Monday 30 November 2009

Help on: Difference in retention times using Agilent and Waters HPLC systems


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"I have a question... I want to know why there is a difference in the retention times observed when using Agilent systems and Waters systems?”

“To answer this question we must first look closely at what exactly is a retention time? It is measured as the time from the injection of the sample to the time the separated component of interest is observed by the HPLC detector. Therefore the total retention time will depend not only on the time taken for the component to travel through the HPLC column but also the time taken to travel through the tubing both before the column and after the column. This time, which is in addition to that spent on the column, will depend on the overall volume of tubing, we refer to this as the ‘extra column volume’.

The amount of extra column volume for different brands of HPLC instruments will be slightly different since they are not all made in the same way. Additionally, the user of the instrument can alter the extra column volume by a variety of actions such as replacing lengths of tubing, cutting off the end of the tubing when removing stainless steel ferrules, installing column switching valves, etc. These factors result in small differences in the retention time observed for different brands of instrumentation.

A feature of the chromatogram which tells us about the extent of the extra column volume is the void volume, t0. This is usually the first disturbance in the baseline and corresponds to the solvent in which your sample was injected. This solvent is unretained and so corresponds to the time required for an unretained component to travel through the column to the detector. If you calculate the capacity factor, k’ you can remove the effects of extra column volume for each peak, this value should be the same for different HPLC systems (assuming all other variables are constant, i.e. same column, same mobile phase, same method etc.).

Capacity factor, k’ = (t – t0)/t0

where t = retention time and t0 = the time taken for non-retained components to elute. The capacity factor is a measure of where the peak of interest is located with respect to the void volume (the elution time of unretained components).

Another reason for a difference in retention times which can lead to larger differences relates to gradient methods. When running these types of method the changes in mobile phase composition are controlled by the gradient proportioning valve (GPV) in the pump (for low pressure mixing HPLC systems, the most common type in use). The consequence of this is that there is a delay between changing the gradient and that effect being experienced at the column. This has to take into account the extra volume between the GPV and the injection port. The total volume from the GPV to the column is called the dwell volume and this is another reason why you may experience different retention times using Instruments from different manufacturers. In my experience I have noticed very little difference when measuring the dwell volumes of Agilent and Waters systems.

To calculate the dwell volume for a particular system, refer to a previous post on interpretation of HPLC methods (Wednesday, 14th October 2009).

In practice we tolerate small differences in retention time on different systems provided there is no negative effect on the observed chromatography but a larger difference may need further investigation.”

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