co-elution

[tilly]

hi fellow chromatographers, i am dealing with a broad 'hump'
at the begining of chromatogram, which is only there in samples not in standards or blank. Am i right to think it must be comming from sample and because it is broader than the rest of the early eluting peaks that it is a coelution? Any thoughts would be kindly appreciated. Especially relating to how it could be possible, if at all, to quantify the co-eluting peaks- if thats what the 'hump' is.

[Uwe Neue]

If it is an isocratic method, it could be a peak from a previous injection.

[tilly]

Hi Newe It is an isocratic method-a pre mixed mobile phase of acn,thf,and water. The broad peak elutes at around the same time in all the samples but not in the 5 standards or blank that are run prior to the samples. Also if a standard is run inbetween samples, the broad peak is not in the standard but appears again in the sample. I therefore didn't think it was a carry over? So if it is from the samples but more than a single band coeluting (as it is so broad early on in the chromatogram) is it possible to quantify the individual bands by some method eg there is purity option in software which has indicated the band is not spectrally homogeneous but can the different bands be quantified?? I thought that perhaps if the mobile phase composition was changed to more water the retention time of the components would increase and there might be more separation of the band. Is this correct? All suggestions will be carefully considered. Many thanks.

[Uwe Neue]

Carry over and a late eluting peak from a previous injection are two different things. I had suggested a late eluting peak. To figure out if this is the problem, double (or triple) the run time.

[tilly]

Sorry in getting your name muddled up Uwe. Thanks for the reply, i will try your suggestion and post the result next week. Thanks

[tilly]

I haven't tried the extended run yet to see if there are any late eluters. I think it would be unlikely that this is why theres a broad peak at the beginning of the chromatograms. i run a blank before my samples and theres nothing there, and after every 5th sample a standard,and theres nothing there. I will however still give it a go and report back. do you have any further ideas on how i can seperate what i think are coeluting peaks out?

[danko]

Hi Tilly,

It might be ionized compounds – from the sample/product matrix………
Or ionized degradation products.
You didn’t describe your mobile phase, but if it isn’t pH controlled, you could try lowering the pH to 2.5 or something like that and see whether the peak/s get separated or at least narrower and more defined.

Best Regards

[Uwe Neue]

Well, it could be a matrix peak... Actually, what are you analyzing?

[tilly]

Uwe, hi I carried out the extended run and there were no peaks eluting after the ones normally, so i think we can now confidetly say the broad peak at the begining is not a late eluter...

Danko, The analysis is of oligomers extracted from polybutylterephthalate, i know there are some acid compounds eluted at the start of the chromatogram.

The mobile phase is ACN/water/THF.
What the PH is i don't know. What would be used to adjust this?Is it safe to use a low ph with all columns?
Thanks for your replies.

[Uwe Neue]

If you may have ionic compounds in your analysis, you are better off using a buffer. What buffer you use, depends on your detection system. If you use UV, and have not used a buffer until now, I woudl use a pH 7 phosphate buffer and see what - if anything - is changing.

[danko]

Danko, The analysis is of oligomers extracted from polybutylterephthalate, i know there are some acid compounds eluted at the start of the chromatogram.

That would be a plausible explanation of what you’re experiencing.

What the PH is i don't know. What would be used to adjust this?

My favorit choice (for a start) would be 0.05 M Sodium Phosphate, pH 2.3 – 2.5 (that is instead of the pure water in your mobile phase).

Is it safe to use a low ph with all columns?

Unless you’re using some kind of very exotic column/stationary phase, it is perfectly (100 %) save!
I’m assuming, the column you’re using is any kind of silica based C18 or C8 or something like that.
In my opinion the low pH buffering offers a number of advantages, especially in the case of acidic compounds separation on silica based reversed phase columns.
BTW, expect longer retention time for this/these peak/s.

Best Regards

[Uwe Neue]

I recommend to stay with pH 7 phosphate buffer first, since this is the closest to your existing condition. If you go to low pH, you change two parameters at the same time (pH and the use of a buffer), and it is more difficult to sort out what is happening.