HI
I was wondering if the light of UV-vis spectrophotometer can enhance the transmitted light (increase the absorption at specific wavelength of a compound when measured over time?
I have this compound that was I am trying to measure the spectra for short time; the compound spectra is measured and shown very nice. but when I measured overtime (exposed to light from the spectrophotometer), the Spectra starts to be enhanced to higher Absorption readings at all wavelengths of the actual spectra.!
as always: SHOW SPECTRA!
This makes discussion much easier and will lead to probable explanations much faster...
Your compound is photosensitive, the light of the photometer induces photochemical reaction in your solution. You should minimize the slits and/or the integration time (accumulation time) of the photometer.
Rise of the absorption over the whole region means increasing of the concentrations, which can be achieved by: a) evaporation of the solvent (as Dr Päch correctly suggested); b) your sample was not fully dissolved and some particles entered in the cell.
PS as well as MP and LA any one may be true. I suggest first to check concentration effect. If your problem does not get resolved then exposure time dependent IR can be helpful.
THANKS ALL for replying. May be I am not really clear about the situation.
I really have increase at most wavelengths that are characteristic of the compound spectra.
In a sample of my substrate with it is modifying enzyme; I blank with my substrate, the enzyme contains FAD; when FAD is reduced (nothing observed for oxidized form) the spectra of FAD have increase at some points of the wavelength range. I also can have decrease at some wavelengths (characterstic of the compound) but it is not as much as the increase that is observed !!!
My compound antibiotic and I know it is photosensitive; but I was not sure for to which extent this can be affected by the spectrophotometer light?? and if it is just the UV light or the visible as well ??!!
Dr. Michael Päch
I have done this in the flow cell inside the stopped flow spectrophotometer. and in a sealed cuvette on the normal spectrophotometer device.
Is it completely dissolved? If you have an increase at all wavelengths like you describe it sounds like it may not be completely dissolved, but an easy way to check it is to just use your compound and a standard spectrophotometer. Prepare identical samples. Take a spectrum of one and wrap it in foil. Then repeatedly take spectra of the other for about an hour. Remove the one in foil and take its spectra again. If they both change then it could be that it's not completely dissolved. The other thing that you may want to consider are changes in pH as your reaction proceeds. Changes in pH can easily result in changes in solubility and/or electronic structure of your compound...both of which will affect your spectrum.
as always: SHOW SPECTRA!
This makes discussion much easier and will lead to probable explanations much faster...
Do the simple experiment as suggested by Respected Jack Carraher. Also check, whether the "increase in absorption" is uniform at all the wavelengths..!
I had another thought as well. I recall some work that I was doing with curcumin several years ago. It wasn't terribly soluble in water so I made stock solutions in acetonitrile. I noticed that when I added my ACN (curcumin) mother solution to my reaction vial that my spectrum changed significantly, but not over time. After some time I finally figured out that I was measuring the rate of conformational changes due to solvent effects. Are your mother solutions in the same solvent? If so, are they at the same pH?
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