BNQ10204

ANALYTICAL AND ORGANIC CHEMISTRY

CHAPTER 10:
BASIC TOOLS AND
UNIT OPERATIONS
By: Dr Nadirul Hasraf bin Mat Nayan
Outline
10.1 Introduction to Unit Operations
in the Biotechnology Industry

10.2 Separation Processes

10.3 Purification Methods

10.4 Reaction Processes

10.1
Introduction to Unit Operations in the Biotechnology Industry
 What is biotechnology?

“Biotechnology is the application of biological organisms,

systems, or processes by various industries to learning
about the science of life and the improvement of the value of
materials and organisms such as pharmaceuticals, crops,
and livestock.

It is a relatively new and fast-developing field that integrates

knowledge from several traditional sciences: biochemistry,
chemistry, microbiology, and chemical engineering.”
- American Chemical Society

4
Products of Industrial biotechnology
Organic acids and
Polymers
Alcohols

Microbial biomass Amino acids

Primary Vitamins and
metabolites Nutraceuticals
Secondary Antibiotics
metabolites
Biopharmaceuticals
Microbial Enzymes
Enzymes
Recombinant
proteins SCPs, Biofuels and
Bioenergy
Biotransformation Fine and specialty
chemicals

New materials
 Bioproducts - Examples

3
Recent breakthrough – vaccine invented by Australians

• The first (preventiveand

therapeutic) cancer vaccine

- Gardasil was developed by

Prof Ian Fraser and Dr Jian Zou in
Australia

- Gardasil is manufactured by Merk in US

-The vaccine protects women against
Virus-like particles as
four HPV strains responsible for 70% vaccines
of cervical cancers

Biochemical engineers play a key role in

making this evolutionary vaccine.
7
 Upstream
process

Fermentation
process
THREE
STAGES

Downstream
process
What do you mean by upstream & downstream process?

❖ The upstream processing in biotechnology involves identifying and extracting the

raw materials. This forms the initial process of fermentation.

Upstream process-it deals with the:

• Inoculum preparation which includes screening or microorganisms and selection of

suitable strain and genetic modification of the organism if needed.
• Preparation of culture media having suitable growth parameters at laboratory scale
• Scale up of the entire process.
• Inoculation.
Downstream process-

❖ When the products are subjected to a series of processes including separation and purification
which are collectively known as Downstream processing.
❖ It is also known as product recovery.
❖ Materials –upstream-finished products

The downstream processing deals with:

• Solid-liquid separation
• Release of intracellular products
• Concentration
• Purification
• Formulation
 DOWNSTREAM PROCESS

5.
FORMULATION

1.
4. DISINTEGRATIO
PURIFICATION
MULT N OR CELL-
DISRUPTION.
I
STAG
E
OPERATIO
2.SOLID-LIQUID
3. N OR
CONCENTRATION CLARIFICATIO
N
10.2
Separation Processes
 INTRODUCTION
Separation is simply the process of dividing material into its
component parts.

Separation techniques are essentially methods of purification.

Homogeneous and heterogeneous mixtures can be separated

into their components by several physical methods

The choice of separation techniques is based on the type of

mixture and difference in the chemical properties of the
constituents of a mixture.
 CLASSIFICATION OF SEPARATION
PROCESS
Based on the nature or physical mechanism of separation, various separation
processes can be classified into,
1) Mechanical separations: separations based on size and/or density
differences of different components in a mixture, for separation of solid
from liquid (e.g. filtration and centrifugation).

2) Diffusional separations (mass transfer operations): separations based on

molecular movement toward a favourable phase, for separation of
dissolved components (e.g. distillation, absorption, extraction). (“Mass
transfer is the transfer of solute molecules from one point to anther or
from one phase to another.”)

3) Membrane separations: use of a semipermeable membrane to separate

molecules with difference in size or some other properties.
VARIOUS TYPES OF SEPARATION PROCESSES ARE:

Separating Funnel
Centrifugation
Evaporation
Crystallization
Magnetic separation
Filtration
Sedimentation
Distillation
Membrane Separations
Chromatography
SeparatingSEPARATING FUNNEL
Funnel:
A separating funnel is used for the separation of components of a
mixture between two immiscible liquid phases. One phase is the
aqueous phase and the other phase is an organic solvent. This
separation is based on the differences in the densities of the
liquids. The liquid having more density forms the lower layer
and the liquid having less density forms the upper layer.
Applications:
To separate a mixture of oil and water.
To separate a mixture of kerosene
oil and water.
Centrifugation:
CENTRIFUGATION
Centrifugation is the process of separation of insoluble materials
from a liquid where normal filtration does not work well. The
centrifugation is based on the size, shape, and density of the
particles, viscosity of the medium, and the speed of rotation. The
principle is that the denser particles are forced to the bottom and
the lighter particles stay at the top when spun rapidly.
Evaporation :
EVAPORATION
The general definition of evaporation is the loss or disappearance
of a liquid due to vaporization. In the process industry,
evaporation process is to concentrate a solution (of a non-volatile
solute) or to separate a volatile solvent from a non-volatile solute,
by vaporizing and removing part of the solvent (mostly water). In
an evaporation process, the liquid solution is usually heated to
boiling by steam.
 MAGNETIC SEPARATION
Magnetic separation is a process in which magnetically
susceptible material is extracted from a mixture using a magnetic
force. This separation technique can be useful in mining iron as it
is attracted to a magnet.
 FILTRATION
Filtration is the mechanical separation of solid particles from a
fluid by passing the fluid through a filtering medium,on which
the solids are deposited. The most common filtering medium is
fabric cloth with strong mechanical properties.

Filtration is any of various mechanical, physical or biological

operations that separate solids from fluids (liquids or gases) by
adding a medium through which only the fluid can pass.
Hot filtration method is mainly used to separate solids from a
hot solution.

Cold Filtration method is the use of ice bath in order to rapidly

cool down the solution to be crystallized rather than leaving it
out to cool it down slowly in the room temperature

Vacuum Filtration technique is most preferred for small batch

of solution in order to quickly dry out small crystals. This
method requires a Büchner funnel, filter paper of smaller
diameter than the funnel, Büchner flask, and rubber tubing to
connect to vacuum source.
Sedimentation
SEDIMENTATION
Sedimentation relies on gravity to separate suspended solids
from fluids. It is accomplished by decreasing the velocity of the
fluid being treated to a point below which the particles will no
longer remain in suspension. When the velocity no longer
supports the transport of the particles, gravity will remove them
from the flow.
 DISTILLATION
Distillation

• Distillation is a process of separating the component or substances

from a liquid mixture by selective evaporation and condensation.
•The application of distillation can roughly be divided in two groups:
laboratory scale, industrial distillation.
The main difference between laboratory scale distillation and
industrial distillation is that laboratory scale distillation is often
performed batch-wise, whereas industrial distillation often occurs
continuously.

Type of distillation
Simple distillation.
Fractional distillation.
Steam distillation.
Vacuum distillation.
Membrane Separations
MEMBRANE SEPARATION
Separation by the use of membranes has been increasingly used in
the chemical and bioprocess industry. In membrane separation, the
membrane acts as a semipermeable barrier which only allows for
certain molecules to pass through it

1. Ultrafiltration
Ultrafiltration (UF) is used for the separation of macromolecules
(polymers) such as proteins, with molecular weights 1000-50,000.
It is a high-pressure membrane process, up to 145 psi (10 bar).
2. Reverse osmosis

Osmosis (as a natural phenomenon) is the flow (diffusion) of water

molecules through a semi-permeable membrane from low-solute
concentration side to high-solute concentration side of the
membrane.

Reverse osmosis is the use of high pressure to force the flow of

solvent (e.g., water) molecules in the reverse direction of osmotic
pressure. Applications of reverse osmosis include: water
purification, sterilization, dewatering and the separation of
components in a mixture
 NOVEL SEPARATION PROCESSES
The separation processes those are not conventional and routine
fall under this category. Therefore, some of the equilibrium and
rate governed separation processes are included this.

Some of the processes are identified as,

(i) Membrane based separation processes
(ii) Chromatographic separation processes
(iii) Supercritical Fluid Extraction
(iv) Electric field assisted separation processes
(v) Ion exchange processes, etc.
 Chromatographic Separation Processes:-

i. Chromatography is an extremely powerful analytical tool for

separating and analyzing complex mixture.
ii. Different types of chromatographic techniques such as column
chromatography, TLC, paper chromatography, and gas
chromatography.

Fig:- Chromatography Separation Technique

Paper chromatography is one of the important
chromatographic methods. Paper chromatography uses paper as the
stationary phase and a liquid solvent as the mobile phase. In paper
chromatography, the sample is placed on a spot on the paper and
the paper is carefully dipped into a solvent. The solvent rises up the
paper due to capillary action and the components of the mixture rise
up at different rates and thus are separated from one another.

Fig:- Paper Chromatography

Supercritical Fluid Extraction

Supercritical Fluid Extraction (SFE) is the process of separating

one component (the extractant) from another (the matrix) using
supercritical fluids as the extracting solvent. Extraction is usually
froma solid matrix, but can also be from liquids.

Carbon dioxide (CO2) is the most used supercritical fluid,

sometimes modified by co-solvents such as ethanol or methanol.
Extraction conditions for supercritical carbon dioxide are above
the critical temperature of 31 °C and critical pressure of 74 bar.

The system must contain a pump for the CO2, a pressure cell to
contain the sample, a means of maintaining pressure in the system
and a collecting vessel.
 Ion Exchange Chromatography

The most popular method for the purification of proteins. IEC can
be divided into two different sub types.

a. Cation exchange chromatography positively charged molecules

are attracted to a negatively charged.

b. Anion exchange chromatography, negatively charged molecules

are attracted to a positively charged.

After the molecule of interest has been adsorbed, the column is

washed to remove any residual unbound species from the solid
phase.
Application :

Used for biopurification

Used in downstream processing platforms.
10.3
Purification Methods
1) Purification of solid organic compounds

• Purification organic compounds is

of importantbecause, organic compound
made in the laboratory contains
impurities.

• The common methods for purification are

crystallisation, sublimation, distillation,
chromatography, etc.
 Crystallization

• Crystallization is the process of forming crystals

Crystallization by Cooling a Hot Concentrated Solution

• In this method the impure substance is dissolved in water or

some other suitable organic solvent such as alcohol, petrol, etc.,
• The solution is filtered to remove any suspended impurities. The
filtrate is heated over a water bath so that the vapors of the
solvent may not catch fire.
• Then the hot solution of the organic compound is cooled, so that
the crystals begin to separate out.
• The crystals are removed by filtration and the impurities are
passed on to the filtrate.
Crystallization by Cooling a
Hot Concentrated Solution
 Recrystallisation
• Recrystallisation: It is a method used to
purify an organic solid.
• A small amount of the solvent is added to a
flask containing an impure solid. The
contents of the flask are heated until the
solid dissolves. Then the solution is cooled. A
more pure solid separates out, leaving
impurities dissolved in the solvent.
 Sublimation
• Sublimation is the direct change of a solid to vapor on
heating without going through the liquid state

• Used to separate volatile organic compounds from non volatile

impurities e.g. Naphthalene, benzoic acid, anthracene, camphor.

• The substances that sublime can be purified by this method, provided

the impurities present does not sublime.
• Many organic compounds directly form vapors, when solid
compounds are heated, without becoming a liquid at any stage. On
cooling the vapors the solid is directly obtained.
 Sublimation

A mixture of two compounds can be

separated by sublimation
2) Purification of Liquid organic compounds
• Organic compounds in the liquid state are purified by
distillation.
• Distillation involves the heating of a liquid to boiling
and then collecting their vapours to condense them in
liquid state.
 Simple Distillation
Simple distillation is designed to evaporate a volatile
liquid from a solution of non-volatile substances; the
vapor is then condensed in the water condenser and
collected in the receiver.
 Fractional Distillation
If the boiling point of the liquids in the mixture are very close to
each other then such mixtures can be purified by fractional
distillation. The difference in boiling points of the mixture is
usually less than 40ºC.
e.g. – acetone(b.p. 56ºC)and methyl alcohol(b.p. 65ºC).
For fractional distillation, a suitable fractionating column is
placed between the flask and the condenser.
Distillation under reduced Pressure (or) Vacuum distillation

• Vacuum distillation- It is used for organic

compounds which decompose at or below
their boiling points.
Example: Glycerol
Distillation under reduced pressure
• The boiling pointof a liquid is the
temperature at which the total
pressure is equal to the vapour
external pressure.
This means that by lowering the pressure the
boiling point of the liquid can be lowered.
 Extraction
• Extraction is a very common laboratory procedure
used when isolating or purifying a product

• In organic laboratory, liquid-liquid extraction is most

commonly used. Liquid-liquid extraction requires two
immiscible liquids known as the organic phase and the
aqueous phase. The aqueous phase is water-based
and the organic phase is an organic solvent.
 Criteria and test for purity
• The following physical properties are
used for checking the purity of substances
(a) Melting point
(b) Boiling point
(d) Density
These properties have long been utilized in
identification and characterisation of organic
compounds
 Criteria for purity
• The purity of an organic compound can be checked
by the following criteria:
(a)Sharp melting point which does not change on
further purification.
(b)In case of solids, mix the solid with known sample
of pure compound and its melting point is noted.
The m.pt should remain the same.
(c) Concordant boiling point
(d) Definite crystalline shape.
10.4
Reaction Processes
Tools of Industrial biotechnology

♣ Genetic Engineering

♣ Protein Engineering

♣ Metabolic Engineering

♣ New developments in Synthetic biology

♣ System biology- Omics and inSilico Approaches

Relation between Biotechnology and Bioreactor
• Biotechnology is a field which deals with obtaining substances
and products which are necessary for humans by using
microorganisms. Biotechnology has contributed to the food
industry, agriculture, medicine, environment protection,
energetics, and chemical industry.

• Bioreactors provide a controlled environment for optimal

growth of microbes. They are used for carrying out
biochemical processes that employ microbes, mammalian cell
systems, plant cells, or fungus for production of biological
products.
 BIOREACTOR

A bioreactor may refer to a device or system meant to grow animal cells or

tissues in the context of cell culture. These devices are being developed for
use in tissue engineering or biochemical engineering.

FERMENTOR

Fermenters are well established for the cultivation of microbes ,proteins

,industrial product(acetic acid, alcohol etc.) under monitored ,controlled
environmental and operational conditions up to an industrial scale.
o FERMENTOR
Closed vessels
Used for fermentation
Production of large scale process
Fermenter is a closed vessels now it is called as bioreactor
Fermenter is a old process used for cultivate the
microorganisms
Fermenter used for cultivate Prokaryotic cells (fungi bacteria)
Bioreactors used for cultivate eukaryotic cells (mammalian
,insects)
 Fermentation is a metabolic
process

convert
sugar

acid gas alcohol

Occur by yeast and bacteria

STRUCTURE OF
FERMENTER
 Components of
Fermenter
Parts Working

Water Circulating the cooling water and remove the heat and
jacket the metabolic activity
generate
Steam inlet Sterilizes the fermenter between
fermentation
Heating plate Raise the temperature
Nutrient inlet Where nutrients enter the
fermenter
Temperature and ph Maintain pH and temperature in neutral
probe level
Stirring paddle Stirs the reaction microorganisms are mixed with the
which stops them from sinking to the bottom and keeps
nutrients,
the temperature well
Harvesting outlet Where the products' are release
Steam outlet Where steam comes out let

Stainless steel Some microorganisms produce acid as waste which

might corrode other metals
Electric motor Connected centre shaft to
rotate
TYPES OF
FERMENTOR

❖ Continuous stirred tank bioreactor

❖ Bubble Colum bioreactor
❖ Air lift flurized bioreactor
❖ Internal loop airlift bioreactor
❖ External loop airlift bioreactor
❖ Tower bioreactor
❖ Photo bioreactor
❖ Cyclone Colum bioreactor
❖ Holl fibre bioreactor
❖ Cylinder conical bioreactor
Classification of Bioreactor process for suspension culture

1. In terms
of process
requirements
they are of
following types-

(i) Aerobic

(ii) Anaerobic

(iv)immobilized
cell bioreactors
2. On the basis of mode of operation, it may be
classified as-

Batch e.g -stirred tank bioreactor

Fed batch. e.g- fluidized bed bioreactor

Continuous -An example of a continuous

bioreactor is the chemostat.
THANK
YOU