https://www.youtube.com/channel/UCTWTCRDLBs2M3EZdhIxGpyw

1. By Prashant Patel
Department of pharmaceutical technology
Indukaka Ipcowala college of pharmacy

2. 
 Calorimetry is the science of heat. It is concerned with how a
given material responds to temperature changes on both the
atomic and macroscopic level. This varies widely from substance
to substance, and reveals important information about the
arrangement and interaction of the atoms.
 MICROCALORIMETRY is an advanced form of Calorimetry.
 Calorimetry of microgram of Sample.
 Power detection limit for micro calorimeter approaches a few
microwatts.
 Temperature change in a microcalorimetric experiment is usually
small, typically <10–3 K
INTRODUCTION

3.  Microcalorimetry works on the principle that all physical and
chemical processes are accompanied by a heat exchange with their
surroundings.
 So when a reaction occurs a temperature gradient is formed
between the sample and its surroundings. The resulting heat flow
between the sample and its surroundings, is measured as a
function of time.
 When any reaction takes place, heat will be generated or absorbed
by the molecules reacting.
 When a calorimeter is calibrated, the calorimetric signal is
standardized by release of an accurately known heat, q, or thermal
power, P = dq/dt. The result of a calibration experiment is usually
expressed in terms of a calibration constant, ε, valid for the
instrument under some specified conditions.
PRINCIPLE

4.  In isothermal microcalorimetry heat input in sample cell
adjusted to keep T constant. So,
 Exothermic reaction will result in negative peaks (less heat
is needed while the reaction proceeds)
 Endothermic reactions will result in positive peaks (more
heat is needed while the reaction proceeds)

5. 
 On the base of heat measurement principles one.
may divide microcalorimeters into three main
groups: adiabatic, heat conduction and power
compensation calorimeters
 adiabatic calorimeter-no heat exchange takes place
between the calorimetric vessel and its surroundings.
The amount of heat that is evolved or absorbed in an
ideal adiabatic calorimeter is equal to the product of
the measured temperature change and the heat
capacity of the vessel, including its content.
CLASSIFICATION OF
CALORIMETERS

7.  heat conduction calorimeter:
 heat released (absorbed) in the reaction vessel is allowed to
flow to (from) a surrounding heat sink, usually consisting of a
metal block.
 A thermopile, positioned between the vessel and the heat
sink, serves as a sensor for the heat flow. The total heat flow
between vessel and the heat sink is proportional to the
temperature gradient over the thermopile and thus to the
measured thermopile potential. Heat flow sensors usually
consist of thermopiles made from semi-conducting materials.

8.  power compensation calorimeter:
 the thermal power from an exothermic process is balanced by
a known cooling power (usually Peltier effect cooling),
alternatively by a decrease of heating power. Endothermic
processes are balanced by a known thermal power released in
a heater or by reversing the Peltier effect current.

10.  There are many commercial types of equipment available;
few of them are as under
 TRONAC solution calorimeter
 LKB Thermal Activity Monitor
 Thermometric 2277 Thermal Activity Monitor
 Characteristics:
 Versatile, four channel system.
 Originally it was marketed as LKB Bioactivity monitor
 More sensitive and accurate
 Four channels can work simultaneously and independently.
 Each channel allows insertion of different measurement
devices.

11.  Four types of inserts are currently available
 Flow mixing cell
 Two liquids may be introduced into the mixing cell with a two-channel
peristaltic pump. It is used to monitor heat changes like heats of mixing,
dissolution, complexation and ligand binding.
 Flow – through cell
 A single liquid is pumped through cell, and the evolution and
absorption of heat as a function of time may be monitored. It is used to
monitor heat changes occurring during bacterial growth, decomposition
or destabilization of solution.
 Ampoule calorimeter
 It is used for stability and compatibility study, bacterial growth, cell
metabolism. It is of greatest importance in pharmaceutical industry in
stability study of pharmaceuticals in solid state which is otherwise a
difficult task to measure the rate of these reactions.
 Perfusion – titration cell
 This cell may be used to monitor the heat flow caused by the interaction
of a fluid (Gas or Liquid) with a solid held stationary in the cell.
Examples include absorption and desorption experiments, mixing of
liquids.

13. APPLICATIONS IN STABILITY STUDIES
 Microcalorimetry is highly useful in following fields,
 Stability testing.
 Studies of powder wettability (by immersion and adsorption).
 Sorption reactions
 Crystal properties.
 Dissolution of tablets and powders.
 Drug-Excipient compatibility.
 To study powder surface energetics.
 Microorganism – Drug interaction
 Cyclodextrin – drug interaction.
 Food-Drug interaction
 Identification of polymorphs

14.  CONVENTIONAL METHODS FOR STABILITY STUDY:
 At present, the standard method used for stability analysis of a
solid state pharmaceutical product is HPLC. In summary, the
concentration of parent compound and/or the concentration of
any daughter compounds produced are determined as a
function of storage time.
 The method has certain drawbacks.
 Often not very sensitive to small changes in concentration.
 It requires a certain degree of method development to establish a
sample preparation and analysis protocol
 It relies on the dissolution of the solid product.
 This last drawback can cause distortions in an assay as a result
of rapid acceleration of decomposition when a compound is in
a solvated state.

15.  For stability study samples are stored under elevated
temperature and humidity after preparation to accelerate the
potential decomposition.
 The samples are then assayed over a period of time that can
range from a few weeks to many months to give reaction
snapshots along the decomposition profile.
 For each storage condition, a rate constant, (k), is calculated. By
plotting lnk against 1/T using the Arrhenius relationship, it is
possible to extrapolate back to ambient temperature and hence
determine the rate constant at that temperature.
 where k is the rate constant, A is the Arrhenius factor or pre-
exponential constant, Ea is the activation energy, R is the gas
constant, and T is the temperature. This technique for the
determination of stability has been accepted as normal practice
for many years.

16. 
LIMITATIONS OF ARRHENIUS EQUATION
 It is assumed that the Arrhenius plot gives a linear
relationship. This may not be true for many reasons.
 If there are two competing reactions occurring
simultaneously, then they will both have an associated
activation energy leading to an incorrect extrapolation &
thus a major error in calculating the ambient rate constant
 if the reaction does not go by a first order reaction, it is
necessary to determine a different rate equation that gives an
improved understanding of the system under study. This is
not always straightforward and for solid state reactions can
be very complex.

17.  When a calorimeter is calibrated, the calorimetric signal is standardized by
release of an accurately known heat, q, or thermal power, P = dq/dt. The
result of a calibration experiment is usually expressed in terms of a
calibration constant, ε, valid for the instrument under some specified
conditions.
 For Adiabetic Colorimeter:
 The quantity of heat evolved or absorbed in an adiabatic calorimetric
experiment is, in the ideal case, equal to the product between the
temperature change, ΔT, and the heat capacity of the calorimetric vessel
(including its contents), C,
 q = C ΔT. (1)
 In practice, there will be normally some heat transfer between the vessel
and the surroundings, and a “practical” heat capacity value, the calibration
constant, is determined in a calibration experiment,
 q = εa ΔT, (2)
 where εa is the calibration constant (sometimes referred to as the “energy
equivalent”). A change in the heat capacity of the content of the calorimetric
vessel (following, for example, injection of a sample) will thus lead to a
change in the calibration value. The thermal power is
 P = εa dT/dt.
MICROCALORIMETRIC DATA

18.  For Heat conduction calorimeters
 Tian equation:
 P = εc [U + τ (dU/dt)], (4)
 Here, εc is the calibration constant, U the measured potential
difference across the thermopile, and τ the time constant
 Under steady-state conditions, for example, during the release
of a constant electrical calibration current, eq. 4 simplifies to
 P = εc U. (5)
 The heat released in the calorimetric vessel is obtained by
integration of eqs. 4 or 5, leading to the simple expression
 q = εc ∫t1t2U dt, (6)
 provided that the initial and final potentials are the same
(normally the baseline value); t1 and t2 are respectively, times
in the fore- and after-periods.

19.  The procedure takes a kinetic equation for a particular reaction, and
modifies it such that it applies directly to microcalorimetric data. This is
achieved by recognition of the fact that the total heat evolved during the
course of a reaction (Q) is equal to the total number of moles of material
reacted (Ao) multiplied by the change in molar enthalpy for that reaction
( H).
Q = A0 H ……………..(1)
 Similarly, the heat evolved at time t (q) is equal to the number of moles of
material reacted (x) at time t multiplied by the change in molar enthalpy for
that reaction.
 q = x H ………………(2)
 Eq. (2) may be substituted into a general rate expression of the form dx/dt to
give an expression of the form dq/dt (or power). For example, the general
rate expression for a simple, first-order, A B process is given by Eq. (3).
…………… (3)
 Substitution of Eq. (2) into Eq. (3) yields,
………. (4)
 This modified rate expression may be used to fit power–time data recorded
using the microcalorimeter.

20.  EXAMPLES
 Few drugs for which stability study can be performed using
microcalorimetry are Aspirin, PAS, and some ß-lactam antibiotics.
 Microcalorimetry is applied to study the thermodynamic stability of
Proteins (Lysozyme, Cytochrome-c and Ribonuclease).
 By using Microcalorimetry, stability study of ampicillin in aqueous
Solutions as a function of conc. of ampicillin, pH & temperature was carried
out.
 Determination of decomposition mechanism of lovastatin by measuring rate
of heat production at different temperature & time was carried out by
microcalorimetry.
 Lovastatin degraded by an auto-catalytic mechanism in presence of
oxygen.
 Microcalorimetry is used to correlate the decomposition rate of several
Cephalosporin in solids & aqueous solution states.
 Testing of physical stability of drug:-
Microcalorimetry has proved to be an effective analytical technique for
characterizing micronized compounds. It can be used to detect the presence of
metastable regions not detectable by X-ray diffraction. Furthermore, the kinetic
of “recrystallization” of these regions can be studied, making a prediction of
physical stability possible. Thus the application of Microcalorimetry in the
pharmaceutical development has a great potential.

24.  DISSOLUTION OF TABLETS AND POWDERS
 For study of dissolution TRONAC solution calorimeter is used.
 It consist of a dewar flask containing the reacting fluid or
solvent, a device for remote delivery of a solid substances, a
stirrer, a device for monitoring the temperature history of the
calorimeter, and a calibration heater.
 The solid sample is contained in a sealed glass ampoule that
may be broken by actuating a remote spring loaded or
solenoid- driven breaking device.
 Heat evolved or absorbed due to a particular dissolution
process is constant.
 In a typical experiment, the system is calibrated by introducing
a known quantity of heat with the calibration heater by means
of a constant current supply and a digital timer.
 After a steady drift rate is re-established, the ampoule is broken
and the change in temperature due to the reaction is measured.

25.  The system may also be calibrated by measuring the change in
temperature incurred by a standard chemical reaction such as a
heat of solution of tromethamine (tris-hydroxymethyl-
aminomethane) in dilute HCl (Exothermic), potassium chloride
in water (endothermic), or a neutralization reaction, for
example, a known quantity of HCl reacting with a solution of
NaOH, for which accurate literature values of H are available.

27.  EXCIPIENT COMPATIBILITY
 Compatibility is an important area in the drug development
pipeline. Conventional compatibility testing methods require
both multiple sample preparation and long storage times in
order to obtain meaningful results.
 It has been reported that a standard method for compatibility
testing of binary mixtures has been developed using isothermal
microcalorimetry. The method involves preparing a binary
mixture, followed by examination in a microcalorimeter after a
period of equilibration.
 If the sum of the heat out put of the compound and the
excipient alone is not equal to the heat output of a binary blend
then there is a potential compatibility issue.

28.  Figure shows a typical response for an excipient, an active
component and a mixture of the two.
 This combination is clearly incompatible as the mixture profile is
very different from the two individual components.
Calorimetric response of a drug, microcrystalline cellulose and a mixture of both.
 Example 1
Microcalorimetry was used to study the effect of menadione
& prednisone on the stability of the micro emulsions. The stability was
not changed in the presence of drugs.
 Example 2
Chemical & physical processes accompanying Cyclodextrin-
drug interactions are usually endothermic or exothermic in nature so
they can be studied by microcalorimetry technique.

29.  The method is only designed as a screen and does not give a
quantification of the amount of degraded active.
 DEGRADATION
 Hydrolysis , oxidation, free radical formation, etc. all have
large heat of reaction.
 Ideally, degradation rates of less then 1% per year can be
predicted in a matter of days.

30.  DETERMINATION OF AMORPHOUS CONTENT IN
CRYSTALLINE SOLID
 Amorphous character in highly crystalline solids can be
difficult to detect using traditional analytical techniques, such
as Powder X-Ray Diffraction (PXRD) and Differential Scanning
Calorimetry (DSC), as the limit of detection is 5–10%. In recent
years, several papers have been published that detail the use of
isothermal microcalorimetry for the quantification of low
levels of amorphous content.
 A typical DSC heating curve of an amorphous substance is
given in Fig. for L-polylactic acid. After the glass transition,
the crystalline substance appears and then melts. The
crystallization can appear spontaneously above the critical
temperature of the glass transition (Tg). For a substance with a
high Tg, crystallization will not occur unless the Tg value is
lowered by the presence of other compounds, such as
impurities or water. The Tg has traditionally been determined
by DSC, but the use of MDSC in this area is increasing.

31.  The glass transition is accompanied by a change in
the heat capacity (change of base line). After this
transition, the crystallization in the crystalline
polymer is accompanied by an exotherm. The
crystalline polymer obtained shows two endotherms:
the first small endotherm is due to rearrangement in
the polymer followed by the melting of the
crystalline polymer.

32.  MICROORGANISM-DRUG INTERACTION
 Growing of microorganism produce heat. This principle may
be used to study the effect of antibiotics on the microbial
growth.
 E.g.
 Microcalorimetric titration used to study the effect of
Vancomycin against gram +ve bacteria.
 Determination of Gibbs energies, enthalpies, entropies and
heat capacities for antibiotic-bacteria binding reactions are
done.

33.  FOOD-DRUG INTERACTION
 The effect of food on the dissolution rate of
Tetracycline hydrochloride was studied using
Microcalorimetry.
 An interaction was observed using Microcalorimetry
between tetracycline and calcium, milk.

34.  SORPTION REACTIONS
 Sorption reactions [i.e., adsorption or absorption of
gases (vapors) and solutes onto solids] are of
fundamental importance in thermochemistry, and
several special microcalorimeters have been designed
for such experiments.
 Sorption reactions, in particular sorption of water
vapor, have recently become one of the most important
practical application areas for microcalorimetry, for
example, in the pharmaceutical industry.

35.  POWDER WETTABILITY
 The combination of Microcalorimetry and
vacuum microbalance techniques allows the
possibility of calculating the thermodynamic
parameters associated with wetting process and in
addition, gives idea about mechanism of wetting.