# microcalorimeter in stability study

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In this applications of Microcalorimeter following by brief introduction are covered.

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### Application of Microcalorimeter in stability study:

Application of Microcalorimeter in stability study By Prashant Patel (M.Pharm sem-1) Department of pharmaceutical technology Indukaka Ipcowala college of pharmacy

### INTRODUCTION:

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

### PRINCIPLE:

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 = d q / d t . 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

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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)

### CLASSIFICATION OF CALORIMETERS :

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

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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.

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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.

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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.

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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.

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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

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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.

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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.

### LIMITATIONS OF ARRHENIUS EQUATION :

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.

### MICROCALORIMETRIC DATA:

When a calorimeter is calibrated, the calorimetric signal is standardized by release of an accurately known heat, q , or thermal power, P = d q / d t . 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, Δ T , and the heat capacity of the calorimetric vessel (including its contents), C , q = C Δ T. (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 = ε a Δ T , (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 = ε a d T / d t . MICROCALORIMETRIC DATA

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For Heat conduction calorimeters Tian equation: P = εc [ U + τ ( d U / d t )], (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 = ε c U . (5 ) The heat released in the calorimetric vessel is obtained by integration of eqs . 4 or 5, leading to the simple expression q = ε c ∫ t 1 t 2 U d t , (6) provided that the initial and final potentials are the same (normally the baseline value); t 1 and t 2 are respectively , times in the fore- and after-periods.

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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 ( A o ) multiplied by the change in molar enthalpy for that reaction (  H ). Q = A 0  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 d x/ d t to give an expression of the form d q / d t (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.

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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.

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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.

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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.

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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.

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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.

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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.

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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 ( T g ). For a substance with a high T g , crystallization will not occur unless the T g value is lowered by the presence of other compounds, such as impurities or water. The T g has traditionally been determined by DSC, but the use of MDSC in this area is increasing.

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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.

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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.

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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.

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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.

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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.