This presentation provides an overview of electrophoresis techniques. It defines electrophoresis as a separation technique where solutes migrate through a conductive medium under an applied electric field. It describes how charges migrate based on their size and charge. It then discusses the different forms of electrophoresis, focusing on capillary zone electrophoresis and gel electrophoresis. For capillary electrophoresis, it explains the concepts of electrophoretic mobility, electroosmotic mobility, and their roles in solute migration. It also outlines the basic instrumentation and processes involved like injection, separation, and detection. For gel electrophoresis, it discusses how it separates biomolecules like DNA and proteins based on size and provides examples of its applications.

1. A Presentationon . . .
Electrophoresis
Haris Saleem
34-M.Phil-Organic Chemistry
Presented to Dr. Sadia Waseem

2. What is Electrophoresis?
• A separation technique based on a solute’s ability to
move through a conductive medium under the
influence of an electric field.
• In the absence of other effects, cations migrate toward the
electric field’s negatively charged cathode, and anions migrate
toward the positively charged anode.

3. How does charges migrate?
• More highly charged ions and ions of smaller size, which
means they have a higher charge-to-size ratio, migrate at
a faster rate than larger ions, or ions of lower charge
• Neutral species do not experience the electric field and
remain stationary
• under special conditions even neutral species and anions
migrate toward the cathode

4. Several forms of Electrophoresis?
• There are several ways in which electrophoresis is
conducted. It can be
• Capillary Zone Electrophoresis
• Gel electrophoresis

5. Capillary Electrophoresis
• In capillary electrophoresis the conducting buffer is
retained within a capillary tube whose inner diameter is
typically 25–75 μm.
• Samples are injected into one end of the capillary tube.
As the sample migrates through the capillary, its
components separate and elute from the column at
different times.
• The resulting electropherogram looks similar to the
chromatograms obtained in GC or HPLC and provides
both qualitative and quantitative information

6. Theory of Capillary Electrophoresis
• In capillary electrophoresis the sample is injected into a
buffered solution retained within a capillary tube. When
an electric field is applied to the capillary tube, the
sample’s components migrate as the result of two types of
mobility
Types of
Mobility
Electrophoretic
Mobility
Electroosmotic
Mobility

7. electrophoretic mobility
• A measure of a solute’s ability to move through a
conductive medium in response to an applied electric
field μep
• Electrophoretic velocity The velocity with which a solute
moves in response to the applied electric field is called its
electrophoretic velocity, νep; it is defined as
• νep = μep E
• where μep is the solute’s electrophoretic mobility, and E is
the magnitude of the applied electric field. A solute’s
electrophoretic mobility is defined as

8. Significance of μep
• The formula for electrophoretic mobility μep is given by
• μ 𝑒𝑝 =
𝑞
6πη𝑟
• Electrophoretic mobility is largest for more highly charge
solutes and solutes of smallest size
• Since q is positive for cations and negative for anions,
these species migrate in opposite directions.
• Neutral species, for which q is 0, have an electrophoretic
velocity of 0.

9. Electroosmotic Mobility μeof
• The movement of the conductive medium in response to
an applied electric field.
• It is observed under normal conditions, however, is that
the buffer solution moves toward the cathode. This
phenomenon is called the electroosmotic flow.
• “Flow of Neutral charge buffer in
presence of Electric Field”

10. How does the buffer migrate?
• Electroosmosis occurs because the walls of the capillary tubing
are electrically charged. The surface of a silica capillary
contains large numbers of silanol groups (Si–OH). At pH levels
greater than approximately 2 or 3, the silanol groups ionize to
form negatively charged silanate ions (Si–O–). Cations from
the buffer are attracted to the silanate ions.
• some of these cations bind tightly to the silanate ions, forming
an inner, or fixed, layer. Other cations are more loosely bound,
forming an outer, or mobile, layer. Together these two layers
are called the double layer. Cations in the outer layer migrate
toward the cathode. Because these cations are solvated, the
solution is also pulled along, producing the electroosmotic flow

11. Illustration diagram of μeof

12. Electroosmotic flow velocity νeof
• The velocity with which the solute moves through the
capillary due to the electroosmotic flow νeof
• νeof = μeof E
• Electroosmotic mobility is defined as
• μeof =
ε ξ
4πη
• where ε is the buffer solution’s dielectric constant, ζ is the
zeta potential, and η is the buffer solution’s viscosity
• zeta potential The change in potential across a double
Layer ζ

13. Significance of μeof in terms of ζ
Potential
• the zeta potential is directly proportional to the charge on
the capillary walls, with a greater density of silanate ions
corresponding to a larger zeta potential. Below a pH of 2,
for example, there are few silanate ions; thus, the zeta
potential and electroosmotic flow velocity are 0.
• As the pH level is increased, both the zeta potential and
the electroosmotic flow velocity increase
Schematic showing a comparison of the
flow profiles for (a) GC and HPLC, and
(b) electrophoresis

14. Total Mobility
• A solute’s net, or total velocity, νtot, is the sum of its
electrophoretic velocity and the electroosmotic flow
velocity thus
• νtot = νep + νeof
• And
• μtot = μep + μeof
• cations elute first in an order corresponding to their
electrophoretic mobilities, with small, highly charged cations
eluting before larger cations of lower charge
• Neutral species elute as a single band, with an elution rate
corresponding to theelectroosmotic flow velocity
• Finally, anions are the last components to elute, with smaller,
highly charged anions having the longest elution time

15. Migration time
• A solute’s total velocity is given by
• νtot =
𝑙
𝑡 𝑚
• where l is the distance the solute travels between its point
of injection and the detector, and tm is the migration
time. Since
• νtot = μtot E = (μep + μeof)E
• We have after rearranging
• 𝑡 𝑚 =
𝑙
μ 𝑒𝑝+ μ 𝑒𝑜𝑓 𝐸
• Finally, the magnitude of the electric field is
• 𝐸 =
𝑉
𝐿
• where V is the applied potential, and L is the length of
the capillary tube

16. Efficiency
• The efficiency of capillary electrophoresis is
characterized by the number of theoretical plates, N, just
as it is in GC or HPLC. In capillary electrophoresis, the
number of theoretic plates is determined by
• N =
μep+ μeof V
2D
• where D is the solute’s diffusion coefficient
• that the efficiency of a capillary electrophoreti separation
increases with higher voltages.

17. Instrumentation
• The basic instrumentation for capillary electrophoresis is
shown and includes a power supply for applying the
electric field, anode and cathode compartments
containing reservoirs of the buffer solution, a sample vial
containing the sample, the capillary tube, and a detector.
Schematic diagram for
Capillary electrophoresis.

18. Capillary Tubes
• Most capillary tubes are made from fused silica coated
with a 20–35-μm layer of polyimide to give it mechanical
strength. The inner diameter is typically 25–75 μm,
which is smaller than that for a capillary GC column, with
an outer diameter of 200–375 μm
Schematic diagram showing cross
section of a capillary column for
capillar electrophoresis.

19. Injecting the Sample
• The mechanism by which samples are introduced in
capillary electrophoresis is quite different from that used
in GC or HPLC
Types of
injection
Hydrodynamic
Injection
Electrokinetic
Injection

20. Hydrodynamic Injection
• An injection technique in capillary electrophoresis in
which pressure is used to inject sample into the capillary
column
• 𝑉inj =
Δ𝑃𝑑4π𝑡
128η𝐿
× 103
• ΔP is the pressure difference across the capillary in pascals
• d is the capillary’s inner diameter in meters
• t is the amount of time that the pressure is applied in seconds
• η is the buffer solution’s viscosity in kg m–1s–1
• L is the length of the capillary tubing in meters.
• The factor of 103 changes the units from cubic meters to liters

21. Electrokinetic injections
• Electrokinetic injections are made by placing both the
capillary and the anode into the sample vial and briefly
applying an electric field. The moles of solute injected
into the capillary, ninj , are determine using
• 𝑛inj = π𝐶𝑡𝑟2 μep + μeof 𝐸
κbuf
κsamp
• C is the solute’s concentration in the sample
• t is the amount of time that the electric field is applied
• r is the capillary’s radius
• κbuf and κsamp are the conductivities of the buffer solution
and sample, respectively

22. Detectors
• Most of the detectors used in HPLC also find use in
capillary electrophoresis. Among the more common
detectors are those based on the absorption of UV/Vis
radiation, fluorescence, conductivity
Schematic diagrams of two approaches
to on-column detection using UV/Vi
absorption spectroscopy

23. UV/ Vis detectors
• UV/Vis detectors are among the most popular. Because
absorbance is directly proportional to path length, the
capillary tubing’s small diameter leads to signals that are
smaller than those obtained in HPLC. Several approaches
have been used to increase the path length, including a Z-
shaped sample cell or multiple reflections

24. Gel Electrophoresis

25. Gel Electrophoresis
• gel electrophoresis is a method to separate
macromolecules DNA or RNA molecules by size
• This is achieved by moving negatively charged
nucleic acid molecules through an agarose matrix
with an electric field
• Shorter molecules move faster and migrate faster
than longer ones .

26. Principle of electrophoresis
• separates molecules from each other on the basis of
• size and/or
• charge and/or
• Shape
• basis of separation depends on how the sample and
gel are prepared

27. Charge
Separation
Size
Separation
Analyze
Identify
PurifyMixture of
Charged
Molecules
Positive Molecules
Negative
Molecules
Schematic Diagram

28. Gel Electrophoresis
Types
of gel
Agar
ose
Polyacry
lamide
Starch

29. Electrophoresis Equipment

30. Gel Casting Trays
• available in a
variety of sizes and
composed of UV-
transparent plastic.
• The open ends of
the trays are closed
with tape while the
gel is being cast,
then removed prior
to electrophoresis.

31. Applied voltage
•  voltage,  rate of migration
• The higher the voltage, the more quickly the gel runs
• But if voltage is too high, gel melts
• The best separation will apply voltage at no more than 5V/cm
of gel length.

32. Buffers
• During electrophoresis water undergoes hydrolysis
H2O  H + OH-
• Buffers prevent the pH from changing by reacting with the
H+ or OH- products
• Most common buffer used is called TRIS
• [tris(hydroxymethyl)aminomethane]

33. Staining of DNA
• To make DNA fragments visible after electrophoresis, the
DNA must be stained
• The favorite—ethidium bromide
• When bound to DNA it fluoresces under ultraviolet light
(reddish –orange colour)
• Convenient because it can be added directly to the gel
• Sensitive—detects 0.1ug of DNA

34. Ethidium bromide
• The standard concentration
used in staining DNA in gels
is 0.5-1ug/mL
• Ethidium bromide is a
fluorescent dye that
intercalates between bases
of nucleic acids and allows
very convenient detection of
DNA fragments in gels.
• Inserting itself between the
base pairs in the double
helix

35. Staining of DNA (cont.)
• UV absorbance maxima at 300 and 360 nm and emission
maxima at 590 nm.
• Detection limit of bound DNA is 0.5-5 ng/band.
• ethidium bromide is mutagenic so care must be taken while
handling the dye.
• Othe alternatives for ethidium bromide :
• Methylene blue
• Syber safe
• xylene cyanol
• bromphenol blue

36. A Comb
• A comb is placed in the
liquid agarose after it
has been poured
• Removing the comb
from the hardened gel
produces a series of
wells used to load the
DNA

37. Sample preparation

38. Method For Electrophoresis
Add running buffer, load samples and marker
Run gel at constant voltage until band separation occurs
Pour into casting tray with comb and allow to solidify
View DNA on UV light box and show results
Preparation of Gel
Melt, cool and add Ethidium Bromide. Mix thoroughly.

39. +-
Power
DNA
How fast will the DNA migrate?
strength of the electrical field, buffer, density of gel…
Size of the DNA!
*Small DNA move faster than large DNA
small
large
gel electrophoresis separates DNA according to size

40. Application of Electrophoresis
• CZE provides effective separations of any charged species
 inorganic anions and cations
 organic acids and amines and
 large biomolecules such as proteins.
• GE used for the separation of
proteins(enzymes, hormones, antibodies)
nucleic acids(DNA, RNA)
• Used to provide genetic information
• Human DNA can be analyzed to provide evidence in
Criminal cases
Diagnose genetic disease
Solve paternity cases

41. Thank You
• Haris Saleem
• Institute of Chemistry
• University of the Punjab, Lahore, Pakistan