Physical properties of Drug Molecules

 Physical properties are properties than can be measured or observed without

changing the chemical composition of the substance.
 The study of physical properties involves the specific interaction between the
molecules and a well-defined form of energy or external influence.
 Example: The concept of weight uses the force of gravity as an external influence
to compare the mass of the objects. Ideally, physical property should be easily
measured or calculated.
 Some physical properties of drug molecules and solvents are:
 Molecular volume
 Dipole moment
 Optical rotation
 Viscosity
 Surface tension
 Refractive index
 Dielectric constant
Applications:
 Identification of drugs
 Determination of percentage composition of drugs
 Drug synthesis
 Elucidation of the structure of molecules
 Formulation of dosage forms
 Evaluation of drug action
 Group contribution methods:
 Several physical properties are measured in the laboratory. These are due to the
contribution of atoms, groups and their arrangement. Hence it is possible to
calculate them theoretically by group contribution method.
 Group contribution method is a method used for calculating the physical
properties theoretically based on contribution of atoms, groups and their
arrangement.
 Basic principles involved in the group contribution methods are given below.
 Colligative
 Additive
 Constitutive

Colligative properties:
 Colligative properties depend mainly on the number of molecules present in a
solution.
 The colligative properties of solutions are osmotic pressure, vapor pressure
lowering, freezing point depression, and boiling point elevation.
 The values of the colligative properties are approximately the same for equal
concentrations of different nonelectrolytes in solution regardless of the species or
chemical nature of the constituents.
 For non volatile solute colligative properties of solid-in-liquid solutions may be
vapor pressure.

Additive properties:
 Additive properties depend on the total contribution of the atoms in the molecules
or on the sum of the properties of the constituents in a solution.
 Example: Molecular weight (because it is the sum of the masses of the constituent
atoms).
 The total mass of the solution is the sum of the masses of the individual
components.
 Consider the case of acetic acid (CH3COOH). Obtaining molecular weight of
acetic acid involves addition of molecular weights of individual atoms that makes
it. Acetic acid contains; C = 2, H = 4 and O = 2.
 So the molecular weight is calculated as;
Molecular weight of acetic acid = C × 2 + H × 4 + O × 2
= 12 × 2 + 1 × 4 + 16 × 2
= 60 g/mol

Constitutive properties:
 Constitutive properties depend on the arrangement and to a lesser extent on the
number and kind of atoms within a molecule.
 These properties give clues to the constitution of individual compounds and
groups of molecules in a system.
 The refraction of light, electric properties, surface and interfacial characteristics,
and the solubility of drugs are at least in part constitutive and in part additive
properties.
 The examples of this type are optical activity, surface tension, viscosity etc.
 Consider the case of lactic acid.
 It exists in two forms namely d-lactic acid and l-lactic acid.
 The specific rotation of d-lactic acid is +3.8° while l-lactic acid shows it as -3.8°.
Refractive index:
The ratio between the speed of light in medium to speed in a vacuum is the
refractive index. When light travels in medium other than the vacuum, the atoms
of that medium continually absorb and re-emit the particles of light, slowing down
the speed light.
Refractive index is defined as “the ratio of the speed of light in a vacuum to
its speed in a specific medium”.
Refractive index is also referred to as refraction index or index of
refraction. The speed of light in a medium depends on the properties of the
medium. In electromagnetic waves, the speed is dependent on the optical density
of the medium.
Optical density is the tendency of the atoms in a material to restore the
absorbed electromagnetic energy. The more optically dense material is, the slower
the speed of light. One such indicator of the optical density of a medium is the
refractive index.

Refractive Index Formula

The refractive index is dimensionless. It is a number that indicates the
number of times slower than a light wave would be in the material than it is in a
vacuum. The refractive index, represented by symbol n, is the velocity of light in
vacuum divided by the velocity of light in a medium. The formula of the
refractive index is as follows:
sin 𝑖
𝑛=
sin 𝑟
Where,
n is the refractive index
c is the velocity of light in a vacuum ( 3 × 108 m/s)
v is the velocity of light in a substance
The vacuum has a refractive index of 1. The refractive index of other materials
can be calculated from the above equation. Higher the refractive index, the higher
the optical density and slower is the speed of light. The table below lists the
refractive indeces of some materials.
Table: Refractive Indices of Some Materials

Snell’s law states that the refractive indes (n) of the liquid is a constant ratio
of the angle of incidence to that of the refraction. It is given by:
sin 𝑖
𝑛=
sin 𝑟
Where, i is angle of incidence
r is angle of refraction
 When monochromatic light passes through a less dense medium such as air
or vacuum and enters a denser medium, the advancing waves at interface are
modified and brought closer together. This leads to decrease in speed and
shortening of wavelength. When light passes the denser medium, a part of wave
slows down more quickly as it passes through interface and makes it bend towards
the interface. This phenomenon is called as refraction. If light passes from denser
medium to less denser medium then it is refracted away from the interface. This
effect observed between mediums is expressed as refractive index (n). The
refractive index is a constant for a given pair of substances under specified
conditions. It can be defined as ratio of speed of light in substance 1 to the speed
of light in substance 2. This is usually written 1n2 and is the refractive index of
substance 2 relative to substance 1. The incident light is in substance 1 and the
refracted light is in substance 2. When the incident light is in a vacuum this value
is called the absolute refractive index of substance.

Figure: Angles of Incidence and Refraction

Refractive indices of most substance are more than air because the velocity
of light in air is greater than in the substance for example, absolute refractive
index of water is 1.330, soda lime glass 1.510. By definition the refractive index
of a vacuum is 1. In practice, air makes little difference to the refraction of light
with an absolute refractive index of 1.0008.

Measurement of Refractive Index:

Figure: Schematic of Abbe’s refractometer

Refractive index is determined by using instrument called refractometer.

Abbes refractometer, immersion refractometer and Pulfrich refractometer are used
for this purpose. Abbes refractometer is commonly used at laboratory scale
because of its advantages over other refractometers. It is most convenient, reliable
and simple instrument with small sample size requirement suitable for range of
substances. Ordinary light source, easy maintenance and economy and easy
determinations are some of the other advantages of this instrument. The
components of Abbes refractometers include light reflection mirror, dispersion
compensator, telescope, and index arm and prism box. Abbes refractometer may
be calibrated with anyone of the liquid specified in Table at temperatures below
25 °C using D-line of sodium.
 Table: Reference liquids for calibration of Abbes’ refractometer

The refractive index varies with varies with temperature and wavelength of
light used and hence it is not a constant property.
Recent research has demonstrated the existence of negative refractive
index. Not thought to occur naturally, this can be achieved with so called
metasubstances and offers the possibility of perfect lenses and other exotic
phenomena such as a reversal of Snell’s law. The real and imaginary parts of the
complex refractive index can be determined as a function of wavelength from an
absorption spectrum of the substance. The refractive index of certain media may
be different depending on the polarization and direction of propagation of the light
through the medium. This is known as birefringence or anisotropy. The strong
electric field of high intensity light for example, a laser, may cause a medium’s
refractive index to vary as the light passes through it, giving rise to non-linear
optics. If the index varies quadratically with the field it is called the optical Kerr
effect and causes phenomena such as self-focusing and self phase modulation. If
the index varies linearly with the field it is known as the Pockels effect. If the
refractive index of a medium is not constant, but varies gradually with position,
the substance is known as a gradient index medium.

Why is high refractive index important for optical polymers?

Optical polymers with high refractive index allow light rays to bend more
within the substance, which helps in lowering the profile of the lens. Also, as the
 refractive index increases, the thickness of the lens decreases, resulting in less
weight.

Applications:
 Since refractive index is a fundamental physical property of a substance it is often
used to analyze and identify a particular substance, confirm its purity, or measure
its concentration.
 Refractive index values are useful in determination of molecular weights and
structures of organic compounds from their molar refraction values.
 Refractive index is used to measure refraction characteristics of solids, liquids,
and gases.
 Most commonly it is used to measure the concentration of a solute in an aqueous
solution.
 For a solution of sugar, the refractive index can be used to determine the sugar
content.
 Similarly alcohol content in bioproduction is also determined from the
refractometry.
 Dielectric constant and molar polarizibility values can be obtained from the
refractive index.
 Refractive index of a substance is the most important property of any optical
system that uses refraction for example, lenses and prisms.

Optical rotation:
 The optical rotation is the angle through which the plane of polarization is rotated
when polarized light passes through a layer of liquid.
 Ordinary light consists of vibrations, which are evenly distributed in all directions
in a plane perpendicular to the direction of propagation, called as unpolarised
 light. When the vibrations of light are restricted to only one plane, the light is said
to be polarized light.

Figure: Ordinary and polarized light

 Some substances rotates the plane of polarized light are called as optically active
substances. This property of optically active substance is measured as angle of
rotation.
 The property in which rotation of plane polarized light is observed is known as
optical activity (or)
 Optical activity is the ability of a compound to rotate the plane of polarized light.
 Optically active substances include organic molecules with a central carbon atom
to which four different groups are attached, making the molecule very asymmetric
(chiral carbon).
 Similarly, laevulose, more commonly known as fructose causes the plane of
polarization to rotate to the left. Fructose is even more strongly leavo rotatory than
the glucose.
 The substance which rotates the plane of polarized light to the right or clockwise
when viewed in the direction of light propagation is called dextro rotatory (d) or
(+) substance.
 The use of one name for glucose, dextrose, refers to the fact that it causes linearly
polarized light to rotate to the right or dexter side. Those optically active
 substances that rotate plane polarized light to the left or counter clockwise are
known as leavo rotatory (l) or (−) substance. Laevulose, more commonly known
as fructose causes the plane of polarization to the left. Fructose is even more
strongly leavo rotatory than the glucose.
 Other examples of optically active substances are lactic acid, tartaric acid, 2-
methyl -1-butanol etc.
 Optical rotation occurs because of optically active substances have different
refractive indices for left and right polarized light. Another way to make this
statement is that left and right polarized light travel through an optically active
substance at different velocities.
 Optical activity is considered to be due to the interaction of plane polarized
radiations with electrons in molecules which shows electronic polarization. This
interaction rotates the direction of vibration of radiation by altering electric field.
 Optically active substances can be categorized in to two types:
1. Those which are optically active only in the crystal state due to their characteristic
crystal structure and becomes optically inactive in the fused or dissolved state, for
example, sodium chlorate, quartz crystal etc and,
2. Those which shows optical activity in all states viz. Crystalline (solid), fused
(liquid) and gaseous state, due to their structural configurations.

Measurement of Optical Activity:

Measurement of orientation of plane polarized light is called polarimetry, and the
instrument used is called a polarimeter. The simplest polarimeterconsists of
monochromatic light source, a polarizer, a sample cell, a second polarizer which is
called the analyzer and a light detector. Polariser and analyzer are made up of
Nicol prisms. When analyzer is oriented 90° to the polarizer no light reaches to
the detector. The polarizer is placed near to the light source while analyzer is
placed between sample cell and the detector. The sample cell of suitable size and
capacity with outward projection at the centre, to trap the air bubble is usually
used. When an optically active substance is placed in the sample cell and beam of
light is passed through, it rotates the polarization of the light reaching the analyzer
so that there is a component that reaches the detector. The angle that the analyzer
must be rotated from the original position is the optical rotation.

Figure: Schematic of Polarimeter

For a pure substance in solution, if the colour and the path length are fixed
and the specific rotation is known, then observed rotation can be used to calculate
the concentration. Optical activity is useful in studying the structure of anisotropic
substances, and for checking the purity and identifying chiral mixtures.
Adulterations in the optically active substances can be determined from the optical
rotation.
For example, optical rotation of honey is opposite to that of sugar due to the
presence of fructose and glucose and hence can be determined from the optical
rotation. Chemical kinetic studies are also carried out by determining
concentration at different time intervals as in case of sugar inversion.
Polarimetry is used in the analysis of various drugs and pharmaceutical
formulations such as Adrenaline Bitartarate, anticoagulant Citrate Dextrose
Solution, Dextran 40 Injection, Dextrose Injection, Sodium Chloride and Dextrose
injection etc.

Dielectric Constant:
A polar molecule can sustain a separation of electric charge either through
the induction by an external electric field or by a permanent charge separation
within a molecule. The separation of charge can be best understood from the
concept called dielectric constant. Consider the example of parallel plate
condenser.

Figure: Parallel Plate Condenser

The parallel plates are separated by some medium across a distance r and
connected to voltage supply source. The electricity will flow across the plates
from left to right through the battery until potential difference of the plates equals
that of the battery which is supplying the initial potential difference. The
capacitance, C, is equal to the amount of electric charge, q, stored on the plates,
divided by V, the potential difference between the plates.
C=q/V
The capacitance of condenser depends on the type of thickness of the
condenser separating the plates. The Co is used as capacitance reference medium
on which to compare other mediums. The Co is the capacitance between the plates
when a vacuum fills the space between the plates. The ratio of capacitance of test
substance (Cx) divided by the capacitance of reference substance is termed as
dielectric constant.
ε = Cx/Co
where, ε is dielectric constant and since it is ration of capacitance it is unit less
quantity. The dielectric constants of some liquids are given in Table.

Table: Dielectric Constants of Some Liquid at 20°C

Liquids Dielectric constants Liquids Dielectric constants
Acetone 21.4 CCl4 2.24
Benzene 2.28 hexane 5.0
Glycols 50.0 Formaldehyde 22.0
Chloroform 4.8 Methanol 33.7
Phenol 9.7 Ethanol 25.7

The polarity of the solvent depends on the dielectric constant as more is the
polar solvent greater is the dielectric constant. Therefore dielectric constant of a
substance affects the solubility of that substance. The highest solubility of caffeine
at 25°C in dioxane – water mixture was found in the dielectric constant range of
20 to 40.

Table: Dipole Moment and Polarizibility of Some Gases

Gas Dipole Polarizibility
moment
HCl 3.6 2.93
 NH3 4.90 2.47
H2O 6.17 1.65
C6H5 0 11.6

If the polar molecules are placed between plates of charged capacitor, the
molecules can undergo an induced polarization. This occurs because of the
separation of the electric charge within the molecules as it is placed in the electric
field between the plates. This polarization is usually temporary and is independent
on the ease with which the molecules can be polarized. This temporary induced
polarization is proportional to field strength of capacitor and induced
polarizibility, αp, which is characteristic property of the particular molecules. The
ease with which a molecule is polarized by any external force (electric field, light
or any other molecule) is known as polarizibility.
The relation between concentration, dielectric constant and polarizibility is
given by Clausius-Mossotti equation as;
(ε − 1) 4
= [ ] [πnαp]
(ε + 2) 3
In the above equation n is the number of molecules per unit volume. The total
polarization is the sum of induced molar polarization and temporary polarization.
P = Pi + Po
Since π = 0, Po is zero. To obtain an induced molar polarization (Pi) the above
equation can be multiplied by the M/ρ on both sides.
(ε − 1) 4 πnMαp
= [ ]Χ
(ε + 2) 3 ρ
4
= [ ] Χπnnαp
3

= pi
 A condition in which electric field strength of condenser (V/m) is unity, π
represents the induced molar polarization.

Dipole moment:
Dipole is a pair of separated opposite electric charges. Electric dipole is an
assemblage of atoms or subatomic particles having equal electric charges of
opposite sign separated by a finite distance. Dipoles are characterized by their
dipole moment, a vector quantity with a magnitude equal to the product of charge
or magnetic strength of one of the poles and the distance separating the two poles.
µ=q×r
where, µ is dipole moment, q is charge on atom and r is distance of separation of
charge. The direction of the dipole moment corresponds for electric dipoles, to the
direction from the negative to the positive charge. The direction of an electric field
is defined as the direction of the force on a positive charge, electric field lines
away from a positive charge and toward a negative charge.

Molecular Dipoles:
Many molecules have dipole moments due to non-uniform distributions of
positive and negative charges on its various atoms. In the case of HCl, the bonding
electron pair is not shared equally rather is attracted towards the more
electronegative chlorine atom due to its higher electro-negativity which pulls the
electrons towards it. It leads to development of positive charge to H atom and
negative charge to chlorine atom. A molecule having positive and negative
charges at either terminal is referred as electric dipoles or just dipole. Dipole
moments are often stated in Debyes. The SI unit is the coulomb meter.
Molecular dipoles are of three types;
 1. Permanent dipoles: These occur when two atoms in a molecule have
substantially different electro-negativity with one atom attracting electrons more
than another becoming more electronegative, while other atom becomes more
electropositive.
2. Instantaneous dipoles: These occur due to chance when electrons
happen to be more concentrated in one place than another in a molecule, creating
a temporary dipole.
3. Induced dipole: These occur when one molecule with a permanent
dipole repels another molecule’s electrons, inducing a dipole moment in that
molecule. In a diatomic molecule, the dipole moment is a measure of the polar
nature of the bond; i.e. the extent to which the average electron charges is
displaced towards one atom. In a polyatomic molecule, the dipole moment is the
vector sum of the dipole moments of the individual bonds. In a symmetrical
molecule, such as tetrafluoromethane (CF4), there is no overall dipole momental
though the individual C-F bonds are polar.

Molecular dipole moments:

In most molecules even though the total charge is zero, the nature of
chemical bond is such that the positive and negative charges do not overlap. These
molecules are said to be polar because they possess a permanent dipole moment.
The example of this type is water molecule. The molecules with mirror symmetry
like oxygen, nitrogen carbon dioxide and carbon tetrachloride have no permanent
dipole moments. Even if there is no permanent dipole moment, it is possible to
induce a dipole moment by the application of an external electric field and is
called as polarization. The magnitude of the dipole moment induced in the
molecules is a measure of the polarizability of that molecular species.
 Permanent dipole moment:
The permanent dipole moment differs from induced polarization in the
sense that it is a permanent separation and it happens only to be in the polar but
not in the non-polar molecules. These charges that separate balance out each other
and therefore have a net charge of zero. The water is example of permanent dipole
moment. The permanent dipole moment is defined as the vector sum of the
individual charge moments within the molecules.

Applications:
 The structure of the molecule can be confirmed from the dipole moment values,
for example, chlorobenzene, benzene, carbon dioxide etc.
 The cis and trans isomers can be differentiated form dipole moment values, for
example, cis and trans dichloroethylene.
 Dipole moments can be used to determine percent ionic characteristic of bond of
the molecule, e.g. H-Cl a covalent bond, ionic characteristic is 17%.
 Permanent dipole moments can be correlated with the biological activities to
obtain information about the physical parameters of molecules.
 The more soluble the molecule the easier it passes the lipoidal membrane of
insects and attacks the insect’s nervous system. Therefore, the lower is the dipole
moment the greater is the insecticidal action.
 For example, p, m and o isomers of DDT show different insecticidal activities due
to their differences in permanent dipole moment as p- isomer shows µ=1.1 and
has predominant toxicity, o-isomer shows µ = 1.5 with intermediate toxicity while
m-isomer shows µ = 1.9 with least toxicity. The variations in activities of different
isomers are due to the greater solubilities in non-polar solvents.

Dissociation constant:
 The fraction of the amount of the electrolyte in solution present as free ions is
called the degree of dissociation.
 Since majority of the drugs are either weakly acidic or weakly basic, their
solutions in water contain ionised and unionised species.
 In other words the drug undergoes dissociation in their aqueous solutions.
 The extent of ionisation depends on the pH of the solution.
 For example a weak acid will ionize mostly in alkaline pH while a weakly basic
will ionize in an acidic medium.
 The unionised drug is more lipid soluble and thus readily absorbed.
 Hence gastrointestinal absorption of a drug can be improved if the extent of
ionisation is reduced.
 The dissociation constants are expressed for weak acids and weak bases.
 The absorption of weakly acidic or basic drugs can be influenced by the following
factors:
 pH at the site of absorption
 Dissociation constant
 Lipid solubility of unionised drugs
 At a particular ph the relative concentration of unionised and ionised species in a
drug solution can be estimated with the help of Henderson-hasselbalch equation;
 For acidic drugs:
pH= pKa + log [ionised drug]/[unionised drug]
 For basic drugs:
pH= pKa + log [unionised drug]/[ionised drug]
 The contents of the stomach have pH ranging from 1-3.
 Weakly acidic drugs having a pKa value less than 4.3 were absorbed faster than
those having pKa value within 2- 4.3, strongly acidic drugs were almost not
absorbed.
 pH of intestinal fluid vary from 5-8.
 In case of basic drugs the absorption is faster for those having pKa values less
than 8.5 than those having pKa values between 9 -12.
 Thus for prediction of the site of absorption of weakly acidic or basic drugs,
knowledge of pKa is very important.
Factors influencing the degree of dissociation
(1) Nature of solute.
(2) Nature of the solvent.
(3) Concentration.
(4) Temperature.
The degree of dissociation of an electrolyte in solution depend on following
factors
Nature of solute:
 Strong acids, strong bases and salts obtained by their interaction are almost
completely dissociated.
 Week acids, week bases and their salts are feebly dissociated.
Nature of solvent:
 Dielectric constant: The capacity of solvent to weaken the force of attraction
between the electrical charges immersed in the solvent.
 The higher the value of dielectric constant the greater is the dissociation of the
electrolyte dissolved in it.
Concentration:
 Dissociation of an electrolyte is inversely proportional to the concentration of its
solution.
 This is due to the fact that in a dilute solution the ratio of solvent molecules to the
solute molecule is large and the greater number of solvent molecule will separate
more molecules of the solute into ions.
Temperature:
 Higher the temperature greater is the dissociation.
 At high temperature the increased molecular velocities overcome the force of
attraction between the ions and consequently the dissociation increases.

Applications of dissociation constant:

 Dissociation constant is incorporated in Henderson-Halsselbalch equation to
calculate the extent of ionization or dissociation, i.e. percent unionized and
ionized forms of drug.
 The absorption of drugs in GIT can be predicted using dissociation constant of the
drug and pH at different sites of GIT.
 The concentration of preservatives such as benzoic acid required to preserve
solutions and emulsions can be predicted.
 The pH of the solution can be calculated. When the concentration of acid is equal
to the concentration of salt, the pH of the solutions is same as the pk a of the drug.
 Ionization constant values can be used to obtain maximal yields in the extraction
of drugs.

Methods of determination of dissociation constant:

 Potentiometric measurements
 NMR spectroscopy
 Capillary electrophoresis
 High performance liquid chromatography
 Hyper rayleigh scattering
 Displacement ELISA
 Calorimetry
 `Partition and distribution coefficient.
 UV spectroscopy [orthogonal method]
 Density functional theory [DFT]
 Isohydric solution principle
 Solvation model
 Solubility data
 Thermal lensing spectroscopy
 Surface tension and interfacial tension
 Voltametry
 Fluorometric method
 Fluorescence polarization method
 Mass spectroscopy
 Surface plasma resonance

Potentiometric measurement:
In a potentiometric titration, a sample is titrated with acid or base using a
pH electrode to monitor the course of titration. The pKa value is calculated from
the change in shape of the titration curve compared with that of blank titration
without a sample present. Potentiometric titration are based on the quantitative
relationship of the E.M.F. of a cell as given by the following equation
E Cell = E ref + E indicator + E junction
A Known volume of the acid to be titrated is kept in a beaker. The
hydrogen half-cell was combined with the reference electrode, half-cell through a
solution bridge. After each addition of titrant into beaker, the E.M.F. is measured.
The potential [Eref] reference electrode is first measured against the standard
hydrogen electrode. The stable E.M.F. of the cell [Ecell] is measured two hours
after the cell had been assembled and the hydrogen ion concentration calculated
from the following equation
pH = [ Ecell - Eref] / 0.0591
The ionization constants for the acids in different solvents are then
calculated from pH = pKa at half neutralization. The equivalence point gets on the
titration curve in the part where there is a relatively large change in pH with a
relatively small change in volume. Therefore it concluded that the change in
electrode potentials of the cell is proportional to the change in pH during titration.
The point where the E.M.F. increases rapidly gives the end point. The different
methods used for calculation of pKa like potentiometry, conductometry have
limited application for determination of second dissociation constant of very weak
acid. The main advantage of this method is applicable for analysis of coloured and
dilute solutions. The apparatus required is generally inexpensive, reliable and
readily available. However, its disadvantages include the requirements to use a
milligram of pure compounds and a mixture of aqueous buffers and to avoid
errors, especially for measurements at neutral-to-high pH, carbonate-free solutions
must be prepared laboriously.

Displacement ELISA
ELISA method is valuable for the measurement of the dissociation constant
of antigen-antibody complexes. ELISA used for determination of plasma HDL
concentration in clinical specimens using monoclonal antibodies A-130 and A-14
reagent.Affinity constant has been measured for monoclonal Ab-Ag pairs using
ELISA methods, but the results tends to be inconsistent.
ELISAs makes possible the determination of dissociation constants of
complexes formed between immobilised antigens and the displacing molecules
without knowing the precise concentration of the antigen. The indirect ELISA is
an effective method for studying the interaction of Ab with Ag; the immobilized
Ag captures the Ab from the test solution and the bound Ab is then revealed by
the enzyme-labelled anti-IgG.

Calorimetry
All calorimetric methods work by the same principle: a physical or
chemical process takes place in a sample and the amount of heat evolved is
measured. For the measurement of pKa values, in Isothermal Titration
Calorimetry [ITC], a regular acid-base titration is carried out inside the
calorimeter while the energy required to keep the temperature constant is
measured. In recent years, the ITC-method used to calculate the dissociation
constants of peptides and the influence of binding on the specific ionizable
groups. This method also calculates the pKa indirectly from a measured enthalpy
change ΔH. By plotting the minima or maxima versus pH, a sigmoid curve is
obtained from which the pKa can be determined from the inflection point.
In isothermal titration calorimetry [ITC], the label-free solution used to
measurement, where the heat change associated with binding is the read-out. ITC
has been termed the ‘‘gold standard’’ for characterizing biomolecular interactions,
even though its main drawback of large amount of sample required for a
measurement. If a ligand binds to a protein with a single-digit nanomolar or
tighter dissociation constant, it is difficult to measure the affinity accurately using
ITC because the titration curve becomes too steep to fit accurately. In such cases,
one can measure the displacement of a weakly binding ligand during titration with
a strongly binding ligand of interest.

Partition and distribution coefficient

 The distribution constant or partition coefficient means if a third substance
is added to a system of two immiscible liquids in equilibrium, the added
component will distribute itself between the two liquid phases until the ratio of its
concentrations in each phase attain a certain value.[58] Nernst concluded in 1891
that the partition coefficient [P] was only a constant if a single substance was
considered. The partition coefficients determination of ionizable compounds
requires the knowledge of the pKa value. W. R. Vezin determined dissociation
constant of phenothiazine derivative based on the pH dependency of partition
coefficient at a range of pH value. Although log P is a relatively simple quantity to
determine, it has been proven that there are still many sources of errors, one of
which is common miscibility of the two phases.

Prepared by:

Sridevi Murugesan

Department of Pharmaceutics

SNS College of Pharmacy and Health Sciences