help to eliminate my ghost peak! [August 11, 2004]
Posted: Sun Aug 29, 2004 6:39 pm
By Curtinuni on Wednesday, August 11, 2004 - 04:53 am:
Dear all,
I am running HPLC with fluorescent detector, isocratic pump, mobile phase is ACN:25mM phosphate buffer pH 3 (4:6), column C18. 1 month ago, it was no problem at all but these days, I have a constant peak at about 6 min (it will interfere my analysis) when I inject any types of solvents: water, ACN, MeOH, mobile phase, the peak area doesn't depent on injection volume. I tried regeneration procedure for my column (wash with MeOH, Acetone, DCM), even tried another similar brandnew column, it still happens. All the solvents I am using are HPLC grade. My HPLC is Agilent with degasser, autosampler. Do you think where this ghost peak come from?What I have to do to eliminate it?
Thank you in advance!
-------------------------------------------------------------------------------------------------------
By MikeS on Wednesday, August 11, 2004 - 10:49 am:
Agilent autosamplers can suffer from carryover due to long-term buildup on the needle seat. To remove, I go to the "Tests" screen and raise the needle. Then wash the needle and seat with appropriate solvent. Place a tissue under the needle seat to prevent a leak error. I have found that this simple procedure completely eliminates any carryover that I've had. Also, it helps to use a wash vial, and PTFE/silicone septa work well to wipe the needle upon withdrawl from vial. Interestingly, I have tried injections from uncapped blank vials, and the second blank inj always had more carryover than the first, since the wet needle dissolved ("loosened? "wetted?") the needle seat crud and the second injection pushed it into the system.
-------------------------------------------------------------------------------------------------------
By Curtinuni on Wednesday, August 11, 2004 - 09:59 pm:
Thank you very much for your suggestion but unfortunately, it is not my reason. I tried to clean as your advice but this ghost peak is still there, evan I tried to inject an air buble in. I don't know how to solve it out now? Do you have any idea more?
-------------------------------------------------------------------------------------------------------
By BT on Thursday, August 12, 2004 - 06:27 am:
The other problem area that I have seen with the Agilent autosamplers is the Injector Rotor Seal. Either clean or replace (I recommend replacing).
Its also possible that the needle seat was just particularly dirty and it may take a couple of wash cycles as described by MikeS. You could also replace the seat.
Since that RT is consistent and your method is isocratic you're almost certainly looking at an injector problem.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, August 12, 2004 - 10:58 am:
Check water for bacteria and also build up on the stone filter. Replace stone filter and water. I've seen build up give ghost peaks.
Just a suggestion...
-------------------------------------------------------------------------------------------------------
By Curtinuni on Thursday, August 12, 2004 - 06:25 pm:
Thanks all,
I even disconect the Agilent injector and connect another manual injector, did several injections but this peak is still there. I did use the HPLC water, different types of ACN and even replaced ACN by MeOH, used different channel of pump but this ghost peak is still consistent. I am totally stumped now! Do anyone have any suggestion more?
-------------------------------------------------------------------------------------------------------
By MK on Friday, August 13, 2004 - 02:18 am:
Check the filter in purge valve
-------------------------------------------------------------------------------------------------------
By JB on Tuesday, August 17, 2004 - 02:27 pm:
If you're running isocratic, a peak can't be coming from the solvent. It has to be from you're syringe, sample, or injector. If you have same problem with autosampler and manual injector, it must be your sample. But if you have injected different solvents and gotten the same results then its not your sample. My guess is you have a dirty filter between the injector and column that has material removed partially on each injection. Change the filter.
-------------------------------------------------------------------------------------------------------
By Curtinuni on Wednesday, August 18, 2004 - 03:41 am:
Thank you MK and JB,
I changed the filter in purge valve, it is dirty. This really helps but can't eliminate all of my ghost peak, it become smaller but still there. I now also suspect that my compound still retain somewhere, maybe pump or detector because before I used this system with preparative column to isolate this compound for identification so I did with huge scale. I trying to wash my system to get rid of it. Any of you have suggestion for washing?
Thank you very much!
PS: Dear JB, the filter you mentioned is the filter in purge valve or another one? Sorry, I am not very good in instrumentation! Could you please clarify it for me? Thanks!
Dear all,
I am running HPLC with fluorescent detector, isocratic pump, mobile phase is ACN:25mM phosphate buffer pH 3 (4:6), column C18. 1 month ago, it was no problem at all but these days, I have a constant peak at about 6 min (it will interfere my analysis) when I inject any types of solvents: water, ACN, MeOH, mobile phase, the peak area doesn't depent on injection volume. I tried regeneration procedure for my column (wash with MeOH, Acetone, DCM), even tried another similar brandnew column, it still happens. All the solvents I am using are HPLC grade. My HPLC is Agilent with degasser, autosampler. Do you think where this ghost peak come from?What I have to do to eliminate it?
Thank you in advance!
-------------------------------------------------------------------------------------------------------
By MikeS on Wednesday, August 11, 2004 - 10:49 am:
Agilent autosamplers can suffer from carryover due to long-term buildup on the needle seat. To remove, I go to the "Tests" screen and raise the needle. Then wash the needle and seat with appropriate solvent. Place a tissue under the needle seat to prevent a leak error. I have found that this simple procedure completely eliminates any carryover that I've had. Also, it helps to use a wash vial, and PTFE/silicone septa work well to wipe the needle upon withdrawl from vial. Interestingly, I have tried injections from uncapped blank vials, and the second blank inj always had more carryover than the first, since the wet needle dissolved ("loosened? "wetted?") the needle seat crud and the second injection pushed it into the system.
-------------------------------------------------------------------------------------------------------
By Curtinuni on Wednesday, August 11, 2004 - 09:59 pm:
Thank you very much for your suggestion but unfortunately, it is not my reason. I tried to clean as your advice but this ghost peak is still there, evan I tried to inject an air buble in. I don't know how to solve it out now? Do you have any idea more?
-------------------------------------------------------------------------------------------------------
By BT on Thursday, August 12, 2004 - 06:27 am:
The other problem area that I have seen with the Agilent autosamplers is the Injector Rotor Seal. Either clean or replace (I recommend replacing).
Its also possible that the needle seat was just particularly dirty and it may take a couple of wash cycles as described by MikeS. You could also replace the seat.
Since that RT is consistent and your method is isocratic you're almost certainly looking at an injector problem.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, August 12, 2004 - 10:58 am:
Check water for bacteria and also build up on the stone filter. Replace stone filter and water. I've seen build up give ghost peaks.
Just a suggestion...
-------------------------------------------------------------------------------------------------------
By Curtinuni on Thursday, August 12, 2004 - 06:25 pm:
Thanks all,
I even disconect the Agilent injector and connect another manual injector, did several injections but this peak is still there. I did use the HPLC water, different types of ACN and even replaced ACN by MeOH, used different channel of pump but this ghost peak is still consistent. I am totally stumped now! Do anyone have any suggestion more?
-------------------------------------------------------------------------------------------------------
By MK on Friday, August 13, 2004 - 02:18 am:
Check the filter in purge valve
-------------------------------------------------------------------------------------------------------
By JB on Tuesday, August 17, 2004 - 02:27 pm:
If you're running isocratic, a peak can't be coming from the solvent. It has to be from you're syringe, sample, or injector. If you have same problem with autosampler and manual injector, it must be your sample. But if you have injected different solvents and gotten the same results then its not your sample. My guess is you have a dirty filter between the injector and column that has material removed partially on each injection. Change the filter.
-------------------------------------------------------------------------------------------------------
By Curtinuni on Wednesday, August 18, 2004 - 03:41 am:
Thank you MK and JB,
I changed the filter in purge valve, it is dirty. This really helps but can't eliminate all of my ghost peak, it become smaller but still there. I now also suspect that my compound still retain somewhere, maybe pump or detector because before I used this system with preparative column to isolate this compound for identification so I did with huge scale. I trying to wash my system to get rid of it. Any of you have suggestion for washing?
Thank you very much!
PS: Dear JB, the filter you mentioned is the filter in purge valve or another one? Sorry, I am not very good in instrumentation! Could you please clarify it for me? Thanks!