DSC high sensitivity

Wissing S, Craig DQM, Barker SA, Moore WD. An investigation into the use of stepwise isothermal high sensitivity DSC as a means of detecting drug-excipient incompatibility. Int J Pharm 2000 199 141-150. 

A significant part of medical and pharmacological research is conducted on the biochemical level. Calorimetric work on such systems usually has the character of thermodynamic measurements and can be considered as part of biophysical chemistry. Two types of experiments currently seem to be the most important studies of binding processes using titration microcalorimetry and investigations of thermal transitions involving high sensitivity DSCs. 

Figure 8 shows a very simple transition curve determined by a high sensitivity DSC. For many proteins much more complex thermograms are obtained. By different deconvolution techniques, the experimental curves can be resolved into several peaks which may be linked to the transition of different cooperative regions in the protein (see Privalov (1989)). 

High sensitivity DSC (HS-DSC) instruments are used to measure small heats of transition. They were developed originally for measuring to heat of denaturation of biopolymers in dilute solution (Hatakeyama and Quinn, 1994). The sensitivity of a heat flux instrument, for example, can be improved by increasing sample size, using multiple serially-connected thermocouples to measure both sample and reference temperatures, and increasing the heat sink capability of the heat flux plate (Hatakeyama and Quinn, 1994). 

Figure 2 A typical high-sensitivity DSC heating thermogram of a multilamellar, aqueous suspension of DPPC which has been annealed at 0-4°C dor 3-5 days prior to commencement of heating. The substransition, pretransition and main phase transition temperatures are denoted by Ts, Tp and Tm respectively.

Figure 3 Typical high-sensitivity DSC heating thermograms of multilamellar, aqueous suspensions of DPPC containing various amounts of incorporated cholesterol. The amount of cholesterol present (in mole %) is indicated near each thermogram.

Figure 5 High-sensitivity DSC heating scans of Acholeplasma laidlawii B eiaidic acid-homogeneous intact ceiis, isoiated membranes and extracted totai membrane iipids dispersed as muitiiameiiar vesicies in water.

Figure 41.1 shows the gel-to-liquid crystalline phase transition temperatures (Tm) of DPPC-cholesterol mixtures as a function of the cholesterol-lipid molar ratio. The Tm of fully hydrated DPPC is 42°C (Crowe and Crowe, 1988 Vist and Davis, 1990 McMullen et al., 1993 Ohtake et al., 2004). Upon the addition of cholesterol, the transition enthalpy decreases continuously imtil it is no longer observable at 50 mol% cholesterol. The disappearance of the melting transition has been attributed to strong interactions between cholesterol and DPPC (McCoimell, 2003). Upon dehydration, the Tm for DPPC increases from 42 to 105°C (Crowe and Crowe, 1988 Ohtake et al., 2004). This Tm increase is caused by the reduction in the spacing between the phospholipids, which allows for increased van der Waals interactions between the lipid hydrocarbon chains (Koster et al., 1994). Between 10 and 70 mol% cholesterol, two endothermic transitions are observed, both lower than the Tm of the pure phospholipid (Figure 41.1). High-sensitivity DSC studies on fully hydrated DPPC-cholesterol systems reported endotherms consisting of two components, suggesting the existence of domains enriched/depleted in cholesterol (Vist and Davis, 1990 McMullen et al., 1993). The two peaks present in our freeze-dried systems also suggest the...

The book opens with the first three chapters devoted to differential scanning calorimetry (DSC), the most commonly used thermal method. These chapters cover the principles, optimal use, and pharmaceutical applications of the method. Subsequent chapters explore modulated temperature DSC, thermogravimetric analysis, thermal microscopy, microcalorimetry, high sensitivity DSC, dynamic mechanical analysis, and thermally stimulated current, all of which have attracted great interest within the pharmaceutical field. Each chapter includes theoretical background, measurement optimization, and pharmaceutical applications. 

The methodology of high-sensitivity DSC and application of the technique to the study of heat-induced changes in proteins have been expounded in several reviews [35,36,39-44], to which the reader can refer. The following is just to outline its main features in order to facilitate subsequent discussion. 

The results of high-sensitivity DSC studies of protein denaturation have greatly helped to clarify the reversibility and the intermediate states issues, as indicated above. They have yielded a detailed analysis of the thermodynamical features of protein unfolding and led to a reassessment of the contributions of the different forces that determine protein stability. 

In parallel to the decrease of the q ratio, aggregation causes a decrease in the ratio r = QD/AH(T)vH, as already mentioned. At low pH, BLG is monomeric and its heat-induced unfolding is a two-state process, with accordingly r very close to 1 as shown by high-sensitivity DSC in the 0.08-0.3% protein concentration range [57]. When... 

The lamellar phase represents the structure of cell membrane lipids under steady-state conditions. However in certain circumstances, particularly in membrane fusion events (e.g. in egg fertilization, or cell infection by some viruses), membrane lipids abandon transiently the lamellar disposition, adopting nonlamellar architectures, of which the best known is the so-called inverted hexagonal , or Hn, phase. Nonlamellar structures are at the origin of the lipid stalk , a structural intermediate that connects two bUayers in the membrane fusion process. Only certain lipids, or lipid mixtures, can undergo the Lo(-Hii thermotropic transition, and the latter can be detected by DSC. Hu, like other nonlamellar phases, has received particular attention lately because of its possible implication in important phenomena such as cell membrane fusion, or protein insertion into membranes. High-sensitivity DSC instruments allow the detection of La-Hn transitions with phospholipid suspensions of concentration 5 him or even less. 

As was mentioned in previous sections, mixing lipids, and particularly sterols, often has the effect of broadening the phase transitions. Intrinsic proteins have the same effect. Thus it is not surprising that cell membranes, containing highly complex mixtures of lipids and proteins, do not usually exhibit measurable thermotropic phase transitions. With a few exceptions, transitions in cell membranes can only be seen with high-sensitivity DSC instruments. 

Low-concentration solutions (< 0.5 wt %) of biological samples are commonly analysed by high-sensitivity DSC. Several hundred milligrams of sample are placed in the sample vessel and an equal amount of the pure solvent is placed in the reference vessel. The sample is heated at a low heating rate (< 2.5 K/min) to avoid thermal interference due to circulating convection currents in the sample vessel. 

---