Kelley Smith
2/2/2012
CHM 339.502
Capillary Electrophoresis
Background
A buffer filled fused-silica capillary, usually 10-100 micrometers in internal diameter and 30-100 cm long
extends between two buffer reservoirs that also hold platinum electrodes. Like the capillary tube used in
GC the outside walls of the fused-silica capillary are usually coated with polymide for durability,
flexibility, and stability. The sample is introduced at one end and detection occurs at the other end. A
voltage of 5-30 kV dc is applied across two electrodes. The polarity of the high voltage can be reversed
to allow rapid separation of anions. High-voltage electrophoresis compartments are usually safety
interlocked for protection. There can be difficulties in sample introduction and detection due to the
small volumes that are involved. Since the volume of a normal capillary tube is 4-5 microliters, injection
and detection volumes should be a few nanoliters or less. The sample is commonly introduced by
electrokinetic injection and pressure injection. For electrokinetic injection one end of the capillary and
its electrode are removed from their buffer compartment and placed in a small cup containing sample. A
voltage is then applied for a measured time, causing the sample to enter the capillary by a combination
of ionic migration and electroosmotic flow. The capillary end and the electrode are then returned to the
regular buffer solution for the duration of the separation process. This method discriminates by injecting
large amounts of the more mobile ion relative to the slower moving ions. For pressure injection, the
sample introduction end of the capillary is also placed in a small cup containing the sample, but here it’s
a difference in pressure that forces the sample solution into the capillary. This pressure difference can
be created by applying a vacuum at the detection end. Microinjection tips constructed from capillaries
drawn to very small diameters allow sampling from picoliter environments. This method can be used to
study amino acids or neurotransmitters.
Detection:
Absorption- Both fluorescence and absorption detectors are widely used in CE, although absorption
methods are more common since they are more generally applicable detection is performed on-column
to keep the volume at the nanoliter level. In this case a small section of the protective polymide coating
is removed from the exterior by burning or etching. That section then works as the detector cell.
Indirect Detection- Indirect absorbances used for species of low molar absorptivity that are difficult to
detect without derivatization. An ionic chromophore is placed in the electrophoresis buffer. The
detector then receives a constant signal due to the presence of the substance. The analyte displaces
some of these ions, so the detector decreases during the passage of an analyte bond through the
detector.
Fluorescence Detection- Yields increased sensitivity and selectivity for fluorescent analytes or
fluorescent derivatives. Laser based instrumentation is preferred to focus the excitation radiation on the
small capillary.
Electrochemical Detection- Two types have been used with CE: conductivity and amperometry. One
problem with this method has been that of isolating the detector electrodes from the high voltage
required for the separation.
Mass Spectrometric- The very small volumetric flow rates of <1 microliters/minute from electrophoresis
make it feasible to couple the effluent directly to the ionization source of a mass spectrometer. The
most common sample introduction and ionization interface for this purpose is electrospray, although
fast atom bombardment, matrix assisted laser desorption-ionization (MALDI), spectrometry, and
inductively coupled plasma mass spectrometry (ICPMS) have also been used.
Procedure
1. Turn the instrument on and allow 30 minutes for it to warm up
2. Open 32 Karat 8.0 software and double click PDA Instrument
3. Open direct window by clicking Control > Direct control > View
(Instrument wizard may pop up. Just click ok)
4. In direct control window click load to bring the buffer tray forward. The tray will be visible
through the clear panel.
5. Lift the cover and insert buffer vials and sample vials in you notebook
Example:
Inlet Buffer Tray (BI)
Outlet Buffer Tray (BO)
0.1 N NaOH
A1
Waste
A1
1 M HCl
B1
Waste
B1
DI water
C1
Waste
C1
Buffer (new)
D1

Do not over fill the vials. The liquid level should be about 1mm below the neck.

SI is the inlet sample tray which is on the left in the back
6. Close the lid.
7. Click File > Method > New
8. Click Method > Instrument Set up
9. Click Initial Conditions and change the temperature of sample storage if desired and leave the
other parameters alone.
10. Click PDA detector initial conditions. Usually a single channel is used. Uncheck Acquistion
Enabled. Check the channel based on your detector wavelength. For example if you set the
detector wavelength at 214 nm check channel 1 then click Apply
11. Click Time Program to build in your method.
12. Click the Event box and use the arrow from the drop down menu select Rinse, Inject, or
Separate.
13. Always rinse and condition the capillary first. Typically the capillary is rinsed first with 1 N NaOH
and then the buffer used for separation. Select Rinse. Typical condition of a rinse is <20 psi
forward pressure. You can change the duration if you want. Change the tray positions to tell the
computer which solutions to use inlet solution from the vial in position B1:A1 outlet vial is in
position BO:A1 and click Ok
14. Select Inject. Injection is typically <0.5 psi forward pressure. The duration can be changed if
desired. If you put the sample in the sample tray, change the inlet tray position to S1:A1. Leave
the outlet tray position intact. Click Ok.
15. Select Separate. Separation is typically <23 kV voltage. The duration can be change if desired.
For voltage to be applied both inlet and outlet electrodes must be immersed in a buffer. Change
tray positions accordingly. Click Ok. On time table type in 0.00 for separation event.
16. Select Auto Zero after the separation begins. Select Stop Data in the end of separation. Rinse the
capillary after each run.
17. To save the method select File > Method > Save as from the menu bar
18. To run the method select Control > Single run from the menu bar to open the single run dialog.

In sample ID put sample name. Click the arrow and select date/time

In Method load the method that was just built and saved.

In Data File copy and paste sample ID and leave the rest intact.
Click Start
19. Data Analysis:
A. If you need to find previous data right click the Data/Graph window. Click Add Multiple
Traces and load a previous data file.
B. To resize right click on Graph>Properties >User defined to change Y maximum
C. To figure out area click Report >View> Area%
D. To print select File> Printer Set up> Print
20. To unload the sample and other solutions select Direct Control> Load and lift the cover and
remove the vials. Keep buffer vials in the tray.
21. Select File> Exit to exit the 32 Karat program. Close the PDA instrument window. Turn off the
instrument and log off.
22. Put your name, date, # of the sample, and condition of the instrument in the log book.