Crystallization
High-Performance Liquid Chromatography
(HPLC)
Crystallization
Crystallization is a separation and
purification process where solid crystals
are formed from a solution.
• It is widely used in chemical,
pharmaceutical, petrochemical, and food
industries
• The process involves controlled
solidification of a solute from a solvent to
form well-defined crystals.
Objectives of Crystallization
• To purify solid compounds by forming
pure crystals
• To separate solids from a solution
• To produce crystals of desire size and
shape(slow cooling or low
supersaturation – big crystals & fast
cooling or high mixing – small crystals)
Crystallization
• Crystallization is a separation and purification process
where solid crystals are formed from a solution
Stages Types
Cooling Evaporative Precipitate Antisolvent
Supersatura Crystal crystallizati crystallizati crystallizati
Nucleation crystallizati
ted Growth on on on
on
A solution Small solid Solute crystals
crystals
contains particles particles form by Two Add a liquid
are formed
more solute start attach to the removing chemicals that makes the
by lowering
than it can forming in nuclei, and solvent react and solute
the
normally the crystals through form a solid less soluble,
dissolve. temperate
supersatura grow larger. evaporation and crystals
te solution. form
It is the first Controlled
It is the conditions Solubility ↓ solute Based on
step where Based on
driving force help form when concentrati solubility
for crystal crystals solubility
begin to pure and Temperatur on &chemical
formation change
appear uniform e↓ increases reaction
crystals
Principle
Solubility: Amount of solute that can dissolve in a
solvent at a specific temperature
Supersaturation: Key driving force; A condition where
a solution contains more solute than it can normally
dissolve at a given temperature
Two key steps:
1. Nucleation - Initial formation of crystal nuclei.
2. Crystal Growth - Growth of nuclei into larger crystals
Types
• Cooling crystallization
• Evaporation crystallization
• Precipitate crystallization
• Antisolvent crystallization
Cooling Crystallization
Cooling crystallization is a process where crystals are formed by
lowering the temperature of a saturated solution, causing the
solute to become less soluble and separate out as crystals.
Principle:
Solubility ↓ when Temperature ↓ (for most solutes).When a hot
saturated solution is cooled, it becomes supersaturated, and
crystals begin to form.
Process Steps:
1. Prepare a hot saturated solution of the solute.
2. Cool the solution gradually.
3. Crystals form as the solution becomes supersaturated.4.
Filter and dry the crystals.
Advantages:
• Simple and cost-effective.
• Suitable for temperature-sensitive materials
• Produces high-purity crystals
Evaporative
crystallization
Evaporative crystallization is a process where crystals form by removing
solvent (usually water) through evaporation, leading to supersaturation of
the solution and crystal formation
Principle:
• When solvent evaporates, the solute concentration increases
• Once it reaches supersaturation, crystals begin to form.
• This method does not rely on temperature drop, but on solvent loss
Process Steps:
1. Prepare a saturated solution.
2. Heat or reduce pressure to evaporate the solvent.
3. As solvent evaporates, supersaturation is reached.
4. Crystals nucleate and grow.
5. Crystals are separated by filtration or centrifugation
Advantages:
Suitable for substances with solubility less affected by temperature, Can be
applied to large-scale continuous processes.
Precipitate
crystallization
Precipitate crystallization occurs when two or more solutions
react chemically and produce an insoluble compound that
forms crystals (precipitate)
Principle:
• Based on the solubility product .
• This is a chemical reaction-based crystallization (not based
on cooling or evaporation).
Process:
1. Two reactants in solution are mixed.
2. A new, insoluble product forms.
3. This product crystallizes out of the solution.
Advantages:
• Useful when cooling is ineffective.
• Suitable for substances with solubility less affected by
temperature
• Can be applied to large-scale continuous processes.
Antisolvent
crystallization
Antisolvent crystallization is a method where a solvent
that does not dissolve the solute (called an
antisolvent) is added to the solution, making the solute
less soluble and forcing it to crystal out
Principle
based on solubility change (solubility decrease, its
solution becomes supersaturated)
Process
1. Prepare a solution of the solute in a good solvent.
2. Add antisolvent slowly under controlled mixing.
3. Supersaturation occurs → crystal formation begins.
4. Crystals are separated by filtration or centrifugation
Advantages:
• Easy to control crystal size
• Purity and shape control
• Scalable and reproducible
HPLC – High Performance Liquid Chromatography
HPLC is an advanced form of liquid chromatography used to
separate, identify, and quantify components in a liquid mixture
with high resolution and efficiency.
Principle
Separation occurs based on differences in interactions (partitioning
or adsorption) between the stationary phase and mobile phase.
Components travel at different speeds depending on their affinity
to each phase
Basic Components:
1. Solvent – Contains mobile phase.
2. Pump – Forces mobile phase
through the system at high
pressure.
3. Injector – Introduces the sample
into the system.
4. Column – Packed with stationary
phase(10-30 cm length,4.6mm
diameter & particle size 3-10um)
5. Detector – detects compounds as
they come out of the column (most
commonly used in UV- Vis Detector)
RT- Retention
time(TRThe
) amount of Depending upon Variable
the chemical depending upon
time compound
composition and the length and
spends in the
orientation the load of the
column after it
retention time is stationary phase
has been
very specific for and diameter of
injected
each compound the column
RRT- Relative Retention Time
• Relative retention times
are a ratio with reference
to another peak in the
chromatogram.
• Relative retention times
are calculated against a
selected reference peak
RF – Response Factor
• RF is a measure of how a detector responds to a specific
compound. It's calculated by dividing the peak area (obtained
from HPLC) by the concentration of that compound in the
sample.
RF = Peak Area / Concentration
RRF – Relative Response Factor
• RRF compares the response of a substance (often an impurity)
to the response of a reference standard (usually the main
active ingredient or API).
RRF = RF of impurity /RF of reference standard (or)
Slope of impurity /Slope of API
Types of HPLC:
Based on Stationary Phase:
• Normal Phase HPLC – Polar stationary phase (e.g., silica).
• Reverse Phase HPLC – Non-polar stationary phase (e.g., C18).
Applications :
• Pharmaceuticals – Drug purity,
assay, stability studies.
• Food Industry – Detect
contaminants, preservatives.
• Environmental – Pollutant detection
in water and air.
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