HPLC 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?



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?  


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. 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 minimized as we 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 LC: 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 V


or V


. 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.  



= hold-up time (also expressed as t


) F


= 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:  


Hold-up time (tM, t0):

The time required for an unretained compound to be eluted, or the time required for one column volume (V


) of mobile phase to pass through the column. In reversed phase HPLC uracil is often used to measure hold-up time and volume. Hold up time will then be used in the equation to determine retention factor of your compound which allows values to be essentially normalized between systems, as the hold-up time will be different for each HPLC system.  

  Two other important definitions that you are likely to encounter later on within CHROMacademy and other HPLC related texts are:  

Dwell volume (VD):

In LC this refers to the volume between the point of mixing of solvents (usually in the mixing chamber or at the proportioning valves in the liquid chromatograph) and the head of the chromatographic column; this is important in gradient elution or when changes in solvent composition are made in isocratic elution so that the column experiences the composition change in the shortest possible time. Low-pressure mixing systems generally have larger dwell volume than high-pressure mixing systems. This is also important when transferring methods between different HPLC systems or transferring methods to fast HPLC conditions so that retention and resolution can be retained.  


Dwell time (tD):

The time equivalent to dwell volume; determined by the product of flow rate times the dwell volume.  



= gradient time  

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