Chromatography Forum

Dear All,

I was trying to determine the retention time of a supposed pure compound (2,3-dinitrophenol) using HPLC but detected and separated out 3 peaks instead.

Is there a way to determine which peak is the one that actually corresponds to 2,3-dinitrophenol instead ?

Thank you so much

Lance

Are you using any solvents? If so, try to run the solvent without putting any sample in and see what happens. Also, is your pure sample "in-date"? What is your sample preparation method?

or your " pure" standard is mixture of isomers?

JM

Thank you all for your suggestions.

The standard was prepared simply by dissolving and diluting in Milliq water. Is it possible to search for the literature value of absorptivity for my dinitrophenol compound and identify the actual dinitrophenol peak in my chromatogram by measuring the absorbance of each peak ?

See if same happens if you dissolve it in an organic solvent. What if you inject less? Does your mobile phase contain any acid? Have you investigated same by GC?

Are all the peaks of a similar size, or is one much larger than the others ?. All else being equal you would expect the largest one to be the compound of interest.

How pure is you compound expected to be ? Is it a standard, a certified standard a synthetic reagent or technical grade ? Are the peak area ratios in line with the expected purity i.e. is one peak 98% of the area for a 98 % pure compound.

The simplest check is to run the same compound from a different source and look for the peaks in common.

Peter

Dear Peter, thanks for your insights. I have a doubt: If my desired compound has a 98% purity, it does not mean my compound peak will be 98% while the impurity peak is 2%, as they have different molar absorptivities. What I am trying to say is that I don't think it's foolproof enough. Or is there some concept I missed?

Hi Lance

You are right about the different absorbtivities, relative peak sizes will be just a guide. If you have to be absolutley certain about compound identity and which peak is the compound you are interested in, you will have to use certified standards (and demonstrate no decomposition during analysis), or use an independent technique such as NMR, MS or GC.

The pressure changes are due to differences in viscosity.

Peter

I am afraid that you have to use LC/MS to find out the identity of those peaks. I really hate to face the same situation when we recieve a reference standard that is not pure to be used as a reference standard in the first place. Good luck.

You might try looking at expected reactants (phenol) and side reaction products (2,4-dinitrophenol) though even if the retention times match, I don't know if that would constitute "proof". By the way, I am not willing to bet the ranch on my suggested reactant or side products.

You could always try to obtain the same standard from another (commerical) souce hoping for higher purity or different impurities...

Another possibility would be, if the selectivity between your samples is "huge" you could try to inject a similar structurally compound... although I wouldn't be very satisfied with the implied assumptions...

you could try to use a diode array detector to verify that you are dealing with 3 isomers. What's the wavelenght?and how big are your peaks?and rt?
in this way you can preliminarly understand if the peaks can be isomers.

Nitrophenols are rather acidic. The UV spectra and retention times are quite pH dependent. For the most consistency, you should be using a buffered or acidified mobile phase. If you have a diode-array, the isomers do have very different spectra, and are easy to distinguish. LC/MS is actually less helpful in this case.