Calibrating Chemicals; Heat Of Protonation Of Tris Base; Sample Preparation; Experiment Setup - TA Instruments Nano ITC Series Getting Started Manual

Calibrating Chemicals

A chemical calibration tests all aspects of the instrument including the calibration constant, the cell vol-
ume, and the injection volume. Heat is released in exactly the same location as occurs during sample titra-
tions, and therefore is the preferred calibration method. (If chemical standards are not available, electrical
pulse calibrations are generally suitable as a second choice.)
There are several standard reactions which are often used in calibrating isothermal titration calorimeters
(see Brigner, L.-E. and Wadsö, I. [1991] Test and Calibration Processes for Microcalorimeters, with special
reference to heat conduction instruments used with aqueous systems J. Biochem. Biophys. Methods 22,
101-118.). Here we will describe one: protonation of Tris base (Tris[Hydroxymethyl] Aminomethane). The
Tris protonation experiment may be used to determine or verify the calibration factor value setting used in
the ITCRun software.

Heat of Protonation of Tris Base

Sample Preparation

It is very important to do a thorough degassing of the water to be used for making the solutions. Use the
lowest ionic content water that is available, such as what is produced by a point-of-use deionized water
system. Degas this water by boiling or stirring under vacuum for a minimum of 45 minutes. Do not degas
the prepared solutions, because this can result in the loss of sample. The solutions can be kept for a short
time in stoppered containers. Use nitrogen or argon to fill the head space in order to exclude ambient air
which contains carbon dioxide.
Prepare a solution of Tris base by dissolving approximately 0.24 g in 50 mL of distilled water. The solution
will be approximately 40 mM, but the exact concentration is not important, as it is well in excess.
A 1.00 mM HCl solution is most readily prepared by pipetting 10 mL of standardized 0.1N HCl into dis-
tilled water and diluting to 1L in a volumetric flask. Alternatively, a standard solution of HCl can be pur-
chased commercially or standardized by acid-base titration (see Skoog, D.A. and West, D.M. [1980]
Analytical Chemistry [Saunders College Publishing], p. 228 ff). Do not degas this solution.

Experiment Setup

Experiment Parameters

Syringe size
Equilibration time
Time between injections
Injection size
Number of injections
1 Rinse the calorimeter cell three times with the Tris solution and then load the cell. The reference cell
may be filled with degassed deionized water. Allow the cells to thermally equilibrate until the heat
reading on the calorimeter is stable.
2 Load the 100 µL syringe with the 1.00 mM HCl solution, making sure to remove any bubbles from the
syringe.
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100 µL (50 µL syringe in the Nano ITC Low Volume)
200 seconds (Hastelloy: 300–400 seconds)
200 seconds (Hastelloy: 400 seconds)
5 µL
20 (10 in the Nano ITC Low Volume)
Nano ITC Getting Started Guide