Frequently Asked Questions C4D

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C4D = capacitively-coupled contactless conductivity detection/detector

You can learn more about C4D by looking at the introduction, C4D products, videos, application notes and manuals.

How do I get the best sensitivity (the lowest limits of detection) from my C4D?

Video for C4D Profiler V2 Software

You need to develop a method for detecting your analyte using contactless conductivity detection, including which background electrolyte to use. It’s best to start by seeing if anybody has analysed your analyte using C4D before, by looking through review research papers such as 1, 2, 3, 4, 5 and 6.

Then, you should optimise the C4D settings for the background electrolyte you are using. The easiest way to do this is to use the C4D Profiler software. The C4D Profiler tests every combination of C4D setting (amplitude, frequency and headstage gain) and shows the detector signal for each combination. You can download it from here, download the manual, look at the instructions and video.

My C4D headstage has two holes in it. Which hole should I use for my capillary/tubing?

C4D headstages have two holes or guide tubes. You can push your capillary/tube into either guide tube, it really doesn’t matter. The guide tube you push your capillary into will act as the detecting tube and the empty one will act as a reference tube. One signal is taken away from the other. This effectively compensates for the baseline conductivity of the background electrolyte; changes in the composition of the background electrolyte are automatically accounted for, as are temperature drifts. A thorough description of the reference guide tube can be found at DOI: 10.1002/elan.201300413

This is NOT a twin detector; You cannot push a capillary into each guide tube and expect to record two different signals. If you need a multi-channel C4D system, you should purchase the ER825 and multiple C4D headstages.

If you wish to obtain the best sensitivity from your headstage (increase the signal-to-noise ratio), and have already perfected your experimental method and optimised the C4D settings (amplitude, frequency and headstage gain), you can try the following: dilute your background electrolyte by 10% (90% background electrolyte and 10% distilled water), fill it into a short length of capillary/tube, and push it into the empty guide tube. Do not use undiluted (100%) background electrolyte, as this will balance the electronic bridge in the headstage and can lead to strange signals.

This has been observed to increase the signal-to-noise ratio. However, this will not improve the signal-to-noise ratio if you are using a capillary will a small inner diameter (like 25 µm ID), or if you are using a background electrolyte with a low to medium conductivity (like MES/His or acetic acid, for example).

You may find it difficult to stop the liquid leaking out of this reference capillary. This procedure adds complication and can be fiddly, so can only be recommended if you really need to boost the sensitivity of your C4D.

Why am I getting a sloping baseline?

If you are doing capillary electrophoresis or microchip electrophoresis, you are applying a high voltage along the capillary or the chip’s channel. This will heat up the background electrolyte, known as Joule heating. This results in a large change in the conductivity of the background electrolyte, which is picked up by the detector and shown as a sloping baseline.

The very small diameter of capillaries and microfluidic channels allows for efficient heat dissipation (much higher voltages can be employed than those used in the lab gel electrophoresis), however, Joule heating can still can problems. As well at resulting in a sloping baseline, which makes it harder to identify your analyte peaks, the increase in temperature and density gradients can reduce separation efficiency. It can even lead to decomposition of thermally sensitive samples or the creation of vapour bubbles in the microchannels.

How to reduce Joule heating?

The power of heating generated by an electrical conductor is proportional to the product of its resistance and the square of the current.

So to reduce Joule heating, reduce the resistance and reduce the current. Catch 22?: Reduce the resistance, so increase the conductivity of background electrolyte. But this will lead to higher current, which you want to reduce!

The heat produced is proportional to the applied high voltage, the current produced and the time.

You can reduce Joule heating by:

  • Choosing a capillary with a smaller inner diameter (leads to a large decrease in current)
  • Choosing a background electrolyte with a lower conductivity
  • Decrease the ionic strength or concentration of the background electrolyte (leads to a proportional decrease in current)
  • Making sure that capillary is being cooled properly
  • Reducing the applied high-voltage along the capillary/chip's channel (leads to a proportional decrease in current)
  • If you are using Chart software to record the C4D signal, you can use the Baseline Adjustment Extension to flatten your baseline without affecting the area of the peaks. Download it from here and see the training video here.

Remember that the above suggestions will reduce Joule heating, but may have detrimental effects on other aspects of the experiment, such as the separation of analytes, the ionic state of the analytes, and the detection of the analytes etc.

References

  • Joule heating effects in capillary electrophoresis - designing electrophoretic microchips, by Witkowski et al, Journal of Achievements in Material and Manufacturing Engineering, vol 37, issue 2, December 2009
  • Joule heating effect on electroosmotic flow and mass species transport in a microcapillary, by Tang et al DOI:10.1016/j.ijheatmasstransfer.2003.07.006
  • "High Performance Capillary Electrophoresis, A Primer" by Agilent Technologies, page 17