Water-Water titrations are a good way to check for possible problems, especially the cleanliness of the ITC and your skills in cell filling. If a water-water titration works well the instrument is in good working order and your cell filling procedure is correct.
During this process, a small amount of water is added to the water-filled cell in several steps and the resulting heat effect is recorded. However, by increasing the amount of water injected (increasing its volume to ten times the usual 1-2 μm), a significantly larger heat effect can be recorded.
I would like to know what effects, presumably resulting from the design of the equipment, are responsible for the abrupt increase in the amount of detected heat.
In addition, if you inject increasing volumes with the same injection time (20 s), the thermal power due to the mixing will increase because of the increased injection flow (heat due to friction/turbulence). Normally, the injection time varies according to the injected volume.
Anyway, if we assume the smallest injection corresponds to 20 μL (not μm), the heat effect is too large.
Just a curiosity, which the cell volume, 200 μL or 1.4 μL?
Thank you for your answers. The dimension is actually microliters just misrepresented in the figure and the cell has a volume of 1.4 mL. I myself suspected turbulence, so if I understand correctly, should the signal decrease with increasing the injection time during constant volume dosing? Of course, I will try this, but confirmation from independent researchers is an important help.
The heat change detected for the water-into-water injections for the ITC (I assume you are using the MicroCal VP-ITC) will depend on injection volume, speed of injection, stirring speed, the cell temperature, and other settings for the ITC experiment. The results can also depend on how clean the ITC cell and syringe are, if the ITC cells are filled properly, the reference cell is also clean and correctly filled, etc.
Adrian Velazquez-Campoy makes an excellent point about the larger injection volumes using the same injection time, resulting in larger heat changes due to turbulence. I recommend an injection speed of 0.5 sec/uL
Another consideration is when you use injection volumes larger than 15 uL with the VP-ITC: 15 uL is the approximate volume that is in the needle of the ITC injection syringe, and this part of the needle is in the ITC cell and filling stem, so this 15 uL of water in the syringe is able to become the same temperature of the sample of the water in the ITC cell prior to the injection. If you have the ITC cell set to 25 deg C and your lab temperature is NOT 25 deg C, you will see a heat change simply due to the difference in temperature with the larger injection volumes. This heat change becomes larger with increasing difference in the lab temperature and cell temperature.
The Malvern Panalytical service team supporting MicroCal ITCs have specific experimental parameters for water-water injections for their testing. They look for reproducible heat changes/injection (within a certain range) as well as measuring the noise between injections. The service team has diagnostic programs that evaluate and "debug" the results (you don't have that program). If the specified test "fails" (non-reproducible heat changes/injection, heat changes much larger than expected, and/or high noise) that suggests one or more issues: the ITC cells and/or syringe could be contaminated, the injection syringe is bent, a possible issue with the injection pipette, or other issues the the ITC system. They can recommend further steps and tests to evaluate your ITC system.
Dear Verna,
Thank you very much for your detailed and careful explanation. Fortunately, our equipment (MicroCal VP-ITC) is working well but now we are trying to develop a new measurement protocol in an industrial collaboration. So this was the reason for using unconventional dosing volume and speed.
Many thanks again for the excellent advice.
Adam Juhasz I am glad to hear the instrument is functioning, and that you are exploring other assays for the ITC.