ghost peak

[ym3142]

I had all samples in diluent of 30 ml MeOH and 60 ml buffer;
my buffer, 10mM KH2PO4 and 10 mM Octanesulfonic acid, ph 2.5;
I injected 5 stand reference, three blank, 5 API1 (0.40mg/mL)and 5 API2(0.025mg/mL), and 5 placebo samples.
Luna c18 150x4.6 column, mobile phase 60% buffer and 40 % MeOH; 30C. Flow 1.5; injection 10uL; run time 17 min; needle wash 50% MeOH aqueous;
started from ~13 min of the third injection I saw the ghost peak; then ~15.5 min of the 4th, end of 5th and 1 min of 6th, ~3.5 min of 7th; ~6 min of 8th and so on.

like a solvent effect this peak started from another smaller negative peak, and it was broad. its area might be 100 times of my API2(which has relative low absorption)

So the ghost was not related to the API's. was this a salt precipitation? But I did not have high concentration buffer or small ratio organic.

any suggestions?

[Uwe Neue]

What was the wavelength at which you were monitoring the c-gram?

[ccyting]

Apparently you are doing pair ion chromatography. I think the "ghost peak" is the API related compound. If you do the calculation, the peak appears at 47, 67, 85, 103, and 123 minutes from the first injection (only your run time is counted). The difference between each run of this ghost peak is around 20 min (pretty constant). I will not call this peak as "ghost peak". My understanding is the ghost peak is floating, sometimes you can detect sometimes you cannot detect it. But after the third run, you can observe this ghost peak in everyrun. When we do the pair ion chromatography, we have to consider the pair ion sites of the analytes. If you have more than one site to do the pair ion, then you could have the second equilibrium for pair ion and will eluate later. Another words, the "ghose peak" could be one of the pair ion products of one of your APIs. We need to examince the chemical structure to conclude the possiblity. One way to eliminate the extra peak is to accelerate the pair ion formation equilibrium duing separation. Good luck.

[syx]

Do you mean that it is late eluted peak? However, the retention time are increased in the next injections.

[ym3142]

my api, phenylephrine, and methscopolamine. Thanks for everyone's input.

[ym3142]

both 220 nm and 254 nm, thanks

[Myriam K]

I've seen late eluter peaks seem to drift in retention time like that. I'd try making a couple of standard injections with a run time of 90 minutes or so (preferrably overnight). That would tell you if there are late eluters. If there are late eluters, you still have to figure out if they are real impurities or artifacts due to the method. That would be the hard part.

[Kostas Petritis]

What you actually see here is either a system peak or a late eluter with an approximate retention time of 52 min.

It is a system peak if you still see these peaks in your 9th, 10th and 11th injection. If you don't see them then it is just a late eluter. If it is a system peak I would anticipate the area of the 9th 10th and 11th injection to be somewhat smaller as the "relaxation" of the system would be less intense in blank injections than when you actually inject something.

From the times you give also it seems that from the moment you finish your analysis at 17 minutes it takes another 2.5 minutes until you inject your next sample (i.e. injection time + any post run time + any software processing time). That is why the retention time of the system peak looks like drifting by in reality it is always there at an approximate retention time of 52 min!

Try it out by injecting and letting your system for about an hour, and you'll see your peak. If ever you change anything relative to your ion-pairing reagent (i.e. concentration, equilibration time -i.e. less than needed- etc) your system's peak retention time will shift.... Injection of (significant) different concentrations of analytes should lead to a system peak with different peak areas...