HPLC Column Dead Volume
A question about column dead volume. If the hold-up volume (time) is the extra-column dead volume expressed in time or volume. What is the column dead volume and how can it be calculated?
Question:
If the hold-up volume (time) is the extra-column dead volume expressed in time or volume, what is the column dead volume and how can it be calculated?
Answer: Dead Volume – Dead volume is extra volume experienced by solutes as they pass through the chromatographic system, in particular any unswept volume exposed to the mobile-phase flow. Excessive dead volume causes additional peak broadening. It can be related to the hold-up volume, which is the volume of mobile phase necessary to elute and unretained compound (i.e. uracil). This should be minimised as we have discussed previously by using the correct length and diameter of tubing, the correct fittings etc.
The following page in CHROMacademy details how to measure column dead volume: Minimizing System Volume >
Hold-Up Volume (VM or V0) – The total volume of mobile phase in the column regardless of where it exists.
In liquid chromatography: The hold-up volume consists of the entire space accessible to a small molecule able to fully permeate all the pores of the packing material. It comprises the interstitial volume and the unoccupied pore volume. It is denoted as VM or V0. The system hold up volume includes the additional volume in the tubing used to connect the injector and detector to the column. The hold-up volume is usually approximated by injecting a small essentially unretained compound. In reversed phase HPLC uracil, acetone, and thiourea are most commonly used.
tM = hold-up time (also expressed as t0)
Fc = column flow rate
The void volume for the column is equivalent to 68% of its volume (i.e. the space between the packing material particles) and is given by:
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Reversed Phase HPLC for the Analysis of Biomolecules
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