Table 19.1.
Typical experimental parameters for an ITC experimenta
| Experimental parameter | Setting | Comments |
|---|---|---|
| Cell temperature | 2–80 °C | See specific concerns regarding high-temperature experiments in text |
| No of injections | 25–35b | Based on single titration method; fewer injections can be used for the two-titration method |
| Injection volume | First Inj.: 1–2 μl; Rest of Inj.: 7–12 μl | First small injection is used to account for syringe backlash; data from this injection should be omitted from data analysis |
| Injection spacing | 250–500 s | Typical spacing is about 300 s; should be lengthened if titration fails to adequately return to baseline prior to commencing the next injection. Spacing can be shortened if long flat baselines are present between each injection |
| Titrate/titrant concentration | Cell: 1.8 μM; Syringe: 42 μM | Can be optimized depending on reaction heat; this example comes from an RNA-RNA experiment with ΔH of ~ 40 kcal/mol and a 1:1 stoichiometry |
| ITC equilibration options | Automatic | |
| Reference power | 25–30 μCal/s | Setting a much higher value than is required may affect the sensitivity of the instrument |
| Initial delay | 60–100 s | Ensure stable baseline prior to first injection |
| Stirring speed | 270–310 rpm | Faster stirring speeds may lead to high-frequency noise in the data |
| Feedback mode | Fast | |
| Filter period | 2 s | For slow reactions, the filter period can be increased to improve data quality |
a
Based on use with a VP-ITC from MicroCal. Specific parameter settings might need to be adjusted for use with other instruments.
b
A terminal ratio of about 3 is obtained for a 1:1 interaction (i.e., the final excess ratio of the titrant over the titrate).