[STM]

Hello everyone,

I need your advice on the above subject matter. I am working with a compound of triphenyl phosphine in which one of the phenyl group has been replaced with a fluorescent chromophore. This parent material is weakly fluorescent. But upon oxidation (with lipid hydroperoxide) it forms an oxidized product which is now strongly fluorescent.

The background fluorescence I am getting from the parent material is quite higher than expected. I am suspecting that:
1. upon storage, the material has auto-oxidized to the form some quantity of the fluorescent product.
2. The oxidized product was present in the supplied stock from manufacturer
3. Dissolution in DMSO has resulted in oxidation of some of the material to the highly fluorescent product form.

Hence, i wish to ask for your opinions particularly regarding the oxidizing ability or tendency of DMSO.

Thank you all

[Dan]

Phosphines and phosphine oxides have characteristically different ³¹P NMR shifts, so you can check for phosphine oxide contaminants in the commercial material easily by ³¹P NMR. Phosphines tend to oxidise to varying degrees (depending on structure) if exposed to air for prolonged periods, and it is not unusual for some commercially supplied phosphines to be significantly contaminated. A colleague of mine had big problems with this in the past.

You can check the possibility that DMSO is oxidising the phosphine by dissolving a sample in DMSO-d₆ and running NMR at a coulpe of time intervals. I would be surprised if DMSO is oxidising a triarylphosphine at room temp - can you smell Me₂S?

[STM]

Dear Dan,

Thank you for your response. I do not perceive Me₂S. Also, my access to 31P NMR is quite limited but I will hold on to that point.

I am thinking if there is a column chromatographic separation method that I can use for the preliminary purification of the starting material to separate it from any pre-formed, more fluorescent, oxidized product. Any idea of such or any reference material that can be helpful. My compound is quite heavy. It is a triarylphosphine linked to a perylene bisimide fluorophore which has a tetraethylene glycol tail.

Thank you

[Dan]

I'm not familiar with these fluorophores, so I'm not sure I can be of much more help.

How does it look on a silica TLC plate? >1 spot?

[Babcock_Hall]

DMSO is an oxidant in the Moffat oxidation, for example:  http://www.synarchive.com/named-reactions/Pfitzner-Moffatt_oxidation  However, I am not sure that this reaction is terribly relevant to the situation that you have, other than to document that DMSO has the potential to be an oxidant.  I agree with Dan that ³¹P NMR would be very helpful.

[MOTOBALL]

I have worked in pharmaceutical R&D, and did once encounter oxidation by DMSO.  Tablets of an experimental formulation were solubilized in DMSO for testing.  HPLC analysis revealed an oxidized impurity at a significant level (5-10% as I recall), and the presence of Me2S was shown by GC-MS;
I believe it was >CHOH to >C=O.

So, it certainly is not unknown.

[Babcock_Hall]

@Motoball, That sounds like a Moffat oxidation.

@OP, Some insight might be gained by looking at the relevant reduction potentials, if they are known.

[pgk]

The given facts are:
1). DMSO is an oxidizing agent but with synergism with a co-reactant.
2). Tiphenylphosphine is used as antioxidant for polymers because it is easily oxidized in air.
3). Any background fluorescence is due to the oxidation or to the conjugation of the perylene moiety.
Tacking in account all above, two possible explanations can be given:
1). Conjugation of the phosphorus free electrons (if directly bounded with perylene in an appropriate substitution) with the amide group through perylene’s aromatic rings. The said conjugation is then, favored in DMSO
2). Or, something like Pummerer type rearrangement on the aryl-diphenylphosphine oxide, followed by perylene conjugation or oxidation.
http://www.gaylordchemical.com/uploads/images/pdfs/literature/105B.pdf
The above hypotheses can be verified by comparison of 1H-NMR,  IR  and UV spectra of the initial perilenyl-diphenylposhine with the 1H-NMR,  IR  and UV spectra of the same compound taken in D6-DMSO and anhydrous DMSO, respectively and the day after samples preparation.

[pgk]

Phosphorus peaks in IR
(If not covered by other bands)
P-Ar      1450-1435 cm-1
P-O-R    1000-1170 cm-1
P-O-Ar   1240-1180 Cm-1
P= O      1350-1175 cm-1
P= O      1250-1150 cm-1 (H-bonding)

[STM]

Woooowww!!! I am so grateful for your response. Thank you so much. Your information have been helpful and enlightening. Unfortunately, the challenge still persist due to my inability to access 31P NMR as suggested. So I am stuck with finding my way around on the basis of Fluorescence.

I have tried dissolving the compound under Nitrogen atmosphere to minimize exposure to air. The background fluorescent reduced but later increased on subsequent days.

According to the authors of the probe, the starting probe is weakly (or non-fluorescent) while the oxidized product is strongly fluorescent particularly in ethanol. I am working in water medium and the fluorescence is expectedly weaker.

Truly, 1 uM of the probe didnt fluoresce in water when I did the 3D- Fluorescence spectrophotometer scan while upon introduction of the hydroperoxide analyte, the oxidized product formed and fluorescence emission was observed.

However, upon trying on HPLC-Fluorescence detector, the probe gave absorption peaks which was significant (about 1 million peak area for the same 1 uM above) but the this area always increase (about 4-5 times higher) when the probe is reacted with the hydroperoxide analyte to form the oxidized product.

Could the peak I observed for the probe be a background fluorescence from the pre-formed product imurity in the probe or could it actually be due to the probe itself (Remembering that 1uM on 3D-Fluorescence scan didnt give any fluorescence signal) ?

Note: Quantum yield of oxidized product is 0.83 while probe 0.03

[pgk]

1). The triarylphsophine probably attracts atmospheric oxygen. Thus, N2 delayed air oxidation but the procedure was not complete. Try Argon which is heavier than air. Imide oxidation might also play a synergistic role.
2). According books, water causes bathochromy up to  8 mμ compared to ethanol, that significantly decreases fluorescence.
3). Compare the IR spectra of the triarylphosphine before and after peroxidation and measure the P=O and N-O (≈ 950 cm-1) peaks (if not covered by perylene peaks) at the absorbance scale which is decimal. It might be helpful and avoiding the 31P-NMR.
Good Luck

[STM]

@pgk: Thanks again.
I strongly agree with comment 1 based on the increased signal intensities I observed upon injecting by the second day.
2. Regarding ethanol, the authors obtained fluorescence measurement in ethanol and it was reported that the triarylphosphine-perylenebisimide probe was non fluorescent while the oxidized product was. For my study, I dissolve the probe firstly in DMSO and then make subsequent dillutions in water. I have observed that dillution in ethanol or ethanol-water ratio (as long as ethanol is present in significant percentage) even enhances the fluoresce signal from both probe and its oxidized product which is quite contrary to expectation. My guess was that perhaps ethanol enhances the dispersibility of the probe in the water medium (Just a guess).

Please can you recommend a very good textbook or materials to me on fluorescence spectroscopy. I really wish to learn more.

Thank you once more

[Corribus]

Lakowicz is the classic text on fluorescence spectroscopy.

[pgk]

A good idea is to use degassed solvents and water by previous treatment in an ultrasound apparatus (ultrasonic bath) and hoping that the water that you use, is not "chlorated".
It might also be possible that the probe is oxidized by DMSO in presence of ethanol/water as co-reactants and perylene excitation as electron source, though not so probable. Can you avoid the DMSO, in order to clarify this?
Please, take a look at the Woodward–Fieser- Kuhn rules in UV spectrometry (not be confused with Woodward-Hoffmann rules in Organic Chemistry), before, in order to have an idea how conjugated double bonds and anulated systems increase the emission wavelength.
http://en.wikipedia.org/wiki/Woodward%27s_rules
Or take a look to the UV –spectrometry chapter in: Williams, Fleming, Spectroscopic Methods in Organic Chemistry, McGraw Hill (prefer an old edition that is inexpensive and this chapter is better written; solvent influence is also cited, thererin).

[STM]

Hello Everyone,

Thanks for your comments so far. They have been so helpful.

Owing to the problem of background fluorescence of my probe on the HPLC system, I switched to the flow injection analysis (FIA) system. Again the challenge of background fluorescence showed up but this time around I was able to overcome it.

I hereby share what I did:

I observed that when I dissolve my probe in DMSO and dillute in water, I was still having background fluorescence upon injection in the FIA. However, in the presence of acidic buffer the background was off. I tried it with the product and it was unaffected. Therefore, I deduced that the acidic buffer preserved the probe from auto-oxidation while still allowing it to undergo reaction with my hydroperoxide analyte to form the product. This observation have been carefully related with blank measurements (acidic buffered-probe + water)and confirmed to be so. The probe dissolved in MQ have been giving increasing background for over 7 days while the same probe dissolved in acidic buffer has not given any peak over the same period of time.

In addition, upon introducing the probe in neutral pH buffer, the background fluorescence was observed.

It thus appear the acidic buffer doesnt affect the product but just the probe although increasing pH favoured the product formation. Hence, I intend to modify my carrier to be a higher pH medium.

I am not sure of the chemistry behind this yet but I am relating it to the concept of preserving Fe (II) in acid. Any ideas or clarifications please?