Download Tutorial 11

Tutorial 11
Modes and Methods in Capillary
Electrophoresis
By the end of the tutorial , students should be able
to
• Define principles of 3 types of capillary
electrophoresis.
•
Discuss principle of CIEF Electrophoresis.
•
Compare different injection mode.
Modes of capillary electrophoresis
1. CZE
Capillary Zone Electrophoresis
2. CGE
Capillary Gel Electrophoresis
3. CIEF
Capillary IsoElectric Focusing
I. CAPILLARY ZONE ELECTROPHORESIS
Briefly describe the mechanism of separation in capillary zone electrophoresis.
What parameters can be changed to optimize separation conditions in CZE?
Separation in capillary zone electrophoresis results from two phenomena, electrophoretic mobility and
electroosmotic mobility.
Electrophoretic flow occurs as a result of movement of charges species in the electric field.
•
cations are attracted to the cathode at the negative end of the capillary, and anions are attracted to
the anode at the positive end.
•
It depends on their charge and their mass , Neutrals are unaffected by the electric field and do not
move as a result of electrophoretic flow.
Electroosmotic flow results from the general flow of solvent (water) toward the cathode end of the
capillary. Cations tend to "drag" solvent along as they move toward the cathode , bulk solvent flow
results, carrying all species (anions, cations, and neutrals) toward the cathode. Elution order is: cations,
neutrals, anions.
Reversed Mobility
2- TOWARD ANODE via reversal of polarity of the wall
by addition of alkyl
ammonium salt:
Normal
Reversed
II. Capillary Gel Electrophoresis CGE
III. CIEF separation principles
A technique where proteins are separated according to their pI
Proteins carry different charges based on pH (+ve, -ve or neutral)
Proteins are +ve charged at pH < pI
Proteins are -ve charged at pH > pI
Proteins at pH = pI
Travel to cathode
Travel to anode
No movement under electric field
Protein in a medium with linear pH gradient / applied field
Move toward electrode of opposite charges
During movement , it will pick up or loose protons , slowing down its
mobility
At pH = pI , it will be uncharged and stop migrating
Proteins focus into sharp bands in the pH gradient zone equal to their pI
Hydrodynamic injection in CE can be performed in two
ways: Describe each of them
Choose the correct answer:
In Isoelectric focusing, proteins:
A. Precipitate out of solution when pH of gradient is above the protein pI
B. Precipitate out of solution when pH of gradient is below the protein pI
C. Precipitate out of solution when pH of gradient is the same as the protein pI
Isoelectric focusing is a technique in which proteins are
separated:
A. By charge alone
B. By size alone
C. By pH and charge
D. By pH and size
The isoelectric point (pI) of a protein is the pH at which:
A. The protein has no net charge
B. The protein has a negative charge
C. The protein has a positive charge
D. A protein migrates best in electrophoresis
In capillary electrophoresis, hydrodynamic (pressure) injection
means:
A. using an injection valve before the capillary
B. using pressure to force the sample into the capillary
C. using a voltage to migrate sample into the capillary
All is true regarding number of theoretical plates (N) achieved in
CZE Except:
A. N Values reach 100,000
B. Larger N means better resolution
C. N value depends on the capillary length
D. Voltage can be increased unlimitedly to improve resolution.
1-
23-
The above chart represents an electropherogram (normal CZE), where X axis is 1- --------------, whereas Y axis is 2- --------------------------------------; where ions appearing at part 1 of
the electropherogram represents 3- ------------------- , ions appearing at part 2 of the
electropherogram represents 4- ------------------, ions appearing at part 3 of the
electropherogram represents 5-
--------------------.
Quiz 3
• Quiz 3 will take place on Wednesday, April 27, 2016
from 12:00-12:30. It will involve lectures 9 and 10 +
tutorials 8 and 9
No make up quiz will be offered.
Kindly attend in your allocated locations as per the next slide.
Hall
H3
H5
H15
C7.01
C7.02
Date / Time
Wednesday
27-04-2016,
12:00-12:30
Lecture 11, PHCM662-SS2016
Groups
1-4
5-8
9-12
13-16
17-20
13