Use of the Spectronic 20™ at a Fixed Wavelength

A Description Turning On The Instrument Adjusting the Wavelength Reading the Meter Preparing a Cuvette Setting Maximum Absorbance Setting Zero Absorbance

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The Spectronic 20™ is an instrument that measures the amount of light absorbed (or transmitted) by a solution.

Light from a lamp is dispersed by a grating into a continuous spectrum of colors. Light of one wavelength (and color) is passed through a sample of solution and than illuminates a phototube detector. The detector produces an electrical signal that depends on the intensity of light. The size of the signal is displayed on a meter. The wavelength is adjusted by rotating the grating so that a small selected region of the visible spectrum passes through the exit slit into the sample.

This diagram is a representation of the inside of a Spectronic 20™

Turn the instrument on using the Power Switch/Zero Control knob, and allow the Spectronic 20™ to warm up for at least 15 minutes. Why should it warm up for 15 minutes?

Video: Turning the Spec 20™ On ( 1.37M )

Which of the following are likely to happen if the instrument has not warmed up for fifteen minutes prior to use? A. Recorded absorbance readings will be inaccurate. B. The sample will be automatically ejected from the sample compartment. C. No light will pass through the sample. Answer

After the instrument warms up, select the desired wavelength by turning the wavelength control knob. Be sure to reset the maximum and zero absorbance values every time you change the wavelength.

The Spectronic 20™ meter displays both absorbance and transmittance.

What is the difference between absorbance and transmittance?

The top scale shows transmittance, and the bottom scale shows absorbance. Read the meter so that the needle obscures its reflection in the mirror on the meter. Correct and incorrect meter readings are shown below.

Correct reading, straight-on: Absorbance = 0.238	An incorrect reading from an angle - note the reflection

Use a clean cuvette that is free of chips and cracks.

Video: How to Prepare a Cuvette ( 3.33M ) Text description

Rinse the cuvette with a small amount of solution, then fill the cuvette at least half full. Check your sample for air bubbles. WHY?

Remember to wipe the cuvette with a laboratory tissue and to align the mark on the cuvette with the mark on the Spectronic 20™. WHY?

Click on the sample below which is appropriate for use in the Spectronic 20™... A B C D E

The maximum absorbance is measured when no light is transmitted to the phototube detector. It is set using the power switch/zero control knob.

Video: How to set the maximum absorbance ( 1.01M ) Text description

Remember, the maximum absorbance is set with the sample compartment empty. WHY?

Video ( 914K ) The Spec 20™ in the video is being set for maximum absorbance. Would any subsequent measurements be accurate? Why or why not? Answer

A reference solution is used to calibrate the scale of absorbance readings. It should be the same as the solution to be analyzed except that it must have zero concentration of the absorbing species. The reference with no absorbing species is used to set zero absorbance.

Place the reference solution in the sample compartment and use the transmittance/absorbance control knob to set the meter to zero absorbance (which is equivalent to 100% transmittance).

Video: How to set the zero absorbance ( 2.05M )

Click on the knob used to adjust the absorbance/transmittance within the picture of the Spectronic 20™ shown below.

The maximum and zero absorbance values must be set each time the wavelength is changed. WHY?

It is a good idea to recheck the maximum absorbance after setting the zero absorbance to verify that the instrument is working properly.

The instrument is now ready for the sample. Place the cuvette containing the sample into the sample compartment and record the absorbance.

A student is measuring the absorbance of a solution of iodine in hexane. Suggest an appropriate reference sample. Answer

Miscellaneous exercises

Video ( 2.66M ) Do you expect the absorbance measured by the student to be higher or lower than the actual absorbance of the solution? Why? Answer

Related module: Spectronic 20™ at Variable Wavelength