New ECD user - current overloading

[smason]

Hello,

I am new to using ECD and we just purchased one for doing norephinephrine assays. To get the sensitivity we need, we will have to be reading in the nanoampere range, but whenever we try it overloads. It works fine to 0.1uA, but we need an LOD of 10pg. (We can only get down to 10ng)

We are using a used Waters 464 detector, glassy carbon vs AgCl reference. The DC scan is at the following:
E = +500mV
Synergi Hydro-RP column
20mM Potassium phosphate buffer, pH at 4.0, flow rate of 1mL/min.

Any advice would be greatly appreciated,
Sam

[folive]

Sam....
Try lowering your applied voltage. The current response curve for NE levels off once you get up to around +175 mV or so, and anything above that won't give you much of an additional response. You should be able to detect 10 pg of NE with this.

What type of matrix are your samples in? plasma? CSF? tissue extracts?

Hope I can help....

Foster Olive
MUSC Dept of Psychiatry

[smason]

We tried lowering the applied voltage, to no avail. This is just our standards in water that are giving us the problem. Could it be the elctrodes? I haven't tried cleaning them yet.

[ravenwork]

It absolutely could be the electrode. Give it a thorough cleaning according to the manufacturer's instructions. ECD electrodes need routine cleaning. The typical biological samples analyzed for catechol amines will tend to cause electrode fouling over time. This can be minimized by operating at smaller potential magnitudes if possible, but even then fouling will still occur.

Your weakly acidic phosphate mobile phase is an excellent medium for microbial growth. I would urge you to flush this system when not in use with pure water, or a weak aqueous solution of methanol if that is possible.

[AA]

For high sensitivity ECD work, along with clean electrodes, you really need a clean LC and, almost as importantly, a clean column.

When I get a new LC (or an old one that has never been used for EC) I start with a flush of 30% H3PO4 in water (a couple of hours) followed by passivation with 6N nitric followed by tons of flushing with water to get rid of all that acid. If you have never done this kind of cleaning and/or passivation before, I would suggest you seek out someone who has done it to help you.

As for columns, I flush my new columns for EC work for 48 hours with 1:1 methanol water at a moderate flow rate (0.5 mL/min depends on column i.d.) followed by 3 or 4 hours with mobile phase before I hook the cell up.