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Usman Yousaf

Introduction to Purification
Many biological processes
require a purification scheme to
reduce the fermentation broth to
its pure final product. Once
product is made in the
production fermenter, the broth
is still highly contaminated.

Factors for deciding the extraction

The value of the final product.
The degree of purity required.
The chemical and physical properties of the
The location of the product in the mixture i.e.
whether it is free within the medium or is cellbound.
The location and properties of the impurities.
The cost-effectiveness of the available alternate
isolation procedures.

Precipitation is a procedure where the addition of a ionic solution
to an ionic fermentation broth forms insoluble particles, where
the desired product is usually contained within those particles.
Ionic fermentation broths usually consist of enzymes or proteins.
The ways to precipitate out a product can vary from simple pH
and temperature changes to chemical reactions involving metal
ions. Precipitation reactions are carried out in reactors,
continuous and batch.

Typical agents used in precipitation to render the compound of
interest insoluble, and these include:
Acids and bases
Salts such as ammonium and sodium sulphate
Organic solvents
Non-ionic polymers
Protein binding dyes

Coagulation and Flocculation

Coagulation is defined for biological processes to be when small

particles directly adhere to each other, while flocculation is when
an agent acts as a bridge that joins particles together.
Coagulation and flocculation techniques are usually applied to
either whole cells, cell debris, or soluble proteins.

Filters use a filter cloth or some porous material along with
applied pressure to push smaller particles through the filter, thus
separating elements of the solution based on size. Filtration for
biological materials is generally completed using batch filtration,
rotary drum filtration, or ultra filtration methods e.g..,
Batch Filtration
Rotary Drum Filtration
Ultra filtration

Centrifugation involves separation of liquids and particles
based on density. Centrifugation can be used to separate cells
from a culture liquid, cell debris from a broth, and a group of

Tubular Bowl Centrifuge

Disc Bowl Centrifuge
Perforate Bowl Basket Centrifuge

Although a centrifuge may be expensive when compared with a filter it
may be essential when:

1. Filtration is slow and difficult

2. The cells or other suspended matter must be
obtained free of filter aids
3. Continuous separation to a high standard of
hygiene is required

purification of products in the

soluble portion

Cell disruption

oMicro-organisms are protected by extremely tough cell walls. In order to release their
cellular contents a number of methods for cell disintegration

Physico-mechanical Method:

Liquid Shear
Solid Shear
Agitation with abrasives

Chemical Methods:
Osmotic shock
Alkali Treatment

Cell Disruption
Liquid Shear

Cell Disruption
Freeze Thawing:
Ball Mill
Solid Frozen cell paste, cells attached to or within a solid
Large scale

Cell Disruption
Challenge: Damage to the product
- Heat denaturation
- Oxidation of the product
- Unhindered release of all intracellular products

Separation of Soluble Products

To separate the solutes based on the different rate of movement
of the solutes in the column with adsorbent materials.
Chromatographic processes involve a stationary phase and a
mobile phase.
Stationary phase can be adsorbent, ion-exchange resin, porous
solid, or gel usually packed in a cylindrical column.
Mobile phase is the solution containing solutes to be separated
and the eluant that carriers the solution through the stationary
Applicable for protein, organics separation.

Summary of separation and

Liquid-Solid Separation
- Filtration: micro- and ultra- filtration
- Centrifugation
Cell disruption
- Mechanical: ultrasonication, milling, homogenization
- Nonmechanical: chemicals, enzyme and osmotic

Summary of separation and

Separation of soluble products
- Precipitation
- Adsorption
- Membrane separation: ultra filtration, dialysis, reverse
- Chromatography
- Electrophoresis
Crystallization and drying