Differential Scanning Calorimetry (DSC)


DSC is a technique which is part of a group of techniques called Thermal Analysis (TA). Thermal Analysis is based upon the detection of changes in the heat content (enthalpy) or the specific heat of a sample with temperature. As thermal energy is supplied to the sample its enthalpy increases and its temperature rises by an amount determined, for a given energy input, by the specific heat of the sample. The specific heat of a material changes slowly with temperature in a particular physical state, but alters discontinuously at a change of state. As well as increasing the sample temperature, the supply of thermal energy may induce physical or chemical processes in the sample, e.g. melting or decomposition, accompanied by a change in enthalpy, the latent heat of fusion, heat of reaction etc. Such enthalpy changes may be detected by thermal analysis and related to the processes occurring in the sample. Thermal analysis encompasses a wide variety of techniques such as :

A modern TA instrument (Gill, 1984) (Fig.3.11) is made up of a furnace for heating (or cooling) the sample at a controlled rate and a selective transducer to monitor changes in the substance. The transducer can be a thermocouple to measure heat flow (DSC or DTA), a balance to monitor weight changes (TG), or a linear variable differential transducer (LVDT) to detect changes in dimension (TMA). The transducer generates a voltage signal which is amplified, stored on magnetic tape or disk along with a direct temperature response from the sample, and recorded on a printer/plotter.

Figure 3.11: Schematic diagram of a modern thermal analyzer. [From Gill, (1984)]


DSC, differs fundamentally from DTA in that the sample and reference are both maintained at the temperature predetermined by the programme even during a thermal event in the sample. The amount of energy which has to be supplied to or withdrawn from the sample to maintain zero temperature differential between the sample and the reference is the experimental parameter displayed as the ordinate of the thermal analysis curve . The sample and reference are placed in identical environments, metal pans on individual bases each of which contain a platinum resistance thermometer (or thermocouple) and a heater (Figure 3.12). The temperatures of the two thermometers are compared, and the electrical power supplied to each heater adjusted so that the temperatures of both the sample and the reference remain equal to the programmed temperature, i.e. any temperature difference which would result from a thermal event in the sample is 'nulled'. The ordinate signal, the rate of energy absorption by the sample (e.g. millicalories/sec.), is proportional to the specific heat of the sample since the specific heat at any temperature determines the amount of thermal energy necessary to change the sample temperature by a given amount. Any transition accompanied by a change in specific heat produces a discontinuity in the power signal, and exothermic or endothermic enthalpy changes give peaks whose areas are proportional to the total enthalpy change (Figure 3.13).

Figure 3.12: DSC Experimental Arrangement.

Figure 3.13: Typical DSC Curve.

In other words, in DSC, the measuring principle is to compare the rate of heat flow to the sample and to an inert material which are heated or cooled at the same rate. Changes in the sample that are associated with absorption or evolution of heat cause a change in the differential heat flow which is then recorded as a peak. The area under the peak is directly proportional to the enthalpic change and its direction indicates whether the thermal event is endothermic or exothermic. For proteins, the thermally induced process detectable by DSC is the structural melting or unfolding of the molecule. The transition of protein from a native to a denatured conformation is accompanied by the rupture of inter- and intra-molecular bonds, and the process has to occur in a cooperative manner to be discerned by DSC (Ma and Harwalkar, 1991). Analysis of a DSC thermogram enables the determination of two important parameters : transition temperature peak (Tp) or maximum (Tmax) or denaturation (Td) temperature, and enthalpy of denaturation (). The denaturation temperatures are measures of the thermal stability of proteins, although they are influenced by the heating rate (Ruegg et al., 1977 ) and protein concentration (Wright, 1984 ). The extrapolated onset temperature ( Tm ) is less influenced by protein concentration and transition temperature at zero heating rate can be obtained by plotting peak temperatures as a function of heating rate (Ruegg et al., 1977). The value, calculated from the area under the transition peak, is correlated with the content of ordered secondary structure of a protein (Koshiyama et al., 1981). The value is actually a net value from a combination of endothermic reactions, such as the disruption of hydrogen bonds determined as 1.7kcal per mole of hydrogen bond (Privalov and Khechinasvili, 1974), and exothermic processes, including protein aggregation and the breakup of hydrophobic interactions (Jackson and Brandts, 1970 ; Arntfield and Murray, 1981). The sharpness of the transition peak can be measured as width at half-peak height (), and is an index of the cooperative nature of the transition from native to denatured state. If denaturation occurs within a narrow temperature range (a low value), the transition is considered highly cooperative (Wright et al., 1977). Heat denaturation of small globular proteins is generally considered reversible in high yield, provided that the reaction is carried out under conditions preventing aggregation., i.e. dilute solution and far from the isoelectric point. This allows indirect thermodynamic evaluation of the process by applying equilibrium thermodynamics and assuming a two-state model, i.e. A (native) --> B (denatured). Under these conditions one can determine the equilibrium constant, K, of the process and subsequently the standard enthalpy change, from the van't Hoff equation:

The standard free energy change, may be obtained from :

and the standard entropy change, , form :

If , made from calorimetric studies and obtained from equilibrium studies are equivalent, it can be deduced that the denaturation process is a two-state, all-or-none process, with minimum intermediate states. At high protein concentrations (5-20%) and heating rates (5-20°C/min), which resemble actual processing conditions, denaturation becomes an irreversible process since extensive intermolecular interactions are favoured with aggregation of the unfolded protein molecules (Biliaderis, 1983). In contrast to denaturation, which is connected with intensive heat absorption, aggregation is generally considered as an exothermic process, therefore, it becomes more difficult to interpret values quantitatively, since they represent the net product of a positive (denaturation) and a negative (aggregation) contributor.

Materials and Methods

Sample preparation

Amylum SWP was hydrated in distilled water at pH 7.0 and diluted as required.


Differential scanning calorimetry studies were carried out on a Setaram microcalorimeter using a sample mass of approx. 0.92g and a scan rate of 0.5 degrees per minute.


Up to 94ºC SWP remained stable with no sign of denaturation or conformational change in its tertiary structure (Figure 3.28).

Figure 3.28: DSC thermogram of 10% SWP at a scanning rate of 0.5ºC/min at pH 7.0