By harald on Sunday, July 11, 2004 - 10:36 pm:

My question is:
Sometimes my Peaks have a strange shape. First there is a baseline then the Baseline gets down and nearly the same hight up then it gets back to baseline. Or it gets up and then down. I have seen Peaks like this in many Chromatograms which were not made by me but no body could explain me the reason for these Peaks. (the Peaks are proportional to the konzentration)

-------------------------------------------------------------------------------------------------------
By Chris Pohl on Monday, July 12, 2004 - 04:00 pm:

This depends a lot on what you're operating conditions are. What electrode and operating voltage are you using? Are you working at constant potential or are you using a pulsed waveform? What is your eluent? What is your column? One possible explanation is a pH effect. Since electrochemical detection is quite pH sensitive, it's possible for analyte bands to be causing a pH disturbance. Such a disturbance can be exhibited in the manner you describe.

-------------------------------------------------------------------------------------------------------
By harald on Wednesday, July 14, 2004 - 10:39 pm:

I am using a RP-18 endcapped column my ph is 4,1
50mM kH2PO4 2,5%AcN 1%MeOH my analyte is a DNA base (8Oxo dG) E=600mV constant Amphoteric detection glassy carbon elektrode ref: Ag/AgCl.
I have watched this effekt under these Conditions at most of the other Bases dG dT dC dA where dG Changes its peake most over a longer time first it starts as a negative Peak then it bekomes like I described and then this Peak gets bigger and bigger (in the positive direction) My analyte is the only peak that stays nearly perfekt (positive peakt) and constant.

-------------------------------------------------------------------------------------------------------
By harald on Wednesday, July 14, 2004 - 10:42 pm:

Are there any rules by selekting pH for elektrochemical detection

-------------------------------------------------------------------------------------------------------
By Chris Pohl on Thursday, July 15, 2004 - 06:05 pm:

Harald,

Based on the fact that you are observing a progressive change in peak characteristics, this suggests that you may well be dealing with an electrode fouling problem. The fact that you don't observe this problem with your analyte is probably connected to the optimum potential for your analyte relative to other bases present. If you need to restore performance, you can accomplish this by exposing the electrode to plus one volt for a few minutes and then minus one volt for a few minutes. If this helps somewhat but doesn't completely restore original performance, you may need to repeat this cycle several times. Alternatively, you can simply polish the electrode to remove the contaminant (of course, this assumes you are using a cell with an electrode which can be polished rather than a coulometric electrode which precludes polishing). Generally, however, it's preferred to clean the electrode electrochemically rather than mechanically since the polishing process can introduce many new variables to worry about.

There are no hard and fast rules regarding optimum pH when using electrochemical detection. It all depends upon the specifics of the electrochemical reaction in question. In some cases, the neutral form of a molecule is more reactive so the pH needs to be adjusted accordingly. But in other cases the ionized form actually enhances detection via electrostatic interaction with the electrode. The best course of action is to read up on the specifics of your analyte and see if there are any publications describing optimum conditions. Otherwise, you may need to resort to empirical studies.

-------------------------------------------------------------------------------------------------------
By harald on Thursday, July 15, 2004 - 10:59 pm:

Is there any good book about elektrochemical detection I have read the manual and what is written in some hplc books. But I am surching for something like "ecd in praktice". Where did you get your knowledge from.

-------------------------------------------------------------------------------------------------------
By ECD on Friday, July 16, 2004 - 12:04 pm:

Try making an injection of air. Are you degassing your mobile phase? If so, Ill bet that's what your are seeing

The peak you describe sounds like an air outgassing peak. Is it small? Always at the same place?

-------------------------------------------------------------------------------------------------------
By harald on Sunday, July 18, 2004 - 09:58 pm:

The Peaks apeare at times where I can see peaks in the UV detektor too and they are proportional to the concentration I am injekting

-------------------------------------------------------------------------------------------------------
By harald on Monday, July 19, 2004 - 10:05 pm:

But I would be interestet what is the reason why air peaks appeare always at the same place when outgassing happens

-------------------------------------------------------------------------------------------------------
By AN on Friday, July 23, 2004 - 07:58 pm:

Air peaks are chromatographic. One can move them to some extent with organic modifier or ion-pair. That is why they elute at the same place at any given set of conditions as they are coming from the injection.

Again, if you inject air (empty vial or syringe), does your peak get larger?

-------------------------------------------------------------------------------------------------------
By harald on Wednesday, July 28, 2004 - 12:44 am:

I am afraid of injekting air because my pump sounds like dont loving it. But I am shure that these Peaks are from sample molekules. The Peaks apeare exaktly at the Positions where I can see them in Pda detector too. And as a konsequence of yoúr advise I have changed the voltage and now the Peaks have a optimal shape.
but back to my starting problem can you explain me what happens when one single comound makes a positive and a negative peak in one Chromatogramm. I am shure that it is no air.

-------------------------------------------------------------------------------------------------------
By harald on Wednesday, July 28, 2004 - 12:45 am:

My mobile Phase is degassed by vakuum in line Degasser

-------------------------------------------------------------------------------------------------------
By Chris Pohl on Friday, July 30, 2004 - 02:23 am:

Harald,

The short answer to question about why you see both positive and negative peak with an electrochemical detector is: an analyte which accelerates electron transport to the electrode will cause positive signal whereas an analyte which inhibits electron transport to the electrode will cause negative signal. The latter may be due to adsorption of the analyte on the electrode surface acting as an insulator thus blocking current flux that would otherwise occur due to oxidation of contaminants in the mobile phase, although it may also due other causes. But negative peaks are a bit of the nature of electochemical detection. Many times you can solve the problem by adjusting the electrode potential (or using a pulsed waveform) but sometimes you just have to live with it.

One common cause of a negative peak in electochemical detection is oxygen dissolved in your sample. Try degassing your sample to see if this minimizes the artifact. As mentioned above, gasses can be retained and eluted in HPLC and since oxygen does typically cause negative peaks, you may be detecting oxygen from your sample.

-------------------------------------------------------------------------------------------------------
By AN on Wednesday, August 4, 2004 - 11:57 am:

Harald, injecting air WILL NOT HURT ANYTHING!! Your pump is BEFORE your injector, correct? Thus I ask, why would you think that injecting a small aliquot of air downstream of your pump would hurt it? Please think about it.

You are degassing your mobile phase and then injecting a solvent that is saturated with air. You are seeing outgassing at the EC electrode. Outgassing gives a peak signature that is a small positive and negative disturbance in regards to the baseline. You inject larger volumes, the peak is larger. It always appears at the same place.

Injecting air will prove/disprove this...and it will take a couple minutes at the most.

-------------------------------------------------------------------------------------------------------
By RH on Wednesday, August 11, 2004 - 09:29 am:

I would not recommend injecting air into the system, because there might well be a problem with PEEK microbore check valves that might cause trouble even with traces of air. This would explain your pump sounding strange, even if in general traces of air are no big problem as they will gas out on the low pressure side of your system, bubbles might get stuck in your DAD cuvette as DAD cuvette designs tend to be more sensitive towards air.
Furthermore the idea with ion pairing agent is not a good one. If you don`t use them you won`t like your column seeing them.