Trying to understand the concept of dwell volume in HPLC?
Dwell volume is defined as the volume between where your gradient is formed to where it touches the column.
It’s just a practical piece of plumbing. There’s a piece of tubing between the pump and the autosampler and that piece of tubing has a certain measurable volume. So it takes a certain amount of time for that new mobile phase to pass through the dwell volume and arrive at the column. What is the significance of the time delay between where the components of the mobile phase are mixed to where that particular strength of the mobile phase actually interacts with the column? It is this: Remember that just because you tell the HPLC, ‘I want 11% acetonitrile’ (or methanol) doesn’t mean that – at that instant – 11% acetonitrile is actually pumping through the column. There is almost always a delay.
I like to compare chromatography concepts to stuff from everyday life. That makes it easier to understand and remember. I do that in all my HPLC and GC courses at Axion Labs, especially in the LC/GC Bootcamp. So here is the analogy that I like to use regarding dwell volume:
Do you ever step into the shower and then turn on the water? No! Why not? I bet you know within a millimeter exactly where to put that handle so that the water is at the perfect temperature, not too hot and not too cold. But you never do it because you know the water is going to be cold even though you told it you want warm water. Why does the water come out cold initially when you turn the handle to where it says ‘warm’? It is because the hot water has to get from the hot water tank to your shower head. There is a volume that the water has to pass through before it gets to your shower head and that takes time. That is the dwell volume of your shower. That’s why it takes time.
Back to HPLC. Some people think in terms of dwell time, some in terms of dwell volume. Now that you know what it is, would you like my advice?
HPLC Method Development and Dwell Volume
Forget I told you anything about it. Just make it disappear. If you follow all my rules about HPLC method development, about looking at where peaks come off, that’s already factored in. Don’t overthink it. The engineer types, you’re going to try and compensate for it. You’ll be like, ’ well if I knew what the volume was I simply subtract and divide by this.’ Nope. For most cases, I recommend against that approach because it invites complication and errors. One reason for that is that even though dwell volume can be measured, it itself is a variable. It can vary based on the pressure.
So don’t overthink this one. Just understand what dwell volume is and why it’s there. Use a consistent approach to method development and optimization and you will not have to worry about it. Some caveats however would be method transfer from one HPLC to another, or other scenarios where precise control over gradient composition is required. Check out this article and video to get some deeper insights.