Determining Transmittance and Absorbance - To assure that spectrophotometer readings indicate only the concentration of the solute we wish to measure, a reading must first be obtained using a blank, a sample that contains all the components of the solution except the absorbing molecule. For instance, if you are using a reagent that changes color when mixed with a certain solute molecule, a blank should contain all the components of the test solution, including the colorimetric reagent, except the substance (solute) to be measured.
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Example A 1-ml sample of substance X is mixed with 5 ml of water and 1 ml of colorimetric reagent to give a volume of 7 ml in a sample tube. A blank is prepared by mixing 5 ml of water with an additional 1 ml of water (as a substitute for substance X) and 1 ml of colorimetric reagent. The volume in the blank tube is 7 ml. Note that the volume of the blank should always be the same as the volume of the sample.
With the blank inserted into the spectrophotometer, the instrument is adjusted to 100% transmittance
(zero absorbance). This step is similar to taring a balance: the transmittance of light through the blank will be less than 100% because of substances (including the colorimetric reagent) present in the blank. However, the instrument can be adjusted to accept this reading as 100% transmittance, so that when the blank tube is replaced by the sample tube to measure absorbance of the sample, the only thing absorbing light will be the sample molecule of interest (the solute that reacts with the colorimetric reagent).
Procedure
- Prepare a sample tube. Place 10 drops of albumin solution into a spectrophotometer tube and add 1 ml of distilled water. Add 5 ml of Coomassie brilliant blue (a colorimetric reagent used to identify protein) and allow the tube to stand until a blue color develops.
- Prepare a blank: place 10 drops of water into a spectrophotometer tube and add 1 ml of distilled water and 5 ml of Coomassie blue.
- Turn the power switch on, and allow a 5-minute warm-up period. The on/off switch is operated by the zero control knob on the left.
- Use the wavelength control knob to adjust the spectrophotometer to any wavelength between 550 and 600 nm. The selected wavelength is indicated on the wavelength readout in the window next to the knob.
- When using the Spectronic 20, the meter must be adjusted to read across its full scale—0% transmittance to 100% transmittance. With no sample tube in the machine, use the zero (left-hand) control knob to set the scale to 0% transmittance (infinite absorbance). (With no sample tube, the light path is automatically blocked, and no light reaches the phototube; thus, 0% transmittance and infinite absorbance are simulated.) Be sure the cover on the sample holder is closed when you perform this step.
- Insert your blank (be sure it is clean and dry on the outside) into the sample holder, and turn the right-hand control knob to set the meter scale to 100% transmittance, zero absorbance. This adjustment regulates the amount of light reaching the phototube in the absence of the absorber. Whenever the wavelength is changed, the 100% transmittance adjustment must be reset. Also, when operating at a fixed wavelength for an extended period of time, periodically check the 100% and 0% transmittance readouts and adjust if necessary.
- If you are beginning an experiment, repeat steps 5 and 6 to make sure the machine is stable.
- Insert the sample tube into the chamber; read absorbance directly on the absorbance scale (lower scale). The reading on the absorbance scale is proportional to the concentration of your sample substrate. Note: The absorbance scale reads from right to left, opposite to the direction of the transmittance scale. Record your data: wavelength nm, absorbance , transmittance . Do not discard your sample and blank tubes!
- The steps that follow provide a
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