So What’s the Big Deal with
Those Little Tubes?
Capillary Electrophoresis
Exercises for MSU Students
Prepared for Chemistry and Biochemistry Fall 2003
Kevin Olsen
Program
•
•
•
•
•
General principles
Safety
Obtaining good results
Example application, CE in drug discovery
Running your samples on the Beckman
Coulter model P/ACE
System Schematic
Applied Electric Field.
1000 Volts / Centimeter
Anode
+
Cathode
Detector (UV, PDA or LIF)
Velocity = (Field Strength) (Electrophoretic Mobility)
Beckman P/ACE System
Electrophoretic Mobility
Z
Uep = --------------6 nr
Where:
Z = Net charge on the analyte
n = Viscosity of the medium
r = Stoke’s Radius
Variables Governing Electrophoretic
Mobility
• Charge
• Radius*
• Mobility
6
5
4
*The Stoke’s radius is
related to molecular
mass. Heavier
molecules will have a
greater radius.
Charge
Radius
Viscosity
3
2
1
0
1
2
3
4
5
Electroosmotic Flow
(EOF)
This flow is a phenomena resulting
when a solution is contained in a
capillary with fixed charges along its
wall. This is also known as the
Electroendosmotic Flow.
Where does the Electroosmotic flow come
from?
Anode
+
Cathode
Detector
Where does the Electroosmotic flow come
from?
Applied Electric Field.
1000 Volts / Centimeter
Anode
+
Cathode
Detector
Where does the Electroosmotic flow come
from?
The interior wall of the capillary contains
charged sites that are created by the
ionization of Silanol groups on the fused
silica.
Anode
+
Cathode
Detector
Where does the Electroosmotic flow come
from?
This is where the Electroosmotic flow
comes from.
What happens to the + cations when we turn on the power?
pH, Silanol Population, and the rate of
EOF flow.
20
18
16
14
12
10
8
6
4
2
0
EOF
2
4
6
8 10 12
• At very low pH, not many
silanols are ionized and
the EOF is slow.
• As pH increases the
number of ionized sites
also increases. The EOF
speed rises steadily.
• At very high pH values, a
maximum number of
ionized sites is reached.
The EOF speed also
reaches a maximum.
The apparent velocity of any analyte
(u) will be a combination of its
electrophoretic velocity and its
movement in response to the EOF.
u = (Uep + Ueo) E
How does apparent velocity help us?
EOF
Analytes with a net positive charge will move faster than EOF
EOF
Analytes with no net charge will move at the same speed as the EOF.
(This is a useful tool that helps us to measure the EOF.)
EOF
Analytes with a net negative charge will move slower than EOF
Separation Efficiency
(apparent mobility) (Voltage)
N = ---------------------------------------2 (diffusion coefficient)
Separation Efficiency (Y) and Diffusion
Coefficient (X)
• Note the very dramatic
drop in separation
efficiency with
increasing diffusion
coefficient.
• This means that in
some cases, there is no
real advantage over
conventional HPLC
for smaller molecules.
Injections
There are two principle methods:
• Pressure differential works by applying a pressure across the
capillary while it it is dipping into the sample solution.
• Electrokinetic injection works by applying a voltage and
allowing ions to migrate into the capillary because of their
charge.
Injection volumes are typically very small:
• Typically if injection volumes exceed 1% of the column
volume, separation efficiency severely suffers.
• Sample volume can be increased by focusing the ions inside
the capillary. This technique uses a combination of
additives to the medium and selectively applied charges.
Preconcentration to Increase Sensitivity
• Attached to front of
column
• Contains a selective
binding agent
• Allows several
capillary volumes to
pass
• Analytes of interest
are then eluted
Pressure and Electrokinetic Injections
+
+
-
-
+ + +
+
+ +
• One additional advantage of electrokinetic injections is that
if appropriate conditions are set, extended injection times
allow analytes to be concentrated without overloading the
column.
Setting up the Capillary Column
• Cut the ends cleanly.
• Load capillary into
the cartridge
• Place the clear
portion in the
detector window.
The Capillary Column’s Cartridge
• Allows the column to
be moved from vial to
vial.
• Contains a cooling
medium.
• Contains gas and
vacuum connections.
• Holds electrodes that
place a charge on the
sample vials.
The Advantages of CE are:
• The number of theoretical plates is typically
in the hundreds of thousands.
• There is no mass transfer between mobile
and stationary phases as with HPLC and
GC, therefore the analytes remain in a
“plug” instead of spreading as a result of
laminar flow. (Peaks can still broaden
however.)
• Altering column conditions allows focusing
or concentration of samples.
Program
•
•
•
•
•
General principles
Safety
Obtaining good results
Example application, CE in drug discovery
Running your samples on the Beckman
Coulter model P/ACE
SAFETY
• Chemical and Biological
• Remember that solvents will be flowing under high
pressure inside an electrically powered device.
• Aerosols may be generated, work in appropriate enclosure.
• Take all normal safety precautions when working with
toxic, pathogenic, or radioactive materials.
• Electrical
• Never remove covers and expose the electronics.
• Under certain conditions the chemist may have to be
grounded for protection against static electricity.
• Mechanical
• The CE unit features a robotic autosampler with many
moving parts and a sharp needle. Keep hands out of the
sample compartment while the unit is running.
Obtaining Reproducible (Good) Results
•
•
•
•
Column condition.
Composition and pH of the medium.
Viscosity of the medium.
Operating temperature.
• Adequate sample volume.
• Use of internal standards.
The pH must be tightly controlled to
obtain reproducible EOF flow.
• Remember that the
percentage of silanols that
are ionized is dependent
on the pH.
20
18
16
14
12
10
8
6
4
2
0
EOF
2
4
6
8 10 12
Column Condition
• As time goes on, certain
molecules will block or
otherwise neutralize the
ionized silanol sites.
This will change the
EOF and alter retention
times.
• It is also very important
to condition the column
properly before use.
Follow the directions in
the published method.
Internal Standards
• The main advantage of an internal standard is
that it is subject to the same conditions as the
analyte.
Program
•
•
•
•
General principles
Safety
Obtaining good results
Example application, CE in drug
discovery
• Running your samples on the Beckman
Coulter model P/ACE
Courtesy of Cetek Corporation
20,000 Compounds Tested per Day
6,000,000 Tested since 1998
Courtesy of Cetek Corporation
Other Applications
• Analysis of molecules that are not suited to
HPLC.
• Chiral separations of enantiomers.
• Determination of drug molecules in biological
fluids.
• Separating bacteria.
• Expect the unexpected.
Program
•
•
•
•
•
General principles
Safety
Obtaining good results
Example application, CE in drug discovery
Running your samples on the Beckman
Coulter model P/ACE
Setting Up Methods
• Use File|Method|New from the menu bar to
create your method.
• When finished use File|Method|Save as from
the menu bar.
Tray Layout
A6
B6
C6
D6
E6
F6
A5
B5
C5
D5
E5
F5
A4
B4
C4
D4
E4
F4
A3
B3
C3
D3
E3
F3
A2
B2
C2
D2
E2
F2
A1
B1
C1
D1
E1
F1
Manipulating Tray Layouts
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
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B1
C1
D1
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D1
E1
F1
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B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
Manipulating Tray Layouts
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
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B1
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D1
E1
F1
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
Blue = Rinse
Green = Multiple use
Orange = Fraction
collection
Dark purple = Sample
Light purple = Other
injection
Red = Selected vial
Yellow = Separation
Manipulating Tray Layouts
• Vials positions can be designated under
“instrument set-up”. Afterwards, you will be
prompted to save the changes to your method.
• Vial positions can also be designated while setting
up the “Timed events” table in your method.
• In either case, the software commands and
procedures are the same.
P/ACE System Control
• Select your method from the drop-dow method
under FILE
• You may then change the tray layouts but all
changes become part of the method.
• In this class, we will have two control options:
1. Direct Control
2. Single Run
THE EXERCISES
• Each team will create one method that will rinse
and condition the column, then inject a sample.
• Each team’s method parameters will be slightly
different.
• Your instructor will string the methods together
for an overnight run.
• When the class meets again, the results will be
compared. Each student will submit a report
comparing and contrasting the results.
Generic Test Solution Method
• Rinse: 0.5 minutes, Regenerator sol’n A, 20 psi.
Destination = waste vial.
• Rinse: 1.5 minutes, Run buffer A, 20 psi.
Destination = waste vial.
• Inject: 10 Seconds, Test mix, 0.5 psi. Destination
= Run buffer A on outlet tray.
• Separate: 7 minutes, Run buffer A, 25 kV. Ramp
time 0.2 minutes. Destination = Run buffer A on
outlet tray.
Team 1, Variation
• Rinse: 0.5 minutes, Regenerator sol’n A, 20 psi.
Destination = waste vial.
• Rinse: 1.5 minutes, Run buffer A, 20 psi.
Destination = waste vial.
• Inject: 10 Seconds, Test mix, 0.5 psi. Destination
= Run buffer A on outlet tray.
• Separate: 7 minutes, Run buffer A, 25 kV. Ramp
time 0.2 minutes. Destination = Run buffer A on
outlet tray.
Team 2 Variation
• Rinse: 0.5 minutes, Regenerator sol’n A, 20 psi.
Destination = waste vial.
• Rinse: 1.5 minutes, Run buffer A, 20 psi.
Destination = waste vial.
• Inject: 10 Seconds, Test mix, 0.5 psi. Destination
= Run buffer A on outlet tray.
• Separate: 7 minutes, Run buffer A, 25 kV. Ramp
time 0.2 minutes. Destination = Run buffer A on
outlet tray.
Team 3 Variation
• Rinse: 0.5 minutes, Regenerator sol’n A, 20 psi.
Destination = waste vial.
• Rinse: 1.5 minutes, Run buffer A, 20 psi.
Destination = waste vial.
• Inject: 10 Seconds, Test mix, 0.5 psi. Destination
= Run buffer A on outlet tray.
• Separate: 7 minutes, Run buffer A, 25 kV. Ramp
time 0.2 minutes. Destination = Run buffer A on
outlet tray.
Team 4 Variation
• Rinse: 0.5 minutes, Regenerator sol’n A, 20 psi.
Destination = waste vial.
• Rinse: 1.5 minutes, Run buffer A, 20 psi.
Destination = waste vial.
• Inject: 10 Seconds, Test mix, 0.5 psi. Destination
= Run buffer A on outlet tray.
• Separate: 7 minutes, Run buffer A, 25 kV. Ramp
time 0.2 minutes. Destination = Run buffer A on
outlet tray.
Team 5 Variation
• Rinse: 0.5 minutes, Regenerator sol’n A, 20 psi.
Destination = waste vial.
• Rinse: 1.5 minutes, Run buffer A, 20 psi.
Destination = waste vial.
• Inject: 10 Seconds, Test mix, 0.5 psi. Destination
= Run buffer A on outlet tray.
• Separate: 7 minutes, Run buffer A, 25 kV. Ramp
time 0.2 minutes. Destination = Run buffer A on
outlet tray.