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Sunday, November 10, 2013

HPLC Dwell Volume (Delay Volume)

Explained By
Dr. Gerard Sharp

B.Sc (Hons) in synthetic organic chemistry from Monash University and a PhD in 
Analytical Chemistry from the University of New South Wales.

  The dwell volume (also called the delay volume) is the volume between the mixing location of the solvents and the start of the analytical column. It is not important in isocratic elution because the mobile phase composition is constant.

This volume however is an important consideration when carrying out gradient programming because the mixing ratio you see on the screen is different to the mixing ratio at the start of the analytical column. This is because there is a defined volume between the mixing chamber and the start of the column and depending on the system flow rate, there will be a lag time before the new mobile phase mix moves through this dwell volume and hits the column.

Depending on your system, the mobile phase mixing is either just before (low pressure mixing) or just after (high pressure mixing) the pump. In a low pressure quartenary pump system, the mixing occurs on the low pressure (solvent bottle) side of the pump and is achieved through a proportioning valve arrangement coupled to the mixing chamber. In a high pressure mixing system, there are actually two pumps (binary pump) and the mixing occurs on the high-pressure side of the pumps. The mixing ratio in this case is determined by the speeds of the pistons of the two pumps.

In the low pressure case with the quartenary pump, the dwell volume includes: 

  • the volume of the connecting tubing from the mixer
  • the pump itself including check valves
  • a damper
  • the injection valve
  • any in-line filters or guard columns
  • all other connecting tubing 
This volume can often be 1-2mL or more thereby introducing a delay time after the mixing chamber. For example, if there is 2.0mL of dwell volume between the mixer and the column and you are pumping at 1.0mL / min, it will take 2.0 minutes for the new mobile phase mix to take effect in the column. The binary pump has less dwell volume because the volume of the pump is eliminated.


If the dwell volume is different from system to system as is often the case, retention times will vary. This can lead to confusion when comparing chromatograms run on different systems of the same mixture and is important when transferring methods and comparing retention times to those listed in a standard method. Also, because the time the mixing ratio of the mobile phase hits the column will be different, selectivity may change. Thus peaks might either move apart or move together causing a loss of resolution and chromatograms obtained from two systems will be different.
Also, this topic has received much attention lately with a focus on Fast and Ultra Fast HPLC using UltraHPLC hardware. With many new systems, there is a capability to use small particle size columns with high back pressures and steep gradients to achieve short run times. If analysis times are targeted at only a few minutes, then having a delay time in the same order is unacceptable. By the time the mobile phase ratio has reached the start of the column, the components have already moved through to the detector.
Even in a conventional system there are modifications which can be made to reduce the dwell volume. The length and diameter of the tubing is the first step. By reducing both the internal diameter and the length, the volume is reduced. A consideration with the diameter however is blockages. A standard diameter might be 0.17mm and this can be reduced to 0.12mm however samples and mobile phase has to be carefully filtered to minimise the chance of a blockage occuring. Even so there is still a chance for a line blockage to occur through wear and tear of pump piston seals and rotor seal degradation. The length though can be controlled to ensure there is no excessive connection length between system modules.

Saturday, March 9, 2013

Practical HPLC Method Development

Lloyd R. Snyder, Joseph J. Kirkland, Joseph L. Glajch

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