SCIEX PA 800 Plus User manual

PA 800 Plus Pharmaceutical Analysis
System
Methods Development Guide
RUO-IDV-05-5330-A
January 2018

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USA

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Contents
CHAPTER 1: About This Guide, 9
Overview, 9
Instructions for Using the Sample Data, 10
Purpose of These Files, 11
Using the Files, 11
CHAPTER 2: Introduction to Capillary Electrophoresis, 13
Overview, 13
General Description, 13
Common Modes of Electrophoresis, 14
Capillary Zone Electrophoresis (CZE), 14
Micellar Electrokinetic Capillary Chromatography (MEKC), 14
Capillary Gel Electrophoresis (CGE), 14
Capillary Isoelectric Focusing (cIEF), 14
Capillary Electrochromatography (CEC), 15
CHAPTER 3: System Overview, 17
Instrument, 17
The Sample Handling System, 19
Capillary Cartridge, 22
Fluid Delivery, Power Supply and Interlock, 24
Syringe Pump, 24
High Voltage Power Supply, 24
LED Indicators, 24
Cartridge and Sample Cover Interlocks, 24
UV Detector Optics, 25
The Photodiode Array (PDA) Detector, 26
Laser-Induced Fluorescence (LIF) Detector, 27
Laser Module, 29
488 nm Laser Module, 29

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CHAPTER 4: 32 Karat Software, 31
Starting the Software, 31
Tool Bar, 32
Menu Bar, 32
Creating and Configuring an Instrument, 32
Creating a New Instrument, 33
Configuring the New Instrument, 33
Auto Configuration, 35
Configuration Options, 37
Analysis Options, 37
PDA, 37
System Suitability, 37
Qualitative Analysis, 38
Caesar Integration, 38
Instrument Options, 38
Standard CE, 38
CEC/LC, 38
Manual Configuration, 39
Manual Configuration of the UV Detector, 39
Manual Configuration of the PDA and LIF Detectors, 40
CHAPTER 5: Direct Control, 41
Introduction, 41
Direct Control Window, 41
Exercise: Conditioning the Capillary, 44
CHAPTER 6: Creating and Editing a Method, 49
Introduction, 49
Creating and Editing a Method, 49
Initial Conditions Tab, 50
Detector Initial Conditions, 52
UV Detector Initial Conditions, 52
Electropherogram Channel, 53
Filter, 53
Relay 1 and Relay 2, 53
Absorbance Signal, 53
PDA Detector Initial Conditions, 54
Electropherogram Scan Data, 54
Filter, 54
Relay 1 and Relay 2, 54
Reference Channel, 55
Absorbance Signal, 55
Electropherogram Channel Data, 55
LIF Detector Initial Conditions, 56

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Electropherogram Channel 1 and 2, 56
Signal, 57
Data Rate, 57
Relay 1 and 2, 57
Time Program, 57
Separate Dialog, 58
Rinse Dialog, 61
Inject Dialog, 61
Autozero, 63
Stop Data, 63
End, 63
Saving the Method, 67
CHAPTER 7: Running the System, 69
Single Run and Sequence Runs, 69
Materials Needed, 69
Single Run, 70
Programming a Sequence, 72
Running the Sequence, 77
Sequence Validation, 78
CHAPTER 8: Integration, 81
Description, 81
Integrating Data, 82
Optimizing Integration, 84
Integration Parameters, 85
Width, 86
Threshold, 87
Integrating a Peak Cluster, 88
Integration Off, 89
Integration Results and Reports, 91
CHAPTER 9: Calibration, 93
Introduction, 93
Analysis Components, 93
Qualitative Analysis, 93
Quantitative Analysis, 93
Creating a Calibration, 94
Developing a Calibration Method, 94
Generating the Calibration Curve, 97
Analyzing Unknowns, 102

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CHAPTER 10: Qualitative Analysis, 103
Data Analysis and Reporting, 103
CHAPTER 11: Mobility, 107
Functions of Mobility, 107
Mobility Markers, 108
Assigning Mobility Values, 108
Mobility Plot, 110
Demonstration: Using the Mobility Features, 111
Peak Identification by Mobility, 113
CHAPTER 12: Creating Reports, 115
Introduction, 115
Creating a Method Custom Report, 116
Modifying a Report Template, 116
Saving a Report as a Template, 119
Saving a Report as a Default Report, 120
Report Templates, 120
Method Custom Report Templates, 120
Sequence Custom Report Templates, 120
Standard Report Templates, 121
Sequence Reports, 121
APPENDIX A: Additional Resources, 123
32 Karat Software Online Help, 123
Installation and Maintenance Guide, 123
SCIEX Service, 123
Other References, 123
APPENDIX B: PA 800 Plus System Calculations, 125
Calculations, 125
CE Calculations, 125
Ohm’s Law, 125
Corrected Migration Time, 125
Corrected Area, 126
Mobility, 126
APPENDIX C: Understanding PDA Data, 129
Overview, 129
Mixed View, 131
Contour Plot, 132

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Spectrum, 132
Electropherogram, 132
3D Plot, 132
APPENDIX D: System Administration, 135
Overview, 135
APPENDIX E: Vial Incrementing, 137
Introduction, 137
Examples of Use, 139
Example 1, 139
Example 2, 142
Summary, 145
APPENDIX F: Sequence Vial Report, 147
Overview, 147
Sequence Vial Increment Preview, 147
Sequence Vial Report-Confirmed, 149
Revision History, 157

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CHAPTER 1
About This Guide
CAUTION
Prior to using the system, refer to the PA 800 Plus Pharmaceutical Analysis System Overview
Guide for detailed information on the safe use and operation of the system.
Overview
The PA 800 Plus Methods Development Guide is a self-paced introduction to the operation of the SCIEX PA 800 Plus
Pharmaceutical Analysis System, which includes the 32 Karat software. It contains a brief introduction to the
technique of capillary electrophoresis (CE), an overview of the PA 800 Plus System hardware, tutorials for the
most frequently used software features, and appendices of useful information.
The Methods Development Guide has been designed to assist users who are new to CE or to 32 Karat software. It
contains step-by-step exercises that allow you to create methods, to generate and analyze data, and to report
results. It does not cover every feature in the system or every aspect of capillary electrophoresis.
The on-instrument activities contain specific instructions for users with ultraviolet (UV), photodiode array
(PDA), and laser-induced fluorescent (LIF) detector systems. The activities are progressive. Each builds on the
previous one. To get the most from this guide, work through all the activities in order.
The activities that teach data reprocessing, calibration, and reporting, are all based on data collected with an
ultraviolet detector system. The steps in these procedures are very similar regardless of the detector type used,
so these exercises are important for users of all detector types. Data files for the post-run exercises are installed
with the 32 Karat software.
Additional information can be found in the 32 Karat Software Online Help. This can be accessed at any time while
using the 32 Karat software. You can access Help through the Help menu located in the menu bar, by pressing the
F1 key while working within the software, or any time a Help button is displayed in an application window.

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About This Guide
Instructions for Using the Sample Data
In some of the exercises in this guide, you are asked to open specific data and method files. These are located in
the data folder, which is a sub-directory of the 32 Karat folder created when your software was installed.
IMPORTANT Make sure to read and understand the following before using the sample data files.
The data folder contains the following files:
• CE Data Sample 1.dat
• CE Data Sample 2.dat
• CE Data Sample 3.dat
• CE Level 1.dat
• CE Level 2.dat
• CE Level 3.dat
• CE Level 4.dat
• CE Level 5.dat
• CE Mobility Data.dat
• CE PDA Data Sample.dat
• CE Unknown 1.dat
• ISTD Calib Level 1 Rep 1.dat
• ISTD Calib Level 1 Rep 2.dat
• ISTD Calib Level 2 Rep 1.dat
• ISTD Calib Level 2 Rep 2.dat
• ISTD Calib Level 3 Rep 1.dat
• ISTD Calib Level 3 Rep 2.dat
• ISTD Calib Level 4 Rep 1.dat
• ISTD Calib Level 4 Rep 2.dat
• multi calibration level 1.dat
• multi calibration level 2.dat
• multi calibration level 3.dat
• multi calibration level 4.dat
• multi calibration level 5.dat
• multi calibration level 6.dat
The methods folder contains the following files:
• CE calibrate.met
• multilevelcalibration.met
• pda estd.met

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About This Guide
Purpose of These Files
The Data folder contains data and method files to supplement the exercises in this guide. The files were created
solely for the purposes of this tutorial. They are intended as training tools only and are not to be used for system
validation or any other purpose.
Using the Files
These files are referred to by name in this guide. For some exercises, you are asked to open one or more of these
files. By using these files, the images you see in the software will match.
IMPORTANT Make copies of these files before use.
Processing these data files and methods in the 32 Karat software causes changes to the files. To keep the original files
unaltered and available for later use in these exercises, follow the steps below.
1 Open Windows Explorer. To open this utility, right-click the Start button in the Windows tool bar and click
Explore.
2 Navigate to the Data folder. It is a sub-directory of the 32 Karat software folder.
3 In the left-hand pane of the Explorer window, click the Data folder to open it.
4 From the menu bar, click File > New > Folder. A new folder is added to the Data folder. The New Folder is
highlighted, which indicates the name may be edited. Type a new name. A good choice is the name of the
individual who is doing the tutorial exercises.
5 Select all of the data files listed above, but do not select the new folder. There are several ways to do a multiple
file select.
Use any one of the following:
• Select the first file in the list by clicking it. Hold down the Shift key and click the last file in the list. All
the files between these two files are selected.
• Select a file by clicking it. Hold down the Ctrl key and click each individual file in the list.
• Using the mouse, you can click and drag a box around all the files to be selected.
6 When all the files are selected (highlighted), click Edit > Copy. Double-click the new folder to open it. Then
click Edit > Paste. Repeat steps 1 through 6 for the methods folder.
You have now created a copy of the sample files in a new folder. When the guide asks you to open a file, open the
copy you have just created, not the original file.
Save all methods and sequences you create as part of the tutorial in this new folder.

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CHAPTER 2
Introduction to Capillary Electrophoresis
Overview
This section describes the common modes of capillary electrophoresis and how these processes are performed by
the PA 800 Plus instrument using the ultraviolet (UV), photodiode array (PDA) and laser-induced fluorescence
(LIF) detectors.
General Description
The PA 800 Plus system separates sample components in a fused-silica capillary, using one of several modes of
electrophoresis. All of these modes are generally referred to as capillary electrophoresis (CE).
In the PA 800 Plus system, the sample is injected into the capillary using either pressure, vacuum, or voltage.
Under the influence of an electric field, sample components migrate differentially through the capillary.
There are two basic methods for detecting samples using capillary electrophoresis. The first is the absorbance of
light. This method is used in UV and PDA detection. As these components pass a window in the capillary, a single
wavelength UV detector or a multiwavelength photodiode array detector (PDA) measures absorbance and
transmits the signal to the computer. The signal can also be transmitted to an external recorder, integrator, or
data system through an analog output. The signal may be plotted graphically in the form of an electropherogram
and analyzed.
The second method is to induce the samples to fluorescence and measure the emitted light. This is done by laser-
induced fluorescence (LIF). Substances in the capillary that fluoresce at the laser wavelength are detected. The
LIF detector measures and records this fluorescence, which appears as a peak on the computer window or printed
electropherogram.
The PA 800 Plus system may be used to separate many different kinds of samples, including peptides, proteins,
nucleic acids, ions, enantiomers, and pharmaceuticals. CE has very low sample requirements relative to other
analytical techniques (50 µL, with the actual injection volume being typically between 5 nL and 50 nL). It provides
a complementary alternative to other separation techniques such as chromatography.

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Introduction to Capillary Electrophoresis
Common Modes of Electrophoresis
Capillary Zone Electrophoresis (CZE)
When voltage is applied to an uncoated fused-silica capillary tube filled with a uniform electrolyte solution,
separation of charged species occurs due to differential migration in the electrical field. This process, in which
charged particles in solution migrate toward an electrode with an opposite charge, is called electrophoresis. If the
inner wall of the capillary is also charged, the fluid in the capillary begins to flow toward the electrode that has
the same charge as the capillary wall. This bulk movement of fluid is termed electroosmotic flow or EOF.
Because EOF is usually of higher magnitude than electrophoresis, analytes of both positive and negative charge
are ultimately carried in the same direction, although at different rates. Electrophoresis separates like-charged,
but otherwise distinct analytes, from one another. In this manner, both positive and negative analyte molecules
can be detected as they are swept past the detector. The direction of EOF can be changed by reversing the charge
on the electrodes, or by changing the charge on the wall of the capillary by chemical means.
The amount of time required for sample molecules to migrate to the detector depends on the length of the
capillary, the electrophoretic mobilities of the particular sample molecules, the specific electrolyte used, the
magnitude of the EOF, and the applied voltage. Molecules having different electrophoretic mobilities are detected
at different times. Other factors to be considered are the size, shape, and charge of the particles, the electrolyte
concentration, the pH of the separation buffer, and the dimensions of the capillary.
Micellar Electrokinetic Capillary Chromatography (MEKC)
In free-zone capillary electrophoresis, neutral molecules travel as a single band. To separate these molecules,
micellar additives such as sodium dodecyl sulfate (SDS) can be added to the electrolyte solution. Neutral
molecules with differing affinities for the charged micelles separate during electrophoresis under these
conditions.
Capillary Gel Electrophoresis (CGE)
In the techniques described above, the analyte molecules are moving in a low viscosity liquid. By increasing the
viscosity of this medium to the point where the migration of molecules is physically impeded, it is possible to
separate different species based on size and shape, as well as charge. A variety of gel solutions can be employed
in this technique.
Capillary Isoelectric Focusing (cIEF)
In cIEF, a mixture of special buffers called ampholytes is used to create a pH gradient along the length of the
capillary. During separation, molecules migrate to the point in the pH gradient at which they have no net charge.
At the end of the process, the various analytes are located in discrete zones in the capillary. In a separate
mobilization step, the bands are moved past the detector for analysis. This technique is most often used for
protein and peptide analysis.

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Introduction to Capillary Electrophoresis
Capillary Electrochromatography (CEC)
CEC uses a capillary packed with porous particles similar to those used in HPLC. Driven by EOF, analytes partition
between mobile and stationary phases as they migrate down the capillary. Under some conditions,
electrophoresis can also participate in the separation. CEC is primarily employed to separate small molecules
such as drugs.

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CHAPTER 3
System Overview
Instrument
The main components of the PA 800 Plus Pharmaceutical Analysis System include trays that hold vials of sample,
buffer, and other solutions, an interface block, a high-voltage power supply and electrodes, a source optics
module and detector, temperature control hardware, and a sample injection mechanism (Figure 3.1, Figure 3.2,
and Figure 3.3).

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System Overview
Figure 3.1 PA 800 Plus System
1. Outer Door (Open Position)
2. UV Detector
3. Two-ended Fiber Optic Cable
4. Clamp Bar and Cable Connection
5. Capillary Cartridge
6. Interface Block

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System Overview
Figure 3.2 PA 800 Plus System Side View
The main power switch is on the lower right side of the front of the instrument. All connections for external
system components are on the upper-left side panel of the instrument, except for the AC inlet and the fuse holder.
Three fans supply cooling air flow for internal system components. Air is exhausted through the vents at the side
and back of the instrument. Keep at least six inches of clearance at each vent to make sure that there is adequate
air flow.
The Sample Handling System
The sample handling system holds four trays; two sample trays (inlet and outlet), and two buffer trays (inlet and
outlet). The sample trays are primarily used for samples; the buffer trays hold the other solutions required for
electrophoresis (for example, buffer and rinse solutions). The trays are on two parallel tracks. Under normal
operating conditions, the trays on the left are inlet trays for sample and buffer; the trays on the right are outlet
trays for sample and buffer (Figure 3.3).
1. Outer Door or Sample Cover (open)
2. Inner Door or Cartridge Cover (open)
3. Indicators
4. Fluid Fill Port
5. Sample Trays
6. Buffer Trays
7. Fluid Bubble Indicator
8. Power Switch

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System Overview
Figure 3.3 PA 800 Plus System Trays
Each buffer tray has slots for 36 universal vials. Sample trays hold either 48 vials or a 96-well plate. Each tray is
assigned a number from the front to the back, starting with the number 1, and assigned a letter from left to right,
starting with the letter A (Figure 3.4).
1. Inlet Sample Tray (48) or 96-Well Plate
2. Inlet Buffer Tray (36 Vials)
3. Outlet Sample Tray (48) or 96-Well Plate
4. Outlet Buffer Tray (36 Vials)