pi-pi cojugation shift

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PRAVEEN PALIWAL
PRAVEEN PALIWAL's picture
pi-pi cojugation shift

Sir ,

I am sending some files along with structure and chromatogram's .
It is basically related to analysis of one sodium salt (disodium 2,2' bipyridine 4,4'-dicrboxylate)
This salt is only solurble in water or water with basic pH. We are analyzing it on ACN(0.1% H3PO4) : Water(0.1% H3PO4): : 20:80 wavelength 230nm. Flow 1.0 ml/min.
We I st dissolve the sample into 0.1N NaOH we got two sahrps peaks both have same UV spectra. II nd ly we dissolve this sample into 0.01N NaOH and analyze it on same conditiion as above we got one sharp peaks which has same UV sepctra which we got earlier .
Again we change the diluting solvent and take 0.1% TEA in  water and analze it on same HPLC condition we got a single peak agin and its UV spectra is also same as Ist one .
I want to know why in the condition Ist we got two peaks of same UV spetra.? As I am thinking It is pi-pi conjugation change in basic pH and that's why we got cis and trans isomer both when we anlyzing it on 0.1N NaOH but when we change the condition (0.01N NaOH and O.1% TEA in water) we got only single peak i.e no swiching in conjugation.
We cant analyze this sample in acidic condition because of precipitate form. It is in soluble form only in basic or neutral condition (pH 7or above)
I want to know why these are happining?

Dr. Analytical
Dr. Analytical's picture
What concentration are you

What concentration are you injecting in each experiment?  What column are you using?

You say that the molecule is insoluble in acidic solution, yet your mobile phase is acidic.  So, this molecule is soluble in acn:water (20:80), or it would never elute from the column.  Under these conditions you are viewing the spectrum of the neutral di-acid, not the sodium salt.

I would try preparing standard solutions in mobile phase.  If you have preciptation, try a smaller concentration of standard or use more acetonitrile (50% or more if necessary). 

Traditional cis and trans isomers are not possible in this molecule, so we must find another explanation.  Perhaps the stronger base (0.01 N NaOH) is causing some decomposition.  You could also try using NaOH solutions between 0.1 and 0.01 N to see if there is a regular change in the relative amounts of the two peaks.

PRAVEEN PALIWAL
PRAVEEN PALIWAL's picture
Sir,

Sir,

I am injecting 100ppm of sample and I am using Inertsil -ODS-3 column. 250 x 4.6mm ID , 5µm.
Yes I am using acidic mobile phase but when my sample solution  is contact with this mobile phase it's pH remains neutral and thus the sample does not get precipitated.
Here on the point if we accept any type o decomposition then how a decompose material give's same UV spectra?
I also tried mobile phase and addition of sammler amout of ACN but solution get hazy.
I hope there is some possibility of some change in conjugative system.
I also tried to change the 0.1N NaoH to 0.01N NaOH and I got one peak  aaginst two.
The most wonder is that the UV-spectra of all three are the same
What exactly happening ? please explain us.

Best regards

praveen

Dr. Analytical
Dr. Analytical's picture
First, the spectra that you

First, the spectra that you are measuring at your detector are the protonated (neutral) form of the molecule.  To see if there are any differences with the ionized form you would have to use a mobile phase with a pH of 6 or greater.

If you want to dissolve the sample in acidic solution, you will have to use a greater concentration of acetonitrile, not a smaller amount..  What else is in your sample that might be precipitating?

I can not explain why you get two peaks from one solution and only one peak from another solution.  A chemical reaction to form similar products (which have similar retention times and similar spectra) is a reasonable explanation, but that is just a general answer.  I do not know what reaction would happen based on the information that you have given me.

Is there anything else that you can tell us?

PRAVEEN PALIWAL
PRAVEEN PALIWAL's picture
Sir,

Sir,

As I think Half part of compound migrate initially quick rather than another part because its pH is high enouh near to 13.
The another part initially converts in to it's acidic form and retain at RT 7.3min.
The Ist part initially migrate quickly due to high pH but it also turns acidic due to acidic enviornment which is surrounding to itself .
So the ist part is also turns to its acidic form but initially it was high basic and migrate fast so it will eluted slight earlier as RT 6.5min.
So as I think both the part of compound or analyte is eluted in it's acidic form and tha's why both has same UV spectra.
What is your though on this?

Best regards
praveen

Dr. Analytical
Dr. Analytical's picture
The injection solvent can

The injection solvent can cause peak shape problems, but usually the peak is just distorted or split.  This effect would usually not cause two nice peaks to appear.

You could test your idea by changing the concentration of the base, or the volume of the injection.

pravishkumar
pravishkumar's picture
Can You Perfrome following

Can You Perfrome following experiment
1) Make 10,000ppm stock solution in 0.1 N NAOH .Inject this solution 10 time less . (say If previously u r incting 20micro liter of 1000PPM,Now inject 2 microlitre of 10,000ppm)
 And see Weather You are getting single peak or double peak,
2) Than make 1000ppm from above stock in water ,than inject 20microlitre of this solution .and see you are getting single peak or Double peak.
 3) If in second Experiment you are getting single peak than it indicate ,Your mobile phase need optimization of pH to separte two peak . 

pravishkumar
pravishkumar's picture
And if you are thinking of

And if you are thinking of sepration due to PI PI conjugation sifting (0.1NNAOH) than it is not possible on C18 column .
 

PRAVEEN PALIWAL
PRAVEEN PALIWAL's picture
I have changed the sample

I have changed the sample preparation method. Now I am preparing sample into 0.01N NaOH. I got a single peak without co-elution.
Actually what was happining , when I am going with 0.1 N NaOH the sample was hydrolize . Haf sludge of sample is ionized immideiately and other half is after some fraction of time and that's why I am getting two peaks , oth peak has same UV spectra also.
Then I have tried with 0.01N NaOH and also tried with 0.1% TEA I got single peaks .