Dear Mingyu Kwak,

From your spectra, you seem to use ethanol as a baseline rather air. This can cause a problem due to the nature of the calculation from the software in the spectrophotometer.

Please be aware of the measurements in the spectrophotometer are the transmission spectra. The absorbance is derived from the transmission spectra using software. For the liquid sample measurements, you should use the baseline of air, which means that when you set the parameters of equipment, you should set transmittance and both sample beam and reference beam are empty to scan the baseline. After that, measure the transmission spectrum of the solvent you use. Then measure your sample in the same solvent.

Finally, you can extract the sample transmission spectra. Then convert the transmission spectra into absorption spectra. In this way, you can gain the nature of your sample absorption spectra.

Dear Mingyu Kwak,

From your spectra, you seem to use ethanol as a baseline rather air. This can cause a problem due to the nature of the calculation from the software in the spectrophotometer.

Please be aware of the measurements in the spectrophotometer are the transmission spectra. The absorbance is derived from the transmission spectra using software. For the liquid sample measurements, you should use the baseline of air, which means that when you set the parameters of equipment, you should set transmittance and both sample beam and reference beam are empty to scan the baseline. After that, measure the transmission spectrum of the solvent you use. Then measure your sample in the same solvent.

Finally, you can extract the sample transmission spectra. Then convert the transmission spectra into absorption spectra. In this way, you can gain the nature of your sample absorption spectra.

Mingyu Kwak

This seems to me basic/fundamental stuff. Are your cuvettes are made of glass, quartz or plastic? Then look at the absorption caused by these materials.

See, for example:

https://handling-solutions.eppendorf.com/sample-handling/photometry/vessels/detailview-vessels/news/which-cuvette-is-the-right-one-glass-vs-plastic-vis-vs-uv-micro-volume-vs-macro-volume/

I have taken the attached figure from this url (Figure 2).

And from:

https://lab-training.com/2016/09/14/select-right-cuvette-material-uv-vis-absorbance-studies/

• 'However, glass absorbs strongly in UV region and its application is not recommended for wavelengths below 340 nm'
• 'Quartz is expensive in comparison with glass and transparent plastics but has the additional benefit of covering both UV and visible regions right from 190 nm'

So you really don't have a peak at 250 nm, IMHO. It's an artifact.

Last, but not least, is this a single or dual beam instrument you are using?

First you should reduce the concentration of your suspensions. Remember that Abs is calculated as -logT, so an absorbance of 3 is a transmission of 0.001. The peaks are saturated, so you don't see their true shape.

Hugh J Byrne

Sir, should absorbance be less than 1? Does Beer-Lambert's law work at lower absorbance values only?

Aarif Shah

Absorbance is logarithmic so a value of 3 is possible - however, this means that the transmission is 10E-3 or 0.001 (0.1%) and thus 99.9% of the light is attenuated.

Aarif Shah

Hi, Absorbance should go from 0 to...... the level at which the signal to noise is no longer acceptable. In most spectrometers, especially in the UV region where the light levels from the lamps are relatively low, a value of 2 is normally the reliable limit.

Remember, Abs = 2 means a Transmission of 0.01, or 1% of your light.

Alan F Rawle

Thank you sir for the reply. I tried to experiment with the UV-Vis spectrophotometer in the lab. Have written the play in next comment.

Hugh J Byrne

Sir, thankyou for the reply. I tried to check the absorbance of a solution by making series of standards (2 ppm to 200 ppm ). What i observed was that the instrument was able to display the absorbance values upto 80 ppm only. Beyond 80 ppm, it displayed 9.999 as the absorbance of all the solutions. What can i infer from this regarding the limit? Sir, could you please share some knowledge regarding this vis a vis Beer Lamberts law

Aarif Shah - you should try to ensure that you have at least 10% (0.01)Transmission through your sample. This means an Absorbance of (A = - log(T)) of 2.

Hugh J Byrne - Thank you sir

Xuhua Wang

Thank you for your answer.

But I can't understand 'both sample beam and reference beam are empty to scan the baseline'.

What means that sample beam and reference beam are empty?

In my understand, the process is as follows.

1. Scan the empty cuvettes transmittance as baseline.

2. Scan the transmittance of the solution and sample.

3. Extract the solution transmittance from the sample's.

4. Convert transmittance to absorption.

Another question is.

Doesn't it matter set the transmittance as baseline and then scan the absorption?If not then when I can use absorption spectrum?

Alan F Rawle

Actually, I use quartz as cuvette, so it does not seem to be a material problem.

But I did not think of the difference due to the cuvette, thank you for your reply.

@Mingyu Kwak,

The empty means that there are no samples presented both for the reference beam and sample beam. In another words, only air is there.

You have to use the transmission mode in a spectrophometer. Do your own calculation to convert into the absorption spectra. If you use the absorption mode, you will find a problem due to the absorbance of your solvent in the UV region as a reference. You will get a artefact in the UV region of absorption spectrum. If you want to know more in details, i may send you my lecture notes. This is related to how the software is used to calculate the absorbance from its transmittance measurements.

Bear in mind that the spectrophometer measures transmission spectra directly, not absorption.

Hope it helps.

Xuhua Wang

Thanks :)

I'll try again. Can I receive your lecture notes?

It would be pleasure if you send it.

This is my mail.

[email protected]