VII Sem. B.Tech., Biotechnology BT-8751 Downstream Processing
VEL TECH HIGH TECH
Dr. RANGARAJAN Dr. SAKUNTHALA ENGINEERING COLLEGE
Department of Biotechnology
SUBJECT : Downstream Processing CODE : BT 8751
BRANCH : B.Tech Biotechnology SEMESTER : VII
UNIT V FINAL PRODUCT FORMULATION AND FINISHING OPERATIONS 9
Crystallization, drying and lyophilization in final product formulation.
Crystallization
Crystallization is an example of a separation process in which mass is transferred from a liquid
solution, whose composition is generally mixed, to a pure solid crystal. Soluble components are
removed from solution by adjusting the conditions so that the solution becomes supersaturated and
excess solute crystallizes out in a pure form. This is generally accomplished by lowering the
temperature, or by concentration of the solution, in each case to form a supersaturated solution
from which crystallization can occur. The equilibrium is established between the crystals and the
surrounding solution, the mother liquor. The manufacture of sucrose, from sugar cane or sugar
beet, is an important example of crystallization in food technology. Crystallization is also used in
the manufacture of other sugars, such as glucose and lactose, in the manufacture of food additives,
such as salt, and in the processing of foodstuffs, such as ice cream. In the manufacture of sucrose
from cane, water is added and the sugar is pressed out from the residual cane as a solution. This
solution is purified and then concentrated to allow the sucrose to crystallize out from the solution.
Various steps involved in crystallization
The crystallization process consists of two major events, nucleation and crystal growth.
Nucleation is the step where the solute molecules dispersed in the solvent start to gather into
clusters, on the nanometer scale (elevating solute concentration in a small region), that becomes
stable under the current operating conditions. These stable clusters constitute the nuclei. However
when the clusters are not stable, they redissolve. Therefore, the clusters need to reach a critical size
in order to become stable nuclei. Such critical size is dictated by the operating conditions
(temperature, supersaturation, etc.). It is at the stage of nucleation that the atoms arrange in a
defined and periodic manner that defines the crystal structure note that "crystal structure" is a
special term that refers to the relative arrangement of the atoms, not the macroscopic properties of
VTHT Department of Biotechnology
1
,VII Sem. B.Tech., Biotechnology BT-8751 Downstream Processing
the crystal (size and shape), although those are a result of the internal crystal structure.
The crystal growth is the subsequent growth of the nuclei that succeed in achieving the critical
cluster size. Nucleation and growth continue to occur simultaneously while the supersaturation
exists. Supersaturation is the driving force of the crystallization, hence the rate of nucleation and
growth is driven by the existing supersaturation in the solution. Depending upon the conditions,
either nucleation or growth may be predominant over the other, and as a result, crystals with
different sizes and shapes are obtained (control of crystal size and shape constitutes one of the
main challenges in industrial manufacturing, such as for pharmaceuticals). Once the
supersaturation is exhausted, the solid-liquid system reaches equilibrium and the crystallization is
complete, unless the operating conditions are modified from equilibrium so as to supersaturate the
solution again.
Many compounds have the ability to crystallize with different crystal structures, a
phenomenon called polymorphism. Each polymorph is in fact a different thermodynamic solid
state and crystal polymorphs of the same compound exhibit different physical properties, such as
dissolution rate, shape (angles between facets and facet growth rates), melting point, etc. For this
reason, polymorphism is of major importance in industrial manufacture of crystalline products.
Advantages of crystallization in bioprocess with applications
High purity of proteins/drugs
Impurities would normally not fit as well in the lattice, and thus remain in solution
preferentially
This gives much better control over crystal size, morphology and consistent crystal
products
The product generally has a somewhat wide crystal-size distribution.
Supersaturation
As mentioned above, a crystal is formed following a well-defined pattern, or structure,
dictated by forces acting at the molecular level. As a consequence, during its formation process the
crystal is in an environment where the solute concentration reaches a certain critical value, before
changing status. Solid formation, impossibile below the solubility threshold at the given
temperature and pressure conditions, may then take place at a concentration higher than the
theoretical solubility level. The difference between the actual value of the solute concentration at
the crystallization limit and the theoretical (static) solubility threshold is called supersaturation and
is a fundamental factor in crystallization dynamics. Supersaturation is the driving force for both
VTHT Department of Biotechnology
2
, VII Sem. B.Tech., Biotechnology BT-8751 Downstream Processing
the initial nucleation step and the following crystal growth, both of which could not occur in
saturated or undersaturated conditions.
Nucleation
Nucleation is the onset of a phase transition in a small region. The phase transition can be the
formation of a bubble or of a crystal from a liquid. Creation of liquid droplets in saturated vapor or
the creation of gaseous bubbles in a saturated liquid is also characterized by nucleation (see Cloud
condensation nuclei). Nucleation of crystalline, amorphous, and even vacancy clusters in solid
materials is also important, for example to the semiconductor industry. Nucleation normally occurs
at nucleation sites on surfaces containing the liquid or vapor. Suspended particles or minute
bubbles also provide nucleation sites. This is called heterogeneous nucleation. Nucleation without
preferential nucleation sites is homogeneous nucleation. Homogeneous nucleation occurs
spontaneously and randomly, but it requires superheating or supercooling of the medium.
Nucleation is involved in such processes as cloud seeding and in instruments such as the bubble
chamber and the cloud chamber.
Primary nucleation
The second category, then, is heterogeneous nucleation. This occurs when solid particles of foreign
substances cause an increase in the rate of nucleation that would otherwise not be seen without the
existence of these foreign particles. Homogeneous nucleation rarely occurs in practice due to the
high energy necessary to begin nucleation without a solid surface to catalyse the nucleation.
Secondary nucleation
Secondary nucleation is the formation of nuclei attributable to the influence of the existing
microscopic crystals in the magma. The first type of known secondary crystallization is
attributable to fluid shear, the other due to collisions between already existing crystals with either a
solid surface of the crystallizer or with other crystals themselves. Fluid shear nucleation occurs
when liquid travels across a Crystal at a high speed, sweeping away nuclei that would otherwise be
incorporated into a Crystal, causing the swept-away nuclei to become new crystals. Contact
nucleation has been found to be the most effective and common method for nucleation.
VTHT Department of Biotechnology
3
VEL TECH HIGH TECH
Dr. RANGARAJAN Dr. SAKUNTHALA ENGINEERING COLLEGE
Department of Biotechnology
SUBJECT : Downstream Processing CODE : BT 8751
BRANCH : B.Tech Biotechnology SEMESTER : VII
UNIT V FINAL PRODUCT FORMULATION AND FINISHING OPERATIONS 9
Crystallization, drying and lyophilization in final product formulation.
Crystallization
Crystallization is an example of a separation process in which mass is transferred from a liquid
solution, whose composition is generally mixed, to a pure solid crystal. Soluble components are
removed from solution by adjusting the conditions so that the solution becomes supersaturated and
excess solute crystallizes out in a pure form. This is generally accomplished by lowering the
temperature, or by concentration of the solution, in each case to form a supersaturated solution
from which crystallization can occur. The equilibrium is established between the crystals and the
surrounding solution, the mother liquor. The manufacture of sucrose, from sugar cane or sugar
beet, is an important example of crystallization in food technology. Crystallization is also used in
the manufacture of other sugars, such as glucose and lactose, in the manufacture of food additives,
such as salt, and in the processing of foodstuffs, such as ice cream. In the manufacture of sucrose
from cane, water is added and the sugar is pressed out from the residual cane as a solution. This
solution is purified and then concentrated to allow the sucrose to crystallize out from the solution.
Various steps involved in crystallization
The crystallization process consists of two major events, nucleation and crystal growth.
Nucleation is the step where the solute molecules dispersed in the solvent start to gather into
clusters, on the nanometer scale (elevating solute concentration in a small region), that becomes
stable under the current operating conditions. These stable clusters constitute the nuclei. However
when the clusters are not stable, they redissolve. Therefore, the clusters need to reach a critical size
in order to become stable nuclei. Such critical size is dictated by the operating conditions
(temperature, supersaturation, etc.). It is at the stage of nucleation that the atoms arrange in a
defined and periodic manner that defines the crystal structure note that "crystal structure" is a
special term that refers to the relative arrangement of the atoms, not the macroscopic properties of
VTHT Department of Biotechnology
1
,VII Sem. B.Tech., Biotechnology BT-8751 Downstream Processing
the crystal (size and shape), although those are a result of the internal crystal structure.
The crystal growth is the subsequent growth of the nuclei that succeed in achieving the critical
cluster size. Nucleation and growth continue to occur simultaneously while the supersaturation
exists. Supersaturation is the driving force of the crystallization, hence the rate of nucleation and
growth is driven by the existing supersaturation in the solution. Depending upon the conditions,
either nucleation or growth may be predominant over the other, and as a result, crystals with
different sizes and shapes are obtained (control of crystal size and shape constitutes one of the
main challenges in industrial manufacturing, such as for pharmaceuticals). Once the
supersaturation is exhausted, the solid-liquid system reaches equilibrium and the crystallization is
complete, unless the operating conditions are modified from equilibrium so as to supersaturate the
solution again.
Many compounds have the ability to crystallize with different crystal structures, a
phenomenon called polymorphism. Each polymorph is in fact a different thermodynamic solid
state and crystal polymorphs of the same compound exhibit different physical properties, such as
dissolution rate, shape (angles between facets and facet growth rates), melting point, etc. For this
reason, polymorphism is of major importance in industrial manufacture of crystalline products.
Advantages of crystallization in bioprocess with applications
High purity of proteins/drugs
Impurities would normally not fit as well in the lattice, and thus remain in solution
preferentially
This gives much better control over crystal size, morphology and consistent crystal
products
The product generally has a somewhat wide crystal-size distribution.
Supersaturation
As mentioned above, a crystal is formed following a well-defined pattern, or structure,
dictated by forces acting at the molecular level. As a consequence, during its formation process the
crystal is in an environment where the solute concentration reaches a certain critical value, before
changing status. Solid formation, impossibile below the solubility threshold at the given
temperature and pressure conditions, may then take place at a concentration higher than the
theoretical solubility level. The difference between the actual value of the solute concentration at
the crystallization limit and the theoretical (static) solubility threshold is called supersaturation and
is a fundamental factor in crystallization dynamics. Supersaturation is the driving force for both
VTHT Department of Biotechnology
2
, VII Sem. B.Tech., Biotechnology BT-8751 Downstream Processing
the initial nucleation step and the following crystal growth, both of which could not occur in
saturated or undersaturated conditions.
Nucleation
Nucleation is the onset of a phase transition in a small region. The phase transition can be the
formation of a bubble or of a crystal from a liquid. Creation of liquid droplets in saturated vapor or
the creation of gaseous bubbles in a saturated liquid is also characterized by nucleation (see Cloud
condensation nuclei). Nucleation of crystalline, amorphous, and even vacancy clusters in solid
materials is also important, for example to the semiconductor industry. Nucleation normally occurs
at nucleation sites on surfaces containing the liquid or vapor. Suspended particles or minute
bubbles also provide nucleation sites. This is called heterogeneous nucleation. Nucleation without
preferential nucleation sites is homogeneous nucleation. Homogeneous nucleation occurs
spontaneously and randomly, but it requires superheating or supercooling of the medium.
Nucleation is involved in such processes as cloud seeding and in instruments such as the bubble
chamber and the cloud chamber.
Primary nucleation
The second category, then, is heterogeneous nucleation. This occurs when solid particles of foreign
substances cause an increase in the rate of nucleation that would otherwise not be seen without the
existence of these foreign particles. Homogeneous nucleation rarely occurs in practice due to the
high energy necessary to begin nucleation without a solid surface to catalyse the nucleation.
Secondary nucleation
Secondary nucleation is the formation of nuclei attributable to the influence of the existing
microscopic crystals in the magma. The first type of known secondary crystallization is
attributable to fluid shear, the other due to collisions between already existing crystals with either a
solid surface of the crystallizer or with other crystals themselves. Fluid shear nucleation occurs
when liquid travels across a Crystal at a high speed, sweeping away nuclei that would otherwise be
incorporated into a Crystal, causing the swept-away nuclei to become new crystals. Contact
nucleation has been found to be the most effective and common method for nucleation.
VTHT Department of Biotechnology
3
