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Is Dead Volume as Scary as it Sounds?

5 Dec 2022

Dead volume can be a scary term and knowing that too much of it can lead to poor peak shape makes it even scarier, but have no fear! There are some easy ways to minimize dead volume in your LC.

Obtaining great peak shape is a top priority for chromatographers. We all know that variables such as temperature, mobile phases, and sample diluent can change the peak shape of different analytes, but sometimes you may run in to peak tailing that is not cleaned up by adjusting one of these variables. This is because peak shape can also be affected by the path the sample takes through your instrument. If there is an excessive amount of dead volume in your instrument, this can lead to poor peak shape, peak broadening, or peak tailing.

Dead volume is the extra volume experienced by solutes as they pass through the system, which is why extra dead volume can cause misshapen peaks. Extra dead volume can usually be found in any connecting tubing, the injector, or the detector.

There are a few ways to ensure that there is minimal dead volume in your LC system. One way is to check all of your fittings and ensure that they are connected properly. If the fittings are not snug or they are not seated properly, this can cause some dead volume in your system. Especially important fittings to check are the column fittings, and any other fittings of the flow path.

Another variable to check is the ends of the PEEK tubing. It is important to always cut PEEK tubing with an appropriate tubing cutter, a guillotine-style cutter. Cutting this tubing with scissors or a razor blade is not recommended because these can cause blockages from crimping in the tubing as well as issues with the connection of the tubing. You want a nice, clean cut in the tubing so that connections and good fittings can be achieved. For further instructions on how to cut PEEK tubing, check out this Restek Tip video.

LC Tubing Cutting & Installation

The tailing in the example below can be pretty scary, especially if you have run samples on the same method in the past and they did not have the tailing before. In the first chromatogram, there is tailing on almost all of the peaks. After this was noticed, all of the fittings were reseated on the instrument to ensure everything was seated correctly. The second chromatogram shows the same analysis after this troubleshooting was complete.

Figure 1: Chromatogram with noticeable peak tailing.

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Figure 2: Chromatogram after a dead volume issue was addressed on the instrument.

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This example shows you how something as small as one fitting being incorrectly seated can cause such a big issue with peak shape for so many analytes. So, if you are having nightmares about misshapen peaks, be sure to keep instrument dead volume in mind and check all fittings and PEEK tubing ends. Some dead volume in the system is inevitable, but it is best to keep it as minimal as possible.