I am using ECD in a HPLC system. It is a amperometric detection. The compound can be run at 1000 mv in DC mode with ACN and Buffer. However the signal is reduced by every injection. RSD for 5 injections is ~ 10%. So, I try pulse mode (E1=1000 mv, E2=1300 mv, E3=100mv). Then, no peak is shown at all. The working Electrode is glass carbon, The reference electrode is Ag/AgCl. I also run UV as a monitor to check the sample is injected. Any suggestions and recommandations are appreciated

I didn't see your add before but anyway...
Are you using ACN Fisher?. I had similar problems with that. Can you describe the buffer composition and pH?
It sounds like a
1) mobile phase problem
2) If you HPLC is a stainless steel one, have you tried passivating th system with HNO3 15% in water (without column!!!)?
regards.

Hi Marcelo!

It has been long time since I post my question. As no one answer it, I almost feel hopeless. That is why I did not look at this forum until today. Now I am so happy that someone can help me on it. I really appreciate you give me some idea.

My mobile phase is ACN/H2O=75/25 (0.05M HClO4, pH 3). When I use mobile phase MeOH/H2O=85/15(0.05M HClO4, pH3), Electrode is fine (no fouling). However for separation reason, I have to use ACN mobile phase. At the begining, the ACN is from Fisher. Now I use the one from Burdick and Jackson (Carbonyl free). When I try mobile phase ACN/MeOH/H2O=70/10/20(0.05M HClO4, pH 3)with new bottle ACN, the electrode was stable for at least 48 hrs. When I switch to ACN/H2O=75/25(0.05M HClO4, pH 3), electrode starts fouling again. By that time, the ACN I used is from the bottle which was opened one week ago. I wonder something happened to ACN. Normally, how long you keep the ACN for ECD use?

I hope you look at this message soon and give me some help.

Thanks again and Happy New Year !!!

OK,
I use acetonitrile Sintorgan that I suppose it is sold only in Argentina (my country). I don't have any problem with it. I keep the prepared mobile phase for 1 month without problems, and opened ACN bottles for more than 1 month.
One of the most important issues is to passivate the stainless steel parts of the HPLC.
Surely, your ECD manual should have the procedure; I use HNO3 15% v/v in water. Flush first the HPLC with water (15 min, 1 ml/min) without column and then the HNO3 solution. Then water again (check pH) to clean.
The other problem is the working electrode surface. I use a HP electrochemical detector 1049A with a glassy carbon electrode and a reference electrode of Ag/AgCl. In my experience the solid state reference electrode doesn't work with high potentials >+0.85 V. I used the ECD to determinate Nifedipine in plasma samples at +1.00V with a C18 column and ACN-KH2PO4 20 mM pH=6 buffer (450:550). And I polished the work-electrode every two-three day as routine (about 150-180 injections). The glassy carbon electrode should not be changed as HP recommends. Polishing the same electrode is better in order to maximize ECD performance. It’s important to keep in mind that ECD is very different from UV detectors, it has a less stable baseline, it requires “personal attention” (I hope you can understand my Tarzan-like English).
Finally ACN is not the best solvent for ECD at high potentials (as you could see). It’s true that MeOH is better, specially when you have more then 50% of organic solvent.
Finally I’d need more information, as for example:
What kind of ECD detector do you have, thin layer or wall jet?
Who is the provider?
What kind of compound are you trying to assay?

If you were using a C18 column, maybe you should think to change it to a less retentive one as C8 or even C4, in order to increase the water proportion of the mobile phase. And you can also use temperature, for instance 50°C to use less ACN.
I’m sending my e-mail (of my Lab.):
phoe200@phoenix.com.ar

Keep us in touch.
Marcelo.

I am using ECD in HPLC as well. It is oxidative coulometric detection. We seem to periodically lose sensitivity. The last time this occurred a solvent peak that was usually off scale (positive)had changed to a negative peak that was close to being off scale. This is using identical potentials (0, 25, 250mV), current ranges (1µA), mobile phase(55/45 v/v(5%ACN/95%50mM NaOAc buffer)/ACN+0.5%HOAc) etc. The only difference we can think of is the temperature of the room in which the detector and HPLC reside (about 16°C presently but has dropped considerably over the last few days), but I'm not sure that this would cause such a drastic change in responses. In another method we also have had trouble trying to get the system to equilibrate at high potentials (950 mV)at a current range of 50 nA. Any ideas or suggestions?

Hi, Jennifer.
Are you using ESA detectors?
How long do you keep AcONa buffer (with 5% of ACN)? AcONa is a good media for microorganism growing and old mobile phases can also suffer pH changes. Moreover, I see you are mixing two solutions of different pH, are you sure that pH keep constant trough differents preparation? In ECD, pH, ion presents and ionic strengh are very important variables. I prefer to preparate the buffer by adjusting the pH first, and then mix it with the organic solvent.
If the sample solvent is different from the mobile phase almost always you will see a strong solvent peak. This is common in trace analysis, for instance, in catecholamines assay from plasma I have had very strong negative solvent peaks while I was using HClO4 0.2M for eluting CA’s from alumina. When I changed to AcOH 0.2M the solvent peak was positive and better in order to resolves it from Noradrenaline. Maybe a little pH or composition change in sample solvent could produce the change in the front peak. Temperature control is important in order to keep repetibility of quantitation but I don't think that can cause stong changes.
In my experience with amperometric detector (both wall jet and thin layer) KH2PO4 buffers were better than AcOH especially at high potentials (i.e. +1.00V vs Ag/AgCl for assaying nifedipine in plasma). Another important issue is the common practice of using EDTA in mobile phase. It doesn’t work at high potentials because amines can be oxidized. The same for amines (TEA. DEA, etc) used for silanol supression. If I had to assay substances that could produce tailing peaks, I’d use a column packed with Luna (Phenomenex) or Purospher (Merck) which do not need any silanol supressing agent like TEA. In my previous message I focused on ACN quality (not always a good ACN for UV is good for EC), water purity (low ppm or ppb of metals like Cu, Fe, Zn, can contaminate the electrode surface, increasin continuosly the background signal), HPLC passivation (very important, Fe from tubing). Now, ultrapure water (>18 Mohms) is the best choice but for EC, double destilled water in glass apparatus (not in stainless steel) should be OK.
I hope to be useful, good luck!