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Chemical separation techniques play a key role in analyzing molecules or compounds used for biological, physical, and chemical research.

They are a powerful tool set to investigate mechanisms of various biological processes.

It is used to separate a mixture into its two or more distinct components, which are used further for the analysis of physical properties from the parent mixture.

This course covers fractionation, fractional distillation (Figure 1), crystallization, osmosis, dialysis, ultrafiltration.

Figure 1.Soxhlet Extractor using distillation process
Figure 1. Soxhlet Extractor using distillation process


Lyophilization is also known as Freeze-drying or cryodessication.

It is a process for removing water from the substance by the process of dehydration under vacuum while substance is in frozen state.

Lyophilization technology is used to freeze-dry products such as biologicals, bacterial cultures, analytical chemistry moieties, and therapeutic molecules (e.g., antibodies, vaccines, drugs, and heat-sensitive proteins).

Candidates for lyophilization include injectable solutions, proteins, peptides, and vaccines.


Osmosis is a movement of water from region of high water concentration to region of low water concentration through a semi-permeable membrane.

The molecules are free to pass across the cell membrane in both directions. As concentration of water is more outside the cell, it enters the cell .

It is diffusion of water molecules across a semi-permeable membrane from a region of high concentration to a region of low concentration until a state of dynamic equilibrium is reached.

Fractional Distillation

Fractional distillation is used to separate mixtures of volatile liquids and miscible liquids. If a liquid is heated in closed container, some molecules present at the surface of the liquid evaporate into the space above the liquid.

After vaporization, some of these molecules present in the vapor condense back into the liquid.

The space above the liquid is occupied by molecules of vaporized liquid, the vapor pressure above the liquid rises until it reaches a certain value.

When the pressure stabilizes, the rates of evaporation and condensation become equal and the system reaches equilibrium.


In chemical science, dialysis is the process of separation of suspended colloidal particles from dissolved ions or molecules by means of their unequal rates of diffusion through the pores of a semipermeable membrane, such as dialysis tubing.

This process was first observed in 1861 by a British chemist, Thomas Graham.

He used this technique for the separation of smaller molecules (sucrose) from the larger ones (gum Arabic) and classified solutes as crystalloids or colloids.

It has been used as tool for protein isolation and purification for long time ago. Lately, dialysis has been used for the modern biophysical techniques and separation procedures.

For example, the dialysis is used concurrently with NMR, or optical dispersion to study antibody peptides structures in solution.


Crystallization is a solid-liquid separation technique, in which mass transfer of solute from liquid solution (parent phase) to a pure solid crystalline phase occurs.

It is used to purify solid compounds. Crystallization is an oldest method for recovering solids from a solution.

More than 80% of the substances used in pharmaceuticals, chemicals, agrochemicals, food, and cosmetics are isolated or formulated in their solid form by this method.

Crystallization is in general last chemical purification step in the production of ingredients.


Fractionation process of breakdown of solid, liquid, solute, or solution into smaller fragments during their phase transition.

Salt Fractionation

Salt fractionation is a method of separation of components of a mixture using salt, such as sodium sulfate, sodium sulfite, sodium chloride, sodium citrate, or ammonium sulfate. It is also known as “salting out”.

This process is based upon the difference in solubility of various protein fractions to different concentrations of salt.

It is a spontaneous process, when appropriate concentration of the salt is reached in solution. This process of separation of organic phase from aqueous phase by the addition of a salt has been known since long time.

Ammonium sulfate precipitation is the most commonly used procedure for salt fractionation.

Application of Salt Fractionation

  • Salt fractionation is most commonly used for protein purification, differential plasma protein estimation, and precipitation of the target protein.
  • It provides analysis of fundamental aspects determining structure and physicochemical properties of proteins.
  • In biochemical research, it has been used to study the chromatin structure and purification of bovine lactate dehydrogenase enzyme.
  • It has been used for constructing genome maps to study physical properties of chromatin in drosophila and extraction of DNA molecule from human nucleated cells.
  • Salting out of cellular proteins is done by dehydration and precipitation of those proteins with a saturated sodium chloride solution.
  • It is used for solvent extraction in organic chemistry laboratory. This process is used in soap industry to manufacture soap.
  • In medicine, salting out is also known as saline abortion or saline amniocentesis. It is second most common method of inducing abortion.