How can i find the maximun uv absorption?

[Newchromatographer]

Hi, guys please help me ,

i have detection wavelength my supervisor gave it to me but i need to find the maximum absorption by my self to confirm what he gives me in real absorption chrmoatogram to say this is my maximum absorption that i set up my detector on it .The proplem is my solvent is methanol ( uv = 205 ) my compound max is ( 210 ) is this ok ? how can i measure it practicaly i mean which concentration can i prepare my analyte on it ( 1 ppm 2 , or what plz ? ) . Is it ok if i measure my analyte max absorption on acetonitrile because i am worry from overlaping between ( 205 and 210 ) or do u thing no proplem exiest ?

thanks guys you are really our mentors

[tom jupille]

Get a hold of a spectrophotometer. Ideally, run the spectra in your mobile phase. If that's inconvenient, run the spectra in both ACN and in MeOH. If your compound is ionizable, be sure to control the pH.

As to what concentration, I have no idea (depends on the extinction coefficient of your compound). Start with a fairly concentrated solution and dilute as necessary.

[JGK]

I would start with a 10 µg/mL STD and, depending on the spectrum you get you can try higher or lower subsequently.

[HW Mueller]

Depending on what type of electronic shifts are involved your max can shift more or less to shorter or to longer wavelength, according to the medium. Therefore, if you really want to be right on the max in your run you would certainly want to check where the max is in the mobile phase. To do this you, of course, have to zero the detector so that the absorption of mobile phase is leveled. It then only interferes indirectly by lowering the light available to the analyte, maybe increasing the noise or even completely blacking out.
Now, I think the best advice I can give here is to get a good book or even better, a good education. A forum is not the correct place to learn basics in science, a forum seems to function best if it is dealing with advanced problems.

[Alex Buske]

If your analyte has its max. absortion around 210 nm and you are bound to methanol as mobile phase, I would determine the S/N ratio of a dilute standard solution at different wavelengths, because that counts.
A huge absorption at 205 nm doesn't help, because it would be overlayed by the solvent absortion.

Alex

[HW Mueller]

Alex, I don´t see your points unless you are assuming that the mobile phase is causing a blackout.

[Alex Buske]

The MP doesn't even has to block UV transmission at the desired WL completely. The S/N ratio goes down even if 1% of the light still goes through th MP. So the optimun detection WL is influenced by the spectrum of the anlyte and the spectrum of the MP.

Alex

[HW Mueller]

How?

[Alex Buske]

Which point you don't understand?

Here are data of a peak without a real chromophore in a MP with methanol at different wavelengths.

Code: Select all

WL    area    S/N
200   3.5      105
205   2.7      215
210   1.7      254
215   0.8      151

The biggest area is observed at 200 nm, the optimun detection would be 205 or 210 or something in between.

Alex

[HW Mueller]

If I understand the original question then Newchr. is trying to find the max. wavelength of the spectrum of his compound. Area and S/N has nothing to do with that, unless the S/N is so bad that it obliterates the lambda max. That could occur near blackout.
Are you trying to say that sometimes it is advantageous to aquire a chromatogram at a wavelength away from the max wavelength of the analyte? (Are you confusing maximum with optimum??).

[lmh]

From Newchromatographer's wording, he talks of finding the maximum so that he can set his detector to it (see very first post of this thread). I think Alex is making the point that perhaps Newchromatographer shouldn't be setting the detector to the measured maximum absorbance, and should concentrate on what he needs for a good method of measurement...

[Alex Buske]

I got it finally.
NC was asking for lambda max to get maximum response in chromatography.
Hans was thinking in spectra, while I was locked in chomatograms.
Sure you can determine the Lambda max for a given compoud. Areas and S/N aren't helpfull in that.
However, detection at lambda max isn't always the the most sensible way in chromatography.
Detection somewhat away from lambda max can give better S/N values. If there was some level region in the spectrum I would consider using that region because of better robustness.
In my example I would go to 215 nm, just to make sure that different methanol qualities can be used without problems.

Alex