Choosing The Run Parameters - MicroCal VP-ITC User Manual

Section 3: Running an ITC Experiment

3.5 Choosing The Run Parameters

Prior to carrying out a ITC run, the experimental run parameters must be entered into the ITC
Controls window. Run parameters may also be loaded from a parameter setup file (*.inj) or a
previous experimental file (*.itc). (Refer to Section 2.4 ITC Controls Window for more
information). Selecting the proper run parameters depend on the sample you will be using. This
section will attempt to provide you with general guidelines for selecting run parameters.
The Total Number of Injections will, in part be determined by your experimental design and the
setting of concentrations. You will need a minimum of 10-15 injections to define a binding
isotherm. There are no negative consequences to 'over-injecting' (titrating beyond the saturation
of binding sites) and doing so will often times provide you with 'control heat' information within
the same experiment.
The Cell Temperature (°C) is determined by your experimental requirements. Typically, the
binding constant and heat of binding will be temperature dependent, but the stoichiometry of
binding will not be. The ITC's operating range is 2° to 80°.
The Reference Power (µCal/sec) setting determines the approximate value that the baseline will
settle at when the system is equilibrated. Measuring large exothermic reactions will require a
'large' reference power of about 30 uCal/sec., while large endothermic reactions will require a
low reference power setting of about 2 uCal/sec. If you have little information about the expected
heats it will be best to use a reference power setting of 15-20 uCal/sec.
The Stirring Speed is typically set at 310 RPM. Faster stir speeds may be necessary if solutions
are more viscous than water, or if particle suspensions need to be maintained. Faster stir speeds
will increase the baseline noise level.
The injection Volume (µl) is generally between 3µl and 15µl. This range ensures high
volumetric accuracy while allowing enough time for the injectant to equilibrate to the temperature
of the cell before injection. The injectant equilibrates to the cell temperature in the stem prior to
reaching the cell. Injections at the high end of this range or larger may result in a reduction in the
repeatability of the injection blank heat. The injection blank heat is the thermal energy associated
with force of the injection and any temperature differences between the cell volume and the
injection volume. A water into water experiment will show the injection blank heat.
The Duration (sec.) of the injection is usually left at the default value, which yields an injection
rate of .5 µl/Second. Very subtle changes in control peak shapes and sizes can be obtained by
varying the injection duration, and is not usually beneficial.
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