MUFFLE

FURNAC
E
Introduction:
Differential scanning calorimetry (DSC) is a thermo
analytical technique in which the difference in the amount
of heat required to increase the temperature of a sample and reference
is measured as a function of temperature. Both the sample and
reference are maintained at nearly the same temperature throughout
the experiment. Generally, the temperature program for a DSC
analysis is designed such that the sample holder temperature increases
linearly as a function of time. The reference sample should have a
well-defined heat capacity over the range of temperatures to be
scanned.
The technique was developed by E. S. Watson and M. J. O'Neill in
1962,[1] and introduced commercially at the 1963 Pittsburgh
Conference on Analytical Chemistry and Applied Spectroscopy. The
first adiabatic differential scanning calorimeter that could be used in
biochemistry was developed by P. L. Privalov and D. R. Monaselidze
in 1964 at Institute of Physics in Tbilisi, Georgia.[2] The term DSC
was coined to describe this instrument, which measures energy
directly and allows precise measurements of heat capacity.

Types of DSC
There are two main types of DSC: Heat-flux DSC which measures the
difference in heat flux between the sample and a reference (which
gives it the alternative name Multi-Cell DSC) and Power differential
DSC which measures the difference in power supplied to the sample
and a reference.
Heat-flux DSC
With Heat-flux DSC, the changes in heat flow are calculated
by integrating the ΔTref- curve. For this kind of experiment, a
sample and a reference crucible are placed on a sample holder
with integrated temperature sensors for temperature
measurement of the crucibles. This arrangement is located in a
temperature-controlled oven. Contrary to this classic design,
the distinctive attribute of heat-flux DSC is the vertical
configuration of planar temperature sensors surrounding a
planar heater. This arrangement allows a very compact,
lightweight and low heat capacitance structure with the full
functionality of a DSC oven.
Power differential DSC
For this kind of setup, also known as Power compensating DSC, the
sample and reference crucible are placed in thermally insulated
furnaces and not next to each other in the same furnace like in Heat-
flux-DSC experiments. Then the temperature of both chambers is
controlled so that the same temperature is always present on both
sides. The electrical power that is required to obtain and maintain this
state is then recorded rather than the temperature difference between
the two crucibles.
Principle of DSC:
To maintain the sample and reference at equal temperature during the
physical transformations of the sample (like phase transitions), either
more or lesser amount of heat is required to be applied on it then that
required for the reference sample or an empty sample pan. This heat
required depends on the nature of the transformation process, that is,
whether it is exothermic or endothermic. For instance, at a constant
heating rate, when a solid sample changes to liquid, more heat is
required to be applied to the sample for increasing its temperature.
This happens because heat is absorbed by the sample while it changes
state from solid to liquid. However, if there is exothermic process in
the sample (for instance, crystallization), less amount of heat is
needed to increase the temperature of the sample. Thus, by calculating
the heat flow differences between the reference and sample, DSC
technique can determine the amount of heat released or absorbed
during a transition process.

Components of DSC

DSC is a commercially available instrument which has two

(2) types: Heat Flux Type and Power Compensation Type.
The above figure shows the block diagram of Heat Flux DSC
as an example.
Heat Flux DSC comprises the sample and reference holder,
the heat resistor, the heat sink, and the heater.
 Heat of heater is supplied into the sample and the reference
through heat sink and heat resistor.
Heat flow is proportional to the heat difference of heat sink
and holders.
Heat sink has the enough heat capacity compared to the
sample. In case the sample occurs endothermic or exothermic
phenomena such as transition and reaction, this endothermic
or exothermic phenomena is compensated by heat sink. Thus
the temperature difference between the sample and the
reference is kept constant. The difference the amount of heat
supplied to the sample and the reference is proportional to
the temperature difference of both holders. By calibrating the
standard material, the unknown sample quantitative
measurement is achievable.

DSC enables the measurements of the transition such as the

glass transition, melting, and crystallization. Furthermore,
the chemical reaction such as thermal curing, heat history,
specific heat capacity, and purity analysis are also
measurable. Recently, with the development of the highly-
functional polymeric material, these thermal properties
analysis needs are increasing dramatically.
DTA and DSC detect the temperature differences between the
sample and the reference; however, DSC can perform the
quantitative measurement of the amount of heat on top.

Working of DSC:
DSC is a thermodynamical tool for direct assessment of the heat
energy uptake, which occurs in a sample within a regulated increase
or decrease in temperature. The calorimetry is particularly applied to
monitor the changes of phase transitions.

DSC is commonly used for the study of biochemical reactions, which is

named as a single molecular transition of a molecule from one
conformation to another. Thermal transition temperatures (Tt;
melting points) of the samples are also determined in solution, solid,
or mixed phases such as suspensions.

In a basic DSC experiment, energy is introduced simultaneously into a

sample cell (which contains a solution with the molecule of interest)
and a reference cell (containing only the solvent). Temperatures of
both cells are raised identically over time. The difference in the input
energy required to match the temperature of the sample to that of the
reference would be the amount of excess heat absorbed or released by
the molecule in the sample (during an endothermic or exothermic
process, respectively). As a result of the presence of the molecule of
interest, more energy is required to bring the sample to the same
temperature as the reference; hence, the concept of heat excess comes
into the picture.

As a powerful analytical tool, DSC is capable of elucidating the factors

that contribute to the folding and stability of biomolecules. Changes in
the Cp are believed to originate from the disruption of the forces
stabilizing native protein structure. For example, this includes van der
Waals, hydrophobic, and electrostatic interactions, hydrogen bonds,
hydration of the exposed residues, conformational entropy, and the
physical environment (such as pH, buffer, ionic strength,
excipients). Therefore, thermodynamic parameters obtained from DSC
experiments are quite sensitive to the structural state of the
biomolecule. Any change in the conformation would affect the
position, sharpness, and shape of transition(s) in DSC scans.
Thermodynamic Terms

In a DSC experiment, thermodynamic parameters are associated with

heat-induced macromolecular transitions. For a typical
macromolecule, the molar Cp is measured as a function of temperature,
subsequently, yielding the following thermodynamic parameters:

The partial Cp of a molecule

DSC measures the partial Cp of a sample. The Cp of the solution

containing a macromolecule is measured with respect to the Cp of
buffer in the absence of macromolecules. Hence, the instrument
measures only part of what could be actually measured, which is the
difference between sample and reference cells. The sample could be a
protein, tRNA, a protein-DNA complex, a protein-lipid complex, or
something else.

The Cp at constant pressure is a temperature derivative of the enthalpy

function [Cp=(ΔH/ΔT)p], and thus, the enthalpy function can be
measured through integration of the Cp [H(T)=∫T/T0 Cp(T)dT+H(T0)]. 2

ΔH, Change in Entropy (ΔS), ΔCp of the Tm

For any biomolecule in aqueous solution, there would be equilibrium

between the native conformation (folded) and its denatured state
(unfolded). Stability of the native conformation is based on the extent
of Gibbs free energy (ΔG) of the system and thermodynamic
relationships between ΔH and ΔS. A negative magnitude of ΔG
represents higher stability of the native conformation than that of the
denatured state. The more negative ΔG, the greater the stability.
During the unfolding process of a protein, forces that play a key role in
stabilization need to be broken. At temperatures where entropy is the
dominant factor, conformational entropy overcomes the stabilizing
forces, leading to unfolding of the protein.

DSC measures ΔH of unfolding as a result of heat denaturation. The

transition midpoint Tm is considered as the temperature, where 50%
of the protein owns its native conformation, and the rest remains
denatured. Higher Tm values would be representative of a more stable
molecule. During the same experiment, DSC is also capable of
measuring the ΔCp. Associated with protein unfolding process,
ΔCp occurs as a result of changes in hydration of side-chains, which are
buried in the native conformation but become exposed to the solvent
in a denatured state.

Calorimetric enthalpy (ΔHcal) means the total integrated zone below

the thermogram peak, which indicates total heat energy uptake by the
sample after suitable baseline correction affecting the transition.

van't Hoff enthalpy (ΔHVH) is an independent measurement of the

transitional enthalpy according to the model of the
experiment. ΔHVH is determined through the shape analysis of an
34

experimental graph of Cpex versus T.

The state of the transition is evaluated by comparing ΔHVH with ΔHcal. If

ΔHVH is equal to ΔHcal, the transition occurs in a two-state mode. In such
processes, meaningful thermodynamic results are determined
through van't Hoff measurements of equilibrium results. When ΔHVH is
more than ΔHcal, the intermolecular cooperation is shown, which is
exposed, for example, as aggregation. Comparison between ΔHVH and
ΔHcal also indicates the cooperative nature of the transition.
Particularly, the ΔHVH/ΔHcal ratio gives an estimation from the fraction
of the structure, which is melted as a thermodynamical value. The
value is also named as the size of the cooperative unit.

Cp for the transitional state is obtained through the difference

between pretransitional and post-transitional baselines of a DSC
process. The curve of Cp against T can be changed to Cp/T versus T
through dividing the raw Cp value by T and drawing the results as a
function of T. By integration, this curve results in the transition
entropy (ΔS), which is expressed as (ΔS) = ∫(Cp/T) dT. Hence, an
individual DSC thermogram can result in ΔH, ΔS, and ΔCp.

After knowing the above data, transition-free energy (ΔG) can be given
at each temperature (T) through the thermodynamic equation ΔG =
ΔH − TΔS. Although ΔS and ΔG can be obtained by DSC results, the
values are more unreliable than the ΔH and ΔCp values determined
directly because of coupling and propagating of errors.

Absolute Cp

Apparent Cp can be obtained by DSC results. It includes the

contribution of water displacement by the protein in the sample cell,
which could have a negative value. Correction for the water
displacement effect and normalization to a mole of protein offer the
absolute Cp. The value is obtained from doing a series of DSC
measurements at different protein concentrations.

Determined absolute heat capacities by DSC could be used to

characterize long-range interactions and cooperative phenomena,
which have been shown to occur in denatured proteins.

Application of DSC
 Determining phase separation (polymer blend, copolymer)
 Estimating percent crystallinity
 Measuring heat capacity
 Determining thermal stability (oxidation induction time)
 Determining effects of additives (blends, fillers, plasticizers,
process aids)
 Measuring residual cure and as a function of cure
temperature/time
 Estimating degree of cure
 Estimating upper use temperature from Tg or melting point
 Analyzing cure or crystallization kinetics

Testing method:
Heat 30°C to 300°C heating rate 10°C/min (Nitrogen atmosphere)
Cool 300°C to 30°C/min (Nitrogen atmosphere)
Heat 30°C to 300°C heating rate 10°C/min (Nitrogen atmosphere)
Results:
1) Glass transition temperature (Tg) = 129.96°C
Onset= 125.55°C
End= 134.46°C

2) Melting point (Tm)= 328.09°C

Area= 284.5828mJ
Delta H= 28.4778J/g
Onset= 323.32°C
End= 332.22°C

3) Crystallisation temperature= 311.02°C

Area= -236.6162mJ
Delta H= -24.6462J/g
Onset= 317.36°C
End= 306.07°C