Written by: Kyle Williams
If you’ve ever analyzed GPC/SEC data collected on your OMNISEC or TDA 305 instrument that included a viscometer detector, you might have observed and wondered about a negative peak that elutes in the viscometer signal long after the sample peak. This peak is normal and expected, and the following information will hopefully help you understand why it exists.
To start, we need to discuss the basics of the viscometer detector’s design. The viscometer detector is comprised of four capillaries, a delay column, and two transducers (DP and IP) arranged in the manner depicted in the figure below.
When the sample enters the viscometer the flow path is split in two. Half of the sample travels through the positive side of the detector (the lower half in this diagram, denoted as such because it corresponds to the positive side of the DP transducer). This portion passes straight through the detector and ultimately out to waste. The other half of the sample, which passes through the negative side of the detector, is held up in the delay column. The delay column serves as a reservoir with a large volume that holds the sample and delays it from eluting. This allows the pressure of the sample in solution on the positive side of the detector to be compared to the pressure of the solvent only since the half of the sample on the negative side of the detector is contained in the delay column. This results in a positive peak in the DP signal, which is the analytical peak used to calculate a sample’s intrinsic viscosity (IV).
A few mL later the sample peak returns to baseline as the sample solution completely exits the positive side of the detector. After 10-15 mL (depending on the volume of the delay column), the remaining half of the sample begins to elute from the delay column. Since the pressure of this sample solution on the negative side of the DP transducer is being compared to solvent only on the positive side of the detector, the resulting peak is an inverse of the positive sample peak. This peak is sometimes referred to as the delay peak, the inverse peak, or occasionally the negative peak. The terms delay peak or inverse peak are more accurate; the later eluting peak is not always negative. The delay peak is typically broader than the sample peak due to the sample diffusion that occurs in the delay column. An example of this is shown in the video and the figure below.
In the following figure, the sample peak elutes between 16-22 mL and then the delay peak elutes between 32-42 mL. Since the sample peak is positive, the delay peak is negative.
The delay peaks for samples are typically negative because the samples usually increase a solution’s viscosity and produce a positive peak. However, the sample is not the only material that produces a delay peak – the dissolution solvent can create one as well. Often, a delay peak for the dissolution solvent is not observed because the dissolution solvent is the same as the mobile phase. However, when the dissolution solvent has a different viscosity than the mobile phase it creates its own “sample” and delay peak. This can happen when a sample is analyzed in a mobile phase containing salt but is dissolved in the pure solvent with no salt. Proteins and other biomaterials dissolved in pure water and analyzed in phosphate buffer or other salt solutions are the most common example of this situation. When the pure water dissolution solvent passes through the positive side of the viscometer detector, it produces a negative peak because the pure water is less viscous, and thus produces lower pressure, than the aqueous mobile phase containing salt. Because the “sample” peak for the water is negative, the corresponding delay peak is positive. This delay peak can cause confusion because it is positive, but really, it’s the inverse of a negative viscometer peak. An example of this is shown below.
The positive sample peak from 5-6 mL produces a broad, negative delay peak at about 22-24 mL. The water that was used as a dissolution solvent produces a negative viscometer peak in the phosphate buffer mobile phase, eluting between 7-8 mL. The resulting delay peak for the negative water peak is a broad, positive delay peak eluting at 24-28 mL.
Typically, sample run times are set so that all the delay peaks have finished eluting before moving onto the next injection. This ensures that the delay peak from the previous sample does not interfere with the sample peak from the subsequent injection.
Hopefully, this has helped you, as a user, better understand how the viscometer detector works and provided insight as to why your GPC/SEC data looks the way it does.
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