By Zetto on Friday, July 23, 2004 - 08:02 am:

Greetings
Here's the sequence of injection
1- 2 injections of the std and sample solvant (to verify cleanliness of the system)
2- 3 injections of the standard (the verify Rt and peak area)(shows a single well defined peak)
3- 2 inj. of the std and the sample solvant (to verify carry over from the std)(Rt = 15 min)
4- 2 inj. of the actual sample (gives fairly clean chromatograms)
5- reinjection as no. 3 for the same reason
6- 3 injections of the standard ==== Gives TWO peaks !!!!!!!!! (there separated by about 2 minutes and about the same size)
So what would explain the splitting of the std peak after injection of the sample ?
I have repeated the same sequence of injections and the problem is reproductible
It seam that no one from the experimented analysts that i've asked can come with an answer !
Thanks

-------------------------------------------------------------------------------------------------------
By DK on Friday, July 23, 2004 - 11:27 pm:

Pls confirm Following things,
1. What is the nature of your sample solution ?? Is it quite different than the standard in terms of pH??

2. Is your standard preparation stable at RT ?? how about injecting freshly prepared standard injection after sample???

3. Do you wash your column before new set of analysis?? if yes with what???

-------------------------------------------------------------------------------------------------------
By zetto on Monday, July 26, 2004 - 04:51 am:

Hi DK
Here's the additional info
1- the sample AND the standard are both prepared in 100% THF.
2- Stable Rt for the std : Not a problem
I have tried freshly prepared std : same problem
3- I inject 3 X 50 ul of THF trough the solvant gradient cycle (THF, water and Ch3CN)
Many thanks
CD

-------------------------------------------------------------------------------------------------------
By Mark on Monday, July 26, 2004 - 07:39 am:

Zetto,
50 uL of THF is quite a large amount of strong solvent to inject into the LC. If it is possible you might try injecting no more than 20 uL or diluting the sample preps to lower the THF conc if possible. That large of a THF injection could be responsible for splitting the peaks.

Regards,
Mark

-------------------------------------------------------------------------------------------------------
By popeye on Thursday, August 5, 2004 - 04:21 am:

Carryover from the sample!!! Your sample is not having 100% of your desired compound, but also has a very strong non-polar compound which elutes after a long time???

-------------------------------------------------------------------------------------------------------
By gina on Friday, August 6, 2004 - 12:48 pm:

Does your sample contain excipients? Could you be creating "channelling" within the column? Have you monitored your pressure over the course of the run? Try adding some of your mobile phase to a vial containing your sample. If it starts to precipitate, you may have found your problem. You may need to leave the mixture to settle for a while since it might take a little while to crystalize.

I've seen something similar. Some of my excipients were soluble in my diluent, but crashed out when they came in contact with my mobile phase during analysis causing my peaks to start shouldering and ultimately split. I must admit yours appears to be a much more abrubt change in peak shape and separation by 2 minutes is extreme!

I agree with the possibility of a carryover peak, however, I would think the peak shape of one of the peaks would be considerably compromised. Not only that, but you wouldn't expect it to be in all the standard injections at the end if it is coming from the sample unless it's retaining through several injections).

To test this, do a single injection of sample and 5-6 blank injections. Either that, or do a single-inject of your sample and ramp up your % organic and hold to see if anything else comes off the column.

Good luck!