Fluorescence Spectroscopy: Emission Spectrum vs Excitation Spectrum

This video is a e-Lecture created for NUS Chemistry CM3292 experiment titled "Fluorescence of Additives in Soft Drinks".
Contents:
Emission Spectrum
Excitation Spectrum
By the end of this e-lecture, students will be able to:
1. Describe the fundamental principles of molecular fluorescence spectroscopy.
2. Compare and contrast the emission spectrum and the excitation spectrum of fluorescent compounds.
Thanks for watching :)
Presenter: Dr Francis Chong
Producer: Dr Francis Chong

[@alexandraproklova6601]

For a long time I have been looking for a similar video...And I am very grateful you now! I needed the explanation of measuring of luminescence not only theoretical information. Thanks a lot! It is the most useful of others videos!

[@FrancisChongYY]

Thank you :) Glad that you find the video helpful

[@woweycowey]

Just realized he's writing everything backwards. Mad impressed with the handwriting.

[@h.p.734]

Sorry buddy he's not writing backwards. He's probably using a mirror or editing software. kzbin.info/www/bejne/m4eygXeHarCMqdE&feature=emb_logo

[@hermitcard4494]

Slow xD

[@sandippaul468]

LOL as if.

[@khanphysics8209]

I have spent my whole day in finding the difference between them. Now finally I found your video and quickly understood the difference. Very best explanation with good writing display... appreciated

[@maxouloulou7183]

I am trying to understand those fluorescence spectrums for 2-3 weeks. You are my hero, thank you very much.

[@sarithaac104]

Really Really useful....There doesn't exist any other video which explains Excitation Spectrum in such a nice way . Thank You ....

[@yonak4963]

This helped me a LOT. My brain could never understand the reason behind excitation spectrum. Now I get it :D

[@FrancisChongYY]

@Yona K, thank you :) Glad that the video helps :)

[@mukheshk.g.1850]

one of the best video I came across

[@vishnuverman4864]

Thank you very much! It was lucidly explained with frequent connections to experimental protocol, going to go run my first emission spectra tomorrow. :)

[@roopamrajvanshi675]

Just wonderful. Was having a hard time understanding the overlap of excitation and emission spectra. This has clarified my basics. Thanks a lot!

[@nancysohlberg8150]

Super clear and helpful, been looking for this explanation for a while, thanks so much for making this!

[@havingicecream]

Oh Man, that was such a great explanation! I had such a hard time understanding this, thank you very much!! also, your drawing and writing is super neat!

[@FrancisChongYY]

@havingicecream, thank you :)

[@JacobSankus]

Great explanation, thanks for the help !

[@mr8Ninja]

Also been searching for this sort of explanation for a while! This video was super helpful, thank you :)

[@FrancisChongYY]

Thanks for watching, @Josh Selfe. Glad that the video was helpful :)

[@MegaPruddy]

thank you so much! this is so neat and straight to the point ❤

[@dyahlangensari4802]

THANK YOU!!! you really help me a lot to passing thru my exam and my prof's class.....

[@mihaelsa]

Thank you for this short and clear presentation. This was very helpfull to me.

[@kiranali6677]

Thanks a lot for this lecture. It was really helpful.

[@shibaprasadpanda1653]

Thank you sir for the clarity 🥳

[@cchem8198]

Thank you very much... great explanation....

[@hamidali5236]

Tremendous one!!!!!! Really well explained and it helped me alot.

[@FrancisChongYY]

Hi @hamid ali, thanks for watching. Glad that the video was helpful :)

[@luluperez4810]

Excellent video!!! So useful, thank you!

[@FrancisChongYY]

Hi @Lulu Perez, you are most welcome! Thanks for watching!

[@user-ko9dq3wm3x]

Thank you very much, I have finally understand the excitation and emission spectra

[@FrancisChongYY]

Thanks for watching. Glad that the video was helpful.

[@hamidali5236]

Tremendous lecture!! It helped me alot Thank you so much sir

[@FrancisChongYY]

Thank you @hamid ali :) Glad that the video helps

[@shameer339]

Good explanation 👍

[@nareshbarik5384]

Thank you ♥️

[@sauravdutta1983]

nice explanation

[@SotosMoud]

Thank you !

[@mohammadramezani7672]

Thank you. it's so useful for me.

[@FrancisChongYY]

@hamed ramezani, thanks for watching. Glad that you find it useful :)

[@dingman149]

Very helpful. Thank you very much!

[@FrancisChongYY]

@Ding Man, thanks for watching. Glad that the video was helpful :)

[@Vikas-eg9tp]

compact and descriptive video...very nice

[@FrancisChongYY]

@Vikas Nain, Thank you very much for your kind words :)

[@princesse9865]

Thank you good video

[@decalogians9321]

Thank you for this

[@pablabra674]

Excellent video. Thank you.

[@FrancisChongYY]

Hi @Pablo Labra Vázquez, thank you very much for your kind words :)

[@gayathris7169]

Thank you for the clear information

[@FrancisChongYY]

@Gayathri S, you are most welcome :)

[@celinrooth2219]

Thank you so much sir for this clear and clean explanation...This video helped me a lot.

[@FrancisChongYY]

Hi @Celin Rooth, thanks for watching. Glad that you found the video helpful :)

[@khurramjaved2580]

Thank you! This is really informative. I was wondering how the photoluminescence (PL) spectrum differs from emission spectra?

[@tashawnawilliams6016]

Wow what an excellent teacher

[@FrancisChongYY]

@TaShawna Williams, thank you for your kind words :)

[@sauravdutta1983]

thanks for such a nice explanation

[@FrancisChongYY]

Thank you. You are most welcome :)

[@javierpadilla007]

Thanks for the video!

[@FrancisChongYY]

@Javier padilla, thanks for watching :)

[@samgaita]

Thanks for the explanation.

[@FrancisChongYY]

Hi @samgaita, you are welcome. Thanks for watching.

[@dinometeourblast4223]

Thank you for the great video. Shouldn't there be multiple peaks for if the fluorescence brings it down to the n=0 energy level, but still has vibrational energy?

[@carolinasalastapia3604]

Just thank you

[@FrancisChongYY]

@Carolina Salas Tapia, thank you

[@pawab]

Hi Francis, thank you for a nice video! A question to you as a specialist: can a sample fluoresce without absorption?

[@serrao4239]

LEGEND

[@samarjitpattnayak6052]

Thank you for this nice work....it helped me a lot...Please suggest me a book to understand fluorescence spectroscopy further

[@paulinafreire3460]

You make my life easier with this video. Could you do a video which explain synchronous fluorescence and synchronous obtained with wavelength deltas ?

[@FrancisChongYY]

Hi Paulina, thank you :) Glad that you find our video helpful :) As of now, we don't have any plan to make a video on Synchronous Fluorescence Spectroscopy (SFS) as it's not included in the syllabus of our course. If you would like to find out more about SFS and how it works, please check out this book chapter here: link.springer.com/chapter/10.1007%2F978-1-4419-9828-6_5#page-1 Li YQ. et al. (2012) Synchronous Fluorescence Spectroscopy and Its Applications in Clinical Analysis and Food Safety Evaluation. In: Geddes C. (eds) Reviews in Fluorescence 2010. Reviews in Fluorescence, vol 2010. Springer, New York, NY

[@sumermeena8388]

thanq sir..

[@FrancisChongYY]

You are welcome, sumer meena :)

[@mariappanm7677]

For a new compound with the UV data available, how to fix the correct excitation wavelength to measure the emission spectra?

[@user-fr6ed1pp3p]

Hi , can you help me please How can I use the synchronous scan in spectrofluorometer

[@cristaylor1362]

Thankyouu

[@richardambrose1827]

Since the emission spectra was made using 350 nm wavelength, is the excitation wavelength in the excitation spectra which gives the max intensity for 450nm emission wavelength, 350nm itself?

[@PremKumar-bc8rq]

my question is which is use to find what? i mean to say exc spectrum is used to find emission or emission is used to find excitation. which to run first?

[@sara7121]

Can I join the two spectra and form Jablonski Diagram for fluorescence? How would it be?

[@williammai7571]

thank you

[@FrancisChongYY]

@Mai Lam Bac, you are welcome :)

[@cutieaadi9599]

So, you mean for different machine we have to find out working excitation and emission wavelength? how much variation could be possible among different machine? I have seen in different publications that 300nm (some 305nm) excitation and 410nm (in some 420nm) emission, thus, my question is, by doing spectra reading in my machine cytation5, I explored higest emmision reading at 350nm and 290nm Excitation....I am confuse with this much variation. is it possible?

[@md.sydulislam4362]

nice

[@Baconguy-mi7jz]

THank you so much! Next time can you teach us how to correct the inner filter effect of fluorescence spectrum! please!

[@FrancisChongYY]

Hi @badamkhatan tuguldur, thanks for watching. The inner filter effect is a secondary absorption caused by the reabsorption of the emission by the analyte species itself or by other species presence in the solution. It occurs when there is an overlap between the emission spectrum and the absorption spectrum. Practically, one quick fix would be to dilute the sample, or change the solvent system.

[@Kaizen_worldview]

Hi sir. May I know how to make a video like this when teaching?

[@dr.p.ramamurthy6458]

Nice presentation. I want to know which device you have used to write . Is it a digital board? How did you include your board as a transparent display. Thank you in advance.

[@FrancisChongYY]

Hi Prof @Ramamurthy Perumal, thank you very much for your kind words. It is not a digital board. I simply wrote the content on a piece of glass with fluorescence markers. You may refer to lightboard.info/ for more information. Hope this helps :)

[@NonymAn0]

So this would mean the excitation peak should be about the same as the UV-Vis peak, if there are no special conversions involved?

[@drivetoperfection112]

@Francis Chong Dear Francis, so the Emission Spectrum it's about Fluorescence and the Excitation Spectrum it's about Phosphorence?? Keep the nice work! Thanks

[@FrancisChongYY]

Hi @Drive To Perfection, thanks for watching. In this video, the emission spectrum we discussed is a fluorescence spectrum, which usually involves a relaxation from a singlet excited state to a singlet ground state. As for phosphorescence, it usually involves relaxation from a triplet excited state to a singlet ground state. The key difference between the two is the occurrence of the inter-system crossing (from singlet excited state to a triplet excited state, in the case of phosphorescence). You may refer to the Jablonski diagram for more information: chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Electronic_Spectroscopy/Fluorescence_and_Phosphorescence

[@Kalyanraj95]

Hi Dr. Chong, I had a question. Why not do the excitation first and find out the excitation maximum and then do find the emissions spectra? Why choose an arbitrary excitation wavelength? Another question: How is the emissions intensity is measured? Does the machine counts the number of photons at each emissions wavelength and the wavelength with the maximum photons is chosen as the emission wavelength.

[@FrancisChongYY]

Hi @Abhishek Bastiray, yes, it is possible to conduct an UV-vis absorption spectrum of the analyte first to have some idea of the absorption maxima of the analyte. If only a fluorescence spectrometer is available, using an arbitrary excitation wavelength will be the next better option. Yes, on the technical aspects of how fluorescence spectroscopy works, I believe you are right. If you are interested in finding out more about the theory behind the emission maxima, this reading may help: application.wiley-vch.de/books/sample/3527316698_c01.pdf Particularly on these two topics: the Franck-Condon Principle and the Born-Oppenheimer approximation.

[@Kalyanraj95]

@@FrancisChongYY Thank you Dr. Chong, this video particularly helped me clearing some of the doubts regarding fluorescence spectroscopy. Again thank you for the sources mentioned I will go through it swiftly.

[@disha6010]

as there is a difference in excitation wavelength and emission wavelength, excitation < emission. how much difference is considerable? is it possible that we give excitation wavelength of 400nm and emission will come in the range of 800-900nm? thankyou in advance

[@latiefmohiuddin6840]

Yes. Thats the typical downconversion behaviour. For broader explanation, Check Upconversion and Dowconversion Phosphors.

[@ashaduladalder1752]

Hello Sir, I have a small question. Why there are two different peaks in excitation spectra?

[@FrancisChongYY]

Hello @Ashadul Adalder, thank you for the question. The small peak is actually a shoulder. They represent the electronic transitions from the ground state (S0) to the first excited state (S1). And the reason why we see two peaks instead of one is because there are many different vibrational energy levels within each electronic energy levels. To learn more about this topic, you may refer to the Jablonski diagram: www.edinst.com/blog/jablonski-diagram/ To learn more about the different types of spectra and practical aspects relevant to the fluorescence of quinine, please check out this paper: pubs.acs.org/doi/abs/10.1021/ed053p191 Thank you, @Ashadul Adalder.

[@ashaduladalder1752]

@@FrancisChongYY thanks a lot sir for answering me.

[@FrancisChongYY]

@@ashaduladalder1752 You are welcome :)

[@princemutshinyalo8684]

Why emission wavelength always higher the excitation wavelength?

[@FrancisChongYY]

Hi @Prince Mutshinyalo, thanks for asking the question. In general, the emission wavelength is usually longer (lower in energy) as compared to that of the excitation wavelength. It is mainly due to the non-radiative relaxation to the lowest vibrational energy level of the excited state before relaxing back to the ground state. You may refer to the Jablonski diagram for more information: chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Electronic_Spectroscopy/Jablonski_diagram

[@SagarMunjalMCY]

why emmission spectrum is gaussion/

[@FrancisChongYY]

Hi @Sagar Munjal (M19CY021), you may refer to the following discussion thread about the question you asked: www.researchgate.net/post/Why_doesnt_the_fluorescence_spectrum_look_like_Gaussian_distribution Hope this helps.

[@samarjitpattnayak6052]

Thank you for this nice work....it helped me a lot...Please suggest me a book to understand fluorescence spectroscopy further