The ratio of the two peaks depends on the environment of the dye. I remember seeing spectra for some Rhodamine labelled molecules, where the two peaks were very similar, so I guess at some conditions the 250 nm peak may be bigger. Incorrect calibration of the instrument can, of course, also distort the ratio of the peaks (incorrect compensation for spectral variations in the light source output and detector sensitivity). This could be easily checked by measuring a blank sample. Otherwise you can test Rhodamine in a plain solvent or check your sample on another instrument to eliminate possible sources of artefacts.

@Radek Machan sir, Thank you sir for your valuable suggestion, I will work on that

By degradation you likely destroy part of the chromophore, i.e. you will have degradation products lost 550 nm peak but not aromatic moiety.

@Titus Sobisch, Thank you, sir, for your answer. but how we can explain the increase in the intensity of the 250 nm peak? please explain sir.

When degrading condensed structures (lower stability against degradation) the more stable aromatic rings are left over.

 thank you, Titus Sobisch sir

Why do you call it peak reversal? It is just reduction in peak intensity of the one bands due to destruction of the chromophore. If my guess is correct the change in intensity of the 250 nm peak may not be appreciable but there is a considerable decrease in peak intensity of 550 nm band.

Sorry sir, it is not the peak reversal, there is reversal of peak intensity (if we follow the general results), please see the attached image 

you are degrading, therefore as expected absorbance of chromophores - conjugated system and aromatic rings drops. Conjugated system is first destructed, therefore this first vanishes. In addition you see an increase in nonspecific absorption/extinction. This may be also turbidity which may develop.

Here are some observations from the image you posted

1. I don't see any scattering signature from your sample.

2. The degradation of the UV absorbing chromophore is leading to formation of a species which absorbs in the entire visible region.

3. If i assume that your baseline is drifting then there is no absorption in the visible region and there is only absorption in the UV only - looks unlikely

4. In your question you mentioned you have used Methylene Blue or Rhodamine dyes and these dyes absorb in the visible region, but I don't see any absorption in the visible region.

    Here could be some possibilities

       Your dye is insoluble in the solvent you are measuring or

        You are working with very low concentration of the dye

5. There is another possibility, you already have a degraded dye at zero time. [Can't say much because I don't know how you are handling the sample and performing the expt].

@Titus Sobisch, @V. N. Ravi Kishore V., thank you, sir's, for your valuable comments

I read the valuable answers of answerers concerning methylene blue with sulfuric acid and this question in the past.

Your 2 questions are too vague  and written in a non scientific manner. WHY?

- you write :"When I am doing UV-Vis experiments with methylene blue or Rhodamine, I am getting the evidence for their degradation,

Do you mean degradation in solution e.g. chemical  or photo or sonodegradation  using  TiO2  p.e.

- please give  what a kind of rhodamine (Rhod. B or 6 G  or another rhod. derivatives.

- What a kind of purity (%)  for rhod dye and MB  and a what a solvent are you using (cut off of the solvent is important).

What a concentration of the above cited dyes are you using (e.g. monomer/dimer ratio).

Please note that MB degrades slowly in the dark according to pH conditions used for the research.

What a kind of spectrometer are you using. ( single beam, dual beam, diode arrays??)

What a kind of catalyst are you using  (TiO2  or another product? Xour sentence : my catalyst ......shows that you are not familiar with posting a good question on RG.

Please use GOOGLE SCHOLAR  and NOT GOOGLE as help for formulate a good question prior posting it on RG.

JRG

An answerer needs all these infos for a good answer.

Sir, following are the related things to this experiment

1. It's a light-induced degradation of the solution

2. It's Rhodamine B, AR grade

3. Solvent is water

4. Dye concentration is 10 (-6) M 

5. spectrometer is dual beam 

Thank you for the fast answer. It is quite perfect!  Why?

- Solvent is water!! What a kind of water (impurity...a.s.o.)

- Rhodamine B AR grade! Well ! What is the counter anion ?Chloride?

- You must check the purity with Thin Layer Chromatography (TLC) prior to use RhodB for your project.

- Is the light induced degradation done with solar   or Hg lamp e.g. UV-Lamp? I send you a picture of Hg lamp spectrum . The HG spikes are bad for your project if you don 't use a filter.

- The degradation will lead at first to a dealkylation of Rhodamine B chloride.

Among them the Rhod 110 will be produced (see the jpg file).

Water has a cut off of 190 nm  if you use quartz cuvette. What a kind of cuvettes are you using. Quartz or plastic??

Your 250 nm peak will be perhaps an artefact belonging to the cuvettes.

JRG

Thank you, sir, for your suggestion

I am using UV light tube (2 X 11 W, 465 nm wavelength) without any filter and solvent is MIlli Q water. Cuvettes are made up of quartz

According to the very good answer of :

V. N. Ravi Kishore V. · 45.45 · Looking for a change

Here are some observations from the image you posted

1. I don't see any scattering signature from your sample.

2. The degradation of the UV absorbing chromophore is leading to formation of a species which absorbs in the entire visible region.

3. If i assume that your baseline is drifting then there is no absorption in the visible region and there is only absorption in the UV only - looks unlikely

4. In your question you mentioned you have used Methylene Blue or Rhodamine dyes and these dyes absorb in the visible region, but I don't see any absorption in the visible region.

    Here could be some possibilities

       Your dye is insoluble in the solvent you are measuring or

        You are working with very low concentration of the dye

5. There is another possibility, you already have a degraded dye at zero time. [Can't say much because I don't know how you are handling the sample and performing the expt].

and your answer to my questions, I think that you don't read my questions correctly. Why? I asked in my first answer : "What a kind of catalyst?". Please answer it!!

If it is TiO2 , please read the pdf joined to my answer  and compare the UV-Vis with Tio2 of this article and your posted spectrum.

Please post a picture of your rhod. B solution in the cuvette in order to see the colour with your concentration 10 -6 M.. I hope you can take a picture and send it as jpg.

What is the name of your dual beam  spectrometer

JRG

Please read the page 41 of the joined pdf article (degradation of Rhod B Cl

@Dr Jean Rene

I read your answers. They are perfect. I have only a question regarding the following

""Please note that MB degrades slowly in the dark according to pH conditions used for the research.""

If MB stock (100 mg/L) with DI  is prepared, the solution can be preserved in dark place. Note that pH is about 5.30. So it will degrades also or not?? If yes, how can we decrease that effect??

Regards

Dear Mahmoud,

prior I answer your question :

If MB stock (100 mg/L) with DI is prepared, the solution can be preserved in dark place. Note that pH is about 5.30. So it will degrades also or not?? If yes, how can we decrease that effect??

I need the following infos:

- The counter anion of MB is ?   e.g. Cl-  or  ZnCl2 or another anion?

- THe name of the Cy selling your MB

- PH 5.3   ! Is it a buffer or an acid solution?

- Have you taken a spectrum of your MB in dest . water  (if yes , please send me the spectrum (UV.Vis) of your stock solution.

- Are you familiar with TLC (Thin Layer Chromatography)? If not , please read a book about TLC. You need TLC for checking the purity of MB.

Please note that you cannot buy pure MB worldwide. It is always a mixture of MB with azures.

JRG