SCIEX Anion Analysis Kit Manual

SCIEX Anion Analysis Kit
For P/ACE™ MDQ and P/ACE™ MDQ plus Capillary
Electrophoresis Systems
Instruction Guide
A49108AE
May 2015

AB Sciex
500 Old Connecticut Path
Framingham, MA 01701
USA
AB Sciex Pte. Ltd and its affiliates disclaims all warranties with respect to this document,
expressed or implied, including but not limited to those of merchantability or fitness for a
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contract, tort, warranty, or under any statute or on any other basis for special, incidental, indirect,
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including but not limited to the use thereof.
For research use only. Not for use in diagnostic procedures.
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owners.
AB SCIEX™ is being used under license and SCIEX Separations is a part of AB Sciex.
© 2015 AB Sciex
Find us on the World Wide Web at www.sciex.com.
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Instruction Guide SCIEX Anion Analysis Kit
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Contents
Chapter 1 Using the Anion Analysis Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Materials and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Storing Kit Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Cleaning Vial Caps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Turning on the UV Lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Cleaning the Capillary Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Installing the Capillary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Conditioning a New Capillary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Storing the Capillary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Preparing the Buffer Trays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Preparing the Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
PCR Vial Setup–P/ACE MDQ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Universal Vial Setup–P/ACE MDQ plus System . . . . . . . . . . . . . . . . . . . . . . .11
Running Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Initial Conditions for All Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Time Programs for Anion Separation Methods . . . . . . . . . . . . . . . . . . . . . . . .13
Time Program for Anion Shutdown Method . . . . . . . . . . . . . . . . . . . . . . . . . .14
Checking System Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Using the Anion Organic Test Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Using the Anion Inorganic Test Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Integration Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Appendix A Filter Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Installing the 230 nm Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Appendix B System Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Configuring the P/ACE™ MDQ or P/ACE™ MDQ plus System . . . . . . . . . . . . .19
Activating Caesar Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

Contents
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Instruction Guide SCIEX Anion Analysis Kit
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Using the Anion Analysis Kit
Introduction
The SCIEX Anion Analysis Kit contains the supplies necessary for the separation and
quantitation of anions, using the SCIEX P/ACE MDQ and P/ACE MDQ plus Capillary
Electrophoresis Systems. Each anion kit yields approximately 500 tests.
This kit permits the analysis of small inorganic anions and organic acids, which are often UV
transparent. For this reason, the separation buffer contains a chromophore, and detection is
achieved in indirect mode.
The separation method is performed under reverse polarity so that the negatively charged ions
migrate toward the anode (the positively-charged electrode). In addition, the capillary is
dynamically coated with a polycation, which reverses the electro-osmotic flow (EOF) toward the
anode, thus reducing the separation time while maximizing migration time reproducibility.
IMPORTANT: The main focus of this application is in the biopharmaceutical market. This product
can also be used for environmental testing and food and beverage markets. This product is for
research use only. It is not for use in diagnostic procedures. No clinical decision or patient
notification may be made based on results using this research assay.
Safety
Refer to the Safety Data Sheets (SDS) information, available at sciex.com/safety-data-sheets,
regarding the proper handling of materials and reagents. Always follow standard laboratory
safety guidelines.
Note: The system must be equipped with a UV detector and a 230 nm filter to perform
this assay.

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Materials and Reagents
Storing Kit Components
Upon receipt, store all components at room temperature and away from direct sunlight.
Table 1-1 Kit Contents (PN A53537)
Component Quantity
Anion Coating 1
Anion Separation Buffer 1
Conditioner — Na 1
Anion Acid Rinse 1
Anion Internal Standard 1
Anion Organic Test Mix 1
Anion Inorganic Test Mix 1
Capillary, 50 cm, 75 µm I.D. 3 pieces
Rinse Solution 1
Ion Analysis Insert 1
Table 1-2 Materials Required but Not Included in This Kit
Description Part
Number
P/ACE System
MDQ MDQ plus
230 nm filter (see note below) 144433 
Adequate pipettes and pipette tips 
PCR vials (100-pack) 144709 
2 mL glass vials (100-pack) 144980 
Red caps for 2 mL glass vials (100-pack) 144648 
PCR vial holders (50-pack) 144657 
PCR vial springs (10-pack) 358821 
Gray caps for PCR vials (50-pack) 144656 
Universal plastic vials (100-pack) A62251 
Blue rubber caps for universal vials (100-pack) A62250 
Note: A 230 nm filter is required for the analysis. This filter is included with the P/ACE
MDQ plus system and is installed at position 4. However, it is not provided for the P/ACE
MDQ system and must be purchased separately.

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Cleaning Vial Caps
1. Using a clean beaker, rinse the vial caps twice with double-deionized (DDI) water.
Do not use soap.
2. Let the caps soak in DDI water for at least one hour, making sure the caps are
completely submerged.
3. Remove the caps from the water.
4. To dry the caps, either place them in an oven set at 55°C for two hours, or allow
them to dry overnight at room temperature covered by clean, lint-free laboratory
tissue.
The vial caps become compressed and lose elasticity during use, which can lead to
pressure failures and current leakage errors. Therefore, reusing caps is not
recommended.
Turning on the UV Lamp
Turn on the UV lamp and allow the system to warm up for at least 30 minutes prior to
experimentation.
Cleaning the Capillary Interface
Carefully clean the system electrodes and interface block as described in the “Maintenance
Procedure” section of the instrument manual. Repeat this procedure after every 24 hours of
operation.
Installing the Capillary
1. Install a 75 µm I.D., 60.2 cm long (50 cm from injection site to detector) fused-silica
capillary into a capillary cartridge using the Capillary Cartridge Rebuild Instructions
(PN 144655).
2. Use an 800 µm aperture in the cartridge. This aperture is labeled with an “8.”
3. After the capillary has been installed in the cartridge, insert the cartridge in the
instrument.
4. Close the cartridge cover and tray cover.
Note: The vial caps may contain impurities that can be detected with the Anion
Analysis Kit, therefore wash the caps before use.

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Conditioning a New Capillary
After installing a new capillary, rinse the capillary for one minute with Conditioner — Na. Wait four
minutes, then rinse for 30 seconds with Conditioner — Na. Rinse for one minute with Rinse
Solution. Use 20 psi of pressure for all rinses.
Storing the Capillary
After use, store the capillary on the instrument or in the original capillary storage box, with both
ends submerged in Rinse Solution. Do not allow the capillary ends to dry, because the capillary
may become plugged.
After a long storage period, or at the start of each day, condition the capillary using the Capillary
Conditioning method described in Running Methods on page 11.
Caution: Do not share capillaries between applications. If the capillary has been used for
cation analysis, do not use it for anion analysis.
Preparing the Buffer Trays
Use the correct vials and caps for your system:
• For the P/ACE MDQ system–use glass vials and red caps
• For the P/ACE MDQ plus system–use universal vials and blue caps
Replace all vials after twenty runs or after 24 hours inside the instrument. The increment option in
the method can be used to automatically increment the vials every twenty runs on both buffer
trays.
1. Fill the vials with equal volumes of each reagent in Tab le 1 -1 and position them in the
buffer trays (refer to Figure 1.1).
Use the correct volume for your system:
• For the P/ACE MDQ system–1.4 mL
• For the P/ACE MDQ plus system–1.5 mL
2. In the Waste position, place a vial partially filled with Rinse Solution.
Use the correct volume for your system:
• For the P/ACE MDQ system–700 µL
• For the P/ACE MDQ plus system–600 µL
3. Close each vial with a clean cap.

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4. Load the Inlet Buffer and Outlet Buffer trays in the instrument.
Note: The rinse solution used in this kit is ultra-purified water specifically for capillary
electrophoresis analysis of ions.
Preparing the Sample
Depending on the concentration of the analytes, the sample should be injected as is or diluted.
Dilution should be done so that the final concentration of the sample anions is between 5 ppm
and 200 ppm. Special care should be taken to verify the pH of the sample, which should be
above pH 5. A 50 mM NaOH solution can be used to dilute the sample and adjust its pH value.
The Anion Internal Standard (I.S.) consists of 30 mM sodium octanoate (sodium caprylate),
which is equivalent to 4296 ppm of octanoate ion. The I.S. can be used in the quantitation of the
sample anions. To use it, dilute the I. S. by a factor of 50 with the sample. For example, mix 4 µL
of I.S. with 200 µL of sample to yield 86 ppm of octanoate ion.
Figure 1.1 Buffer Tray Configuration for Anion Analysis
Note: When analyzing fluoride and/or phosphate ions, fill a vial with Anion
Acid Rinse and place it in position F1 of the Inlet Buffer Tray. The use of the
Anion Acid Rinse is required when running the Anion Inorganic Test Mix to
avoid peak tailing.

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PCR Vial Setup–P/ACE MDQ System
Fill a PCR vial with 200 µL of test or sample mix. Make sure there are no air bubbles at the
bottom of the PCR vial. Air bubbles can affect the sample injection. If bubbles exist, centrifuge
the vials for 2 minutes at 1000 x g and repeat if necessary. Place the PCR vial in a PCR holder
equipped with a vial spring (Figure 1.2). Seal the PCR vial with a clean gray cap and place it in
the inlet sample tray.
Figure 1.2 PCR Vial Setup–P/ACE MDQ System
Item Description
1 Vial cap (PN144656)
2 PCR vial (PN 144709)
3 PCR vial spring (PN 358821)
4 PCR vial holder (PN 144657)
1
4
3
2

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Universal Vial Setup–P/ACE MDQ plus System
Fill a PCR vial or micro vial with 200 µL of test or sample mix. Make sure there are no air bubbles
at the bottom of the vial. Air bubbles can affect the sample injection.If bubbles exist, centrifuge
the vials for 2 minutes at 1000 x g and repeat if necessary. Place the vial into the universal vial
and seal with a blue cap (Figure 1.3).
–P
p
Running Methods
Three methods are required for performing anion analysis:
• Anion Capillary Conditioning
• Anion Separation
• Anion Shutdown
Save all three methods, with their respective names, in the 32 Karat folder.
Figure 1.3 Universal Vial Setup–P/ACE MDQ plus System
Item Description
1 Universal vial cap (PNA62250)
2 PCR vial (PN 144709)
3 Universal vial (PN A62251)
4 Micro vial inside of universal vial
Note: These methods can be downloaded from sciex.com/products/capillary-
electrophoresis-instruments/p/ace-mdq-plus (click Resources).
2
1
34 3

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Initial Conditions for All Methods
All three methods utilize the same Initial Conditions (Figure 1.4) and UV Detector Settings
(Figure 1.5).
Note: When analyzing samples containing inorganic anions or organic acids that
absorb in the low UV (such as nitrate and azide), the detection wavelength (Figure 1.5)
should be set at 254 nm for optimal detection. Save this method with a different name,
such as “Anion Separation Method at 254 nm”.
Figure 1.4 Initial Conditions for Anion Capillary Conditioning, Anion Separation, and Anion
Shutdown Methods
Figure 1.5 UV Detector Initial Conditions for Anion Capillary Conditioning, Anion
Separation, and Anion Shutdown Methods

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Time Programs for Anion Separation Methods
There are two Anion Separation methods: one for the P/ACE MDQ system and the other for the
P/ACE MDQ plus system. The difference between the two methods is the duration of the Anion
Coating rinse (step 1 in the Time Program):
• For the P/ACE MDQ system–0.5 min (Figure 1.7)
• For the P/ACE MDQ plus system–1.0 min (Figure 1.8)
Figure 1.6 Time Program for Anion Capillary Conditioning Method
Figure 1.7 Time Program for Anion Separation Method–P/ACE MDQ System
Figure 1.8 Time Program for Anion Separation Method–P/ACE MDQ plus System

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Time Program for Anion Shutdown Method
Checking System Performance
Using the Anion Organic Test Mix
To check system performance, run the Anion Organic Test Mix after performing the Capillary
Conditioning method. Compare the electropherogram obtained with the one shown in
Figure 1.10. The electrical current during the separation should be stable around -53 µA. A
negative value indicates that reverse polarity was used in the separation.
In Figure 1.10, the concentration of each ion in the test mix is approximately 20 ppm, with the
exception of azide, which is about 10 ppm.
Figure 1.9 Time Program for Anion Shutdown Method
Figure 1.10 Anion Organic Test Mix–Typical Electropherogram

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Using the Anion Inorganic Test Mix
To check system performance, run the Anion Inorganic Test Mix after performing the Capillary
Conditioning method. Compare the electropherogram obtained with the one shown in
Figure 1.11. The electrical current during the separation should be stable around -53 µA. A
negative electrical value indicates that reverse polarity was used in the separation. Make sure to
use the Anion Acid Rinse in the separation to prevent the tailing of the fluoride and phosphate
peaks.
In Figure 1.11, the concentration of each ion in the test mix is approximately 20 ppm, with the
exception of azide, which is about 10 ppm.
Integration Parameters
The integration parameters in the analysis method should be optimized for each sample. As a
starting point, use the values in Figure 1.12. These values will successfully integrate both Anion
Test Mixes.
Figure 1.11 Anion Inorganic Test Mix–Typical Electropherogram
Figure 1.12 Recommended Integration Parameters and Initial Values

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The parameters have the following effects on the integration:
•Integration off sets time intervals in the electropherogram that are not integrated.
•Width sets the sensitivity of the peak detection regarding changes in the baseline.
•Threshold determines how high a peak must rise above the baseline noise before it
is recognized as a peak.
•Shoulder sensitivity enables the detection of shoulders in large peaks. Its value
specifies the slope value for splitting a peak.
• (Optional, not shown) Minimum Cluster Distance can be used to split peaks when
shoulder sensitivity does not provide proper integration. It specifies the distance
between non-baseline separated peaks so that they are not identified as one peak.
Additional help is available from the 32 Karat Software Online Help.
Troubleshooting
Problem Possible Cause Corrective Action
Unstable current Problem with capillary Replace capillary with new one
No peaks Wrong polarity in method Use reverse polarity in method
No sample vial or sample at wrong
location
Check sample vial position
No stable migration
time
Buffer depletion Replace all buffer vials after every
20 runs
Presence of ghost
peaks
Contaminated buffer vials Replace all buffer vials after every
20 runs
Vial caps are wet Replace caps with clean, dry caps
Vial caps are dirty Always use clean caps
Fluoride and
phosphate peaks are
tailing
Using Rinse Solution instead of
Anion Acid Rinse.
Make sure that a vial filled with
Anion Acid Rinse is placed at
position F1 and successive
incremental positions in the Inlet
Buffer Tray
Anion peaks have
longer migration
times.
Rinse time with Anion Coating is
too short.
Increase Anion Coating rinse time
from 0.5 min to 1.0 min in the
separation method when using the
P/ACE MDQ plus system.

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Filter Installation
Installing the 230 nm Filter
1. Before installing the 230 nm filter, check the condition of the filter as instructed in the
appropriate guide for your system.
• For the P/ACE MDQ system–“Installation UV detector wavelength filters” in
the P/ACE MDQ Installation and Maintenance Guide (PN A36419).
• For the P/ACE MDQ plus system–”Install Wavelength Filters for the UV
Detector” in the P/ACE MDQ plus System Maintenance Guide (PN B54955).
2. Set the buffer trays to the load position in the Direct Control window.
3. Lift the cartridge cover door and allow the coolant to drain from the capillary
cartridge.
4. Turn off the instrument.
5. Loosen the two thumb screws and lift the insertion bar.
6. Remove the capillary cartridge.
7. Loosen the thumb screws and remove the optics source assembly.
8. Wearing clean gloves, remove the filter wheel access cover and rotate the filter
wheel to the correct position for your system:
• For the P/ACE MDQ system–position 6
• For the P/ACE MDQ plus system–position 4
9. Place the 230 nm filter at the appropriate position with the reflective side facing
inward (toward the back of the instrument). Do not touch the filter with your hands.
10. Reinstall the filter wheel cover on the optics source assembly.
11. Replace the optics source assembly and tighten the two thumb screws.
12. Place the cartridge inside the instrument, lower the insertion bar, and tighten the two
thumb screws.
13. Close the cartridge cover door.
14. Turn on the instrument.
15. Follow the instructions in Configuring the P/ACE™ MDQ or P/ACE™ MDQ plus
System on page 19 to configure the 32 Karat software for performing anion analysis.
Note: A 230 nm filter is required for the analysis. This filter is included in the P/ACE
MDQ plus system and is installed at position 4. However, it is not provided for the P/ACE
MDQ system and must be purchased separately.

Filter Installation
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System Configuration
Configuring the P/ACE™ MDQ or P/ACE™ MDQ
plus System
IMPORTANT: Make sure that the system is turned on, and that the UV detector has been
installed.
1. Open the 32 Karat software.
2. Right-click in the right pane of the Enterprise window.
3. Select New > Instrument.
A new icon that looks like a question mark appears.
4. Right-click the question mark icon and select Rename.
5. Rename this icon Anion.
6. Right-click the Anion icon and select Configure.
7. Select P/ACE MDQ CE as the instrument type and click Configure.
A new window opens.
8. Click the UV detector icon on the left.
9. Click the Green arrow. The UV detector icon should now be on the right side under
Configured Modules.
10. Double-click the UV Detector icon to display the configuration settings.
Figure B.1 P/ACE MDQ CE Configuration for Anion Analysis

System Configuration
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If necessary, edit the settings to match the appropriate figure.
• For the P/ACE MDQ system–refer to Figure B.2.
• For the P/ACE MDQ plus system–refer to Figure B.3.
11. Click OK to accept the detector configuration.
Figure B.2 Anion Analysis Settings–P/ACE MDQ System
Figure B.3 Anion Analysis Settings–P/ACE MDQ plus System