Use of the Spectronic 20 Use of the Spectronic 20 SpectrophotometerSpectrophotometer

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 ControlsOverview Spectronic 20 Controls

Turn POWER ON with Power/Zero control (left) knob.

Red light will go on.

Allow 15 min

warmup time.

MeterMeterAbsorbance and % Transmittance Scales

Sample Holder

Power/ZeroControl

100% Trans/LightControl

WavelengthWavelengthControlControl

WavelengthWavelengthScaleScale

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 WavelengthSet 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 MeterReading the Meter

Slide 4-Reading the MeterSlide 4-Reading the Meter

The meter simultaneously reads:

Percent Transmittance--the portion of incident light the portion of incident light passing through the samplepassing through the sample--on the top scale, and

Absorbance--the portion of incident light absorbed the portion of incident light absorbed by the sampleby the sample--on the bottom scale.

Reading the MeterReading the MeterTop scale is read to the right.Top scale is read to the right.

Bottom scale is read to the left.

Slide 5a-Reading the MeterSlide 5a-Reading the Meter

• The top scale (%T)(%T) is divided into increments of constant size and must be read from left to left to rightright.

This linear scale is easy to read.

• The bottom scale (Absorbance)(Absorbance) has increments increments between tick marks that vary across the scalebetween tick marks that vary across the scale and must be read from right to leftright to left.

This nonlinear scale is more difficult to read accurately.

Reading the MeterReading the MeterAvoid Parallax ErrorAvoid Parallax Error

Position your head directly in front of the meter.

Adjust viewing angle until the needle and itsshadow on the mirror are aligned.

Reading the MeterReading the MeterAvoid Parallax ErrorAvoid Parallax Error

Calibrating the InstrumentCalibrating 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% TransmittanceSetting 0% Transmittance

With an EMPTY sample chamber adjust the LEFT knobuntil the meterreads 0% transmittance.

Setting 100% TransmittanceSetting 100% Transmittance

With a cuvette containingthe BLANK solution (water) adjust the RIGHT knobuntil the meter reads 100% transmittance.

Sample HandlingSample 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 CuvettesRinse 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 SolutionUse Correct Volume of Solution

Filled properly. Not filled enough.

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 BubblesRemove 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 KimwipesClean 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 HolderSample Holder

Sample Sample HolderHolder

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 Inserting and Aligning the Cuvette in the Sample Holderin the Sample Holder

Vertical Index MarkVertical Index Mark

NubNub

Align the Vertical Index mark on the cuvette with the raised nub on the front of the sample holder.

Insert the cuvette into the sample chamber by gentlygently pushing it into position.

Hard pushing could damage the instrument.

To ensure reproducible positioning in the sample holder alignalign the Vertical Index mark on the Vertical Index mark on the cuvettecuvette with the raised nubraised nub on the front of the on the front of the sample holdersample holder..

17a--Inserting and Aligning the Cuvette in the Sample Holder

Final Step--Take the ReadingFinal 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 MeasurementSummary--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.