Other Separation

Methods

UNIT 12 ELECTROPHORESIS
Structure
12.1

Introduction
Objectives

12.2
12.3
12.4
12.5

Electroosmotic Flow
Basic Principle and Operation
Different Forms of Electrophoresis
Slab Electrophoresis
DNA Gel Electrophoresis
SDS-PAGE Gel Electrophoresis
Two-Dimensional SDS-PAGE Gel Electrophoresis

12.6

Capillary Electrophoresis
Capillary Zone Electrophoresis
Capillary Gel Electrophoresis
Capillary Isotachophoresis
Capillary Isoelectric Focusing

12.7
12.8
12.9
12.10

12.1

Capillary Electrochromatography
Summary
Terminal Questions
Answers

INTRODUCTION

We know that positively charged ions (cations) move towards cathode and negatively
charged ions (anions) move towards anode. Thus, charged particles can move towards
respective electrodes and the direction is decided according to their charge.
Electrophoresis is a separation technique based on the migration of ions in an electric
field. It is one of the most important techniques in analytical chemistry. The
positively charged ions migrate towards a negative electrode and the negatively
charged ions migrate towards the positive electrode. Ions have different rates of
migration depending on their total charge, size, and shape and can, therefore, be
separated by this technique. This separation technique was developed by Swedish
chemist Arne Tiselius and he was awarded Nobel prize in the year 1948 for his
valuable contributions.
This versatile technique is now-a-days applied to separate several species like drugs,
inorganic ions, carbohydrates, amino acids, peptides, proteins, nucleic acids, etc. In
fact, there have been innumerable applications of this technique in the
biotechnological research and industry.
This unit deals with principle, classification and instrumentation used in
electrophoresis. Various analytical applications of this technique have also been
highlighted.
In electrophoretic separation technique, a small amount of sample is allowed to flow
through a paper or a semisolid gel media under a dc potential. These media could be
porous and are immersed into an aqueous buffer solution. At a particular potential
gradient, there is a differential movement of ions. The velocity of migration in the
electric field is proportional to the charge-to-size ratio.

Objectives
After studying this Unit, you should be able to

36

explain electroosmotic flow,

discuss the basic principle of electrophoresis and operation, and

describe the classification of electrophoresis and the instrumentation used, and

some typical applications

12.2

Electrophoresis

ELECTROOSMOTIC FLOW

When a high potential is applied across a capillary tube made of silica containing
buffer solution, the positively-charged ions migrate towards the negative electrode and
carry solvent molecules in the same direction. As shown in Fig. 12.1, the electric
double layer is developed on the interface of silica and solution. The surface of the
silicate glass capillary contains negatively-charged functional groups (formed due

Fig. 12.1: A charge distribution in capillary during electroosmotic flow

to the presence of Si-O-H ) and they attract positively charged counterions. This
overall solvent movement is called electroosmotic flow. During a separation, the
uncharged molecules move at the same velocity as the electroosmotic flow (with very
little separation). The positively-charged ions move faster and negatively-charged ions
move slower.

SAQ 1
Why does the internal wall of the capillary tube attract positively charged ions?
...
...

12.3

BASIC PRINCIPLE AND OPERATION

From the foregoing discussion, it should be clear that electrophoresis is a separation

method based on differential rate of migration of charged species in a buffer solution
across which a dc electric field is applied. An electrophoresis separation is achieved by
injection of a small volume of sample into an aqueous buffer solution which is
contained on a porous support medium like paper or semi-solid gel or a narrow tube.
A simple schematic diagram of gel electrophoresis is shown in Fig. 12.2.

Fig. 12.2: A simple schematic diagram of gel electrophoresis

37

Other Separation
Methods

The potential applied causes the species to migrate towards the one or the other
electrode. The rate of migration of a given species depends upon the charge and also
the size. Thus, the separations are based upon the differences in charge to size ratios of
the various species. The larger is this ratio, the faster an ion migrates under the
influence of the electric field.

12.4

DIFFERENT FORMS OF ELECTROPHORESIS

Now-a-days electrophoresis is one of the most important techniques for molecular

separation because this powerful technique is reasonably easy and inexpensive.
Electrophoresis can be one-dimensional (1D) meaning one plane of separation or
two-dimensional (2D) meaning two planes of separation. One-dimensional
electrophoresis is used for most routine protein and nucleic acid separations.
Two-dimensional separation of proteins is used for finger printing, proteomics studies,
etc.
Broadly, different types of electrophoresis are categorised as follows:
i)

Slab electrophoresis

ii)

Capillary electrophoresis

Let us now study these in detail.

12.5

SLAB ELECTROPHORESIS

In slab electrophoresis, separations are carried out on a thin flat layer (slab) of porous
semi-solid gel containing an aqueous buffer. In general, slab has dimensions of a few
centimeter on a side and is capable of separating more than one sample
simultaneously. Samples, as small as few L, are introduced as bands or spots on the
slab and finally the separated species are detected by staining. Typical examples are
DNA gel electrophoresis, SDS-PAGE gel electrophoresis and two-dimensional SDSPAGE gel electrophoresis, etc. You will now learn about their principle, operational
aspects and applications.

12.5.1

DNA Gel Electrophoresis

In this technique, DNA samples are run through agarose gel with the help of a
potential and differential mobility of different DNA samples is responsible for their
separation.
The following instruments and reagents are important for the preparation and running
of DNA gel electrophoresis:

Gel casting trays: These are available in a variety of sizes and are composed of
U-shaped UV-transparent plastic. The open ends of the trays are closed with
tape while the gel is being cast in between the A and B walls. The tape is
removed before electrophoresis.

Fig. 12.3: Gel casting tray

38

Combs: These are plastics combs around which molten agarose is poured to
form sample wells in the gel. Once the gel is cast, comb is easily removed and

Electrophoresis

the wells are made in the gel-slab. The size of the comb determines the volume
of the well produced.

Electrophoresis buffer: The buffer which is used for the DNA gel
electrophoresis usually is Tris-acetate-EDTA (TAE) or Tris-borate-EDTA
(TBE).

Loading buffer: This buffer is important not only for the loading of the
samples in the wells of the DNA gel but also to impart a color for the tracking of
the samples. The components of the loading buffer are glycerol and dye.
Glycerol helps to allow the sample to go into the sample wells and dyes help
visual monitoring of the sample and the extent the electrophoresis has
proceeded.

Ethidium bromide: This is used for the detection of the DNA inside the gel.
Ethidium bromide is a fluorescent dye giving fluorescence when intercalated
inside the DNA and is used for staining nucleic acids. Ethidium bromide is
carcinogenic and should be handled as a hazardous chemical and one should
wear gloves while handling it.

Transilluminator: Transilluminator is an ultraviolet light source which is used

to visualize ethidium bromide-stained DNA in gels.

After DNA gel electrophoresis is run, the whole slab is kept over transilluminator.
The spots of DNA in the agarose slab can easily be found by the fluorescence of
intercalated ethidium bromide (Fig. 12.4).

(a)

(b)

Fig. 12.4: Slab electrophoresis separating DNA samples. Rectangle boxes are the wells
generated by comb in agarose slab. (a) and (b) represent the gel-slab before
and after run, respectively. Well M contains standard marker to examine the
mass of the DNA.

Among linear and supercoiled DNA, the later will move faster in agarose slab as
compared to the former because of the difference of surface area. Hence, a plasmid
DNA could easily be examined whether it is linear or supercoiled or a mixture of two.
In fact, it is easily understandable that a nicked plasmid will move slower in DNA gel
than the native one.
Pulse-Field Gel Electrophoresis (PFGE) allows investigators to separate much
larger pieces of DNA than conventional agarose gel electrophoresis. In conventional
gels, the current is applied in a single direction (from top to bottom). However, in
PFGE, the direction of the current is altered at regular intervals. Initially, the agarose
gel is prepared and samples are loaded into the wells after mixing with blue loading
buffer. Then the current is turned on and the direction of the current is changed in a
regular pattern. This is repeated until the loading dye reaches near the end of the gel.

39

Other Separation
Methods

The gel is then soaked in a solution containing ethidium bromide which fluoresces
orange when bound to DNA.

SAQ 2
What is the importance of loading buffer in DNA gel electrophoresis?
...
...
...
...
...

SAQ 3
Why does plasmid DNA show two bands in gel electrophoresis ?
...
...
...
...

12.5.2

SDS-PAGE Gel Electrohpresis

In this technique, protein samples are run through page-gel with the help of a potential
and differential mobility of different protein samples is responsible for their
separation according to the mass of the protein. Polyacrylamide gels are formed from
the polymerization of two compounds viz. acrylamide and N,N-methylene- bisacrylamide. Bis acrylamide is a cross-linking agent for the gels. The polymerization is
initiated by the addition of ammonium persulphate along with either
-dimethylamino-propionitrile (DMAP) or N,N,N,N,- tetramethylethylenediamine
(TEMED).
Gels are caste in between two glass-plates and combs are also used to prepare the
wells. Like DNA-gel casting, combs are removed after polymerisation of the polymer.
The gels are neutral, hydrophillic, three-dimensional networks of long hydrocarbons
crosslinked by methylene groups. Acrylamide (CH2=CH-CO-NH2) and
methylenebisacrylamide (CH2=CH-CO-NH-)2CH2 react in presence of ammonium
persulphate (APS), (NH4)2S2O8. APS is the sulphate free radical generator and crossed
linked polymeric slab is produced.
CONH2

CONH2 CONH2

CH2-CH-CH2-CH-CH2-CH-CH2-CHCONH-CH2-CONH2
CH2-CH-CH2-CH-CH2-CH-CH2-CHThe structure of the polymer

The instruments and reagents important for PAGE-Gel are gel casting apparatus,
loading buffer and running buffer. Casting of gel is done in such a way that the whole
slab will have two portions. One portion (at the top) is called stacking gel and the rest
part (the bottom one) is called the running gel, (see Fig. 12.5(a)).

40

When the detergent SDS (sodium dodecyl sulphate) is added to the proteins during
the sample preparation, proteins become negatively charged by their attachment to the
SDS anions. When separated on a polyacrylamide gel, the procedure is abbreviated as
SDS-PAGE (for Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis).
These negatively charged proteins are allowed to run through the polyacrylamide gel
towards positive electrode. The technique is a powerful tool for the identification of
the protein as well as determination of mass of a protein. A known marker protein
mixture is used to compare and determine the mass of a protein.

Electrophoresis

Fig. 12.5: (a) Schematic diagram of PAGE-gel electrophoresis and (b) A typical PAGE
gel after Coomassie staining

SAQ 4
Explain why ammonium persulphate is used for the page-gel preparation.
...
...
...

12.5.3

Two-dimensional SDS-PAGE Gel Electrophoresis

Each amino acid has its own isoelectric point i.e., pI. pI is the pH at which the
mobility of the amino acid is zero. Hence, proteins which comprise amino acids, do
have a characteristic pI value. In two-dimensional gel electrophoresis, proteins are
separated according to their pI and then they are allowed to mix with SDS. Denatured
proteins are then allowed to enter a polyacrylamide gel. The passage of the proteins
by potential ultimately separates the proteins according to their masses.

41

Other Separation
Methods

Fig. 12.6: Spots found after staining in 2D gel electrophoresis

SAQ 5
Why is 2D PAGE-gel electrophoresis important in proteomics study?
...
...
...
...
...
...

12.6

CAPILLARY ELECTROPHORESIS

Capillary electrophoresis is a more sensitive technique and with the similar principle,
it could detect samples as small as few nL. Besides being more sensitive, this
separation technique has high-speed and high resolution. Instead of staining of the
samples as in slab electrophoresis, there is one quantitative detector at the end of the
capillary. The capillary can also be filled with a gel, which eliminates the
electroosmotic flow (vide infra). Thus, the separation is accomplished as in
conventional gel electrophoresis but the capillary allows higher resolution, greater
sensitivity and on-line detection.
A buffer filled fused silica capillary that is typically 10 to 100 m in internal diameter
and 40 to 100 cm long, extends between two buffer reservoirs that also hold platinum
electrodes. Sample introduction is performed at one end and the detection at the other.
The polarity of the high-voltage power supply can be as indicated in Fig. 12.7 or can
be reversed to allow rapid separation of anions. Although the instrumentation is
conceptually simple, there are significant experimental difficulties in the sample
introduction and detection due to the very small volume involved. Since the volume of
a normal capillary is 4-5 L, injection and detection volumes must be of the order of a
few nano litres or less.
Let us now study more about sample introduction and detection.

42

Electrophoresis

Fig. 12.7: A schematic representation for capillary electrophoresis

a)

Sample Introduction
The most common sample introduction methods are as follows:

Electrokinetic injection

Pressure injection

With electrokinetic injection, one end of the capillary and its electrodes are
removed from their buffer compartment and placed in a small cup containing the
sample. A potential is then applied for a period of time causing the sample to
enter the capillary by a combination of ionic migration and electroosmotic flow.
The capillary end and electrode are then placed back into the regular buffer
solution for the duration of the separation. This injection technique discriminates
by injecting larger amounts of more mobile ions relative to the slower moving
ions.
With pressure injection, the sample introduction end of the capillary is also
placed momentarily into a small cup containing the sample and a pressure
difference is then used to drive the sample solution into the capillary. The
pressure difference can come from applying a vacuum at the detector end by
pressurizing the sample or by elevating the sample end. Pressure injection does
not discriminate due to ion mobility but cannot be used in gel field capillary. For
both the injection procedures, the volume injected is controlled by the duration
of the injection. Injections of 5-50 nL are common, and the injection of volumes
below 100 pL has been reported. For a buffer with density and viscosity near the
values for water, a height differential of 5 cm for 10 s injects about 6 nL with a
75 m inside diameter capillary.
Microinjection tips constructed from capillaries pulled down to very small
diameters allow sampling from picolitres environments such as single cells or
substructures within single cells. This technique has been employed to study
amino acids and neurotransmitters from single cells.
b)

Detection
There are several ways one can detect the samples after capillary
electrophoresis. Examples are absorbance methods, fluorescence methods, mass
spectrometry, conductivity measurements, potentiometry, amperometry,
radiometry, electrochemical detection, etc.

12.6.1

Capillary Zone Electrophoresis

Capillary Zone Electrophoresis also known as Free-Solution Capillary

Electrophoresis (FSCE), is the simplest form of Capillary Electrophoresis. It can be

43

Other Separation
Methods

used to separate ionic species by their charge and frictional forces. The separation
mechanism is based on differences in the charge-to-mass ratio of the analytes. In
traditional electrophoresis, electrically charged analytes move in a conductive liquid
medium under the influence of an electric field. Fundamental to CZE are
homogeneity of the buffer solution and constant field strength throughout the length of
the capillary. Introduced in the 1960s, the technique of capillary electrophoresis (CE)
is designed to separate species based on their size to charge ratio in the interior of a
small capillary filled with an electrolyte. This technique is used for the separation of
small ions as well as for the separation of molecular species. Following are the
applications of this technique.

12.6.2

Capillary Gel Electrophoresis (CGE)

Capillary gel electrophoresis is generally performed in a porous gel polymer matrix,

the pores of which contain a buffer mixture in which the separation is carried out. In
early slab electrophoresis studies, the primary purpose of the polymeric medium was
to reduce analyte dispersion by convection and diffusion and to provide a medium that
could be conveniently handled for detection and scanning. It subsequently developed
that this type of medium could provide a molecular sieving action that retarded the
migration of analyte species to various extents depending upon the pore size of the
polymer and the size of the analyte ions. This type of sieving action is particularly
helpful in separating macromolecules such as proteins, DNA fragments, and
oligonucleotides that have substantially the same charge but differ in size. Currently,
most macroscale electrophoresis separations are carried on a gel slab. Some capillary
electrophoretic separations of species that differ in size are also performed in gels
contained in capillary tubes.
The most common type of gel used in electrophoresis is a polyacrylamide polymer
formed by polymerizing acrylamide ( -CH2 = CH CO NH2) in the presence of a
cross linking agent (vide infra). The pore size of the polymer depends upon the ratio of
monomer to cross linking agent. An increase in the amount of cross linking agent
results in smaller pore size. Other gels that have been used for capillary gel
electrophoresis include agarose, a polysaccharide extracted from a marine alga,
methyl cellulose and polyethylene glycol.

12.6.3

Capillary Isotachophoresis (CITP)

Isotachophoresis (ITP) is a focusing technique based on the migration of the sample

components between leading and terminating electrolytes. Solutes having mobilities
intermediate to those of the leading and terminating electrolytes stack into sharp,
focused zones. Although it is used as a mode of separation, transient ITP has been
used primarily as a sample concentration technique.

12.6.4

Capillary Isoelectric Focusing (CIEF)

This technique allows amphoteric molecules, such as proteins, to be separated by

electrophoresis in a pH gradient generated between the cathode and anode. A solute
will migrate to a point where its net charge is zero. At the solutes isoelectric point (pI),
migration stops and the sample is focused into a tight zone. In CIEF, once a solute has
focused at its pI, the zone is mobilized past the detector by either pressure or chemical
means. This technique is commonly employed in protein characterization as a
mechanism to determine a protein's isoelectric point.

12.7

CAPILLARY ELECTROCHROMATOGRAPHY

Capillary Electrochromatography (CEC) is a hybrid separation method that couples

the high separation efficiency of CZE with HPLC and uses an electric field rather than
hydraulic pressure to propel the mobile phase through a packed bed. Because there is

44

minimal backpressure, it is possible to use small-diameter packings and achieve very

high efficiencies. Its most useful application appears to be in the form of on-line
analyte concentration that can be used to concentrate a given sample prior to
separation by CZE.

Electrophoresis

Electrochromatography, a hybrid of capillary electrophoresis and HPLC, offers some

of the best features of each of the two techniques. Two types of capillary
electrochromatography have been developed since the early 1980s: packed column and
micellar electrokinetic capillary. To date, the later has found more widespread
applications.
Capillary electrochromatography appears to offer several advantages over either of the
parent techniques. First, like HPLC, it is applicable to the separation of uncharged
species. Second, like capillary electrophoresis, it provides highly efficient separations
of microvolumes of sample solution without the need for a high- pressure pumping
system. In electrochromatography, a mobile phase is transported through a stationary
phase by electroosmotic-flow pumping rather than by mechanical pumping; thus,
simplifying the system significantly. In addition, electroosmotic pumping leads to the
plug flow profile rather than the hydrodynamic profile. The flat face of plug flow leads
to less band broadening than the hydrodynamic profile. This results in greater
separation efficiency.

12.8

SUMMARY

Electrophoresis is an important technique in analytical chemistry. The basic process is

to run a charged macromolecular species against a potential through a thick semi-solid
media called gel. The species are separated according to their mass, size and charge.
There are mainly two types of electrophoresis techniques used now-a-days and they
are slab electrophoresis and capillary electrophoresis. DNA electrophoresis, SDSPAGE gel electrophoresis and two-dimensional SDS-PAGE gel electrophoresis are
typical examples of slab electrophoresis whereas Capillary Zone Electrophoresis
(CZE), Capillary Gel Electrophoresis (CGE), Capillary Isotachophoresis (CITP),
Capillary Isoelectric Focusing (CIEF) are the applications of capillary electrophoresis.
Both the techniques are used for the separation of DNA proteins and other
macromolecules.

12.9

TERMINAL QUESTIONS

1.

What is meant by electrophoresis?

2.

What is the role of ethidium bromide in DNA gel electrophoresis?

3.

How are samples introduced into the capillary electrophoresis?

4.

How are samples detected in the capillary electrophoresis?

5.

What is the basic principle behind pulse field gel electrophoresis?

12.10 ANSWERS
Self Assessment Questions
1.

The internal surface of the silicate glass capillary contains negatively-charged

functional group due to the presence of Si-O-H moiety. These groups attract
positively-charged counterions.

2.

During electrophoresis, DNA samples are mixed with loading dye and then
loaded in the wells of the gel electrophoresis. The loading dye contains
something dense glycerol to allow the sample to "fall" into the sample wells.

45

Other Separation
Methods

DNA is colorless and tracking of the movement of DNA is important in slab

electrophoresis. For that besides glycerol, other important component is tracking
dyes, which migrate in the gel and allow visual monitoring or how far the
electrophoresis has proceeded.
3.

Plasmids are circular and double-stranded and have two forms : one is
supercoiled one and the other form is not-supercoiled. The supercoiled one
moves faster due to less surface area as compared to the other form and hence,
two bands for plasmid DNA are seen when DNA gel electrophoresis with
plasmid is performed.

4.

For the preparation of polyacrylamide gel acrylamide (H2C=CH-CONH2) and

methylenebisacrylamide are used as precursor. Persulphate is used as a radical
generator and hence produces free radical. The sulphate free radical helps
initatiates the polymerization.

5.

Proteomics is the large-scale study of proteins, particularly their structures and

functions. Once we handle a large number proteins, it is very difficult to
separate them according to their masses only which is generally done in protein
gel electrophoresis. In two-dimensional gel electrophoresis, proteins are
separated according to their pI first and then according to their mass. This gives
rise to a better separation of the proteins and the analysis becomes simpler.

Terminal Questions
1.

Electrophoresis is a technique used to separate and sometimes purify

macromolecules. When charged molecules are placed in an electric field, they
migrate towards either the positive or negative pole. Proteins and nucleic acids
differ in size, charge or conformations. Hence, they are analyzed by following
the movement of proteins and nucleic acids driven by a potential gradient in a
thick media. This technique is widely-used techniques in biochemistry and
molecular biology.

2.

Ethidium bromide is a fluorescent dye having following structure:

NH2

H2N

CH3

Br
This compound intercalates between bases of nucleic acids. Outside DNA, it
does not have fluorescence whereas intercalated ethidium bromide shows
fluorescence at the visible region. This phenomenon allows a convenient
detection of DNA fragments in gels.

46

3.

Most common sample injection for capillary electrophoresis are done in two
ways-electrokinetic injection and pressure injection. For details, refer to
Sec.12.5.

4.

There are several ways of detection for the samples after capillary
electrophoresis. Examples are absorbance methods, fluorescence methods, mass
spectrometry, conductivity measurements, potentiometry, amperometry,
radiometry, electrochemical detection, etc.

5.

Whenever there are large number of DNA samples, it is very difficult to

separate them. Pulsed field electrophoresis is a technique in which the direction
of current flow in the electrophoresis chamber is periodically altered. This
allows fractionation of pieces of DNA ranging from 50,000 to 5 millon bp,
which is much larger and cannot be resolved on standard gels.

Electrophoresis

Further Reading
1.

Principles of Instrumental Analysis, By Douglas A. Skoog, F. James Holler,

Timothy A. Nieman, Thompson, Singapore, 2004.

2.

Instrumental Methods of Analysi, 7th Edition, By H. H. Willard , L. L. Meritt, J.

A. Dean, F. A. Settle (Jr), CBS Publishers and Distributers, New Delhi.

3.

Lehlinger Principles of Biochemistry, Fourth Edition, By David Nelson Michael

M. Cox. W. H. Freeman and company, New York, 2007.

4.

Biochemistry, Third Edition, By Lubert Stryar W. H. Freeman and company,

New York, 2007.

47

Other Separation
Methods

INDEX
Active transport 17
Analysis 31
Applications of membrane separation 28
Analysis 31
Ultrafiltration 31

Desalination 28
Hemodialysis 28
Ion selective membrane electrode 29
Glass membranes 30
Heterogeneous membranes 30

Particulate contamination analysis 30

Production of table salt 28
Protein recovery 28
Specific gas probes 30
Water treatment 28

Arne Tiselius 36
brine 25
Bulk liquid membrane (BLM) 12
Capillary electrochromatography (CEC) 44, 45
Capillary electrophoresis 38, 42, 43
Schematic representation 43
Sample intrduction 43
Electrokinetic injection 43
Pressure injection 43
Detection 43

Capillary electrochromatography (CEC) 44, 45

Electrochromatography 45
Micellar electrokinetic capillary 45
Packed column 45

Capillary gel electrophoresis (CGE) 44

Capillary isoelectric focusing (CIEF) 44
Capillary isotachophoresis (CITP) 44
Capillary zone electrophoresis 43
Capillary electrophoresis 43
Free-solution Capillary Electrophoresis (FSCE) 43

Chemical potential 19
Co- transport 12
Coion transport 12
Coion transport 26
Concentration Polarization 23
Combs 38
Coulomb efficiency (h) 26
Counterion transport 12
Coupled transport 12
Critical pore diameter 15
Detergent SDS 41
Desalination 28
Dialysate 24, 25
Dialysis 23
Dialysate 24
Diffusive solute flux (JD) 24
Intrinsic membrane resistance (RM) 25
Membrane diffusive permeability (PM) 24
Partition coefficient 24
Solute diffusion coefficient 24

Diffusive solute flux (JD) 24

Diffusion 14
DNA gel electrophoresis 38

48

Combs 38
Electrophoresis buffer 39
Ethidium bromide 39
Gel casting trays 38
Loading buffer 39
Pulse-field gel electrophoresis (PFGE) 39
Transilluminator 39

Electrophoresis

Donnan effect 15, 16

Electrical ion fluxes (J) 27
Electrophoresis 36
Different forms of 38
Capillary electrophoresis 38
One dimensional 38
Slab electrophoresis 38
Two dimensional (2D) 38

Electrophoresis buffer 39
Electrochromatography 45
Electrodialysis 25
brine 25
coion transport 26
free electrolyte diffusion 26
Coulomb efficiency (h) 26
Dialysate 24, 25
Electrical ion fluxes (J) 27
Electroosmotic water transport 26
Ficks first law 27
Limiting current density (ilim) 27
Osmosis 26
Perselectivity (P) 26
Rate of counter ion transfer flux ( j ) 26
Steady state 27
Transport processes 25

Electroosmotic Flow 37
Electrophoresis 36
Basic principle 37

Electroosmotic water transport 26

Ethidium bromide 39
Facilitated Diffusional Transport 16
Ficks first law 27
Free electrolyte diffusion 26
Free molecular diffusion 16
Free-solution Capillary Electrophoresis (FSCE) 43
Gas separation 10

Gel casting trays 38

Gel electrophoresis 37
General aspects of membrane process 6
Permeance 7
Selectivity 7
Selectivity coefficient 7
Solute retention 7
Solute selectivity 7
Transmembrane flux 7

Gibbs free energy 19

Glass membranes 30
Hemodialysis 28
Heterogeneous membranes 30
Intrinsic membrane resistance (RM) 25
Immobilised liquid membrane 12
Ion selective membrane electrode 29
Isoelectric point 41

49

Other Separation
Methods

Knudsen flow 16
Limiting current density (ilim) 27
Liquid membrane processes 11
Bulk liquid membrane (BLM) 12
Coion Transport in 12
Immobilised liquid membrane 12
liquid surfactant membrane 12
Permeability coefficient 12
Supported liquid membrane 12

Liquid surfactant membrane 12

Loading buffer 39
Mechanisms of separation 14
Active transport 17
Donnan effect 15, 16
Donnan exclusion 16

Facilitated diffusional transport 16

Schematic representation 17

Knudsen flow 16
Free molecular diffusion 16

Preferential sorption -capillary flow 15

Critical pore diameter 15

Surface flow 16
Membranes 14
Macroporous 7
Mechanisms of separation 14
Mesoporous 7
Microporous 7
Nonporous 7
Permeation 15

Membrane processes 5, 8
Dialysis 9
Electrodialysis(ED) 10
Schematic representation of electrodialytic process 10

Gas separation 10
General aspects of 6
Liquid membrane processes 11
Bulk liquid membrane (BLM) 12
Immobilised liquid membrane 12
liquid surfactant membrane 12
Permeability coefficient 12
Supported liquid membrane 12
Transport in 12

Mechanisms of separation 14
Sieving 14
Solution - diffusion 14

Microfiltration (MF) 9
Nanofiltration (NF) 9
Pervaporation 11
Schematic representation 11

Reverse Osmosis (RO) 8

Schematic representation 7, 8

Schematic representation
Ultrafiltration (UF) 9

Membrane diffusive permeability (PM) 24

Membrane separation
Application of 28

Micellar electrokinetic capillary 45

One dimensional 38
Osmosis 18
Osmotic phenomena 18
Chemical potential 19
Gibbs free energy 19

50

Osmosis 18
Osmotic pressure ( ) 18
Raoults law 20
Standard chemical potential 18
Thermodynamic activity 19
vant Hoff factor 20
Osmotic pressure ( ) 18

Electrophoresis

Packed column 45
Particulate contamination analysis 30
Partition coefficient 24
Permeation 15
Permeation constant (B) 22
Permeability coefficient 12
Perselectivity (P) 26
Permeance 7
Pervaporation 11

Polyacrylamide gel 41
Preferential sorption -capillary flow 15
Production of table salt 28
Protein recovery 28
Pulse-field gel electrophoresis (PFGE) 39
Raoults law 20
Rate of counter ion transfer flux 26
Reverse osmosis process 21
Basic equations 21
Permeation constant (B) 22
Solute flux, (JS) 22
Solution-diffusion mechanism 22

Concentration Polarization 23
Solute separation, R 21
Water flux, JW 21
Water recovery, Y 21

Running gel 40
SDS-PAGE 41
Selectivity 7
Selectivity coefficient 7
SDS-PAGE gel electrohpresis 38, 40
Ammonium persulphate (APS) 40
Detergent SDS 41
Running gel 40
SDS-PAGE 41
Sodium dodecyl sulphate polyacrylamide gel electrophoresis 41
Stacking gel 40

Two dimensional SDS PAGE gel electrohpresis 41

Isoelectric point 41
Polyacrylamide gel 41

Two dimensional SDS-page

gel electrophresis 38

Sieving 14
Slab electrophoresis 38
Sodium dodecyl sulphate polyacrylamide gel electrophoresis 41
Solute diffusion coefficient 24
Solute flux, (JS) 22
Solute retention 7
Solute selectivity 7
Solute separation, R 21
Solution - diffusion 14
Solute diffusion coefficient 24
Solution-diffusion mechanism 22
Specific gas probes 30

51

Other Separation
Methods

52

Standard chemical potential 18

Stacking gel 40
Steady state 27
Supported liquid membrane 12
Surface flow 16
Thermodynamic activity 19
Transport processes 25
Transmembrane flux 7
Transilluminator 39
Ultrafiltration 31
vant Hoff factor 20
Water flux, JW 21
Water recovery,Y 21
Water treatment 28