Isothermal titration calorimetry instrumentation

Instrumentation. H and NMR spectra were recorded on a Bruker AV 400 spectrometer (400.2 MHz for proton and 100.6 MHz for carbon) at 310 K. Chemical shifts (< are expressed in ppm coupling constants (J) in Hz. Deuterated DMSO and/or water were used as solvent chemical shift values are reported relative to residual signals (DMSO 5 = 2.50 for H and 5 = 39.5 for C). ESl-MS data were obtained on a VG Trio-2000 Fisons Instruments Mass Spectrometer with VG MassLynx software. Vers. 2.00 in CH3CN/H2O at 60°C. Isothermal titration calorimetry (ITC) experiments were conducted on a VP isothermal titration calorimeter from Microcal at 30°C. 

Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC) have become standard techniques in the field of thermodynamic investigation of natural membranes and model membrane systems. Due to the new developments of more sensitive DSC instruments, studies of lipid-protein systems liave become feasible, which could previously not be perfonued because of the limitations on amount of material. It is to be expected that DSC methods will again make a large progress because of these improvements in sensitivity. 

Recently there has been considerable interest on the subject of chemical test reactions for isothermal microcalorimeters. Chemical test reactions allow a user to check if an instrument is functioning correctly because they reflect more accurately the processes under study in a real experiment. Indeed, the ideal case would be to have a universally accepted chemical test reaction for each type of experiment (perfusion, titration, etc.) one may wish to investigate. Examples of systems that have been proposed as chemical test reactions include 18-crown-6/barium sulfate (2) for titration calorimetry... 

As mentioned above, titration methods have also been adapted to calorimeters whose working principle relies on the detection of a heat flow to or from the calorimetric vessel, as a result of the phenomenon under study [195-196,206], Heat flow calorimetry was discussed in chapter 9, where two general modes of operation were presented. In some instruments, the heat flow rate between the calorimetric vessel and a heat sink is measured by use of thermopiles. Others, such as the calorimeter in figure 11.1, are based on a power compensation mechanism that enables operation under isothermal conditions. 

In this way, we produce a binding titration just like we did from an NMR or a UV/ vis study. This allows us to determine K., and AH°. From we get AG°, and then we can solve for AS°. As promised, a single titration has produced all the quantities of interest. Isothermal calorimetry is thus a very powerful technique for evaluating binding. It does require fairly sophisticated instrumentation that is dedicated to such measurements. Also, fairly large quantities of material are sometimes needed, especially for hydrophobic binding interactions which can have fairly small AH° values, and thus release or absorb relatively little heat. 

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