[CZE] Question

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Dear Fellow chromatographists,

I was wondering if someone could help me with this capillary zone electrophoresis problem.
I'm running a CZE method to separate iso-flavones. These are the parameters of the method:
Run buffer: 150mM Boric Acid pH 10.5
Capillary: Uncoated fused silica 50µm ID, 50cm length
pre-injection steps: 3min wash at 45psi with 0.1M NaOH, followed by 10min wash with run buffer at 45psi.
Separation: 23kV normal polarity

Here is the thing. After 2-3 injections that run smoothly, the following happens: The current will spike to a normal level for a few seconds and then it falls down to between 0.5 and 2 µAm.
Any idea what is happening? Is the capillary insufficiently filled with run buffer, or is something else happening?

Thanks for your time and advice
Maurice
Sounds like you may have had a bubble in the capillary.
plz check by changing the sample injection pressure and varying with new capillary of same dimensions..
Thanks for your answers guys.

I've had limited succes with changing the rinse pressure to a lower pressure. I say limited, because it didn't really work on the long run.
Next week I will try to play around with the injections pressures or different capillaries. You think that prehaps due to incorrect injection I don't get a current running?

Thanks again
Maurice
That's right. If liquid is not continuous, the current cannot flow.
MauriceS wrote:
Dear Fellow chromatographists,

Here is the thing. After 2-3 injections that run smoothly, the following happens: The current will spike to a normal level for a few seconds and then it falls down to between 0.5 and 2 µAm.
Any idea what is happening? Is the capillary insufficiently filled with run buffer, or is something else happening?


Hi Maurice,
It is always hard to troubleshoot on paper. Questions that come up are:
Did this application ever run properly, or have you had these problems all the time?
As suggested by others, at the look of it, there seems to be air or gas in your system, or some blocking. What is the sample matrix? Anything in it that could behave strange with borate or at high pH? Are the capillary ends cut straight? The rinsing times seem long enough. What is your current if everything goes well, and how does you current profile look then? IF it is a high current, you might be generating more heat than you'd like. At the pH you are working on, some gas may get out of the system when you apply the voltage and heat up. Do other, well-known applications work well? What water do you use for the BGE. we once had a problem with our MilliQ system in the lab, without knowing it. The resistance of the water was still OK, no extra currents, but the water turned out to be saturated with air, which can out in tiny bubbles once in the CE (30 degrees at that time, 10 degrees above lab temperature), giving all kind of weird problems we never had before. Degassing (He, boiling) helped.
It's been some time since you posted this, so I wondered whether you still have this problem, or whether it "just disappeared"?
/Cari Sänger
www.kantisto.nl
It is typically outgassing problem of running electrolytes inside the capillary. I guess using such alkaline background electrolyte would give you way high current and in turn tremendous temp raise which results in bubble formation inside the capillary. Better lower the temp, say by lowering the pH, reducing the applied voltage, using smaller id or longer capillary, etc would help.

Hubert
CZE is notorious for bubble formation. First rule of thumb, check for bubbles in your solution before you load the vials to inject. Depending on how the sample is dispenses into them, it may be easy for bubbles to form there. Also check the volume in your vials, if it runs low you are more likely to get a bubble. That makes it risky to inject multiply times from the same vial.

Back when I was doing this, our manager did some replumbing so that the flow went into the pump on the underneath side and came out the top on our ABI. This did help considerably. It's been a few years so I don't remember many details. I do remember fighting the bubbles though!
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