Polymorphism

When the crystalline structure of the same chemical compound (and atomic formula) exhibits
two or more patterns of the repeated unit cells, these crystalline structures are called
polymorphs and the phenomenon is referred to as polymorphism. Polymorphism is an
exclusively solid-state phenomenon. Polymorphs have distinct:
(i) Crystal structures (mutual arrangements of molecules, atoms, or ions).
(ii) Physical and chemical properties.
E.g.,
Friedrich Wöhler and Justus von Liebig in1832 observed that the silky needles of freshly
crystallized benzamide slowly converted to rhombic crystals.
Carbon: Diamond in a cubic (tetrahedral lattice arrangement); Graphite in a sheet of a
hexagonal lattice.

Types of polymorphism:

1. Conformational polymorphism: Each of the isolated crystal forms contains a different

conformation of the same molecule.
E.g., Venlafaxine
2. Tautomeric polymorphism: Crystal forms of tautomers are often considered
polymorphs because the tautomers generally equilibrate in solution at the temperature
at which the solid forms are isolated.
E.g., Omeprazole
True polymorphs can be classified into two different types

1. Enantiotropic: One polymorph can be reversibly changed into another one by varying
the temperature or pressure. i.e., one form is stable over certain pressure and
temperature range, while the other polymorph is stable over different pressure and
temperature ranges.
E.g., Sulfur.
2. Monotropic: The change between the two forms is irreversible. That is only one
polymorph is stable at all temperatures below the melting point, with all other
polymorphs being unstable.
E.g., Glyceryl stearate, chloramphenicol palmitate

Phase transition: It is the process of transformation of one polymorph into another

Pseudopolymorphism

The phenomenon in which solvent molecules get incorporated into the crystal lattice of a solid
is known as solvates. These solvates exist in different crystal forms called pseuodopolymorph
and the phenomenon is called as Pseudopolymorphism, also known as a hydrate when water is
solvent.

E.g., A novel dihydrate of levodopa is reported. The structural characterization of the novel
dihydrate shows water molecules lying within a cage-like structure. The novel dihydrate
transforms into an anhydrous form.

Properties:

➢ Polymorphs differ in their morphologies, solubility, color, melting or sublimation

temperatures, densities, thermal or electrical conductivities and other physical properties.
They have the ability to exist in different arrangements or conformations in the crystal
lattice.
➢ Polymorphs show the same properties in the liquid or gaseous state but they behave
differently in the solid-state.
➢ At a certain temperature, only one crystalline form can be stable. Depending upon their
relative stability, one of the several polymorphic forms will be physically more stable than
the others.
• Stable polymorph represents the lowest energy state, has the highest melting point and
least aqueous solubility.
• The metastable form represents the higher energy state, has a lower melting point and
high aqueous solubility. The metastable form is converted to the stable form due to its
higher energy state. The metastable form shows better bioavailability and is therefore
preferred in formulations. Only 10% of the pharmaceuticals are present in their
metastable form.

Examples of polymorphic drugs

1. Ribavirin & Ritonavir (antiviral)

2. Ranitidine & Famotidine (H2 receptor antagonist)
3. Acetaminophen: 2 polymorphic forms, the marketed one is the stable Form I having
monoclinic crystals
4. Loperamide (inhibits GI motility)
5. Risperidone (atypical antipsychotic)

Relevance of the crystalline polymorphism in the industry

• New polymorphic forms may have better stability

• Have longer shelf-life
• Easier method of production
• Can be handled better
• Possess better formulation properties and have better bioavailability.

Factor affecting polymorphism:

1. Temperature and Humidity:

Storage conditions affect physicochemical reactions which are accelerated at a higher

temperature.

Humidity acts as a catalyst on the solid surface.

E.g., In Zanoterone the solid degradation rate of form IV is found to be greater than that of
form III at 40 C/ 25 %RH and 40 C/75% RH. At 40 C/ 25 % RH, the rate of degradation is 4-
fold higher for form IV than for form III

2. Photostability:

Generally, light-sensitive drugs are protected from photolytic degradation by packing them
suitable in a light-resistant container.
The stable crystalline form resists photochemical degradation and does not require a light-
resistant system.

E.g., Acetametacin alpha, beta - stable, gamma - unstable.

3. Effect of solvent:

Solvents can bring a dramatic change in growth mechanism and morphology. Two important
factors determined the kinetic of crystal growing from solution.

a. degree of molecular roughness

b. nature of absorption of the solvent from the surface.

4. Effect of grinding:

The grinding process reduces particle size, so increasing specific surface area and that has a
direct effect on dissolution rate and bioavailability of the formulation.

During the process, solid-state polymorphic transformation into noncrystalline or metastable

form is caused by mechanical action.

E.g., The dihydrate form is more stable than the anhydrous form. With increasing grinding time
compounds become unstable because grinding weakens bonding crystals and water molecules.

5. Effect of tablet compression:

The stability and compaction behavior of the polymorphic form of the drug is important.

E.g., Phenylbutazone in which form 3 converted to 2 forms at >2000kg/cm.

Ostwald’s law of stages:

• At high supersaturation, the first form which crystallizes is the thermodynamically least
stable (more soluble) form
• This form subsequently dissolves and transforms into a more stable one & the cycle
continues until the thermodynamically stable (less soluble) polymorph remains.

E.g., Acetyl salicylic acid was first prepared in 1897. Its crystalline structure was determined
in 1964 and the second polymorphic form was discovered in 2004 in hot acetonitrile which is
stable only at 100 K and at ambient temperature recrystallizes into form I.
Methods for preparation of polymorphic forms:
1. Sublimation

• The process of the transition of a substance from the solid phase to the gas phase without
passing through an intermediate liquid phase is called sublimation.
• The polymorphic modification is affected by the sublimation temperature. Unstable
crystals are preferentially formed at lower temperatures, while at higher temperatures,
stable forms are obtained.
• The sublimation technique is used only for thermally stable compounds.
• In this technique, the compound undergoes a phase change from solid to gas and then
back to solid. E.g., Form I of 9, 10-anthraquinone-2- carboxylic acid, obtained as
needle-like crystals above 250 °C.
• This technique has also been used for purification. E.g., The purification of polymorphs
of theophylline is obtained by this technique.
• Vacuum sublimation has been reported to produce new stable morphs of 1, 3-
dimethyluracil and malonamide.

2. Single solvent crystallization

• The crystallization technique is designed using parameters such as temperature, solvent

range, concentration, etc.
 • In this method, a compound is dissolved in a single solvent and a clear solution is
obtained at room temperature or higher. The compound is then crystallized using
gradual cooling (the hot crystallizing solution is brought to room temperature slowly
and then cooled) or sudden cooling (the crystallizing solution is cooled rapidly).
• If crystals do not grow upon cooling, then crystallization is induced by scratching with
a glass rod or the addition of a seed. The solvent is selected with care and those that are
likely to be encountered during formulation or processing are preferred.
• Various factors that affect crystallization by this technique are (a) solvent polarity, (b)
concentration, (c) temperature (including cooling rate), (d) seeding and (e) agitation.
• The main reason for the variation in crystallization in different solvents is the difference
in polarity of these solvents.
• Different types of hydrogen-bonded aggregates tend to form when a molecule is in
solution.
• The concentration of the solution also plays an important role in the generation of
crystals where the better the concentrate, the better the crystallization.
• Isolation of the desired morph often uses seeding; E.g., Seed crystals always require
obtaining the pure morph of Form I of atorvastatin calcium. Mixing ethanol and water
makes the system polar, whereas mixing ethanol and ethyl acetate makes the solvent
system less polar.
• The quantity of seed added varies from compound to compound, ranging from a pinch
up to 1 %.
• Agitation also affects crystallization, where fast agitation results in metastable morphs
with smaller particle sizes and slow agitation results in thermodynamically stable
morphs with larger particle sizes.

3. Multi-solvent crystallization

• If the compound is not soluble in a single solvent or the desired polymorphic

transformation is not obtained a mixture of solvents can be used.
 • This method employs the dissolution of the compound in a mixture of two or more
solvents, followed by crystallization through gradual or sudden cooling.
• This system usually depends on the solubility of the compound in the different solvents.
Often a solvent system is selected in which the solute is more soluble in the component
with higher vapor pressure. Because the two solvents evaporate at different rates, as the
volume of solution is reduced, the composition of the solvent mixture changes.
• Crystallization can be designed by choosing a variety of solvent systems in which the
compound is soluble, thereby increasing or decreasing the polarity of this solvent
system. Mixing ethanol and water makes the system polar, whereas mixing ethanol and
ethyl acetate makes the solvent system less polar.
• E.g., Kitamura et al., illustrated that Form A and Form B of L-histidine was crystallized
using an ethanol-water mixture with a volume fraction of 0.2 and 0.4, respectively.

4. Complete removal of Solvent

• This technique involves the complete removal of solvent from solution by evaporation
at normal pressure or under vacuum and is commonly used to obtain the amorphous
form.
• If the solvent occupies channels in the crystal structure, the structure remains intact
upon evaporation and a solvate is formed.
• Amorphous powder is formed, when the solvent is strongly bonded to the solute
molecules and the structure collapses upon evaporation. The amorphous form is
obtained upon complete removal of solvent. However, the problems, such as the
presence of organic volatile impurities (solvents), have to be dealt with, according to
FDA guidelines.
• This technique has been used to obtain anhydrous raffinose from pentahydrate,
erythromycin from dihydrate and tranilast from monohydrate.

5. Grinding
• An established method named “Grinding” is also used for polymorphic
transformations.
• Grinding has been used for sulfathiazole, barbital, cephalexin, indomethacin,
phenylbutazone and chloramphenicol.
• Although grinding of compound results in an amorphous substance, different
polymorphic forms can be obtained as reported in the case of chloramphenicol where
the metastable Forms B and C were transformed into the stable Form A upon grinding
at room temperature.
6. Lyophilization

• Lyophilization or freeze-drying, involves freezing the material followed by reducing

the surrounding pressure to allow the frozen water in the material to sublimate.
• This technique is particularly useful for compounds that decompose in the presence of
moisture but are stable as dry solids.
• This technique results in the complete removal of solvent, especially when water is used
to obtain the amorphous form.
• Lyophilization usually has three stages, freezing, primary drying and secondary drying.
• To obtain amorphous materials, rapid freezing is employed to avoid crystallization.
• E.g., Rapid freezing of a methyl p-hydroxybenzoate solution containing α-cyclodextrin
and benzoate/cyclodextrin in a ratio of 0.33 gives the amorphous form after freeze-
drying. β-Cyclodextrins have been isolated in the amorphous form by lyophilization.

7. Vapour Diffusion
 • This technique is particularly useful for the preparation of single crystals for
crystallographic analysis.
• In this method the substance is dissolved in a solvent and this solution is kept in another
larger vessel containing a small amount of a miscible and volatile anti-solvent. The
larger vessel is a desiccator and so it is then tightly closed. As a solvent equilibrium is
approached the antisolvent diffuses through the Vapour phase into the solution and
saturation or supersaturation is achieved.
• E.g., Crystals of human serum albumin have been successfully grown in a variety of
gels using crystallization conditions equivalent to those utilized in the popular hanging-
drop Vapour-equilibration method.

8. Crystallization from the Melt

• According to Ostwald’s rule, “cooling the melts of polymorphic substances often yields
the least stable modification first, which subsequently rearranges into stable forms.”
• Super-cooling is necessary for crystallization as the metastable form has a lower
melting point. The system must be super-cooled below the melting point of the
metastable form after melting and at the same time, the crystallization of the more stable
form or forms must be prevented.
• Quench cooling of the melt results in the amorphous form, which is usually obtained
by cooling the melt so quickly that crystallizing nuclei do not have sufficient time to
grow. The liquid remains a fluid below its normal freezing point. As cooling continues,
the viscosity of the liquid continues to increase and finally a glassy stage is obtained
which is the amorphous form.
• E.g., Caffeine Form I is obtained by heating Form II at 180 °C for 10 h.
Method to identify polymorphism:

1. Optical crystallography:
• Use in the identification of polymorphs crystals exists in isotropic and anisotropic
forms.
• In the isotropic crystal, the velocity of light is the same in all directions.
• Anisotropic crystals have 2 or 3 different light velocities or refractive indices.
• Video recording system and polarizing microscope fitted during heating and cooling
stage for investigating polymorph.

(a) Molecule of ROY, (b) Polymorphs of ROY: (1) Red Prisms—R, (2) Yellow Prisms—Y, (3) Orange
Needles—ON, (4) Orange Plates—OP, (5) Yellow Needles—YN, (6) Orange Red Plates—ORP.

Application:

1) To study the degree of stability of metastable form.

2) Transition temperature, Melting point, rate of transition under various thermal and
physical conditions.
3) Whether to peruse polymorphism as a route to an improved dosage form.

2. Hot stage microscope:

• Silicon oil stage microscopy is used for the detection of pseudopolymorph.
• The sample is viewed using some form of optical microscopy while a hot stage is
employed to control the temperature of the sample.
Application:

1) In the study of solid-state active pharmaceutical ingredients (APIs), excipients and

pharmaceutically relevant polymers and lipids.

3. X-ray diffraction method:

E.g., X-ray diffraction patters of silica polymorphs in silicified wood.

• It provides the most complete information about the solid-state.

• This method is based on the scattering of x-ray by crystals.
• By this method, one can identify the unit cell dimensions & conclusively establish the
crystalline lattice system & provide specific differences between crystalline forms of
a given compound.
 • In an X-ray diffraction measurement, a crystal is mounted on a goniometer and
gradually rotated while being bombarded with X-rays, producing a diffraction pattern
of regularly spaced spots known as reflections.
• It is tedious hence it is not used or unsuitable for routine use.

Application:

1) To study materials that form crystals such as salts, metals, minerals, semiconductors,
as well as various inorganic, organic and biological molecules.

4. Differential Thermal Analysis (DTA):

• The advantage is that the sample size required is only 2-5mg.

• DTA measures the tempt difference between sample and reference as a function of
temperature or time when heating at a constant rate.
• A DTA consists of a sample holder comprising thermocouples, sample containers and
a ceramic or metallic block, a furnace, a temperature programmer and a recording
system.
• The key feature is the existence of two thermocouples connected to a voltmeter. One
thermocouple is placed in an inert material such as Al2O3, while the other is placed in
a sample of the material under study.
• As the temperature is increased, there will be a brief deflection of the voltmeter if the
sample is undergoing a phase transition.

5. Differential Scanning Calorimetric (DSC):

• DSC is also like DTA except that the instrument measures the amount of energy
required to keep the sample at the same temperature as the reference i.e. it measures the
enthalpy of transition.
• When no physical or chemical changes are occurring within the sample then there is
neither a temperature change nor the need to input energy to maintain an isotherm.
• Samples that may be studied by DSC or DTA are powders, fibers, single crystals,
polymer films and semi-solids.
 • DSC measures endothermic and exothermic transitions as a function of temperature
(Endothermic heat flows into a sample, Exothermic heat flows out of the sample).

E.g., In the following example, 2.09 mg of paracetamol was heated twice from room
temperature to 200°C in aluminum crucibles and a nitrogen atmosphere. The cooling step in
between was also performed at 10 K/min. In the 1st heating step, an endothermal effect is
visible with an extrapolated onset temperature of 169°C. This correlates well to the melting
point of form I. During the subsequent controlled cooling step (not shown here), no
crystallization takes place. This means that the paracetamol is still amorphous at the beginning
of the 2nd heating step. In the 2nd heating, an exothermal effect (with a peak temperature of
82°C) appears first which is related to a cold or post crystallization process. But with increasing
heating, it turns out that there is not the same modification present as before because the
corresponding melting effect is shifted to lower values. The extrapolated onset temperature of
157°C is characteristic for form II. Thus, the metastable form II was formed during post
crystallization.

Applications of DTA / DSC in preformulating studies:

1) To determine the purity of a sample

2) To determine the number of polymorphs and to determine the ratio of each polymorph
3) To determine the heat of solvation.
4) To determine the thermal degradation of a drug or excipients.
5) To determine the glass-transition temperature(tg) of a polymer.

6. Thermo Gravimetric Analysis (TGA):

• It is a type of testing that is performed on samples to determine changes in weight in

relation to changes in temperature.
• Such analysis relies on a high degree of precision in measurements: weight and
temperature change.
 • As many weight loss curves look similar, the weight loss curve may require
transformation before results may be interpreted.

E.g., TGA curves of different polymorphs of Efavirenz

Application:

1) To determine degradation temperatures, absorbed moisture content of materials.

2) The level of inorganic and organic components in materials, decomposition points of
explosives and solvent residues.
3) It is also often used to estimate the corrosion kinetics in high temperature oxidation.

Applications of Polymorphism:

1) For improvement of therapeutic activity of the drug.

2) To prevent loss of raw material
3) For better bioavailability of the drug.

Significance of studying polymorphism:

• Different polymers exhibit different solubility, therapeutic & stability.

• The desire forms consistently can be manufactured.
• The effect of pharmaceutical manipulations is understood. E.g., Granulation, milling &
compression.
• The effect of storage condition on the dosage form can be evaluated & predicated. E.g.,
Crystal growth in suspension, cream.
• The knowledge of solid-state properties in an early stage of drug development helps to
avoid manufacturing problems, fine-tune the performance of drugs and provide space
for innovations. E.g., Famotidine which is an excellent histamine H2 receptor
antagonist is also found to exist in two different polymorphic forms, metastable
polymorph B and stable polymorph A.
• For improvement of the therapeutic activity of the drug.
 • To prevent loss of raw material.
• For better bioavailability of the drug.
• Influence on a variety of API properties including flowability, tableting, dissolution
rate, solubility, stability and even biological performance including efficacy and
toxicity. E.g., Six polymorphs of cocoa butter and all the polymorphs have significantly
different melting points
• Enhance the shelf-life of the product & will help to maintain the potency, as
planned/desired
• Metastable form shows better bioavailability and therefore preferred in formulations.

Reference:

1) Fegley's book Practical Chemical Thermodynamics for Geoscientists

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