What is Dead Volume?
- It is also called the extra-column volume, which is somewhat clearer. It comprises the volume of an HPLC system between the point of injection and the point of detection, but excludes the part of the column that contains the packing.
- Therefore it includes the injection volume, the volume of the injector, the volume of the connection tubing before and after the column, the volume in the end fittings of the column, including the frits, and the detector volume. Actually, to be precise, it includes half the injection volume and half of the detector volume.
- It causes extra-column bandspreading. Bandspreading means that the peaks become broader as they flow through the extra-column volume. This is undesirable since it may destroy some of the separation achieved in the column.
ALSO READ: Troubleshooting for UV Detector of HPLC
What is Dwell Volume?
- The dwell volume is responsible for the time delay for a gradient. It is the volume of a gradient HPLC system between the mixing chamber and the column inlet. This volume does of course also exist under isocratic elution, but in that case, it has no impact on the separation.
- It includes the volume of the gradient mixer, the connecting tubing to the pump, the pump head and check-valves, the tubing between the pump and the injector, the injector itself, and the tubing between the injector and the column inlet.
- When initiating a gradient, the column is not subjected to the change in eluent composition until the gradient has passed the dwell volume.
- During that time, the column is operated under isocratic elution. Attention has to be given when transferring a gradient method from one instrument to another. If the dwell volume differs, the retention times will likely differ as well, despite an identical method and column.
The dwell volume is equal to the time between the injection and half height of the detector trace, multiplied by the flow rate.
How to measure the dwell volume?
Disconnect the column from system. Then run a step-gradient from methanol to methanol with 10 mg/L propyl paraben, using a UV detector. The UV detector will detect an S-shaped trace. Then measure the time delay from the point at which started the gradient to the point when half the height of the step is reached. Multiplying this time with the flow rate gives the gradient delay or dwell volume.
How to measure the extra-column volume ?
- The total extra-column volume and extra-column bandspreading can only be measured using special equipment. It includes the column end-fittings and the frits in the column, which are not accessible by the user. So just have to trust that the column manufacturers have done a good job and minimized that part of the extra-column volume. What we can do easily though is measure the extra-column volume associated with the HPLC system. To that purpose we simply disconnect the column and replace it with a "zero dead-volume" union.
- Then we inject a small volume of sample and record the detector response. An example of the response is shown in figure.
- From this "chromatogram" we can determine the extra column volume and the extra-column bandspreading. The extra-column volume is strictly speaking the distance from the point of injection to the center of the gravity of the peak.
- To determine it we need to compute the first moment of the peak. However, we do not have to be overly precise and can for simplicity use the distance between the point of injection and the peak maximum as our measure of system dead volume.
How to measure the extra-column bandspreading?
- We can use the standard deviation of the peak as a measure of the system band-spreading. The standard deviation of the peak can be computed from the second moment of the peak. An easier way is to measure the width of the peak.
- Since this peak is highly asymmetric, it is best to measure the width as close to the baseline as possible to get a good measure of extra-column bandspreading. For example the width of the peak at 4.4 % of the height of the peak corresponds to a width of 5 standard deviations.
Tags
Chromatography
