I am insterested in determining experimentally and mathematically the dead volume (volume from the injector to the head of collumn). I saw several formula on the internet, however, I found a confusion among "dwell volume", "dead volume" and "void volume" concepts.
Could you please help me with that?
Thanks in advance!
According to compendial documents (eg. Pharmacopoeias) the volume from the injector to the head of column is called "dwell volume". It is recommended to know/determine the dwell volume of your HPLC system as it can affect separation in case of gradient elution.
European as well as USP pharmacopoeia describe how to measure it - usually a gradient should be applied (without the column installed) formed between phases of different absorption e.g water and 0,1%acetone in water (det 260 nm). The delay of background signal increase in relation to "pump time" recalculated with flow rate dertermines the dwell volume (it should be expressed in ml)). From my practice, I would suggest to apply step gradient instead of ramp as it gives sharper signal rise.
Hi Andre,
You need to know the internal diameter, as well as the length of the tubing from your injector to the column. I am not sure of the difference between dwell/void/dead volumes but assume that it is the volume of liquid displaced before the sample hits the column.
In any case, once you know the length and internal ID of the tubing, you can calculate the volume of liquid between the injector and column. Since you have a flow rate (eg., ml/min or ul/min or nl/min) you can work out how long it will take for the volume to reach the column..If it is very long, you can imagine shortening the tubings "en route", to make the system more efficient. This is not trivial if you have stainless steel tubings. With capillary tubings, it is relatively easy to shorten the liquid path by cutting the tubing and reassembling the fittings.
Best,
Hediye
Hi everyone! Thank you so much for your help! All your intel helped me a lot!
I hope I can contribute some day with your work! You can count on me.
Have a nice weekend!
Terminology needs to be clear to answer your true question (I think you have your terms all mixed up). "dead" volume or "Dwell" volume of an HPLC system are very different than what you may be asking about. If you use HPLC, then you will need to know what the column dead volume (or dead time) is to calculate the column void time. Really no need to measure the entire HPLC, just certain parts of the flow path.
Here are two articles and a table that will help you estimate, calculate and MEASURE Column Void Volume and System Dwell Volume. *Always measure it, but start by estimating the value before you begin (this is how we begin all HPLC methods).
The calculation of Column Void Volume is critical to performing HPLC analysis (you can not perform HPLC w/o understanding what it is).
- "Determination of HPLC COLUMN Dead Volume / Dead Time (T zero)"; http://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html
*System DWELL volume is the volume from the pump head (or mixing valve) to the head of the column and affects the gradient delay only. Dwell volume is useful when transferring or comparing gradient methods between systems.
- "Determine the HPLC System Dwell Volume (Gradient Delay Volume)"; https://hplctips.blogspot.com/2017/02/determine-hplc-system-dwell-volume.html
If you need to calculate the tubing volume from one point to another (i.e. the volume from the injector to the column inlet fitting, then measure the tubing used to make these connections).
- "HPLC Capillary Tubing Connection Volumes": https://hplctips.blogspot.com/2012/10/capillary-tubing-connection-volumes.html
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