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Capillary Electrophoresis (CE)
Standard Operating Procedure
INSTRUMENT:
Beckman Coulter P/ACE Capillary Electrophoresis
INSTRUMENT START-UP:
A. Turn on the instrument. The switch is located on the front panel of the instrument.
B. Log on to the computer
1. username administrator
2. password gold
C. Open the 32 Karat 8.0 software and select double-click PDA instrument. The window of
PDA instrument will pop up.
D. The window of Instrument wizard may pop up, click OK.
E. Let the instrument warm up for 30 minutes before running any experiment. While you
are waiting, you may create your method and prepare your samples.
------------------------------------------------------------------------------------------------------------------------------PREPARING SAMPLES:
A. Use the 1.5 mL glass vials and red caps for your buffer, solutions, NaOH, HCL, and
distilled water solutions
1. Using a micropipette, add 1.4 mL of each solution into the respective vials
2. LABEL your vials
3. DO NOT over fill the vials! (if the liquid is above the shoulder of the vial, it is too full)
B. Use the small conical plastic vial and blue cap for your sample
1. Using a micropitette, add 1 mL of your sample to the vial
2. LABEL the vial
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CREATING A METHOD:
A. From the Control menu, select Direct Control, the select View
B. In Direct Control window, click Load to bring the buffer tray and sample to the front.
Look through the clear cover on top of the instrument, you will see the buffer trays and
sample trays are lowered and brought to the front.
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C. Lift the clear cover on the top front of the instrument, insert buffer vials and sample
vials.
D. Record the position of each vial on your notebook. This will be important when the
method is created.
Buffer Inlet Tray (BI): Front Left
A1
B1
C1
D1
0.1 N
1.0 M
DI Water Buffer A
NaOH
HCl
Buffer Outlet Tray (BO): Front Right
A1
B1
C1
Waste
Waste
Buffer A
(empty)
(empty)
Sample Inlet Tray (SI): Back Left
A1
B1
C1
D1
Sample Outlet Tray (SO): Back Right
A1
B1
C1
Waste
--(empty)
Mix B
--
--
--
1. BI: The front tray on the left is the “Buffer Inlet tray” where rinsing solution and
buffer vials are placed. A1, B1, C1, D1 represent the position of each solution.
2. BO: The front tray on the right is called “Buffer Outlet tray” where the waste is going
to be collected. The electrodes and capillary tips on both sides must be immersed in the
liquid. The waster container in the outlet tray must contain some buffer to begin with.
3. SI: The back tray on the left is the “Sample Inlet tray” where the sample vials are
placed.
4. SO: The back tray on the right is the “Sample Outlet tray” where the sample waste
vials are placed.
E. Close the top cover of the instrument.
F. In the PDA instrument window, select File  Method  new. Then select Method 
Instrument set up.
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G. From the Initial Conditions menu, make sure the mobility channel is unchecked. You
may change the sample storage temperature if necessary. DO NOT change anything
else on this screen.
H. From the PDA detector initial conditions menu, check that the following parameters are
selected:
Acquisition Enabled: Unchecked
Channel 1 (214 nm): Checked
Note: The detector wavelength
may be changed based upon the
experiment.
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I. From the Time Program menu, click on Event and use the options to start building your
method.
Note: There are three main parts of the method: 1) Rinse 2) Inject 3) Separate. You always want
to rinse and condition the capillary in the beginning. Typically, the capillary is rinsed first with
1.0 N NaOH and then the buffer used for separation.
1. Select Rinse and make sure the following parameters are selected, then click OK:
Pressure Type: Pressure Pressure: 20.0 psi
Inlet:
BI:A1
Duration: 1.00 min
Outlet: BO:A1
Pressure Direction: Forward
Note: The duration of the rinse may be changed. Change the
tray positions to tell the computer which solutions to use.
Refer to the sample positions in your notebook.
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2. Select Inject and make sure the following parameters are selected, then click OK:
Injection Type: Pressure
Pressure: 0.5 psi
Pressure Direction: Forward
Duration: 10 sec
Inlet:
SI:A1
Outlet:
BO:A1
Note: The duration of the injection may be changed. If you
put the sample in the sample tray, position A1, you should
change the inlet Tray position to S1:A1. Leave the outlet tray
position intact.
3. Select Separate and make sure the following parameters are selected, then click OK:
Separation Type:
Voltage
Voltage: 25 KV
Note: The duration of the separation may change based
on your method. For voltage to be applied, both inlet and
outlet electrodes must be immersed in a buffer solution.
Change Tray positions accordingly. A time of 0.00 min
must be entered on the time table for separation to occur.
4. Select Auto zero after the separation even in the time table.
5. Select Stop Data to end the data collection. The time will be based upon your
method.
6. Rinse the capillary after each run
J. To save the method, select File  Method  Save As from the menu bar. Give the
method a name with a tile and a date for future reference. Click Save. The default path
at installation is: C:\32Karat\Projects\Default\Methods. The method is now ready to
run.
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Sample Time Program for Test Mixture B:
Buffer Inlet Tray (BI): Front Left
A1
B1
C1
D1
0.1 N
1.0 M
DI Water Buffer A
NaOH
HCl
Buffer Outlet Tray (BO): Front Right
A1
B1
C1
Waste
Waste
Buffer A
(empty)
(empty)
Sample Inlet Tray (SI): Back Left
A1
B1
C1
D1
Sample Outlet Tray (SO): Back Right
A1
B1
C1
Waste
--(empty)
Mix B
Time
(min)
1
2
3
4
5
6
7
8
9
0.00
1.50
7.00
--
--
Event
Value
Rinse
Rinse
Rinse
Rinse
Rinse
Injection
20 psi
20 psi
20 psi
20 psi
20 psi
0.5 psi
separation 25 KV
Auto zero
Stop data
--
Duration
(min)
1.00
1.00
2.00
1.00
2.00
10 sec
Inlet
vial
B1:D1
B1:B1
BI:A1
BI:B1
BI:C1
S1:A1
Outlet
vial
BO: A1
BO:A1
BO:A1
BO:A1
BO:A1
BO:A1
Summary
Comments
Forward
Forward
Forward
Forward
Forward
Forward
7.00
B1:C1
BO:C1
0.17 min ramp
1.0 M HCl
DI Water
0.1 N NaOH
DI Water
Run Buffer A
Testing
mixture B
Run Buffer A
------------------------------------------------------------------------------------------------------------------------------RUNNING AN EXPERIMENT:
A. To run the method, Select Control | Single Run from the menu bar to open the Single
Run dialog.
B. In sample ID, input sample name click the arrow and select date and Time.
C. In method: Load the method you just built and saved.
D. In data file, copy and paste sample ID. Leave the remaining items unchanged.
E. Click Start.
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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 value.
C. To figure out area, select Report, view, Area%.
D. To print, select file, printer set up, print
------------------------------------------------------------------------------------------------------------------------------INSTRUMENT SHUT-DOWN:
A. To unload your sample and other solutions, select Direct Control→ Load. Lift the cover
and remove the vials. Place the buffer solution vials BI: A1 and BO:A1 and close the lid.
The capillary must be immersed in the buffer A solution, otherwise it will dry out and
clog.
B. Select File, Exit to exit 32 Karat 8.0 program. Close PDA instrument window. Turn off
the instrument, and log off the computer.
C. Put your name, date, number of sample, and condition of instrument on the log book.
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WASTE CONSIDERATIONS FOR INSTRUMENTAL CHEMISTRY COURSE:
A.
B.
C.
D.
E.
Adjust the pH of the standards to a pH between 5-8 using 0.1 M NaOH or 0.1 M HCl
Use the pH paper provided to test the pH of the solutions
Once the pH is adjusted, standards and samples may be disposed of in the sink
Samples may be disposed in waste container B in the waste hood
Container is denoted by Red Label tape
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