Electrophoresis and electrodialysis by yansee_maria_jiaxuan

1. Electrophoresis & Electrodialysis
Koh Jia Xuan
Maria Pogodajeva
Phang Yan See

2. Abstract
Most microbial products, such as antibiotics, organic acids, solvents, amino acids, and
extracellular enzymes, are soluble and extracellular. Electrophoresis and electrodialysis are
some downstream processing methods which have been developed over half a century ago to
recover such soluble products. This report first describes how electrodialysis and
electrophoresis originated and developed to become widely used separation methods. Next, the
theory and mechanism of both methods will be explored. The importance of electrophoresis and
electrodialysis in the biotechnology process will be emphasized and proved by several
examples. Lastly, from the findings, it can be concluded that electrophoresis and electrodialysis
are both useful but distinct methods for separation of soluble products. In our opinion,
electrophoresis is similar to an identification method like chromatography while electrodialysis is
a purification for the stream to eliminate unwanted ions or concentrate target ions for further
processing.
Background
Electrodialysis
Electrodialysis technology was first developed in 1950s by Juda and McRae to demineralize
brackish water. Synthesizing ion exchange membrane paved the way to its industrial
application. Major developments were made since then, concentrating mostly on water
desalination. Electrodialysis Reversal (EDR) was commercially introduced into the market in
1967 by Ionics. It was well developed both economically and technologically and was a reliable
process for twenty years. Advantages of the process included slime reduction on membrane
surfaces, automatic cleaning of electrodes, polarization scale prevention and reduced cost of
hydraulics.​ ​
New technology was developed based on ion-exchange resin beads filling desalting
compartment to later be introduced into electrodeionization (EDI) process. First commercial
system we introduced in 1991. EDI resulted in higher levels of water purity, thus, stagnating the
EDR technology production market.
Nowadays, electrodialysis has a wide array of applications. Major part of the application is
brackish water desalination and boiler feer and process water treatment. However, both
processes present costs problems. Waste treatment also has a big commercial use as well as
table salt production. Demineralization of food products shows a good potential use, although it
comes with high membrane selectivity.
Electrophoresis
Electrophoresis started its history in 1930 when Tiselius published his thesis, “The Moving
Boundary Method of Studying the Electrophoresis of Proteins”. In 1948, Tiselius won the Nobel
Prize for the development of the moving boundary method and chromatographic adsorption
analysis. Moving boundary electrophoresis according to Tiselius, was almost the only

3. commercially available type of electrophoresis apparatus so it ​spread slowly until the advent of
effective zone electrophoresis methods in the 1940s and 1950s.​ Electrophoresis in aqueous
solutions in the spaces of filter-paper as a supporting medium, paper electrophoresis, became a
success from about 1950 thanks to contributions by Wieland, Fischer and others. Tiselius
apparatus was expensive and a lot of space was required for the equipment. In addition,
complete separation of proteins was difficult to achieve and many milligrams were required,
unlike paper electrophoresis. ​New electrophoresis methods were also beginning to address
some of the shortcomings of the moving boundary electrophoresis of the Tiselius apparatus. By
the 1960s, increasingly sophisticated gel electrophoresis methods made it possible to separate
biological molecules based on minute physical and chemical differences, helping to drive the
rise of molecular biology.​ ​Since then, electrophoresis methods have diversified considerably,
and new methods and applications are still being developed.The main fields of application are
biological and biochemical research, protein chemistry, pharmacology, forensic medicine,
clinical investigations, veterinary science, food control as well as molecular biology.
Theory
Electrodialysis
Electrodialysis is a process that uses electrical potential difference to move ions in solution
through ion exchange membranes. The process includes the use of several selective
membranes that can only pass through either anions or cations as a stack. The membranes are
separated from each other by inert spacers which provide mechanical stability and turbulisation.
This results in increase of mass transfer coefficient and decrease in concentration polarization
at membrane surface.
Inlet feed containing ions is fed to the electrodialysis cell which contains parallel semipermeable
membranes charged with electrical potential.
Electrochemical reaction takes place in order to create an electric field and introduce electric
current into the system. As a result, oxygen is formed at the anode and hydrogen at the
cathode.
Reactions​ ​at the cathode is
2e​−​
​+ 2 H​2​O → H​2​ ​(g) + 2 OH​−
and at the anode,
H​2​O → 2 H​+​
​+ ½ O​2​ ​(g) + 2e​−​
​or 2 Cl​−​
​→ Cl​2​ ​(g) + 2e​−

4. Electrical field is applied to the cell forcing the movement of ions. Cations permeate across
cation membranes but cannot pass through anion membranes, while anions go through anion
membranes but cannot pass through cation membranes. This results in three types of streams:
● Dilute stream, or product stream
● Concentrate stream, or brine, which becomes concentrated in ions
● Electrode stream, which passes over the electrodes
Figure 1. Electrodialysis cell scheme. Source: Joyce River High Tech.
http://joyceriver.com.sg/?p=98
Specific application of ED and EDR requires a certain configuration of the membrane stack. The
membranes are produced in the form of foils composed of fine polymer particles with ion
exchange groups anchored by polymer matrix. Impermeable to broth under pressure,
membranes are reinforced with synthetic fiber which improves the mechanical properties of the
membrane. The two types of ion exchange membranes used in electrodialysis are:
● Cation transfer membranes which are electrically conductive membranes that allow only
positively charged ions to pass through. Commercial cation membranes generally
consists of crosslinked polystyrene.
● Anion transfer membranes, which are electrically conductive membranes that allow only
negatively, charged ions to pass through. Usually, the membrane matrix has fixed
positive charges from quaternary ammonium groups (-NR3 +OH-) which repel positive
ions.

5. Both types of membranes shows common properties: low electrical resistance, insoluble in
aqueous solutions, semi-rigid for ease of handling during stack assembly, resistant to change in
pH from 1 to 10, operate temperatures in excess of 46ºC, resistant to osmotic swelling, long life
expectancies, resistant to fouling and hand washable.
It depends on the manufacturer but usually each membrane is 0.1 to 0.6 mm thick and is either
homogeneous or heterogeneous, according to the connection way of charge groups to the
matrix or their chemical structure.
The spaces between the membranes represent the flow paths of the demineralized and
concentrated streams formed by plastic separators which are called demineralized and
concentrate water flow spacers respectively. These spacers are made of polypropylene or low
density polyethylene and are alternately positioned between membranes in the stack to create
independent flow paths.
Electrophoresis
Gel Electrophoresis​ ​is the most common electrophoresis method for bioprocessing technology
and a separation technique that is used to separate proteins, RNA or DNA fragments of different
size, shape and charge. An electric field (electrophoretic field) is created from a battery or
electrical source inside the gel, and one end of the gel is positively charged (anode) while the
other is negatively charged (cathode). The negatively charged particles will travel to the anode
while the positively charged particles move to the cathode. For example, in DNA, the phosphate
groups in it cause it to be negatively charged, hence they move towards the anode. However,
the DNA fragments of different sizes will move at different speeds to the anode due to the
molecular weight. This movement of charged particles towards the opposite charge is called
migration. The gel behaves like a permeable sieve that allows particles to move through it but at
a restricted speed, so that the differently sized particles move at different speeds and separate
out after some time. Hence, the gel can also be seen as a sieve that filters the particles of
different speeds. This sieve or gel complex with internal holes, while allowing particles to pass
through, will cause the heavier and larger molecules to move more slowly across the gel, while
the lighter and smaller molecules pass through quickly.
After a suitable period of time, similar to chromatography, the particles show up in distinct bands
across the gel according to their molecular size and how fast they travel along the gel, showing
that they have separated. Each gel has a number of openings or sample wells on one side that
accepts the initial solution of particles pipetted inside for later comparison between different
samples. Dyes are added beforehand to trace the movement of the particles and prevent the
samples from floating out of the cells. The smaller molecules move quickly and have a larger
distance from the starting point or the comb while the larger molecules move slowly and are
nearer to the wells. Figure 2 below shows the gel electrophoresis setup on the left and a sample
result of the electrophoresis on the right.

6. Figure 2: Illustration of an electrophoresis cell setup (left) and a sample result (right). Source:
Britannica Encyclopedia. https://media1.britannica.com/eb-media/72/47672-004-4E16B61F.jpg
These visible bands are very similar to the concept of chromatography, where bands of different
substances show up along the chromatography paper after a certain period of time. However,
the difference is that chromatography relies on liquid movement, diffusion and capillary action
while the primary driving force of electrophoresis is the difference in electric potential between
the two ends of the polymer gel that moves the charged protein particles.
There are many types of gel that can be used in electrophoresis, depending on the types of
protein fragments to be seperated and other conditions. Some examples include different types
of starch and SDS-Page (sodium dodecyl sulphate-polyacrylamide gel electrophoresis), but the
most popular gel used is agarose. Agarose is able to separate relatively large DNA fragments
that are larger than 50 base pairs, while SDS-Page has smaller openings that allow smaller
protein fragments to be separated. A DNA ladder is usually loaded in a separate well in the
same agarose gel along with the samples as the DNA ladder has specific and defined number
of base pairs that can be easily identified (e.g. 4 bands of 50, 100, 200 and 400 base pairs).
These act as markers for comparison for the samples as one can check where each sample
band is closest to the band of the DNA marker, for example a sample band that is the same
perpendicular distance from the starting point as the 100 bp DNA ladder will have approximately
100 bp as well. Between samples, bands that are adjacent to each other also signify a possible
identical component in two different samples.
After explaining the principles of electrophoresis, the steps of gel electrophoresis are as follows.
The diagram below shows how the apparatus looks like for easier understanding.

7. Figure 3: Electrophoresis tank with the electrodes, power supply, gel, buffer solution and sample
wells for introducing the samples. Source: Wellcome Genome Campus.
http://www.yourgenome.org/facts/what-is-gel-electrophoresis
In the first step, sample preparation is done by importing the DNA or protein fragments required
from separation. It could have arrived from a purification step in the previous process. The
solution concentration is optimized and a suitable buffer is added. In the second step, the gel
and buffer is prepared based on the characteristics of the proteins involved and the goals of the
separation experiment. Usually, agarose polymer is added with water and heated, and
subsequently cooled down to form the gel for electrophoresis. The percentage of agarose used
is about 1-2%, and the higher the percentage of agarose, the smaller the pores which allows for
the separation of smaller proteins or DNAs with lower number of amino acids or base pairs. The
agarose gel casting tray is set up along with the sample comb that creates holes or wells for
samples to be added. After the gel has cooled and solidified, the buffer solution and gel is
added into the electrophoresis cell. In the third step, the samples are loaded by pipetting it into
small holes or wells of the gel and the optimum running conditions are selected before
electrophoresis commences. Figure 4 below shows how the DNA samples are loaded to the
wells on the left side, closer to the cathode since the negatively charged DNA moves to the
positive anode.

8. Figure 4: An illustration of how the DNA molecule samples are pipetted to sample wells adjacent
to each other before the experiment starts. Source: Nicerweb.
http://bio1151.nicerweb.com/Locked/media/ch20/20_08GelElectrophoresis.jpg
In the fourth and final step, the protein is analysed through an appropriate staining technique
and imaging equipments to determine the composition of the solution. An example of a sample
result is shown below in Figure 5. Assuming that the samples were pipetted from the top, the
lower the bands, the lighter the protein or DNA fragments were and vice versa.
Figure 5: An example of a result of electrophoresis of 6 different samples in 6 different sample
wells. Source: Wellcome Genome Campus.
http://www.yourgenome.org/facts/what-is-gel-electrophoresis

9. Finally, it is useful to note that there are actually many types of electrophoresis developed, like
paper, gel, thin layer, and cellulose acetate electrophoresis. These are classified as zone
electrophoresis, where the protein particles move under an electric field but generally do not
have interactions with a boundary layer of the medium. The particles is able to move all the way
to the end terminal where it has an opposite charge with the electrode. In this report, we only
focus on gel electrophoresis as it is mostly commonly used in biological applications. On the
other hand, moving boundary electrophoresis is more complicated as the molecules will interact
with both the electric field and the boundary to settle at an equilibrium position where it settles
down. Examples include capillary, isotachophoresis, isoelectric and immunoelectrophoresis. All
electrophoresis methods utilize an electric field to separate charged particles in the apparatus.
Importance & Case Studies from Bioprocess Technology
In bioprocess technology, we strive to obtain large amounts of desirable products from using
complete living cells and harvesting their primary or secondary metabolite in the fermentation
process. Electrophoresis can be useful for both upstream and downstream processes for
identifying proteins and DNA. For instance, in pre-fermentation, the bacteria used often has new
genetic material introduced into their genetic code to produce a new metabolite that they do not
usually produced, which is referred as Recombinant DNA Technology. Electrophoresis can be
used after ​Polymerase Chain Reaction (PCR) where millions of plasmid bacterial DNAs with the
new gene are replicated and this can be sent for testing in electrophoresis alongside a backup
sample of the identical plasmid bacterial DNA so that it can be verified that the polymerase
chain reaction was successful when the bands appearing are adjacent to each other and
identical in the number of base pairs. For post-fermentation, if some protein metabolite are
harvested from the reactor, it is useful to carry out an electrophoresis test with a pure sample of
the required protein to ensure that the yield and purity (not too much contaminants) of the
purified solution after fermentation has a good amount of the essential protein for harvestation.
Electrodialysis is an important separation process from environmental, economical and
technical standpoints. It is used for recovery of different organic acids which nowadays are used
in biodegradable, “green” products. The system compounds can be reused several times, such
as membrane stacks, because of the effluent removal through the reversal process, thus,
making the process more sustainable. Electrodialysis requires relatively little maintenance, it
works at lower pressure, thus, reducing noise pollution. Not only is it used in bioprocessing but it
plays a big role in water desalination. A well known example of the use of ED in bioprocess
technology is lactic acid recovery from fermentation. The process has been studied and
revealed to be an efficient process in regards to environmental aspect. Yield of lactic acid can
be reached to 151 g/l while keeping the energy consumption at 1.5 kWh/kg.

10. Conclusion
In conclusion, electrodialysis and electrophoresis are two useful separation methods that allow
us to separate charged particles using an electric field in the apparatus. Electrophoresis is more
of an identification method rather than a large scale separation method, where it allows us to
identify the size and type of proteins, DNA or RNA in the samples and compare it between
different samples or a standard DNA ladder sample. This identification process is a bit similar to
chromatography where bands will appear in the gel where it signifies the position of the various
differently sized substances after their movement along the gel. Electrophoresis allows us to
identify charged particles and proteins and can be used alongside with other identification
methods to improve the accuracy of analysis. However, it is not used to separate large amounts
of substances between two flows like electrodialysis. In electrodialysis, instead of large protein
and DNA molecules, soluble and smaller charged ions migrate between concentrated and dilute
flows through two distinct and oppositely charged membranes that only allow for a single type of
ion migration (positive or negative). As a result, one can obtain dilute streams with small
amounts of ion, and two types of concentrate streams with the positive and negative ion
respectively. Electrodialysis is more useful for purification of a stream like the treating of
wastewater where an unnecessary ion can be removed, or an essential ion can be separated
into the concentrated stream for further processing. It is a modern process that is competitive to
reverse osmosis and is gaining attention from the pharmaceutical and food industries. It has a
number of important advantages such as preservation of nutritious properties of the product and
lower energy consumption.
References and Image Credits
● Electrodialysis: From an Idea to Realization. V. D. Grebenyuk and O. V. Grebenyuk
Ionics Inc., 65 Grove Street, Watertown, MA, USA
● Sims, K., Zhang, L., and Elyanow, D., Proc. of 53rd Annual Int. Water Conf., Pittsburgh,
1992, p. 19
● Electrodialysis, a mature technology with a multitude of new applications H. Strathmann
● Electrodialysis Němeček M., Kratochvíla J., Kodým R., Šnita D
● Electrodialysis Technology. Theory and Applications. Fernando Valero, Angel Barceló
and Ramón Arbós Aigues Ter Llobregat (ATLL). Spain
● Modern Method of Lactic Acid Recovery from Fermentation Broth. Vera Habova, Karel
Melzoch and Mojmir Rychtera. Czech Republic
● Vesterberg, Olof (1989). "History of Electrophoretic Methods", ​Journal of
Chromatography, volume 480, pp. 3–19
● YourGenome, ​What is gel electrophoresis?, G. Electrophoresis, Editor. 2016, Wellcome
Genome Campus: United Kingdom
● Britannica, ​Diagram of electrophoresis. 2007, Encyclopedia of Britannica.

11. ● Guerrero, A., ​Gel electrophoresis | Chemical processes, K.A. MCAT, Editor. 2013, Khan
Academy: YouTube. Available from: https://www.youtube.com/watch?v=mN5IvS96wNk
● YourGenome. ​What is gel electrophoresis? Illustration of an electrophoresis cell. 2016
25-01-2016 [cited 2016 5th December]; Available from:
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