HPLC Products

Gradient dead volume (also known as Dwell Volume) is the internal volume of an HPLC system from the point at which the gradient is actually created through to the top of the column, where the separation starts. This includes the loop, so it is important that the valve is left in the Inject Position when measuring it.

The significance of the Gradient Dead Volume is that although the gradient actually starts when you make an injection, the gradient does not even reach the column until sometime later, when this dead volume has been swept through the system. For example, if you have a 4 ml Gradient Dead Volume, and are pumping at 1ml/min, the gradient starts in the column 4 minutes later than it is mixed in the pump. If you were working with a narrow bore column, with a flow of only 0.1ml/minute, the gradient would hit the column 40 minutes after the run start, possibly after the gradient pump had reached the end of its programme! Typically the Gradient Dead Volume will be between 1-5ml, but it has been known to exceed 10ml on some systems. So here is a method for measuring it on your system.

Solvents required:

A:           HPLC Water

B:           0.2% Acetone in HPLC Water (v/v) (eg 2ml in 1 litre water)

Remove the column and connect the tubing from the autosampler or valve directly to the detector with a union. If necessary, join the two pieces by inserting a length of 0.25mm id (or 0.17mm) tubing in place of the column.

Set the UV detector to 265nm, and then test each solution by pumping 100% through the flowcell to check its absorbance. Solution B will have a higher absorbance, and it is important that neither solution causes the signal to go off scale.

Ensure the valve is set to the Inject position.

Set up a gradient profile to run a 0-100%B gradient at 2ml/min over 10 minutes and hold at 100%B. The profile will look similar to:

Time                    Flow                    %A                       %B

0                          2ml/min               100                      0

10                        2                          0                          100

15                        2                          0                          100

The resultant chromatogram will look similar to the one shown below. It should show a linear slope with minimal curvature at the ends, followed by a plateau where the system equilibrates at 100%B.

Now to the calculation of the Gradient Dead Volume:

Locate the midpoint of the gradient. If you measure the absorbance at 100% A and 100%B, the midpoint is exactly half this absorbance value. At this midpoint, identify the time from the x axis.

Subtract half the gradient time from this time. ie if the midpoint occurs at a time of 7.2 minutes, and the gradient is over 10 minutes, half the gradient time is 5 minutes so the calculation is 7.2 – 5 = 2.2minutes. This is the Gradient Dead Time under these conditions

To calculate the Dead Volume, multiply the Dead Time by the flow rate, (in this case 2ml/min) giving 2.2min  x 2ml/min = 4.4ml (in this case!)

If you find you have more than 5ml dead volume, this may well cause problems for your methods, and you may like to give us a call to discuss what can be done. Otherwise keep this Dwell Volume with the equipment. It will be the same for all gradients regardless of the solvent composition, provided that you do not change the loop size. Should you do any computer modelling to optimise a gradient, it will require you to enter the Gradient Dead Volume so it can calculate the effect of changes. And if you publish a method, remember to specify the Dwell Volume of the system on which the results were obtained!