The Road to Calorimeter Master Vol.3 Isothermal Titration Calorimeter iTC Measurement and Analysis Points!

by Malvern Panalytical, Tuesday, 1st September 2015

The characters and story appearing here are fiction.
In technical matters, we have received guidance from Professor Fukada, a visiting researcher at Osaka Prefecture University.

[Story So Far]
Mr. Nakakura from Maruban Pharmaceuticals was instructed by his boss, Mr. Tanaka, to set up a calorimeter, but it was one challenge after another. To deepen his understanding, he headed to Professor Fukada with the content he wanted to confirm.
Tackling each issue one by one, he will deepen his understanding of the ITC phenomenon and its reasons.

Professor Fukada, I would like to try the operations, so please guide me!

Yes, understood. Let’s do it.

The system remains on. How long before should the power be turned on?

Well, I tend to leave it on continuously. The system stabilizes better that way. However, since each lab may have its rules, you may want to set up the temperature a day in advance or at least an hour before. If you want the baseline to stabilize, make sure to power it on in advance. Also, don’t forget to set the measurement temperature!

Understood. Next, replace the ultrapure water in the reference. This operation should be done weekly, right? What if the system won’t be used for more than a week? Do we still need to replace it?

If it’s not going to be used, you don’t need to change it unnecessarily. However, even water might breed unknowns if left unchecked, so let’s replace the ultrapure water in both the sample and reference cells once a month. Important point! Always ensure fresh ultrapure water is in the cell before finishing the measurement. If the cell dries, any remaining dirt might stick if left unremoved.

Got it! Next is system cleaning. According to the manual, use the control software’s command. You can follow the instructions on the screen by selecting Cell and Syringe Wash.

That’s right. Following the instructions should be exactly fine. But if you want a more thorough cleaning, you can also use other commands appropriately. Their meanings should be in the manual, so please check them out.

On page 42 of the manual.

If you need a manual, please contact us separately.

For me, every time after a sample measurement, I clean the cell with detergent, specifically 14% Decon90 or 20% Contrad 70. After removing the post-measurement sample, I make sure to rinse with ultrapure water using a gas-tight syringe for precaution. Then, I fill it with detergent using a gas-tight syringe. It tends to foam, so be careful. If the sample seems particularly dirty, you might let it sit for a few minutes after adding detergent. After removing the detergent, repeat the Cell Water Rinse (Long) several times to ensure it’s fully removed. How well you can maintain a clean cell is pivotal for obtaining good data.

I see. Now, are there any tips for syringe cleaning?

In standard cleaning, after the drying step, ensure no water droplets are left on the glass part of the syringe or in the clean position by eye. If droplets remain, drying might be insufficient, and methanol might mix into the syringe sample. For example, seeing a large exothermic reaction during water-water measurement indicates methanol might be involved. The main reason could be insufficient fitting of the fill port adapter.

I’m worried about this fill port adapter until I get used to it.

That’s possible. But you should get the hang of it after using it two or three times! Another point on syringe cleaning: even without any methanol residue, if an exothermic reaction happens in water-water measurement, a dirty titration syringe may be to blame. This is also recorded in the manual, so try cleaning with detergent based on that.

Page 42, right. It looks like syringe drying has finished, so let’s fill the sample. No water droplets. The sample needs to be pre-equilibrated to room temperature to prevent air bubbles. If bubbles are present at this point, it’s better to degas, right?

For iTC200, basic degassing is unnecessary. But that’s a different story if visible bubbles are present. Light centrifugation at room temperature should remove most bubbles.

I get it, some ingenuity is needed. Now, fill the sample cell with EDTA and the titration syringe with CaCl₂. No bubbles between the plunger tip either! Parameters set! Start! Once the DP stabilizes, titration will begin.

That’s right. Let’s watch the process.

This time, Mr. Nakakura pre-set the jacket temperature, which he forgot last time, so the syringe began rotating immediately. Note that if the set temperature differs significantly from the actual temperature, the syringe won’t start immediately.

By the way, professor. The baseline stabilization decision is left to the system, but the manual says it can be done manually…

Software baseline stability determination may sometimes start measurements even if there’s slight drift. While iTC200 has a good S/N ratio, and the drift might not significantly affect measurement results, it’s essential to understand why the baseline becomes unstable.

You need to be careful about environmental influences like air conditioning when setting up the system, right?

Yes. There are other causes too, though. Nearby systems generating strong magnetic fields can influence it as well. When air conditioning or magnetic fields affect, a patterned wave-like drift may be observed. Additionally, if air bubbles are mixed in the cell, drift occurs. This can happen from initial bubble inclusion or cell dirt causing bubble formation. If bubbles are in the sample cell, as the syringe sample is titrated, the bubbles may be expelled, resulting in a pattern where the baseline gradually slopes upward, or suddenly rises once the bubble escapes.

Initially, the DP at measurement start needs to be within ±1 µcal/sec of the set value, correct?

Indeed. But ±1 µcal/sec seems slightly lenient as a criterion. What’s crucial is staying as close as possible to the set value. Is the baseline stable by now?