UV vis Spectroscopy – How to Use a Spectrophotometer

UV vis Spectroscopy – How to Use a Spectrophotometer

In this article we will show you how to use a UV visible spectrophotometer. Instrument: Shimadzu UV-1800 UV Spectrophotometer.

These are the double beam UV visible spectrophotometers in the instrument room in the general teaching laboratory at the National University of Singapore.

Spectrophotomeer from NUS Lab

Take note that the on/off switch is at the right side of the spectrophotometer.

Spectrophotometer Switch

Here are all the apparatus that you need for this setup: a solvent bottle, a waste beaker, a pair of quartz cuvettes, and a volumetric flask containing your solution.

Apparatus for Spectroscopy

The sample will be placed in the cuvette for measurement.

Spectrophotometer Cuvette

There are three types of cuvettes: plastic, glass and quartz.

Plastic Quartz Glass Cuvettes

A common feature of our cuvettes is that each one has two opposite opaque sites and two opposite transparent sites.

You will hold a cuvette binder to opaque size the transparencies are meant for the pathway of the UV or visible light.

Cuvette fro Spectrophotometer

This is the UV visible spectrophotometer:

Uv vis Spectrophotometer

To conduct a scan, you will need a pair of cuvettes:

Spectrophotometer Cuvettes

For the first run we have to do a blank solution scan, meaning that both the “reference” and “sample” contain just the solvent.

We should rinse the cuvettes with the solvent being used. Here we are using water. (Wearing gloves is optional)

Rinse the Cuvette

Fill it with the solvent, put on the cap, shake it and dispose the liquid into the waste beaker.

Discard Cuvette Solution

You can rinse it again.

Now use the Kimwipe to clean the transparent sides of the cuvette.

Clean the Cuvette Transparent Sides

We have to make sure that there are no fingerprints, grease, paper lints, droplets, on the transparent size of the cuvette because light is passing through here and we don’t wish the light rays to be refracted.

Clean the Cuvette

Open the lid of the sample chamber, insert the cuvette in the way that the transparent sites are in the east-west direction, that’s where the light is coming from.

Place Cuvette in the Sample Holder

There are two cuvettes holders, labeled blank and sample, so place them accordingly.

Cuvette Sample

Close the lid and do the auto zeroing. There is a manual placed next to the machine you may refer to it anytime.

Spectrophotometer Instruct Manual

Switch on the machine and wait for it to initialize. So on the control panel we press the button “2” to access the spectrum.

Start the Spectrophotometer

On the screen we will see the scan range and we should assign it as 800 nm – 400 nm. The absorbance value we use is from 0 to 1.5A. Make sure these two rows data are correctly imputed.

Set Spectrophotometer Spectrum Range

Following the manual press Auto zero. You know it’s done when the spectrophotometer beeps a sound.

Take out the sample cuvette, discard the solution, and we are ready to run our sample solution. Remember that the cuvette still contains some Auto zero solvent, and we need to rinse it again with our sample solution a couple of times to ensure that the solution we are measuring is not diluted.

Take note for quantitative analysis, it is vital not to let solvent remnants to dilute and affect our results.

Now slowly pour in some of our samples into the cuvette. Put on the cap, rinse it and dispose it into the waste beaker. Repeat the cycle again.

Fill in Sample Cuvette

Now we can fill the cuvette to 4/5 of the height with the solution. Use the kimwipe to clean the transparent sides and place it into the sample holder, and close the lid.

Place Cuvette in the Testing Chamber

Press start on the control panel. Notice how the graph is forming. The lines are tracing from 800 nm to 400 nm.

And we have a peak, the maximum absorbance at about 600 nm.

Run Spectrophotometer

To zoom press F1, and then press F3 to autoscale. Now the spectrum looks fuller.

Autoscale The Spectrum Curve

Press return to change the tabs. Then press F2 for data processing, followed by “3”.

Data Processing Spectrophotometer

You can see the maximum absorption wavelength for this compound is at 591 nm and the absorbance value is 0.425. Press F2 to print the spectrum.

So this is a datasheet nicely printed and you see the peak the maximum absorption and absorbance value corresponding to it.

Print the Data

You’re good to go!


We hope you have learned well and do master this technique in the laboratory.

Demonstration: Dr. Hoang T.G.

Video/Presentation: Mr. Fung F.M.

Source: National University of Singapore

Link: https://www.youtube.com/watch?v=s5uIVQGFDE4

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