Chromatography Forum

Hi Chromatography Forum,

Its been a while since I posted anything here, so hope everyone is having a good start to the New Year.

Below is a chromatogram and I'm trying to resolve the two peaks that are eluting, but I am encountering some difficulty:



The method conditions are as follows:
Mobile phase: 0.1% phosphoric acid in water (100%)
Flow: 1.2 mL/min
Isocratic for 20 min
Column: Waters Atlantis T3 250 x 4.6 (5um particle size)
Sample is dissolved in deionized water
UV detection, 190 nm

I have tried running a gradient with acetonitrile to no avail, I'm not sure if I am starting the gradient too late or not soon enough or if I shouldn't use ACN at all.

I'm kind of under pressure to figure this one out, so any help would be appreciated. Thanks,

Tim.

I'm assuming automated injection, and what is the size of injection and/or the concentration of the analyte?

I'm assuming automated injection, and what is the size of injection and/or the concentration of the analyte?

Forgot that bit of info. 5.0 uL injection and the sample size is 1.0 mg/mL.

What happens if you prepare the sample in the mobile phase and inject that?

do you get a similar chromatogram?

With a wavelength of 190 nm the analyte mustn't have much of a chromophore. Could you not try somenthing like ELS detection instead?

What happens if you prepare the sample in the mobile phase and inject that?

do you get a similar chromatogram?

With a wavelength of 190 nm the analyte mustn't have much of a chromophore. Could you not try somenthing like ELS detection instead?

You're correct, we don't have that much of a chromophore, and unfortunately UV is all I have to work with. (They've seriously squashed budgets and the hopes of getting an ELSD are zilch at this point).

The analyte in the above chromatogram is a carbodiimide, which converts to a urea compound in acidic conditions. I posted about a method I was working to improve a while back for analysis of that Urea product. (worked beautifully) This is essentially the opposite. The more I delved into it, the more I think the acidic mobile phase won't work.

Perhaps these two peaks are showing that conversion? I do believe the peaks at 2.1 and 4.0 are from the carbodiimide and the peak at about 7min is the hydrolized form (the urea).

So I don't think it would make sense to dilute the sample in mobile phase.

So now do I change the pH of the mobile phase and go to a dibasic potassium phosphate buffer at a pH of 6.8? What will that do at such a low wavelength?

Thanks.

First, dissolve the sample in your mobile phase with the phosphoric acid. If nothing is changing, then we should consider a conversion of your analyte during migration through the column. Could be degradation (less likely) or interconversion of stereoisomers.

TimB,

Enter "Fronting, Cause and Remedy" in the search box.

You will find a discussion from Dec. 2005 regarding peak fronting that may well be very similar to yours, i.e. chemical interconversions (and how to modulate them).

Please let us know if temp. UP/DOWN makes a difference. In any case, tell us what works/does not work so that we can learn from you.

Good Luck,

JMB

The idea that the peak at 4 converts to the one at 7 seems the best (otherwise I would have trouble explaining the sharp rise at 4 and the sharp decline at 7). The stuff is stable at pH = 6.8?

Thanks for all the suggestions. I did what Uwe and JGK suggested and dissolved the sample in the mobile phase. I managed to separate the peaks, but could only see the second peak after subsequent injections. This convinced me that all of the carbodiimide was being converted to urea. I went back to my literature and racked my brain even more and thats when I realized two things:

1. In aqueous media at acidic pH (2-4 roughly), carbodiimides will hydrolyze to form the corresponding Urea. However, at neutral pH, carbodiimides are stable in solution. So the use of a 0.1% phosphoric acid mobile phase is completely out.

2. I also noticed that when I managed to separate out the compounds, peak one had a higher lambda max (214 nm as opposed to 190.6).

So after changing the wavelength, and changing the mobile phase to water/acn, I got a nice peak at about 3 min. I don't like that the peak elutes so soon, but I managed to run a few concentrations and get a nice linearity with an r2=0.9999.

Now I just have to play around with the mobile phase composition and solve some peak fronting issues, and I can finally look at this compound. Once again, my deepest gratitude for everyone on this board for the suggestions.

Tim