Bahir Dar University

Bahir Dar Institute of Technology

Faculty of Chemical and Food Engineering
MSc. Program in Industrial Biotechnology
Assignment for the course Bioprocess Engineering
(ChEg 6204) on
Chromatography and Electrophoresis as separation
techniques

Submitted to: Dr. Metadel K.

Submitted by: Henok Mesfin
ID No.: BDU1401510
: Kaweso Kasho
ID No.: BDU1401515
: Demelash Demeke
ID No.: BDU1401518
Submission Date: August 17, 2022
Bahir Dar, Ethiopia
Purification
After a product is isolated using processes like extraction and adsorption during the recovery /
concentrating stage of downstream processing, it may need to be purified further. The purification
can be accomplished by numerous techniques such as Chromatography, Electrophoresis, and
Ultrafiltration. We will see the first two techniques in detail.
A. Chromatography
Chromatography is a group of methods for separating very small quantities of complex mixtures,
with very high resolution. The ability of the modern analytical chemist to detect specific
compounds at ng/g or lower levels in such complex matrices as natural waters or animal tissues is
due in large part to the development of chromatographic methods. The basis of all types of
chromatography is the partition of the sample compounds between a stationary phase and a mobile
phase which flows over or through the stationary phase, Chromatographic methods are specially
applied for separating proteins and take advantage of properties that vary from one protein to the
next, including size, charge, and binding properties.
The most powerful methods for fractionating proteins make use of column chromatography, which
takes advantage of differences in protein charge, size, binding affinity, and other properties. A
porous solid material with appropriate chemical properties (the stationary phase) is held in a
column, and a buffered solution (the mobile phase) percolates through it. The protein-containing
solution, layered on the top of the column, percolates through the solid matrix as an ever-expanding
band within the larger mobile phase. Individual proteins migrate faster or more slowly through the
column depending on their properties.
Chromatography has been used to purify proteins and peptides from complex liquid solutions
extensively on the laboratory scale. When the chromatographic processes can be scaled up from a
laboratory scale to a larger scale, the diameter rather than the height of a column should be
increased. In this way, only the flow rate is increased without changing the retention time and other
operating parameters. For large-scale chromatography, a stationary phase has to be selected which
has the required mechanical strength and chemical stability properties. A modern refinement in
chromatographic methods is HPLC or high-performance liquid chromatography. HPLC makes use
of high-pressure pumps that speed the movement of the protein molecules down the column, as
well as higher-quality chromatographic materials that can withstand the crushing force of the
pressurized flow. By reducing the transit time on the column, HPLC can limit the diffusional
spreading of protein bands and thus greatly improve resolution.

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The three chromatographic methods used in protein purification are:
Ion-exchange chromatography
Exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH.
The column matrix is a synthetic polymer containing bound charged groups; those with bound
anionic groups are called cation exchangers, and those with bound cationic groups are called anion
exchangers. Ion-exchange chromatography on a cation exchanger is shown here. The affinity of
each protein for the charged groups on the column is affected by the pH (which determines the
ionization state of the molecule) and the concentration of competing free salt ions in the
surrounding solution. Separation can be optimized by gradually changing the pH and/or salt
concentration of the mobile phase so as to create a pH or salt gradient as illustrated in the figure
below.

Size-exclusion chromatography
Also called gel filtration separates proteins according to size. In this method, large proteins emerge
from the column sooner than small ones, a somewhat counterintuitive result. The solid phase
consists of beads with engineered pores or cavities of a particular size. Large proteins cannot enter
the cavities, and so take a short (and rapid) path through the column, around the beads. Small

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proteins enter the cavities, and migrate through the column more slowly as a result. This is
illustrated in the diagram shown below.

Affinity chromatography
Separates proteins by their binding specificities. The proteins retained on the column are those that
bind specifically to a ligand cross-linked to the beads. (In biochemistry, the term "ligand” is used
to refer to a group or molecule that binds to a macromolecule such as a protein.) After proteins
that do not bind to the ligand are washed through the column, the bound protein of particular
interest is eluted (washed out of the column) by a solution containing free ligand as illustrated in
the figure below.

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Application of chromatography
It is widely used in the pharmaceutical industry for analyzing and identifying the presence of any
sorts of trace amounts of elements and chemicals in the given sample.

It also plays a vital role in the development of newer drugs. For example, any presence of
impurities and other unknown compounds are detected in the sample of drugs by using
chromatography.

It plays a crucial role in the food industry for the determination of the shelf life of several food
substances through helping in the analysis of the point wherein the food tends to spoil.

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It plays a major role in in chemical industry by detecting the presence of toxic contaminant in the
oil or pesticides.

It is used in molecular biology for purifying enzymes, insulin and proteins.

B. Electrophoresis
Electrophoresis is a technique that is used to separate biomolecules based on the particle charge,
particle size, and particle shape. The migration of the molecule, known as electrophoretic mobility,
depends on the type of polymer/gel used, its pore size, the voltage provided, running time, and the
surface-to-volume ratio.

Types of electrophoresis
Paper electrophoresis
It is probably the simplest type of electrophoresis wherein the sample is applied on a strip of filter
paper moisturized with a buffer solution. The end of the strip is dipped into separate tanks that
have a buffer solution and different electrodes. A current is applied which will make the sample
move towards the electrode with opposite polarity. The strip is dried and checked under the
detection system.

It is an inexpensive method and requires only micro-quantities of protein. Paper is a popular

support medium as it is easy to handle, less expensive, and readily available. The paper contains
98% of cellulose. Paper electrophoresis has potential limitations. The greatest problem is the

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thickness and large pore size of the paper. The separation of proteins by paper electrophoresis
takes a longer time which limits its use.

Agarose gel electrophoresis

It uses an agarose gel to separate fragments of DNA or RNA of varying lengths. It observes the
movement of negatively charged RNA or DNA molecules from the negative electrode to the
positive one. The molecules are differentiated according to their size of molecules.

Agarose is one of the several components that can be separated from agar. The major source of
agar is certain species of seaweed. Agarose is a linear polymer made up of alternating units of
galactose and 3,6- anhydrogalactose. Agarose gels are completely transparent when cooled to
room temperature

In Agarose gel electrophoresis, DNA or RNA molecules can be separated based on their size. This
is achieved by the movement of negatively charged nucleic acid molecules through an agarose
matrix in horizontal electrophoresis. Molecules with smaller sizes move faster and migrate farther
as compared to longer ones. The distance between DNA or RNA bands of a given length is
determined by the percentage of agarose in the gel.

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Poly Acryl Amide Gel Electrophoresis
PAGE is a type of electrophoresis that uses a polyacrylamide gel. This gel has a higher resolving
power than other gels, making it ideal for separating small molecules. For example, PACE is often
used to separate DNA and RNA molecules.

Benefits of PAGE

It has a higher resolving power than other gels, making it ideal for separating small molecules.
It is less likely to produce artifacts, such as smearing or banding than other methods.
This method can separate a wide variety of molecules, including DNA, RNA, and proteins.
Drawbacks of PAGE

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The method can be expensive, particularly if you need to purchase the polyacrylamide gel chamber
and the equipment required to run the test. This type of electrophoresis can be time-consuming.

Capillary Electrophoresis
CE is a type of electrophoresis that uses capillaries to separate the molecules. This method is often
used for small molecules, as it has a high resolution and can be performed quickly. It uses a thin
capillary tube made of plastic, quartz, or glass. The tube is filled with the required buffer for
agarose work. Capillary electrophoresis is used for analytical work such as genetic analysis,
counter-ion analysis, pharmaceuticals with enantiomers, and characterization of protein.

Benefits of Capillary Electrophoresis

CE has a high resolution, making it ideal for separating small molecules.

This method is quick, making it a good choice if you are short on time.
Capillary electrophoresis is less likely to produce artifacts, such as smearing or banding, than other
methods.
Drawbacks of Capillary Electrophoresis
Capillary electrophoresis can be challenging to use if you are inexperienced.

Applications of electrophoresis
1. To separate complex biological molecules- many complex biological
molecules like vitamins B12.

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 2. Antibiotics, protein and can be separated efficiently by electrophoresis-
This is possible due to the charge difference among the mixtures.

3. For analysis of nucleic acids like RNA and DNA studies- This helps to
determine the size of breaks in the DNA or RNA molecule.

References
1. 11th Biochemistry: Chapter 10: Biochemical Techniques

2. https://www.medialab.com/electrophoresis.aspx

3. Lehninger PRINCIPLES OF BIOCHEMISTRY Fourth Edition David L. Nelson

(University of Wisconsin–Madison) Michael M. Cox (University of Wisconsin–Madison
4. Biochemical engineering (James M.Lec)