Frequently Asked Questions C4D
C4D = capacitively-coupled contactless conductivity detection/detector
- 1 How do I get the best sensitivity (the lowest limits of detection) from my C4D?
- 2 Which C4D settings should I use?
- 3 What is the Headstage Gain?
- 4 My C4D headstage has two holes in it. Which hole should I use for my capillary/tubing?
- 5 Why am I getting a sloping baseline?
- 6 Why are my peaks negative?
- 7 K raw and delta K
How do I get the best sensitivity (the lowest limits of detection) from my C4D?
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. See "Which C4D settings should I use?" below.
Which C4D settings should I use?
The C4D settings (amplitude, frequency and headstage gain) refer to the frequency and amplitude of the sine wave applied to the transmitter electrode in the headstage or platform. This is shown in the Contactless Conductivity Introduction page.
It’s important to select the best 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.
What is the Headstage Gain?
The Headstage Gain is a 5x amplification that occurs in the headstage/platform when it is turned on in the software. It is generally used when using a background electrolyte with low conductivity, in order to boost the signal. It amplifies both the signal and any noise present. It should not be used if it causes the signal to exceed 2V, as above 2V the signal will not be linear and will overload the detector.
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.
- 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
Why are my peaks negative?
C4D measures conductivity. The detector is continually measuring the conductivity of the background electrolyte, and then the conductivity of the analytes when they are in the detector. If the conductivity of the analyte is less than the conductivity of the background electrolyte (as is the case for the cations in the eDAQ EC20 Standard Test Solutions) then you get a negative peak.
When the background electrolyte is in the detector, you have hydrogen cations (proton H+) and acetic anions. When the sodium analyte is in the detector, you have sodium cations (K+) and acetic anions. The conductivity of the proton H+ is greater than the conductivity of the sodium K+; the charge is +1 for both, but because the H+ is smaller than the K+, the charge-to-size ratio is greater, so it’s conductivity is larger.
If you use a different background electrolyte with a different counter ion, the peaks could be positive.
You can confirm all this by using PeakMaster. PeakMaster software can predict the electropherogram, if you enter the background electrolyte, analytes and experiment conditions. PeakMaster software can be downloaded for free from this website.
You can download File:K Na Li in MES-Histidine buffer.zip and open it in the PeakMaster software. Click Calculate, to see the predicted electropherogram, with negative peaks. Hover the mouse over the peaks in the electropherogram to see which peak is which; a great way of determining which peaks are which when you are analysing a sample.
K raw and delta K
K raw: K raw is the raw signal that the detector collects. It should be kept under 2.0V during experiments because above 2.0 V, the signal from the detector will not be linear. Signals above 2.5V will overload the detector; it will not be able to record a signal above 2.5V (the signal stays stuck at 2.5V even if the voltage is actually higher) and the hardware unit will give a beeping sound (on most hardware models).
K raw can be kept under 2.0V by selecting C4D settings (amplitude, frequency and headstage gain) which are low enough. Higher amplitudes, higher frequencies, turning on the Headstage Gain and using a background electrolyte with high conductivity will all increase K raw.
You can check the K raw value is under 2.0V by making sure there is no zero applied: the offset value in the C4D Amplifier window should be at about 0V; if it isn't, click on the % number under the word “Offset” and this will clear the offset.
Delta K: Delta K is the signal given by the detector after it has been zeroed. Delta K is equal to K raw when there is no zero applied (when the offset is zero). This is the signal shown on the main graph in PowerChrom or Chart software during the experiment.
Why zero the signal? In most capillary electrophoresis and microchip electrophoresis experiments, the background electrolyte will produce a background conductivity signal which may be at around 1V or 1.5 V, depending on the conductivity of the liquid. The analyte peaks might only be 5 or 30 mV high. In order to make it easier for the detector to detect these small peaks on top of the large background, it is usual to zero the signal with the background electrolyte inside the capillary or the chip’s channel. This enables the user to use a small recording range, usually about 50 mV, so that the software can record the analyte peaks with a high resolution.