Help , ghost peak appeared in the blank run!

[bily200677]

RECENTLY, we meet a puzzled problem in validation work. there are existe many ghost peaks in blank run (no injection ), but at the first time ,we use this colum do test , the blank run and every thing is normal . When we intend to use the same method to analyse another very alike substance , when inject the diluent solvent to the chromatography, we fond the gost peak come forth, moreover , increse in response gradually with the injection times increasing . FIRSTLY , we doubt the chromatogranphic sytem and column were contaminated by somthing , so we fulsh the chromatogranphic sytem and column . Then do this test used another instumnt , but the result is same , the blank run also have many ghost peak at 26~30min . these ghost peaks have bad shape ! I think this column may be contaminated badly (BUT we also use guard column normally), any advices for me , thank you gratefully!

Note: chomaotogranphic condition we use listed in the follwing:
column：Ultimate Prime C18 , 250×4.6 mm, 5µm ,
mobile phase--
mobile phase A：10 mM dipotassium hydrogen phospha , add 1.0ml
triethylamine (TEAS) ,rhen djust the pH to 7.1 with phosphoric acid .
mobile phase B:Acetonitrile
flow rate：1.0ml/min
injetion volume：10µl；
injetion temperature：5℃
colum temperature：25℃
wavelength：230nm；
run time：55min；

gradient eluent table :
time（min） mobile phase A（v/v) mobile phase B（v/v）
0 90 10
10 60 40
20 45 55
35 30 70
45 30 70
46 90 10
55 90 10


Diluent solvent ：methanol :wanter=50:50(V/V)

[Noser222]

Are you sure your triethylamine is good?

[bily200677]

Are you sure your triethylamine is good?

I have someone do this test , use the water repalce the buffer to try, hope the result!
thank you sincerely again!

[Kostas Petritis]

Another thing you may want to test is to do some more blank runs where you change the equilibration time. If the peak areas change almost lineary to the equilibration time then there is high likelihood that your problem is mobile phase contaminants...

[bily200677]

Another thing you may want to test is to do some more blank runs where you change the equilibration time. If the peak areas change almost lineary to the equilibration time then there is high likelihood that your problem is mobile phase contaminants...

Sorry, I made a mistake in mobile phase A( Add 1.0ml triethylamine to 1000ml purified water, ,then djust the pH to 7.1 with phosphoric acid which was we used the buffer solution) .
the gradient eluent table :
time（min) milbe phaseA（v/v） mobile phse B（v/v）
0.0 85 15
5.0 75 25
25.0 35 65
42.0 35 65
42.1 85 15
55.0 85 15



In addition，this method have been validated a week ago, the abornoaml thing just appear these several days！ in the past one month, every thing is good, why now we could not repeat the test ?
Kostas Petritis give a good advice to check the mobile phase the purity , let us to ry !
thank you !
happy new year to every one in here! ha

[aicmen]

In my opinion,this problem came from column , It is probably that column
has been contaminated.
It may be solved If you wash the column with water　(about ph4　adjusted by phosphonic acid)&acetonitrile，certainly, It is better to use a
gradient elution procedure.

[AA]

9 times out of 10, problems like this are related to contaminated water. Remember it only take a very minor contamination of the water to show up, you concentrate the organic on the head of the column. A previous poster mentioned how to diagnose this (extend re-equil time and see if unwanted peaks increase in size). To isolate the problem to the water you may have to do some experiments without buffer components. Buffer components can also add unwanted material to the MP.

[bily200677]

ha , we have found out the cause for this problem, the TEA reagent was contaminated,when we use the new opend TEA reagent , the ghost peaks disappeared!
thank you every for your advices!

[mohan_2008]

Recurring issues like contamination can occur, if improperly rinsed pipettes are used to aliquot TEA portions into the mobile phase.

It appears that someone might have used a pipette contaminated with your compound (improperly rinsed) into the TEA bottle, subsequently which gave rise to the ghost peak, exactly at the peak of interest. This is magnified if your compound is more sensitive to the wavelength used.

Apparently, from your discussion, I was pretty sure it was a contamination and not a carry over since you had a pretty strong gradient clean up incorporated in your gradient.