Ghost peak in Gradient programm only

[pat_val]

Hi Guys,

Right now, i am working on one HPLC method. My mobile phase is as follow.

Mobile Phase A: Purified Water (pH 2.5 with H3PO4)
Mobile Phase B: Acetonitrile

It's Gradient program.
0.0 min to 2.0 min Mobile Phase B Acetontirle (100%)
2.0 min to 10.0 min Mobile Phase A 0% to 60% and Mobile Phase B 100% to 40%

My peak of interest is at 8.6 min. At 8.5 min i am getting one sharp peak which merged with peak of interest. This peak shows up in diluent (methanol), Water, std and sample. If i run Mobile phae A 100% up to 10 minutes, ghost peak is not there. Same way, i rum mobile phase B 100%, the ghost peak is not there. I don't understand why it shows up in gradient only... Pls help me....i have tight time line for this...

Thanks in advance.

[seamoro]

it only shows up when you use the gradient cause probaly the contamination is being stored at the front of the column and only when the gradient kicks in does it elute. The contamination can be from a number of scources...glassware...buffer....but im sure ur not stupid and you have checked these!. otherwise it could be coming from ur HPLC system itself!!. we had the same problem...try running 6N nitric acid through ur system for a while....should remove any contamination (remember to take off ur column before u do this)...hope this helps.

[pat_val]

Thanks for your prompt reply. Tomrrow i will try with different system and hopefully i will get another new column by tomorrow. Any other toughts from anyone else?

[tom jupille]

I think seamoro nailed it. It's a *very* common problem, to the point where we've put together a 15-minute "mini seminar" covering the causes and diagnoses:
http://www.lcresources.com/more_resourc ... hp?f=3&t=5

You will have to register on the LC Resources site (it's free) in order to view the seminar.

[Kimico]

I would advise to take a look at the 15 minute presentation by LC Resources, you will learn a lot about ghost peaks. Also, I would probably leave running the 6N HNO3 as a last resort; passivation of your LC system is probably not the problem. I'd suggest you search the archives on LC-GC for a 2 article series on passivation of stainless steel before you attempt to do this. Passivation requires a series of steps involving degreasing , passivation and flushing the system.

Also, do not discard the organic modifier as the source, I already had that experience thinking that it was my buffer, but it was a bad lot of methanol with "hydrophobic contaminants".

Good luck!

[Uwe Neue]

I am sure that all the advice on the ghost peaks for RP will be applicable. However, this is NOT a RP gradient and therefore you are not using a RP column. So let us know what the column is, and what your detector settings are. The solution may be different...

[pat_val]

Thanks all for your valuable advises. I will go th' this seminar. My column is Luna, SCX 5 micron, 250 x 4.6 mm.

[Uwe Neue]

Hmmh...
So you have an ion-exchange column, and you are running a HILIC gradient...
You still have not yet told us how you detect...

[pat_val]

Oh I forgot to mention....i am using 276 nm and PDA detector

[Uwe Neue]

It could be the ligand...

Is it worse when you start up in the morning?