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In-class Questions: Molecular Luminescence

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    112630
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    I provide a very brief introduction on the meaning of luminescence and indicate that the process of fluorescence – a subcategory of luminescence – involves emission of radiation from a species that has first been excited by light, the details of which we will develop through a series of questions. I also describe how a fluorescent light works.

    Energy level diagrams for organic molecules

    1. Draw an energy level diagram for a typical organic compound with \(\pi\) and \(\pi\) * orbitals and indicate which orbitals are filled and which are empty.

    Given our prior discussion of UV/VIS absorption, groups can immediately write the answer to this question.

    1. Now consider the electron spin possibilities for the ground and excited state. Are there different possible ways to orient the spins (if so, these represent different spin states).

    The groups can usually see that there is only one way to write the ground state. They often see that in the excited state it is possible to have the spins of the electrons paired or parallel. It is worth indicating that for the situation where the excited state has paired spins, it does not matter which one is spin-up and spin-down as these are identical so long as the spins are paired.

    1. Do you think these different spin states have different energies?

    Many students just make the intuitive guess that they do have different energies, although they really may not yet understand why this is.

    1. Which one do you expect to be lower in energy?

    I generally find that students get this wrong and think that the excited state with the spins paired would have the lower energy. Even if they think the state with the parallel spins is lower, they rarely if ever provide the proper justification for this answer. I point out that they actually learned something in general chemistry while doing atomic structure that would allow them to answer this question and justify their answer. If this doesn’t lead them to the proper answer, I direct them to consider the following:

    Think back to atomic structure and remember what happened when two electrons were put into a set of p-orbitals.

    They do remember that the electrons go into separate orbitals with parallel spins. We can then discuss why this was the case – it takes energy to pair electrons, therefore parallel spins in degenerate orbitals will be the lower energy of the two possibilities.

    Do you think the energy state with parallel spins in non-degenerate \(\pi\) and \(\pi\)* orbitals will be lower in energy than paired spins?

    At this point they decide that the state with the parallel spins will be lower in energy.

    1. If the spin state is defined as (2S + 1) where S represents the total electronic spin for the system, try to come up with names for the ground and possible excited states for the system that are based on their spin state.

    They may need to be reminded about the values of electron spin quantum numbers. Recognizing that the ground state and excited state with pair spins have an S of zero and a spin state of one is obvious to them. I point out that they only need to use +1/2 for the electron spins for the case where both have parallel spins and they arrive at the number three for the spin state. With those numbers, they usually can come up with the name singlet and triplet or something close to that to distinguish the two states.

    1. Draw a diagram of the energy levels for such a molecule. Draw arrows for the possible transitions that could occur for the molecule.

    With the knowledge from the previous section of the course on UV-Vis absorption spectroscopy, the students are usually able to draw a reasonable first approximation for the energy level diagram needed to discuss fluorescence. They know about the first and second excited states, and that there are vibrational and rotational levels superimposed on each. They can usually locate the first excited triplet state, and I usually point out that we often write this off to the right side of the first excited singlet state to make the diagram a bit clearer.

    As they start to put transitions onto the diagram, they are usually quick to realize there will be absorption into the excited singlet states because that was discussed earlier in the unit on UV-Vis spectroscopy. If not, I may prompt them to show possible absorption transitions on their diagram. They also usually draw an absorption transition to the T1 state, and I point out to them that absorption transitions that involve a spin flip are forbidden by selection rules (if the idea of selection rules has not been discussed yet in the course, this is a good time to discuss that or remind them how selection rules were important in determining something like the molar absorptivity).

    Now that the electron is excited, what are its various options for getting back to the ground state?

    They can usually determine that the excited species can either lose the extra energy as heat (I indicate that this is something we can denote on the figure with a squiggly line) or lose it as radiation (I indicate that this is something we can denote on the figure with a solid line). We can then illustrate both transitions for a system in the S1 state and I indicate that the loss of energy as heat is referred to as radiationless decay and the lost of energy as radiation is referred to as fluorescence. I remind them that fluorescence specifically refers to a system that has been excited by absorbing light (as distinct from being excited by absorbing heat), and that the emission of light must be a transition that goes from one singlet state to another.

    What process would promote radiationless decay?

    They can usually reason out that collisions of the excited molecule with surrounding compounds in the matrix will promote radiationless decay.

    What can happen to an electron excited into the S2 state (or higher vibrational level within the S2 state)?

    They usually think it is possible to have both radiationless decay and fluorescence, and I indicate that only on a few rare occasions have people discovered molecules that fluorescence from the S2 state.

    At this point, it is useful to mention that it is important to consider the lifetimes of the different states.

    Can you guess the lifetime of an electron is a first excited singlet stage, second excited singlet state, and vibrational state?

    They usually propose that the lifetime of the second excited singlet state is probably less than that of the first excited singlet state. I then have them take guesses and play a game of warmer and colder as we hone on in the values. With the lifetimes now at their disposal, they can appreciate why systems excited to higher energy states than the S1 state will quickly undergo radiationless decay.

    It is now necessary to discuss the process of internal conversion that allows systems in the S2 state to convert into the S1 state and to indicate these on the diagram.

    Is there only one possible fluorescence transition from S1?

    Since they are already familiar with the fact that molecules can be excited into higher vibrational levels of electronic states, they usually reason out that molecules can perhaps undergo fluorescent transitions into higher energy vibrational states of S0. We then discuss how fluorescence from a molecule can have a variety of different wavelengths. Also that there will be different probabilities for each of the fluorescent transitions and that the S1-S0 may or may not be the most probable one.

    At this point we have Figure 3.4 from the text complete except for a consideration of the T1 level.

    Would it be possible for a molecule to undergo a transition from S1 to T1?

    They remember that we just said transitions that involved spin flips are forbidden, but they usually suspect an S1 to T1 transition can happen because we have just spent so much time developing the idea of the triplet state. We then discuss what may account for the intersystem crossing process to occur. We also discuss how the transition can occur into an upper vibrational level of T1 and then undergoes radiationless decay into the T1 state.

    1. What do you expect for the lifetime of an electron in the T1 state?

    They usually realize that the only way for the electron to get out of the T1 state is to first undergo a spin-flip, and this will take some time. We play another guessing game of warmer-colder to get the range of lifetimes of systems in the T1 state.

    When prompted they usually think the only possible route to lose the extra energy is through an intersystem crossing back into a higher vibrational level of S0 followed by radiationless decay. But I also indicate that even though emission of a photon from T1 to S0 is “forbidden”, in some systems it actually happens, and we call this phosphorescence. I point out examples of where they have seen phosphorescence (glow in the dark substances, their TV screen for a few seconds after they turn it off).

    1. Why is phosphorescence emission weak in most substances?

    They can usually rationalize out that, in most compounds, intersystem crossing is weak to begin with (I use this as an occasion to point out the fact that processes like radiationless decay, fluorescence, and intersystem crossing are all in competition with each other), which is one reason for the weak intensity of phosphorescence. Similarly, they can usually reason out that the long time the system spends in the T1 state means more collisions will occur.

    What could you do to a sample to enhance the likelihood that phosphorescence would occur over radiationless decay?

    Usually someone in each group can come up with the idea of freezing the sample into a solid to reduce the number of collisions.

    1. Which transition (\(\pi\)*-\(\pi\) or \(\pi\)*-n) would have a higher fluorescent intensity? Justify your answer.

    Think back to our unit on UV-Vis spectroscopy – we discussed at least one important reason that will help answer this question.

    Some of the students usually remember that the molar absorptivity for n-\(\pi\)* transitions is lower than that for \(\pi\)-\(\pi\)* transitions so that there will be fewer excited molecules and fewer to undergo fluorescence.

    I then tell them that the lifetime of the n-\(\pi\)* excited state is longer than that of the \(\pi\)-\(\pi\)* excited state, and ask how this will influence fluorescence intensity.

    With that information, they realize that there will be more collisional decay for the molecule in the n-\(\pi\)* state and therefore lower fluorescence.

    Instrumental considerations for luminescence measurements

    1. What would constitute the basic instrumental design of a fluorescence spectrophotometer?

    The students think of things like a light source, monochromator, sample holder and detector. They rarely seem to be able to think it through to the point of realizing that fluorescence spectrophotometers will need two monochromators, one for the excitation beam and one for the emitted light. I almost always have to remind them of the process – radiation is used to excite the molecule, light is emitted by the molecule – before someone realizes that there are two sources of light to consider and they will need a monochromator for each.

    Where would you position the emission monochromator relative to the excitation monochromator?

    Some of them realize that the molecules will emit the light in all directions such that the emission monochromator can be placed somewhere else besides at 180o to the excitation monochromator.

    Would a 90o placement have any advantage over a 180o placement?

    The groups can usually determine that the 180o placement is less favorable since the source light will strike the detector and that at 90o only the fluorescence will be measured.

    1. What would be the difference between an excitation and emission spectrum in fluorescence spectroscopy?

    They can usually figure out that one involves scanning the excitation and the other the emission monochromator. However, I often need to point out that when scanning the emission monochromator, the excitation monochromator needs to be set to a known excitation wavelength, and when running an excitation spectrum, the emission monochromator needs to be set to a known emitting wavelength.

    1. Draw representative examples of the excitation and emission spectrum for a molecule.

    The students are usually stumped by this question and I need to provide some prompts to get them to think it through. The first is to redraw the energy level diagram on the board showing the possible absorption and fluorescent transitions. The second is to draw two sets of axes on the board, one on top of the other, where the y-axis is fluorescent intensity and the x-axis is wavelength. I indicate that the wavelength scale on the two axes is identical and ask them to draw this diagram in their notes.

    Draw the excitation spectrum on the top.

    After all groups have a satisfactory representation of an excitation spectrum, I then instruct them to:

    Draw the emission spectrum on the bottom set of axes.

    Some of the students finally realize that the two spectra will be mostly offset from each other except for the S0-S1 transition. If not, I may prompt them by asking:

    Are there any transitions that have exactly the same energy in the excitation and emission spectrum?

    Once both are on the board and understood, I ask them which one most resembles an absorption spectrum and they immediately realize it is the excitation spectrum.

    1. Describe a way to measure the phosphorescence spectrum of a species that is not compromised by the presence of any fluorescence emission.

    They are usually perplexed by this problem, often remembering back that phosphorescence occurs from solids, but now knowing whether that is useful. Also, they want to think of a way to promote intersystem crossing to get all of the excited-state molecules into the T1 state so that no fluorescence occurs.

    Can they remember any significant differences that exist between the S1 and T1 energy states?

    Someone usually brings up the difference in lifetimes of the excited states, which leads them to ask whether there is some way to excite the compound, turn off or block the excitation source, and then build a delay into the measurement of the emission. I then discuss the idea of using a pulsed source, and the concepts of having a delay and gate time.

    1. If performing quantitative analysis in fluorescence spectroscopy, which wavelengths would you select from the spectra you drew in the problem above?

    Because we have discussed wavelength selection in UV-Vis spectroscopy, they usually know to select the \(\lambda\)max for both the excitation and emission spectrum.

    Can they think of any problem with setting both the excitation and emission monochromator to the S0-S1 transition?

    Some are able to think of the possibility of scattering taking place. We then discuss how scatter will occur in all directions and be indistinguishable from fluorescence.

    1. Which method is more sensitive, absorption or fluorescence spectroscopy?

    Before giving them this question, I do a brief lecture on the meaning of a quantum yield. I define the meaning of the term (ratio of the number that fluoresce relative to the number that were excited).

    Identify all of all the processes that compete against fluorescence from the S1 state.

    With this decided, we then write the expression for the quantum yield using the rate constants for the different processes. We then examine how the fluorescent quantum yield will range from 0-1 and that it will be rare for a molecule to have a quantum yield of 1. I indicate that molecules with fluorescent quantum yields of 0.01 or higher are useful for analysis by fluorescence spectroscopy.

    I then provide them with the question about relative sensitivity of absorption and fluorescence spectroscopy and provide the following additional prompt:

    Think about the instrumental setup that is used for absorption and fluorescence measurements and consider exactly what is measured or compared. Also think about each technique when a very low concentration sample is being measured.

    At this point, they are able to reason that the values of P0 and P in absorption spectrophotometry will be quite close in magnitude whereas the fluorescence measurement is just a small signal.

    I now ask them to consider an observation with their own eyes where they would examine the difference in intensity between a 99- and 100-Watt light bulb (comparable to absorption spectrophotometry) compared to the difference between a 1-Watt light bulb and darkness.

    Which difference would be easier to detect?

    They immediately say it would be easier to distinguish the 1-Watt bulb over darkness than the difference between a 99- and 100-Watt bulb. We then discuss how electronic devices can also distinguish this better and fluorescence (or any emission method) has an inherent sensitivity advantage over an absorption method.

    Variables that influence fluorescence measurements

    1. What variables influence fluorescence measurements? For each variable, describe its relationship to the intensity of fluorescence emission.

    I often preface this question by suggesting that the students think back to things we already considered in our unit on UV/Vis absorption spectroscopy. Students come up with the obvious one of concentration, and usually realize that the path length and molar absorptivity will influence this as well. Some even realize that the source power is important in fluorescence as well. This then allows us to write the equation for fluorescence intensity.

    What would the curve look like at high concentration?

    Recognizing the negative deviation that occurred in Beer’s law, they usually realize that a negative deviation would be expected as well in fluorescence spectroscopy. I then introduce the idea of self-absorption that can occur at high concentrations in emission methods.

    What would you do with a sample to insure that it was not at a concentration range where self-absorption was occurring?

    They almost immediately recommend diluting the sample.

    Are there other variables that influence the magnitude of the quenching?

    The importance of collisions and its contribution to radiationless decay has been discussed earlier so some realize that this is likely an issue. Most then realize that temperature is an important variable and can determine that higher temperatures should reduce fluorescent intensity.

    Many also suggest that solvent is a variable, and we revisit some of the discussion we had in UV/Vis spectroscopy on the effect of solvent on \(\lambda\)max for \(\lambda\)-\(\lambda\)* and n-\(\lambda\)* transitions. Many also suggest that pH is a variable.

    I then explain how paramagnetic substances promote intersystem crossing.

    Can you think of any important paramagnetic substances that might be present in a sample?

    Usually each group has someone who identifies paramagnetic metal ions and dissolved oxygen as possibilities.

    Can you think of a way to eliminate dissolved oxygen from a sample?

    Some are familiar with using a ultrasonicator to degas a solution and sometimes a student even comes up with the idea of purging the solution with a gas that is not paramagnetic.

    We discuss how removing paramagnetic metal ions from a sample is a more difficult problem.

    1. Consider the reaction shown below for the dissociation of 2-naphthol. This reaction may be either slow (slow exchange) or fast (fast exchange) on the time scale of fluorescence spectroscopy. Draw the series of spectra that would result for an initial concentration of 2-naphthol of 10-6 M if the pH was adjusted to 2, 8.5, 9.5, 10.5, and 13 and slow exchange occurred. Draw the spectra at the same pH when the exchange rate is fast.

    2-naphthol_dissociation.png

    This is a complex question with several parts to it, although we did cover some background for this in the unit on UV/Vis spectroscopy. If they have forgotten this, I prompt them to focus on the two extremes and ask them first to describe where the reaction lies at a pH of 2. The groups realize this will be virtually all of the protonated form. I then ask them to describe where the reaction lies at a pH of 13 and they realize it will be virtually all of the deprotonated form.

    Do you expect any difference in the emission spectrum of the two species in these two forms, and if so, what might the difference be?

    Intuitively, they expect the two forms to have a different emission spectrum – if not, why are we even talking about this problem! After giving them a bit of time to talk about it in their groups and hearing some of the things they are thinking about – rarely do they come up with something that is the actual explanation for why the deprotonated form will fluoresce at a longer wavelength than the protonated form.

    Draw the resonance forms for the two species.

    Some are rusty at this but others quickly realize and explain to their group members that the naphtholate ion will have more resonance forms than the naphthol species.

    What does the presence of more resonance forms do to the energy of the excited state?

    Some propose that it may lower the energy of the excited state and therefore the energy gap such that the transition shifts toward the red.

    At this point, we can draw spectra for the two extremes.

    Consider the point where the pH equals the pKa, and think about the difference that would be observed between fast and slow exchange.

    Usually the groups can reason out what would occur for the two different situations and we can fully draw and discuss the spectra represented in Figure 3.11 of the text.

    Which to you think actually happens – fast or slow exchange?

    I prompt them to think back about UV/Vis spectroscopy and lifetimes of states as well. Groups arrive at the conclusion that slow exchange is likely to occur on the time scale of fluorescence spectroscopy.

    Questions always arise about whether the two species would have the same intensity or not and we have some discussion about that question.

    1. Devise a procedure that might allow you to determine the pKa of a weak acid such as 2-naphthol.

    We have examined a similar question in UV/Vis spectroscopy so the students usually hone in on the procedure that would be done.

    I then use this as an opportunity to talk about the complication that arises if this experiment is actually performed using fluorescence spectroscopy – namely that the calculated pKa values at each pH will be different because the pKa value of the excited state of the compound is different than the pKa value of the ground state.

    1. Which compound will have a higher quantum yield: anthracene or diphenylmethane?

    anthracene_diphenylmethane.png

    Students usually want to answer this question by considering the relative extent of conjugation of the system rather than the role of collisional deactivation for the two compounds.

    Would collisional deactivation be different for the two molecules?

    Not all the students will immediately realize that anthracene will exhibit less collisional deactivation than diphenylmethane (they might propose that since it’s bigger, they will collide into each other with more force – if they give this response, it is important to remind them that most of the collisions actually occur with solvent).

    Which would come out worse in a collision – a Greyhound bus or a car towing a boat on a trailer?

    They usually realize that the collisions with diphenylmethane will lead to more radiationless decay than those with anthracene.

    I then discuss how relatively few compounds exhibit intense fluorescence – that fluorescence spectroscopy is far more selective than UV/Vis absorption spectroscopy – and that derivatization of compounds with fluorescent chromophores is sometimes done for the analysis because of the enhanced sensitivity of fluorescence.

    Finally, I present to them a brief discussion of topics like chemiluminescence, bioluminescence and triboluminescence. We also talk briefly about the process that takes place in glow sticks.


    This page titled In-class Questions: Molecular Luminescence is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Thomas Wenzel.