettes need to be filled about ¾ full.

Remove Trapped Air Bubbles Tapping to remove bubbles

14a-Remove Trapped Air Bubbles Trapped air bubbles can be removed by tapping the bottom of the cuvette to dislodge the them. Filling the cuvette with a Pasteur pipette reduces the chance of trapping bubbles in the sample solution.

Clean Cuvettes with Kimwipes

15a--Clean Cuvettes with Kimwipes 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 absorbance readings. Wipe the cuvette first with a damp Kimwipe and then with a dry one. After cleaning handle cuvettes only by their tops.

Sample Holder Sample Holder

16a--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 20. It is fitted with a cover which must be closed before taking readings.

Inserting and Aligning the Cuvette in the Sample Holder Align the Vertical Index mark on the cuvette with the raised nub on the front of the sample holder. Vertical Index Mark Nub

17a--Inserting and Aligning the Cuvette in the Sample Holder Insert the cuvette into the sample chamber by gently pushing it into position. Hard pushing could damage the instrument. To ensure reproducible positioning in the sample holder align the Vertical Index mark on the cuvette with the raised nub on the front of the sample holder.

Final Step--Take the Reading Carefully close the cover to the sample chamber. Stray light can enter and give false readings. Record the % Transmittance or Absorbance value. Remove the cuvette from the sample holder as soon reading has been completed.

Summary--Making a Measurement Calibrate the Spec 20 with an empty chamber by turning the left dial to 0% T. Place a cuvette ¾ filled with distilled water into the chamber. Using the right dial, adjust the Spec 20 to 100%T. Remove the cuvette, rinse with sample and fill to ¾ with sample solution. Read % Transmittance and/or Absorbance of the sample carefully.