Calorimetry: differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC)

Transcription

1 Calorimetry: differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) Dr. Yin Li Department of Biophysics, Medical School University of Pecs

2 Thermal Analysis IUPAC definition - a group of techniques in which a physical property is measured as a function of temperature, while the sample is subjected to a controlled temperature programme (heating, cooling or isothermal). A range of techniques e.g.: Differential Thermal Analysis (DTA) temperature Differential Scanning Calorimetry (DSC) energy Isothermal Titration Calorimetry (ITC) energy Thermogravimetric Analysis (TGA) mass Thermomechanical Analysis (TMA) dimensions Dielectric Analysis (DEA) dielectric/electric properties

3 Basic Principles of Thermal Analysis Modern instrumentation used for thermal analysis usually consists of the following parts: sample holder/compartment for the sample sensors to detect/measure a property of the sample and the temperature an enclosure within which the experimental parameters (temperature, speed, environment) may be controlled a computer to control data collection and processing temperature control (furnace) sample sensors PC

4 Differential Scanning Calorimetry (DSC) DSC measures the heat absorbed or released during the various transitions in the sample due to temperature treatment Differential: sample relative to reference Scanning: temperature is ramped Calorimeter: measures heat DSC measurements are both qualitative and quantitative and provide information about physical and chemical changes involving: Endothermic processes sample absorbs energy Exothermic processes sample releases energy

5 Principles of DSC Analysis Power Compensation DSC Voltage to keep ΔT = TS TR= 0 vs. Time; The resulting power difference is proportional to heat flow. Heat Flux DSC Fast measurement Low sensitivity Noisy baseline ΔT = TS TR 0 vs. Time the signal is converted to heat flow through the equation q = ΔT/R where R= the well defined thermal resistance of the thermocouple. Slow measurement High sensitivity Flat baseline

6 Typical Features of a DSC Curve ^exo Exothermic upwards Endothermic downwards CRYSTALLISATION DESOLVATION GLASS TRANSITION MELTING 20 mw H 2 O DECOMPOSITION Y-axis heat flow X-axis temperature (and time) temperature [ o C]

7 Information obtained from DSC Melting points crystalline materials Desolvation adsorbed and bound solvents Glass transitions amorphous materials Heats of transitions melting, crystallisation Purity determination contamination, crystalline/amorphous phase quantification Polymorphic transitions polymorphs and pseudopolymorphs Processing conditions environmental factors Compatibility interactions between components Decomposition kinetics chemical and thermal stability

8 Influential factors Sample mass (3-15 mg) and particle size a: big amount b: small amount Heating rate If the particle size is too large, it can somehow inhibit the heat transfer between sample and the atmosphere. If the particle size is too small, the degree of crystallization could be lowered down. a: Too large; b: appropriate; c: too small Heating rate can influence the peak position and area. If the heating rate is high, the shape of peak become sharper. If it is too high, the system does not have enough time to achieve thermo equilibrium, the peak area cannot reflect the amount of heat it requires. It also can result in baseline drift. If the heating rate is low, we will obtain broad and shallow peaks. Baseline drift is not significant. But the measurement takes a long time. And it requires a high sensitive detector.

9 Atomsphere and its pressure Influential factors Some samples are susceptible to the oxygen, inert gas such as N 2, Ne, He would be necessary to be purged into the system. Since enthalpy depends on the atmospheric pressure, the pressure will also influence the measured heat. Reference To obtain a stable baseline, the reference must be stable have no transition within the studied temperature range. Try the best to choose a reference which has a similar heat capacity, thermo-conductivity and particle size to the sample. Common references: MgO, quartz sand.

10 Isothermal titration calorimetry (ITC) What is ITC? A direct measurement of the heat generated or absorbed when molecules interact.

11 What can ITC do? Study interactions between: Protein-small molecule Enzyme-inhibitor Protein-protein Protein-DNA Protein-lipid Protein-carbohydrate Etc.

12 How Do ITCs Work? Power difference is measured between the reference and sample cells. ITCs always try to maintain the temperature difference between reference and sample around zero.

13 Performing an ITC experiment Ligand in syringe Macromolecule in sample cell Heat of interaction is measured Parameters measured from a single ITC experiment: Affinity (Binding Constant) - K Energy (Enthalpy) - DH Number of binding sites - n

14 Titration begins: First injection Ligand in syringe Macromolecule in cell Macromolecule-ligand complex As the first injection is made, all injected ligand is bound to target macromolecule. The signal returns to baseline before the next injection.

15 Second Injection Ligand in syringe Macromolecule in cell Macromolecule-ligand complex Signal again returns to baseline before next injection. As a second injection is made, again all injected ligand becomes bound to the target. Signal again returns to baseline before next injection.

16 As injections continue As the injections continue, the target becomes saturated with ligand, so less binding occurs and the heat change starts to decrease.

17 As injections continue As the injections continue, the target becomes saturated with ligand so less binding occurs and the heat change starts to decrease.

18 End of titration When the macromolecule is saturated with ligand, no more binding occurs, and only heat of dilution is observed.

19 Experimental results 1) Integrate the area under each peak (gives you total heat released at each titration point). 2) Calculate the running quantity of ligand or molar ratio of ligand to macromolecule (in this case normalized to volume to give ligand concentration; x-axis).

20 Advantages & Disadvantages of ITC Advantages: Based on a single experiment you can get binding constant, Gibbs free energy, enthalpy and entropy Accurate stoichiometry Do not need to label anything Disadvantages: A lot of sample is needed. (2 ml, mm concentration) Intrinsic sensitivity of the detector.