Capillary Electrophoresis Setup

Discussions about CE, MEKC and related topics

2 posts Page 1 of 1
Hi all,
I am new to CE and am trying to setup an Agilent CE using DAD detector. All the parts are working well and I have no trouble connecting to the software and controlling the set points and samples vials etc. I have set up my method for detection of Anions using the Agilent method notes. I have a nice baseline on the DAD detector but I am not getting any peaks for the Anions. I'm confident the machine is working as the DAD passes the calibration and all the lifts etc work fine. The install time of the bulb is over the recommended time limit but I have no idea if the last user reset it when the bulb was installed. I think it's a good one though. I have a couple of questions about the capillary etc....

1. What are some common causes of flat baseline (when DAD lamp/system appears to be working)?
2. Why is the polyimide removed from ends of capillary column? And do I need to trim the column ends or leave them as they are out of the box? (I'm using an uncoated fused silica column (a tiny bit shorter than the method notes mention but I think it should only elute the analytes a bit faster...) and have conditioned it with 1N NaOH (how do you determine there is flow through the column?)
3. According to the method notes I am running a negative 30kv setting and the setpoints do achieve this but my uA is -12.1 and there are -0.4watts... please let me know if the uA or Watts may be off what you would expect to see. I have tried both inlet and outlet loading of sample with no luck.
4. I am currently not using the regeneration options but am loading the proper buffers, samples, and waste vials where they belong. The buffer is a bit old - could this be the issue? The Anion sample is new.
5. I have tried both Pressure sample loading and Electrical sample loading with no luck. I'm using the internal pressure pump and not the external. Any thoughts?
6. The DAD is set to the recommended settings and the software reads back as though all is well...I believe my column is installed correctly with the clear etched part lined up in the detection cell and both ends are clearly under the sample/solvent level in both inlet and outlet vials.

Thanks for your help in advance!
Hi!

I'll try and provide some answers, see below squeezed between your questions.

1. What are some common causes of flat baseline (when DAD lamp/system appears to be working)?
If the baseline is really, totally flat, even if you zoom in, there is no signal coming in from the detector. You write it is an old lamp. Did it ignite properly? Could something be blocking your window, e.g. is the alignment interface clean, is the polyimide coating properly removed in the window etc. It is hard from here to determine whether you have a mechanic problem or an application problem. Usually, one would go back to a very known application and see if everything looks normal then. If OK, you'd replace the capillary and application you've been working with when the problem appeared, and check whether it is still there. If so, you'd check point by point and replace with fresh parts, putting the old back if this didn't help. Seeing your questions below, you might want to check whether the capillary is blocked, e.g. because of an improper end.

2. Why is the polyimide removed from ends of capillary column? And do I need to trim the column ends or leave them as they are out of the box? (I'm using an uncoated fused silica column (a tiny bit shorter than the method notes mention but I think it should only elute the analytes a bit faster...) and have conditioned it with 1N NaOH (how do you determine there is flow through the column?)
The polyimide is removed for a number of reasons. It reduces carry-over, and makes that you can check if the end is straight (do check this). If you have solvents in you BGE, polyimide might swell and block the end. It is also been said that there can be some space at the end between polyimide and the silica, creating a perfect hide-out for everything you don't want to stick there.
And yes, you should trim the ends and check that they are straight. You can do this with a ceramic cutter or with a diamond cutter, whatever you prefer. Do not apply too much pressure while cutting. With the ceramic one: don't saw, just make one jape with the cutter angled about 30 degrees. You can also check Polymicro Technologie's website for more tips.
A tiny bit shorter shouldn't matter, as long it isn't more than a tiny bit and you have to stress the capillary through the cassette to make the proper length at the end.
1 N NaOH is been done. Do check what is in the application note for that method, though. I hardly ever use 1N, usually 0.1N, but also time and temperature play a role. However, capillary conditioning should be part of method development. The nice thing of the Agilent equipment is that you can follow the signal even when there is no run. NaOH absorbs low wavelength UV light, so compared to water you should see the baseline go up dramatically at 200-210 nm.

3. According to the method notes I am running a negative 30kv setting and the setpoints do achieve this but my uA is -12.1 and there are -0.4watts... please let me know if the uA or Watts may be off what you would expect to see. I have tried both inlet and outlet loading of sample with no luck.
The application should give you an indication of the current you should get. The current trace is a very powerful troubleshooting tool. A current trace should be part of your method SOP as well.

4. I am currently not using the regeneration options but am loading the proper buffers, samples, and waste vials where they belong. The buffer is a bit old - could this be the issue? The Anion sample is new.
5. I have tried both Pressure sample loading and Electrical sample loading with no luck. I'm using the internal pressure pump and not the external. Any thoughts?
6. The DAD is set to the recommended settings and the software reads back as though all is well...I believe my column is installed correctly with the clear etched part lined up in the detection cell and both ends are clearly under the sample/solvent level in both inlet and outlet vials.

In order to help you better from here, I'd need to see the application note, you programmed method and some e-grams and current traces you got.
Since I only just subscribed to this forum, and you question was posted almost 3 months ago, I'd curious to get an update on what you've achieved in between. Feel free to contact me through the details on my website, http://www.kantisto.nl (sorry for the roundabout way, I hope it keeps the spam limited).
/Cari Sänger
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