By Prashant PatelDepartment of pharmaceutical technologyIndukaka Ipcowala college of pharmacy
Calorimetry is the science of heat. It is concerned with how agiven material responds to temperature changes on both theatomic and macroscopic level. This varies widely from substanceto substance, and reveals important information about thearrangement 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 fewmicrowatts. Temperature change in a microcalorimetric experiment is usuallysmall, typically <10–3 KINTRODUCTION
Microcalorimetry works on the principle that all physical andchemical processes are accompanied by a heat exchange with theirsurroundings. So when a reaction occurs a temperature gradient is formedbetween the sample and its surroundings. The resulting heat flowbetween the sample and its surroundings, is measured as afunction of time. When any reaction takes place, heat will be generated or absorbedby the molecules reacting. When a calorimeter is calibrated, the calorimetric signal isstandardized by release of an accurately known heat, q, or thermalpower, P = dq/dt. The result of a calibration experiment is usuallyexpressed in terms of a calibration constant, ε, valid for theinstrument under some specified conditions.PRINCIPLE
In isothermal microcalorimetry heat input in sample celladjusted to keep T constant. So, Exothermic reaction will result in negative peaks (less heatis needed while the reaction proceeds) Endothermic reactions will result in positive peaks (moreheat is needed while the reaction proceeds)
On the base of heat measurement principles one.may divide microcalorimeters into three maingroups: adiabatic, heat conduction and powercompensation calorimeters adiabatic calorimeter-no heat exchange takes placebetween the calorimetric vessel and its surroundings.The amount of heat that is evolved or absorbed in anideal adiabatic calorimeter is equal to the product ofthe measured temperature change and the heatcapacity of the vessel, including its content.CLASSIFICATION OFCALORIMETERS
heat conduction calorimeter: heat released (absorbed) in the reaction vessel is allowed toflow to (from) a surrounding heat sink, usually consisting of ametal block. A thermopile, positioned between the vessel and the heatsink, serves as a sensor for the heat flow. The total heat flowbetween vessel and the heat sink is proportional to thetemperature gradient over the thermopile and thus to themeasured thermopile potential. Heat flow sensors usuallyconsist of thermopiles made from semi-conducting materials.
power compensation calorimeter: the thermal power from an exothermic process is balanced bya known cooling power (usually Peltier effect cooling),alternatively by a decrease of heating power. Endothermicprocesses are balanced by a known thermal power released ina heater or by reversing the Peltier effect current.
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 measurementdevices.
Four types of inserts are currently available Flow mixing cell Two liquids may be introduced into the mixing cell with a two-channelperistaltic 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 andabsorption of heat as a function of time may be monitored. It is used tomonitor heat changes occurring during bacterial growth, decompositionor destabilization of solution. Ampoule calorimeter It is used for stability and compatibility study, bacterial growth, cellmetabolism. It is of greatest importance in pharmaceutical industry instability study of pharmaceuticals in solid state which is otherwise adifficult task to measure the rate of these reactions. Perfusion – titration cell This cell may be used to monitor the heat flow caused by the interactionof a fluid (Gas or Liquid) with a solid held stationary in the cell.Examples include absorption and desorption experiments, mixing ofliquids.
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
CONVENTIONAL METHODS FOR STABILITY STUDY: At present, the standard method used for stability analysis of asolid state pharmaceutical product is HPLC. In summary, theconcentration of parent compound and/or the concentration ofany daughter compounds produced are determined as afunction 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 asample preparation and analysis protocol It relies on the dissolution of the solid product. This last drawback can cause distortions in an assay as a resultof rapid acceleration of decomposition when a compound is ina solvated state.
For stability study samples are stored under elevatedtemperature and humidity after preparation to accelerate thepotential decomposition. The samples are then assayed over a period of time that canrange from a few weeks to many months to give reactionsnapshots along the decomposition profile. For each storage condition, a rate constant, (k), is calculated. Byplotting lnk against 1/T using the Arrhenius relationship, it ispossible to extrapolate back to ambient temperature and hencedetermine 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 gasconstant, and T is the temperature. This technique for thedetermination of stability has been accepted as normal practicefor many years.
LIMITATIONS OF ARRHENIUS EQUATION It is assumed that the Arrhenius plot gives a linearrelationship. This may not be true for many reasons. If there are two competing reactions occurringsimultaneously, then they will both have an associatedactivation 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 isnecessary to determine a different rate equation that gives animproved understanding of the system under study. This isnot always straightforward and for solid state reactions canbe very complex.
When a calorimeter is calibrated, the calorimetric signal is standardized byrelease of an accurately known heat, q, or thermal power, P = dq/dt. Theresult of a calibration experiment is usually expressed in terms of acalibration constant, ε, valid for the instrument under some specifiedconditions. For Adiabetic Colorimeter: The quantity of heat evolved or absorbed in an adiabatic calorimetricexperiment is, in the ideal case, equal to the product between thetemperature 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 vesseland the surroundings, and a “practical” heat capacity value, the calibrationconstant, is determined in a calibration experiment, q = εa ΔT, (2) where εa is the calibration constant (sometimes referred to as the “energyequivalent”). A change in the heat capacity of the content of the calorimetricvessel (following, for example, injection of a sample) will thus lead to achange in the calibration value. The thermal power is P = εa dT/dt.MICROCALORIMETRIC DATA
For Heat conduction calorimeters Tian equation: P = εc [U + τ (dU/dt)], (4) Here, εc is the calibration constant, U the measured potentialdifference across the thermopile, and τ the time constant Under steady-state conditions, for example, during the releaseof a constant electrical calibration current, eq. 4 simplifies to P = εc U. (5) The heat released in the calorimetric vessel is obtained byintegration 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, timesin the fore- and after-periods.
The procedure takes a kinetic equation for a particular reaction, andmodifies it such that it applies directly to microcalorimetric data. This isachieved by recognition of the fact that the total heat evolved during thecourse of a reaction (Q) is equal to the total number of moles of materialreacted (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 ofmaterial reacted (x) at time t multiplied by the change in molar enthalpy forthat reaction. q = x H ………………(2) Eq. (2) may be substituted into a general rate expression of the form dx/dt togive an expression of the form dq/dt (or power). For example, the generalrate 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 recordedusing the microcalorimeter.
EXAMPLES Few drugs for which stability study can be performed usingmicrocalorimetry are Aspirin, PAS, and some ß-lactam antibiotics. Microcalorimetry is applied to study the thermodynamic stability ofProteins (Lysozyme, Cytochrome-c and Ribonuclease). By using Microcalorimetry, stability study of ampicillin in aqueousSolutions as a function of conc. of ampicillin, pH & temperature was carriedout. Determination of decomposition mechanism of lovastatin by measuring rateof heat production at different temperature & time was carried out bymicrocalorimetry. Lovastatin degraded by an auto-catalytic mechanism in presence ofoxygen. Microcalorimetry is used to correlate the decomposition rate of severalCephalosporin in solids & aqueous solution states. Testing of physical stability of drug:-Microcalorimetry has proved to be an effective analytical technique forcharacterizing micronized compounds. It can be used to detect the presence ofmetastable regions not detectable by X-ray diffraction. Furthermore, the kineticof “recrystallization” of these regions can be studied, making a prediction ofphysical stability possible. Thus the application of Microcalorimetry in thepharmaceutical development has a great potential.
DISSOLUTION OF TABLETS AND POWDERS For study of dissolution TRONAC solution calorimeter is used. It consist of a dewar flask containing the reacting fluid orsolvent, a device for remote delivery of a solid substances, astirrer, a device for monitoring the temperature history of thecalorimeter, and a calibration heater. The solid sample is contained in a sealed glass ampoule thatmay be broken by actuating a remote spring loaded orsolenoid- driven breaking device. Heat evolved or absorbed due to a particular dissolutionprocess is constant. In a typical experiment, the system is calibrated by introducinga known quantity of heat with the calibration heater by meansof a constant current supply and a digital timer. After a steady drift rate is re-established, the ampoule is brokenand the change in temperature due to the reaction is measured.
The system may also be calibrated by measuring the change intemperature incurred by a standard chemical reaction such as aheat of solution of tromethamine (tris-hydroxymethyl-aminomethane) in dilute HCl (Exothermic), potassium chloridein water (endothermic), or a neutralization reaction, forexample, a known quantity of HCl reacting with a solution ofNaOH, for which accurate literature values of H are available.
EXCIPIENT COMPATIBILITY Compatibility is an important area in the drug developmentpipeline. Conventional compatibility testing methods requireboth multiple sample preparation and long storage times inorder to obtain meaningful results. It has been reported that a standard method for compatibilitytesting of binary mixtures has been developed using isothermalmicrocalorimetry. The method involves preparing a binarymixture, followed by examination in a microcalorimeter after aperiod of equilibration. If the sum of the heat out put of the compound and theexcipient alone is not equal to the heat output of a binary blendthen there is a potential compatibility issue.
Figure shows a typical response for an excipient, an activecomponent and a mixture of the two. This combination is clearly incompatible as the mixture profile isvery different from the two individual components.Calorimetric response of a drug, microcrystalline cellulose and a mixture of both. Example 1Microcalorimetry was used to study the effect of menadione& prednisone on the stability of the micro emulsions. The stability wasnot changed in the presence of drugs. Example 2Chemical & physical processes accompanying Cyclodextrin-drug interactions are usually endothermic or exothermic in nature sothey can be studied by microcalorimetry technique.
The method is only designed as a screen and does not give aquantification of the amount of degraded active. DEGRADATION Hydrolysis , oxidation, free radical formation, etc. all havelarge heat of reaction. Ideally, degradation rates of less then 1% per year can bepredicted in a matter of days.
DETERMINATION OF AMORPHOUS CONTENT INCRYSTALLINE SOLID Amorphous character in highly crystalline solids can bedifficult to detect using traditional analytical techniques, suchas Powder X-Ray Diffraction (PXRD) and Differential ScanningCalorimetry (DSC), as the limit of detection is 5–10%. In recentyears, several papers have been published that detail the use ofisothermal microcalorimetry for the quantification of lowlevels of amorphous content. A typical DSC heating curve of an amorphous substance isgiven in Fig. for L-polylactic acid. After the glass transition,the crystalline substance appears and then melts. Thecrystallization can appear spontaneously above the criticaltemperature of the glass transition (Tg). For a substance with ahigh Tg, crystallization will not occur unless the Tg value islowered by the presence of other compounds, such asimpurities or water. The Tg has traditionally been determinedby DSC, but the use of MDSC in this area is increasing.
The glass transition is accompanied by a change inthe heat capacity (change of base line). After thistransition, the crystallization in the crystallinepolymer is accompanied by an exotherm. Thecrystalline polymer obtained shows two endotherms:the first small endotherm is due to rearrangement inthe polymer followed by the melting of thecrystalline polymer.
MICROORGANISM-DRUG INTERACTION Growing of microorganism produce heat. This principle maybe used to study the effect of antibiotics on the microbialgrowth. E.g. Microcalorimetric titration used to study the effect ofVancomycin against gram +ve bacteria. Determination of Gibbs energies, enthalpies, entropies andheat capacities for antibiotic-bacteria binding reactions aredone.
FOOD-DRUG INTERACTION The effect of food on the dissolution rate ofTetracycline hydrochloride was studied usingMicrocalorimetry. An interaction was observed using Microcalorimetrybetween tetracycline and calcium, milk.
SORPTION REACTIONS Sorption reactions [i.e., adsorption or absorption ofgases (vapors) and solutes onto solids] are offundamental importance in thermochemistry, andseveral special microcalorimeters have been designedfor such experiments. Sorption reactions, in particular sorption of watervapor, have recently become one of the most importantpractical application areas for microcalorimetry, forexample, in the pharmaceutical industry.
POWDER WETTABILITY The combination of Microcalorimetry andvacuum microbalance techniques allows thepossibility of calculating the thermodynamicparameters associated with wetting process and inaddition, gives idea about mechanism of wetting.