CESI 8000 Plus High Performance Separation-ESI Module
User Manual
RUO-IDV-05-3897-C March 2020 This document is provided to customers who have purchased SCIEX equipment to use in the operation of such SCIEX equipment. This document is copyright protected and any reproduction of this document or any part of this document is strictly prohibited, except as SCIEX may authorize in writing. Software that may be described in this document is furnished under a license agreement. It is against the law to copy, modify, or distribute the software on any medium, except as specifically allowed in the license agreement. Furthermore, the license agreement may prohibit the software from being disassembled, reverse engineered, or decompiled for any purpose. Warranties are as stated therein. Portions of this document may make reference to other manufacturers and/or their products, which may contain parts whose names are registered as trademarks and/or function as trademarks of their respective owners. Any such use is intended only to designate those manufacturers' products as supplied by SCIEX for incorporation into its equipment and does not imply any right and/or license to use or permit others to use such manufacturers' and/or their product names as trademarks. SCIEX warranties are limited to those express warranties provided at the time of sale or license of its products and are the sole and exclusive representations, warranties, and obligations of SCIEX. SCIEX makes no other warranty of any kind whatsoever, expressed or implied, including without limitation, warranties of merchantability or fitness for a particular purpose, whether arising from a statute or otherwise in law or from a course of dealing or usage of trade, all of which are expressly disclaimed, and assumes no responsibility or contingent liability, including indirect or consequential damages, for any use by the purchaser or for any adverse circumstances arising therefrom. (GEN-IDV-09-10816-B) For Research Use Only. Not for use in Diagnostic Procedures. Trademarks and/or registered trademarks mentioned herein are the property of AB Sciex Pte. Ltd., or their respective owners, in the United States and/or certain other countries. AB SCIEX™ is being used under license. © 2020 DH Tech. Dev. Pte. Ltd.
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CHAPTER 1: Safety, Notices and Labels, 13 Overview, 13 Alerts for Warning, Caution, Important, and Note, 14 Instrument Safety Precautions, 15 Moving Parts or Sharp Objects, 16 Electrical Safety, 16 Laser Safety (for Optional Laser Device), 17 Class 1 Laser Caution Label, 17 Chemical Precautions, 17 Safety Symbols and Labels, 18 High Voltage Electric Shock Risk Symbol, 18 Attention Safety Symbol, 18 Sharp Object Label, 18 Mobile Cart Caution Label, 19 Cancer and Reproductive Harm Label, 19 RoHS Notices, 19 China RoHS Caution Label, 19 Other Instrument Labels, 20 Recycling Label (WEEE), 20 Disposal of Devices Containing Mercury Components, 21 CE Mark Label, 21 RCM Mark Label, 21 CSA Mark Label, 21 章 2: 安全、通知和标签 , 23 综述 , 23 警告、注意、重要事项以及注释的提示 , 24 仪器安全防护措施 , 25 活动部件或尖锐物 , 26 电气安全 , 26 激光安全 （适用于可选激光设备） , 26 1 类激光小心标签 (1 级激光警告标签 ), 26
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化学品注意事项 , 27 安全符号和标签 , 27 高压电击危险符号 , 27 注意安全符号 , 27 尖锐物标签 , 28 流动车警告标签 , 28 致癌和生殖危害标签 , 28 RoHS 通知 , 28 中国 RoHS 警告标签 , 29 其他仪器标签 , 29 环保标签 (WEEE), 29 处理包含加汞组件的设备 , 30 CE 标志标签 , 30 RCM 标志标签 , 30 CSA 标识标签 , 30 CHAPITRE 3: Sécurité, Consignes et étiquettes, 31 Présentation, 31 Alertes « Avertissement, Mise en garde, Important et Remarque », 32 Précautions de sécurité pour l'instrument, 33 Pièces mobiles ou Objets tranchants, 34 Sécurité électrique, 34 Sécurité laser (pour les appareils avec laser en option), 35 Étiquette de mise en garde : produit laser de classe 1, 35 Précautions chimiques, 36 Symboles et étiquettes de sécurité, 36 Symbole « Haute tension, risque de choc électrique », 36 Symbole de sécurité Attention, 37 Étiquette Objet tranchant, 37 Étiquette de mise en garde chariot mobile, 37 Étiquette concernant les risques de cancer et de malformations congénitales, 37 Consignes RoHS, 38 Étiquette de mise en garde RoHS (Chine), 38 Autres étiquettes de l'instrument, 38 Étiquette de recyclage (WEEE), 39 Élimination des appareils contenant des composants au mercure, 39 Étiquette marque CE, 39 Étiquette marque MRC, 40 Étiquette marque CSA, 40 CHAPTER 4: System Overview, 41 About This Manual, 41
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Optional Detectors, 41 Introduction, 42 Instrument, 43 Sample Handling System, 44 Syringe Pump, Power Supply, LEDs and Interlock, 46 Syringe Pump, 46 High Voltage (HV) Power Supply, 46 LED Indicators, 46 Cartridge and Sample Cover Interlocks, 47 OptiMS Cartridge, 48 CHAPTER 5: Integrating the CESI 8000 Plus System with a Mass Spectrometer, 51 CESI-MS Integration Procedures, 51 CESI 8000 Plus System: Installing OptiMS Cartridge, 52 OptiMS Cartridge Installation, 52 Thermo Scientific Mass Spectrometer: Integration with the CESI 8000 Plus System, 56 Physical Integration with the CESI 8000 Plus System, 56 OptiMS Cartridge Sprayer Tip Installation into a Thermo Scientific NanoSpray II Ion Source, 59 Establishing Communication Between the CESI 8000 Plus Controller and the Thermo Scientific Mass Spectrometer, 65 Aligning the CESI 8000 Plus System with a Thermo Scientific Mass Spectrometer, 66 SCIEX TripleTOF® 5600 Mass Spectrometer: Integration with the CESI 8000 Plus System, 71 Physical Connections with the CESI 8000 Plus System, 71 OptiMS Cartridge Sprayer Tip Installation into the SCIEX Nanospray® III Source, 74 Establishing Communication Between the CESI 8000 Plus Controller and the SCIEX Mass Spectrometer, 82 Aligning the CESI 8000 Plus System with the SCIEX TripleTOF® 5600 Mass Spectrometer, 83 CHAPTER 6: 32 Karat™ Software Overview, 89 Overview of 32 Karat™ Software, 89 System Administration, 89 Controller and Instrument Start Up, 90 Controller/Network Login, 90 License Key, 91 Demo Mode, 91 Launching 32 Karat™ Software, 91 Instrument Start Up, 91
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Instruments and Projects, 92 Configuration, 92 Online Versus Offline, 92 Using the Direct Control Window to Load the Cartridge and Samples, 93 TripleTOF® 5600 Mass Spectrometer Using AAO, 95 Methods and Sequences, 96 Creating and Editing a Method, 97 Instrument Setup Window, 97 Instrument Setup — Initial Conditions, 97 Instrument Setup — Time Program, 99 Separate Dialog, 100 Rinse Dialog, 102 Inject Dialog, 102 Stop Data, 104 End, 104 Saving a Method, 104 Overview of Creating a Sequence, 105 Single Run and Sequence Runs, 105 Programming a Sequence, 106 Sequence Table, 110 Sequence Validation, 111 CHAPTER 7: CESI 8000 Software, 113 Overview of the CESI 8000 Software, 113 Launching the CESI 8000 Software, 114 Login and Logout, 115 Lock and Unlock, 116 About Dialog, 116 Help, 116 Users, 116 Run Application, 117 Application Selection, 117 Samples and Vials, 120 Samples/Vials Dialog, 120 Insert Option, 122 Sequence Dialog, 123 Sequence Table Rows, 125 Fill Down Option, 129 Display Options, 130 Vial Legend, 131 Tray Detail View, 131 Vial Preview, 132
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Run Acquisition, 133 SCIEX TripleTOF® 5600 Mass Spectrometer Using AAO, 135 Graph Options, 136 Sequence Transfer Overview, 137 Sequence Transfer, 137 Describe Sequence, 143 Sequence Verification, 146 Sequence Display Options, 146 CHAPTER 8: Using a SCIEX TripleTOF® 5600 Mass Spectrometer with the CESI 8000 Plus System, 149 Introduction, 149 Stock Reagents and Other Consumables Required, 150 Preparation of Reagent Solutions and Test Sample, 151 Preparation of 10% (v/v) HAc—Background Electrolyte (BGE), 152 Preparation of 20% (v/v) Acetic Acid (HAc)—For LE pH Adjustment, 152 Preparation of 200 mM LE (Leading Electrolyte) Buffer, 152 Reconstitution of 1 μM Beta-Galactosidase Digest Solution (Beta-Galactosidase Stock Solution), 153 Preparation of Beta-Galactosidase Test Sample, 153 Preparation of Beta-Galactosidase for Auto-calibration, 154 Buffer Tray Setup, 154 Sample Tray Setup, 155 Procedure for Installation of Adapter and OptiMS Cartridge, 156 Methods for the CESI 8000 Plus System, 157 CESI-MS Beta-Galactosidase System Performance Test, 161 Capillary Conditioning, 164 Establishing a Stable Spray and Determining Optimum ESI Voltage, 167 Running CESI-MS, 174 Create a Sequence in the Analyst® TF Software, 175 Start a Sequence in the Analyst® TF Software, 178 Load Sample and Reagents on the CESI 8000 Plus System, 179 Sample Tray Setup, 179 Create a Sequence in the CESI 8000 Software, 181 Start a Sequence in the CESI 8000 Software, 185 Automatic Rinse Feature, 186 Beta-Galactosidase Analysis, 187 CHAPTER 9: Using a Thermo Scientific Mass Spectrometer with the CESI 8000 Plus System, 191 Introduction, 191
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Stock Reagents and Other Consumables Required, 192 Preparation of Reagent Solutions and Test Sample, 193 Preparation of 10% (v/v) Acetic Acid (HAc)—Background Electrolyte (BGE), 193 Preparation of 20% (v/v) Acetic Acid (HAc) — For LE Buffer pH Adjustment, 194 Preparation of 200 mM LE (Leading Electrolyte) Buffer, 194 Reconstitution of 1 μM Beta-Galactosidase Digest Solution (Beta-Galactosidase Stock Solution), 195 Preparation of Beta-Galactosidase Test Sample, 195 Sample Tray Setup for System Performance Test, 195 Procedure for Installation of Adapter and OptiMS Cartridge, 196 Capillary Conditioning, 197 Buffer Tray Setup, 197 Capillary Conditioning Procedure, 198 Establishing a Stable Spray and Determining Optimum ESI Voltage, 200 Tuning, 204 Running CESI-MS, 205 Create a Method with the Xcalibur Software, 206 Create a Sequence with the Xcalibur Software, 209 Start a Sequence with the Xcalibur Software, 210 Load Sample and Reagents on the CESI 8000 System, 211 Create a Sequence in the CESI 8000 Software, 213 Automatic Rinse Feature, 214 Start a Sequence in the CESI 8000 Software, 215 Beta-Galactosidase Analysis, 216 CHAPTER 10: Shutdown Procedures and Decoupling Instruments, 219 Shutdown and Decoupling Overview, 219 Cartridge Removal and the CESI 8000 Plus System Shutdown, 219 Cartridge Removal and Instrument Shutdown, 219 Decoupling the CESI 8000 Plus System, 224 Decoupling from the Thermo Scientific Mass Spectrometer, 224 Decoupling from the SCIEX Mass Spectrometer, 227 APPENDIX A: Specifications, 231 CESI 8000 Plus System, 231 Validated Controller Configuration, 232 Cart Specifications, 232 Sample Temperature Control, 233 Capillary Temperature Control, 233 Pressure and Vacuum System, 233
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Detector Specifications, 234 UV Detector, 234 Laser Induced Fluorescence (LIF) Detector (Optional), 235 Photo Diode Array (PDA) Detector (Optional), 235 APPENDIX B: Nominal Network Configuration, 237 Overview, 237 Switching from the CESI 8000 Plus System to Different Mass Spectrometer Systems, 238 Switching to the CESI 8000 Plus System from Different Mass Spectrometer Systems, 238 Switching from a SCIEX Mass Spectrometer to Another Mass Spectrometer, 239 Switching to a SCIEX Mass Spectrometer from Another Mass Spectrometer, 241 Advanced Networking and Computer Configuration for the Analyst® TF Software 1.7, 243 CE-MS Networking, 243 PC Configurations, 243 Network Sharing Setting for the Crossover Connection, 264 Setting the Mass Spectrometer Type, 266 Curtain Gas Patch Installation, 267 CE-MS Driver Installation, 269 Saving Network Configurations, 271 Analyst® TF Software Hardware Profile Configuration, 272 To configure the Mass Spectrometer Hardware Profile, 272 Curtain Gas Setting and Verification, 278 Curtain Gas Setting, 278 Curtain Gas Verification, 280 APPENDIX C: TripleTOF® 5600 Mass Spectrometer Manual and Auto-Calibration Methods, 283 Overview, 283 Required Reagents, 283 Reagent and Sample Preparation, 283 Manual Calibration Before Auto-calibration, 284 Create a Reference Table for CE-MS Calibration, 284 Manual Calibration of the Mass Spectrometer, 287 Manual Calibration in TOF Mass Spectrometer Mode, 288 Manual Calibration in Product Ion Mode, 295 Methods for Auto-Calibration, 298 CE Method for Auto-Calibration, 298 MS Method for Auto-Calibration, 299 Executing Auto-Calibration, 302 Create a Sequence in the Analyst® TF Software, 302
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Start a Sequence in the Analyst® TF Software, 305 Load Sample and Reagents on the CESI 8000 System, 307 Sample Tray Setup, 307 Create a Sequence in the CESI 8000 Software, 308 Start a Sequence in the CESI 8000 Software, 312 Automatic Rinse Feature, 314 Auto-Calibration Data Analysis, 314 Troubleshooting Auto-Calibration Failure, 319 Auto-calibration Troubleshooting Table, 321 APPENDIX D: Auto Vial Increment Feature, 323 Overview, 323 Setting Auto Vial Increment on a Rinse Step, 323 Setting Auto Vial Increment on an Injection, 324 Setting Auto Vial Increment on a Separation Step, 326 Auto Vial Increment in a Method, 328 Reviewing Vial Positions before Running a Sequence, 329 APPENDIX E: Conductive Liquid Capillary Conditioning, 331 Conductive Liquid Capillary (CLC) Conditioning Procedure, 331 APPENDIX F: Using Vials and Hardware Maintenance, 333 Overview, 333 Using CESI-MS Vials and Caps, 334 CESI-MS Vials and Micro Vials, 334 Filling the Micro Vials, 335 Hardware Maintenance Procedures, 336 Capillary Cleaning Procedure (Short Term Storage), 336 Capillary Storage Procedure, 337 Removing and Re-installing OptiMS Interface Plate, 338 Opening Levers, Electrodes, and the Interface Block, 340 Clean Electrodes, Opening Levers, and the Interface Block, 343 Refilling Coolant, 345 Replacing Quad Rings, 346 Replacing Fuses, 348 APPENDIX G: Troubleshooting and Mobile Cart Homing Procedure, 351 Overview, 351 Troubleshooting and Flow Diagrams, 351 Sample Preparation Considerations for Analysis with CESI- MS, 352 Properties of a Method or Sequence Cannot be Modified or Saved, 352 Blocked Sprayer Tip, 353 Possible Cause, 353
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Possible Solution, 353 Electrospray Cannot Be Established Between the CESI 8000 Plus System and Mass Spectrometer, 355 Electrospray is Detected at Zero Ion Spray (IS) Voltage while Applying CE Voltage, 356 Unstable Electrospray while Applying CE Voltage and IS Voltage, 357 Droplet at Capillary Tip Disappeared Indicating that Electrospray has Started, but No Mass Spectrometer Signal is Detected, 358 Signal of Total Ion Current (TIC) is Lower Than Usual and/or Background Signal Intensity Fluctuates More Than 40%, 359 Unstable CE Current, 360 No Analyte Signals Detected by Mass Spectrometer During a CE- MS Separation, 361 Some Analyte Peaks are Missing or Smaller Than Expected, 362 CE Current Gradually Decreases During CE-MS Separation, 363 The Iron-Acetate Cluster m/z 537 is Suppressing the Sample Signals, 364 High Voltage System Unable to Deliver the Required Current, 365 Fine Tuning Sprayer Tip Position for Thermo Scientific Mass Spectrometer, 366 Fine Tuning Sprayer Tip Position for SCIEX Mass Spectrometer, 368 CESI 8000 Plus Mobile Cart Homing Procedure, 370 Contact Us, 371 Customer Training, 371 Online Learning Center, 371 Purchase Consumables, 371 SCIEX Support, 371 CyberSecurity, 372 Documentation, 372
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Overview
A description of the symbols and labels that are used on the SCIEX CESI 8000 Plus High Performance Separation-ESI Module, or that are shown in this manual, can be found in this section.
Do not attempt to perform any procedure before carefully reading all instructions. If in doubt as to how to proceed in any situation, contact your SCIEX representative.
SCIEX urges its customers and employees to comply with all national health and safety standards such as the use of barrier protection. This may include, but is not limited to, protective eyewear, gloves, and suitable laboratory attire when operating or maintaining this or any other automated laboratory instrumentation.
WARNING If the equipment is used in a manner not specified by SCIEX, the protection provided by the equipment may be impaired.
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Alerts for Warning, Caution, Important, and Note
All warnings and cautions in this document include an exclamation point in a triangle or a triangle with an icon for a specific type of hazard.
The exclamation point symbol is an international symbol which serves as a reminder that all safety instructions should be read and understood before installation, use, maintenance, and servicing are attempted.
WARNING WARNING indicates a potentially hazardous situation which, if not avoided, could result in death or serious injury. It may also be used to indicate the possibility of erroneous data that could result in an incorrect diagnosis. May be used to indicate the possibility of severe instrument damage.
CAUTION CAUTION indicates a potentially hazardous situation, which, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. May be used to indicate the possibility of erroneous data that could result in an incorrect diagnosis.
IMPORTANT Used for comments that add value to the step or procedure being performed. Following the advice in the Important adds benefit to the performance of a piece of equipment or to a process.
NOTE Used to call attention to notable information that should be followed during installation, use, or servicing of this equipment.
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Instrument Safety Precautions
WARNING Risk of operator injury if: • All doors, covers and panels are not closed and secured in place prior to and during instrument operation. • The integrity of safety interlocks and sensors is compromised. • You contact moving parts. • You mishandle broken parts. • Doors, covers and panels are not opened, closed, removed and/or replaced with care. • Improper tools are used for troubleshooting.
To avoid injury: • Keep doors, covers and panels closed and secured in place while the instrument in use. • Take full advantage of the safety features of the instrument. Do not defeat safety interlocks and sensors. • Acknowledge and act upon instrument alarms and error messages. • Keep away from moving parts. • Report any broken parts to your SCIEX representative. • Use the proper tools when troubleshooting.
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CAUTION System integrity could be compromised and operational failures could occur if: • This equipment is used in a manner other than specified. Operate the instrument as instructed in the product manuals. • You introduce software that is not authorized by SCIEX into your computer. Only operate your system’s computer with software authorized by SCIEX. • You install software that is not an original copyrighted version. Only use software that is an original copyrighted version to prevent virus contamination.
CAUTION If you purchased this product from anyone other than SCIEX or an authorized SCIEX distributor, and, if it is not presently under a SCIEX Service Maintenance Agreement, SCIEX cannot guarantee that the product is fitted with the most current mandatory engineering revisions or that you will receive the most current information bulletins concerning the product. If you purchased this product from a third party and would like further information concerning this topic, contact your SCIEX representative.
Moving Parts or Sharp Objects
WARNING Risk of personal injury. To avoid injury due to moving parts, observe the following: • Never attempt to exchange labware, reagents, or tools while the instrument is operating. • Never attempt to physically restrict any of the moving components of the instrument. • Keep the instrument work area clear to prevent obstruction of the movement.
Electrical Safety To prevent electrically related injuries and property damage, properly inspect all electrical equipment prior to use and immediately report any electrical deficiencies. Contact a SCIEX representative for any servicing of equipment requiring the removal of covers or panels.
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Laser Safety (for Optional Laser Device)
WARNING This product may contain a laser module. The laser (optional) is designated as “Class 3B.” The “3B” classification means that “direct intrabeam viewing of this type of laser is always hazardous to personnel.” The laser and several other integral components are contained in a sealed housing that together comprise the laser assembly. The laser assembly has no user serviceable parts. Service of the laser assembly is restricted to qualified SCIEX Field Service Employees (FSE). Therefore, the overall laser classification of the CE Instrument is “Class 1,” defined as “lasers which are safe under reasonably foreseeable conditions of operation.” To prevent users from potentially harmful laser light, observe all safety warnings and NEVER REMOVE THE OUTER CASING OF THE LASER ASSEMBLY.
IMPORTANT The laser markings noted above can vary depending on the type of device, and will be described in the documentation provided with the module.
Class 1 Laser Caution Label If the instrument contains a laser system, a label reading “THIS PRODUCT CONFORMS TO APPLICABLE REQUIREMENTS OF 21 CFR 1040 AT THE DATE OF MANUFACTURE” is found near the Name Rating tag. The laser light beam is not visible.
CLASS 1 LASER PRODUCT THIS PRODUCT CONFORMS TO APPLICABLE REQUIREMENTS OF 21 CFR 1040 AT THE DATE OF MANUFACTURE.
MANUFACTURED:
726024-C
Chemical Precautions
• Determine which chemicals have been used in the system prior to service and regular maintenance. Refer to Safety Data Sheets for the health and safety precautions that must be followed with chemicals. • Work in a well-ventilated area. • Always wear assigned personal protective equipment, including powder-free neoprene or nitrile gloves, safety glasses, and a laboratory coat. • Follow required electrical safe work practices.
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• Avoid ignition sources when working with flammable materials, such as isopropanol, methanol, and other flammable solvents. • Take care in the use and disposal of any chemicals. Potential risk of personal injury if proper procedures for handling and disposing of chemicals are not followed. • Avoid skin contact with chemicals during cleaning and wash hands after use. • Comply with all of the local regulations for the storage, handling, and disposal of biohazardous, toxic, or radioactive materials.
• The lamp in this product contains mercury. Do not put in the trash. Recycle or dispose of according to local, state, or federal laws.
Safety Symbols and Labels
High Voltage Electric Shock Risk Symbol This symbol indicates that there is high voltage and there is a risk of electric shock, and the operator should use care when accessing this area.
Attention Safety Symbol This symbol calls attention to important information to read, or is accompanied by another symbol indicating a particular safety hazard. The information is located either on the label with the symbol or in the CESI 8000 Plus documentation.
Sharp Object Label This symbol indicates that there are sharp objects, and the operator should use care when accessing this area.
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Mobile Cart Caution Label A label cautioning users when moving the cart or adjusting the height of the cart can be found on the CESI 8000 Plus mobile cart.
The CESI 8000 instrument is situated on the mobile cart. The OptiMS cartridge spray tip on the CESI 8000 instrument has to be in very close proximity to a mass spectrometer when the system is used. To avoid damage to the OptiMS cartridge, remove it from the MS adapter before adjusting the height or moving the cart.
Cancer and Reproductive Harm Label This label indicates there is risk of cancer and reproductive harm to the operator. This warning is called for by the California Safe Drinking Water and Toxic Enforcement Act of 1986, commonly referred to as Proposition 65, enacted by the State of California. A full list of harmful chemicals is available at www.P65Warnings.ca.gov.
RoHS Notices
These labels and materials declaration table (the Table of Hazardous Substance’s Name and Concentration) are to meet People’s Republic of China Electronic Industry Standard SJ/T11364- 2006 “Marking for Control of Pollution Caused by Electronic Information Products” requirements.
China RoHS Caution Label This label indicates that the electronic information product contains certain toxic or hazardous substances. The center number is the Environmentally Friendly Use Period (EFUP) date, and indicates the number of calendar years the product can be in operation. Upon the expiration of
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the EFUP, the product must be immediately recycled. The circling arrows indicate the product is recyclable. The date code on the label or product indicates the date of manufacture.
Other Instrument Labels
This section provides information for some labels and symbols appearing on the CESI 8000 instrument housing. These labels and symbols may be associated with user-serviceable procedures. Individual hazards associated with a specific procedure in this manual may use these labels and symbols, and are included in Warnings or Cautions within the procedures for that task.
Recycling Label (WEEE) The symbol of a crossed-out wheeled bin on the product is required in accordance with the Waste Electrical and Electronic Equipment (WEEE) Directive of the European Union.
The presence of this marking on the product indicates:
• That the device was put on the European Market after August 13, 2005 and • That the device is not to be disposed via the municipal waste collection system of any member state of the European Union. Follow local municipal waste ordinances for proper disposal provisions to reduce the environmental impact of WEEE (waste, electrical, and electronic equipment). To safely dispose of this equipment, contact a local Customer Service office for complimentary equipment pick- up and recycling.
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Disposal of Devices Containing Mercury Components This product may contain a mercury-added part. Recycle or dispose of according to local, state, or federal laws. It is very important that you understand and comply with the safe and proper disposal of devices containing mercury components (switch, lamp, battery, relay, or electrode). The mercury component indicator label can vary depending on the type of device.
CE Mark Label A “CE” mark indicates that a product has been assessed before being placed on the market, and has been found to meet European Union safety, health, and/or environmental protection requirements.
RCM Mark Label The RCM mark is intended for use on products that comply with Australian communications Media Authority (ACMA) EMC Requirements.
CSA Mark Label The CSA symbol on the CESI 8000 instrument indicates that the instrument has been certified by a Nationally Recognized Testing laboratory (NRTL) to applicable Laboratory Equipment Safety Standards for the United States and Canada.
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综述
用于 SCIEX CESI 8000 Plus 高性能分离系统或在本手册中显示的符号和标签的描述可在 本节中找到。 仔细阅读所有说明之前，请勿尝试执行任何操作。在任何情况下如果不知如何处理， 请与您的 SCIEX 代表联系。
SCIEX 强烈要求其客户和员工遵守所有国家健康和安全标准，如防护的使用。此标准 包括但不限于下列事项：操作或维护本仪器或任何其他实验室自动仪器时，请佩戴防 护眼镜、手套和合适的实验室装备。
警告 如果设备的使用未能按照 SCIEX 公司所指定的方式进行，该设备所具备的 保护性能可能受损。
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警告、注意、重要事项以及注释的提示
本文档中的所有警告与注意都包含感叹号，设在三角形中。 感叹号符号是国际通用符号，用于提示在安装、使用、维护和维修前应阅读并理解所 有的安全说明。
警告 “警告”指可能的有害情况，若未加以避免，则会导致死亡或严重伤害。也 可用它来说明有可能出现可能导致错误诊断的错误数据。还可用它来说明 仪器严重损坏的可能性。
注意 “小心”是指如果未能避免可能导致轻微或中度伤害的潜在危险情况。可用 于警示不安全操作。可能用于说明有可能出现可能导致错误诊断的错误数 据。
重要 用于对正在执行的步骤或程序进行有价值的备注。遵循“重要事项”中的建议有助于改善 某件设备或某个程序的性能。
注释 用于提醒在设备安装、使用、或维修过程中应遵照的重要信息。
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仪器安全防护措施
警告 如果出现以下情况，操作员会有受到伤害的危险： • 操作仪器过程中或之前没有关闭所有仪器门、盖板和面板，并确保其安 全到位。 • 安全联锁和传感器的完整性受到损害。 • 接触到活动部件。 • 对破碎的部件处理不当。 • 没有小心地打开、关闭、移去和/或更换仪器门、盖板和面板。 • 使用不正确的工具进行故障排除。
要避免造成伤害，请遵循： • 在使用仪器时保持仪器门、盖板和面板关闭，并确保其固定到位。 • 充分利用该仪器的安全特性。请勿破坏安全联锁装置和传感器。 • 确认仪器警报和错误消息并进行相应的处理。 • 远离活动部件。 • 向 SCIEX 业务代表报告所有破碎的部件。 • 使用正确的工具来进行故障排除。
注意 以下情况会破坏系统完整性并可能导致操作失败： • 未按操作要求使用本设备。请按照“产品手册”中说明的方式操作仪器。 • 在计算机中安装了未经 SCIEX 授权的软件。请在系统的计算机上仅运 行 SCIEX 授权的软件。 • 安装的软件并非是具有原始版权的版本。请仅使用具有原始版权的软 件，以防止病毒感染。
注意 如果是从 SCIEX 或 SCIEX 授权分销商之外的另一方购买的本产品，同时 目前也不在 SCIEX 服务维护协议的范围内，则 SCIEX 不能担保该产品具 有最新的强制性工艺修订，也不能担保用户可获得有关该产品的最新信息 公告。如果您从第三方购买了此产品且想要了解这方面的更多信息，请联 系您的 SCIEX 代表。
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活动部件或尖锐物
警告 可能导致人身伤害。为避免遭到活动部件的伤害，请遵守以下操作提示： • 仪器运转时，请勿调换实验室器具、试剂或工具。 • 不得手动限制仪器上任何组件的运动。 • 保持仪器工作区清空，以防限制组件的活动。
电气安全 为避免电气相关的伤害和财产损失，使用前请正确检查所有的电气设备并及时报告所 有的电气缺陷。维修设备时如需拆除盖板或面板，请联系 SCIEX 代表。
激光安全 （适用于可选激光设备）
警告 此产品可能包含一个激光模块。激光 （可选）指定为“3B 类”。“3B”类表明 “直视此类型激光会造成人身伤害”。 激光装置由激光和其它构成部分组成，储备在一个密封的外壳里。激光装 置没有用户维修的部件，维修服务仅限于合格的SCIEX维修员工。 系统正常操作情况下，用户不会接触到激光。因此，CE 仪器的整体激光分 类属于“1 类”，定义为“合理且可预测操作情况下的安全激光”。 为了避免用户受到可能的激光伤害，请遵守安全警告，并且注意任何情况 下不能移除激光装置的外壳。
重要 上述激光标识会根据设备的类型存在差异，因此将会在模块提供的文档中对激光进行描 述。
1 类激光小心标签 (1级激光警告标签) 如果仪器包含激光系统, 应该能够在名称评级标签附近找到一个标签：此产品生产时符 合 21 CFR 1040 的适用要求。激光束 不可见。
CLASS 1 LASER PRODUCT THIS PRODUCT CONFORMS TO APPLICABLE REQUIREMENTS OF 21 CFR 1040 AT THE DATE OF MANUFACTURE.
MANUFACTURED:
726024-C
A016350L EPS
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化学品注意事项
• 在维修和定期维护前，首先确定系统中已经使用了哪些化学品， 然后请参阅化学 品安全技术说明书实施有关健康和安全预防的措施。 • 在通风良好的区域工作。 • 一定要穿戴指定的个人防护设备，包括无粉氯丁橡胶或丁腈手套、防护眼镜和实 验室外套。 • 遵循所要求的电气安全工作实践。 • 当工作中用到易燃材料，如异丙醇、甲醇和其他易燃溶剂时，请避免火源。 • 要小心地使用和处置任何化学品。 如果不遵循处理和处置化学品的适当程序，就 会存在人身伤害的潜在风险。 • 清洗过程中应避免皮肤接触化学品，使用后洗手。 • 请遵守关于生物危害性、有毒或放射性物质的存储、处理和处置的所有当地法 规。 • 此产品的灯含汞。 请勿混入垃圾。 请遵照当地、州/省或联邦法律进行回收或处 置。
安全符号和标签
高压电击危险符号 此符号表示存在高电压和电击危险，并且操作员进入此区域时应小心操作。
注意安全符号 此符号提示您注意阅读重要信息，或者与另一符号结合表明存在特定的安全隐患。信 息位于符号标签上，或者在 CESI 8000 Plus 文档中。
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尖锐物标签 此符号表示存在尖锐物，并且操作员进入此区域时应小心操作。
流动车警告标签 在移动手拉车或调整手拉车的高度时，请注意在 CESI 8000 Plus 流动手拉车上的用户警 告标签。
CESI 8000 仪器位于移动手拉车上。使用系统时，CESI 8000 仪器上的 OptiMS盒式喷射头 ( 消费品）必须十分接近质谱仪。为了避免损坏OptiMS盒，在调整高度或移动手拉车之 前，请将它从 MS 适配器上移除。
致癌和生殖危害标签 此标签表示对操作员存在致癌和生殖危害风险。此项警告由 1986 年的 《加州安全饮 用水和有毒物质强制法令》(California Safe Drinking Water and Toxic Enforcement Act) 提 出 （通常称为 65 号提案 (Proposition 65)，加利福尼亚州颁布）。有害化学物质的完整 列表请访问： www.P65Warnings.ca.gov.
RoHS 通知
这些标签和材料声明表 （有害物质的名称和含量表）必须符合中华人民共和国电子行 业标准 SJ/T11364-2006 《电子信息产品污染控制标识要求》中的要求。
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中国 RoHS 警告标签 该标签表示电子信息产品包含某些有毒或有害物质。中间数字为环保使用期限 (EFUP) 日期，表示产品可运行的年数。EFUP 到期后，必须立即回收产品。环形箭头表示产品 可回收。标签或产品上的日期代码为制造日期。
其他仪器标签
本节提供了出现在 CESI 8000 仪器外罩上的一些标签和符号信息。这些标签和符号可能 与用户自行执行的操作过程有关。本手册中，与特定操作过程相关的各种风险可能使 用这些标签和符号，详见该任务操作过程中的“警告”或“注意”部分。
环保标签 (WEEE) 按照欧盟报废电子电气设备 (WEEE) 指令要求，产品上必须标有带叉的有轮垃圾桶符 号。
产品上出现该标志，说明：
• 该设备是在 2005 年 8 月 13 日以后投放欧洲市场的，并且 • 设备将不通过欧盟的任何成员国的市政废物收集系统进行处置。 遵循当地城市废物法规条例中的合适处理规定，减少 WEEE （废电子电机设备）对环 境的影响。 为了安全地处理设备，请联系当地的客户服务部进行免费的仪器上门回 收。
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处理包含加汞组件的设备 此产品中可能包含加汞组件。应依据当地/国家的或联邦法律循环使用或处理。您理 解并遵守对含加汞组件 （开关、灯、电池、继电器或电极）设备的安全正确处理规定 至关重要。加汞组件指示器标签可能发生变化，具体取决于设备的类型。
CE 标志标签 标志– “CE”标志表示产品上市前经过评估，并已被认定符合欧盟安全、健康和/或环境 保护要求。
RCM 标志标签 RCM 标 记用于符合澳大利亚通信媒体管理局 (ACMA) EMC 要求。
CSA 标识标签 CESI 8000 仪器上的 CSA 符号表明，该仪器被国家认可测试实验室 (NRTL) 认证为符合适 用的美国和加拿大实验室设备安全标准。
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Présentation
Dans cette section, il est possible de trouver une description des symboles et des étiquettes utilisés sur le Module de séparations hautes performances SCIEX CESI 8000 Plus, ou qui sont indiqués dans ce manuel.
N’essayez pas d’exécuter une procédure avant d'avoir lu attentivement toutes les instructions. En cas de doute sur la manière de procéder dans une situation donnée, contactez votre représentant SCIEX.
SCIEX incite fortement ses clients et ses employés à respecter toutes les consignes nationales de santé et de sécurité telles que l'utilisation d'équipements de protection personnelle. Cela peut inclure, sans y être limité, le port de lunettes de protection, de gants et d’un vêtement de laboratoire approprié lors de l’utilisation ou de l’entretien de cet instrument ou de tout autre instrument automatisé de laboratoire.
WARNING Si l'équipement est utilisé d'une manière non spécifiée par SCIEX, la protection qu'il fournit risque d'être rendue inefficace.
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Alertes « Avertissement, Mise en garde, Important et Remarque »
Tous les avertissements et mises en garde de ce document comprennent un point d'exclamation entouré d'un triangle.
Le point d'exclamation est le symbole international qui permet de rappeler que toute consigne de sécurité doit être lue et comprise avant que l'installation, le fonctionnement, la maintenance et l'entretien ne soient entrepris.
WARNING AVERTISSEMENT indique une situation potentiellement dangereuse qui, si elle n’est pas évitée, peut entraîner la mort ou des blessures graves. Elle peut aussi être utilisée pour indiquer la présence éventuelle de données erronées qui peuvent entraîner un diagnostic incorrect. Peut être utilisée pour indiquer la possibilité de dommages importants sur l'instrument.
CAUTION MISE EN GARDE indique une situation potentiellement dangereuse qui, si elle n’est pas évitée, peut entraîner des blessures mineures ou modérées. Cette mention peut aussi être utilisée pour alerter sur des pratiques dangereuses. Peut aussi être utilisée pour indiquer la présence éventuelle de données erronées qui peuvent entraîner un diagnostic incorrect.
IMPORTANT est utilisée pour les commentaires qui complètent une étape ou la procédure concernée. En suivant le conseil prodigué, l’opérateur peut améliorer le fonctionnement de l’appareil ou le déroulement du procédé en question.
NOTE Sert à attirer l’attention sur des informations importantes dont il convient de tenir compte lors de l’installation, l’utilisation ou l'entretien de cet équipement.
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Précautions de sécurité pour l'instrument
WARNING Risque de blessures corporelles si : • Tous les capots, panneaux et portes ne sont pas fermés et fixés solidement avant et pendant l’utilisation de l’instrument ; • L’intégrité des verrouillages et des capteurs de sécurité n’est pas assurée ; • Vous entrez en contact avec des pièces mobiles ; • Vous ne maniez pas avec précaution des pièces cassées ; • Les portes, les capots et les panneaux ne sont pas ouverts, fermés, retirés et/ou replacés avec précaution ; • Des outils inadaptés sont utilisés lors du dépannage.
Pour éviter toute blessure : • Gardez toutes les portes, tous les capots et panneaux fermés et fixés solidement pendant l’utilisation de l’instrument. • Faites usage de toutes les fonctions de sécurité de l’instrument. Ne rendez pas inopérants les verrous et les capteurs de sécurité. • Tenez compte des alarmes et des messages d’erreur de l’instrument. • Tenez-vous à distance des pièces mobiles. • Signalez toute pièce cassée à votre représentant SCIEX. • Utilisez des outils adaptés lors du dépannage.
CAUTION L’intégrité du système peut être compromise et des pannes risquent de se produire si : • Cet équipement est utilisé d’une manière autre que celle spécifiée Utilisez cet instrument conformément aux instructions des manuels du produit. • Vous installez un logiciel non autorisé par SCIEX sur votre ordinateur. N’utilisez l’ordinateur de votre système qu’avec des logiciels agréés par SCIEX. • Vous installez un logiciel qui n’est pas une version d’origine protégée par droits d'auteur. N’utilisez que des logiciels qui sont des versions d’origine protégées par droits d’auteurs afin d’éviter toute contamination par virus informatique.
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CAUTION Si vous avez acheté ce produit ailleurs que chez SCIEX ou un distributeur SCIEX autorisé, et s’il ne fait pas l’objet d’un contrat de maintenance SCIEX, SCIEX ne peut garantir que le produit a bénéficié des toutes dernières révisions techniques obligatoires ou que vous recevrez les bulletins d’information les plus récents concernant le produit. Si vous avez acheté ce produit à un tiers et souhaitez obtenir d’autres informations à ce sujet, contactez votre représentant SCIEX.
Pièces mobiles ou Objets tranchants
WARNING Risque de blessure corporelle. Pour éviter toute blessure provoquée par des pièces mobiles, veuillez suivre les consignes suivantes: • Ne jamais tenter de remplacer des fournitures, des réactifs ou des outils pendant le fonctionnement de l'instrument. • Ne jamais tenter de restreindre physiquement les composants mobiles de l’nstrument. • Maintenir la instrument zone de travail dégagée pour éviter toute obstruction de mouvement.
Sécurité électrique Afin d'éviter toute blessure ou dommage matériel liés à l'électricité, inspectez convenablement tous les équipements électriques avant utilisation et signalez immédiatement toute défaillance électrique. Contactez a SCIEX representative pour tout entretien de l'équipement nécessitant le retrait des capots ou des panneaux.
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Sécurité laser (pour les appareils avec laser en option)
WARNING Il se peut que ce produit contienne un module laser. Le laser (en option) est désigné comme « Classe 3B ». La classification « 3B » signifie que « regarder directement dans le faisceau de ce type de laser est toujours dangereux pour le personnel ». Le laser (en option) et d’autres composants intégraux sont enserré dans un boîtier étanche dont l’ensemble constitue le bloc laser. Le bloc laser ne contient aucune pièce dont l'entretien peut être effectué par l’utilisateur. Les procédures d'entretien du bloc laser sont limitées aux employés de terrain qualifié de SCIEX. La lumière laser n’est pas accessible à l'utilisateur au cours du fonctionnement normal du système. Par conséquent, la classification laser globale de l'instrument CE est de « classe 1 », c’est-à-dire des « lasers qui ne présentent pas de danger dans des conditions de fonctionnement raisonnablement prévisibles ». Pour éviter que les utilisateurs soient exposés à une lumière laser éventuellement nuisible, respectez tous les avertissements de sécurité et NE RETIREZ JAMAIS LE BOÎTIER EXTÉRIEUR DU BLOC LASER.
IMPORTANT Les marquages laser cités ci-dessus diffèrent en fonction du type de l'appareil et seront décrits dans la documentation fournie avec le module.
Étiquette de mise en garde : produit laser de classe 1 Si l'instrument comporte un système laser, une étiquette indiquant « CE PRODUIT EST CONFORME AUX EXIGENCES APPLICABLES DE LA NORME 21 CFR 1040 À LA DATE DE FABRICATION » se situe à proximité de l'étiquette signalétique. Le faisceau laser n'est pas visible.
CLASS 1 LASER PRODUCT THIS PRODUCT CONFORMS TO APPLICABLE REQUIREMENTS OF 21 CFR 1040 AT THE DATE OF MANUFACTURE.
MANUFACTURED:
726024-C
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Précautions chimiques
• Déterminez quels sont les produits chimiques qui peuvent avoir été utilisés dans le système avant sa mise en service et son entretien régulier. Consultez les fiches de données de sécurité pour les précautions d'hygiène et de sécurité qui doivent être suivies avec les produits chimiques. • Travailler dans un endroit bien aéré. • Portez toujours l’équipement de protection individuelle attribué, comprenant des gants en néoprène non poudrés ou des gants nitrile, des lunettes de sécurité et une blouse de laboratoire. • Suivez les pratiques sécurisées pour les travaux d'électricité. • Éviter les sources d'étincelles lors de l'utilisation de matériaux inflammables, comme l'isopropanol, le méthanol, et autres solvants inflammables. • Utilisez et éliminez les produits chimiques avec précaution. Risque potentiel de blessure corporelle si les procédures adéquates de manipulation et d'élimination des produits chimiques ne sont pas respectées. • Évitez tout contact des produits chimiques avec la peau pendant le nettoyage et lavez-vous les mains après utilisation. • Conformez-vous à toutes les réglementations locales pour le stockage, la manipulation et la mise au rebut des déchets biologiques, toxiques ou radioactifs. • La lampe de ce produit contient du mercure. Ne pas mettre au rebut. Recycler ou mettre au rebut conformément à la législation locale, étatique ou fédérale.
Symboles et étiquettes de sécurité
Symbole « Haute tension, risque de choc électrique » Ce symbole indique la présence de haute tension et d'un risque de choc électrique, et l'opérateur doit faire preuve de précautions lors de l'accès à cette zone.
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Symbole de sécurité Attention Ce symbole attire l'attention sur des informations importantes à lire, ou est accompagné d'un autre symbole indiquant un danger particulier pour la sécurité. Les informations se trouvent soit sur l'étiquette avec le symbole ou dans la documentation du CESI 8000 Plus.
Étiquette Objet tranchant Ce symbole indique la présence d'objets tranchants et l'opération doit faire preuve de précautions lors de l'accès à cette zone.
Étiquette de mise en garde chariot mobile Vous trouverez une étiquette avertissant les utilisateurs lors du déplacement du chariot ou réglant la hauteur du chariot sur le chariot mobile CESI 8000 Plus.
L'instrument CESI 8000 est situé sur le chariot mobile. La pointe de la cartouche de pulvérisation OptiMS de l'instrument CESI 8000 doit se trouver très près d'une spectromètre de masse lors de l'utilisation du système. Pour éviter les dommages sur la cartouche de OptiMS, retirez-le de l'adaptateur MS avant de régler la hauteur ou de déplacer le chariot.
Étiquette concernant les risques de cancer et de malformations congénitales Cette étiquette indique si l'appareil présente des risques de cancer et de malformations congénitales pour l'opérateur. Cet avertissement s'applique en vertu du décret de 1986 en vigueur dans l'état de Californie sur la sécurité de l'eau potable et les substances toxiques,
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communément appelé Proposition 65. La liste des produits chimiques dangereux est disponible sur www.P65Warnings.ca.gov.
Consignes RoHS
Ces étiquettes et ce tableau de déclaration des matériaux (Tableau des noms et des concentrations des produits dangereux) répondent aux exigences de la norme de l'industrie électronique de la République populaire de Chine SJ/T11364-2006 « Marquage destiné au contrôle de la pollution causée par les produits informatiques ».
Étiquette de mise en garde RoHS (Chine) Cette étiquette indique que ce produit informatique renferme certains éléments toxiques ou dangereux. Le nombre au centre indique la date de fin de période d'utilisation sans risques pour l'environnement (EFUP) et indique le nombre d'années le produit peut être utilisé. Une fois cette date dépassée, le produit doit être immédiatement recyclé. Le cercle de flèches indique que le produit est recyclable. La date sur l'étiquette ou le produit correspond à la date de fabrication.
Autres étiquettes de l'instrument
Cette section fournit des informations sur certaines étiquettes et certains symboles qui apparaissent sur le boîtier de l'instrument CESI 8000. Ces étiquettes et symboles peuvent être associés aux procédures exécutables par l’utilisateur. Les risques individuels associés à une procédure spécifique décrite dans ce manuel peuvent être indiqués par ces étiquettes et symboles et sont précisés dans les Mises en garde ou les Avertissements dans les procédures décrivant la tâche.
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Étiquette de recyclage (WEEE) Le symbole représentant une poubelle barrée sur le produit est obligatoire conformément à la directive Déchets d'équipements électriques et électroniques (DEEE) de l'Union européenne.
La présence de ce symbole sur le produit indique que : • l'appareil a été mis sur le marché européen après le 13 août 2005, et que • l'appareil ne doit pas être éliminé par le système municipal de collecte des déchets d'aucun des États membres de l'Union européenne. Suivre les ordonnances municipales sur les déchets pour la mise au rebut en vue de réduire l'impact environnemental des DEEE (déchets d'équipements électriques et électroniques). Afin d’éliminer cet équipement en toute sécurité, contacter un bureau du service à la clientèle local pour bénéficier de l’enlèvement et du recyclage gratuits de l’équipement.
Élimination des appareils contenant des composants au mercure Il se peut que ce produit contienne des composants avec du mercure ajouté. Recycler ou éliminer conformément aux lois en vigueur. Il est très important de comprendre et de respecter les procédures appropriées d'élimination des appareils contenant des composants au mercure (interrupteur, lampe, batterie, relais ou électrode). L'étiquette indiquant un composant au mercure diffère en fonction du type d'appareil.
Étiquette marque CE Marque CE - La marque « CE » indique qu’un produit a été évalué avant d’être placé sur le marché et qu’il a été jugé comme répondant aux exigences de l’Union européenne en matière de sécurité, de santé et/ou de protection de l’environnement.
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Étiquette marque MRC Le marquage MRC est destiné aux produits conformes aux exigences de l’ACMA (organisme national australien chargé de la réglementation des télécommunications) en matière de compatibilité électromagnétique.
Étiquette marque CSA Le symbole CSA sur l’instrument CESI 8000 indique que l’instrument a été certifié par l’organisme Nationally Recognized Testing laboratory (NRTL - laboratoire national d’essai reconnu) aux Normes sur la sécurité de l’équipement de laboratoire pour les États-Unis et le Canada.
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About This Manual
This manual provides information on how to use the SCIEX CESI 8000 Plus High Performance Separation-ESI Module to perform CESI-MS experiments. In addition to the systems described here, additional adapters for other mass spectrometers are available from SCIEX:
• OptiMS Waters MS Adapter for NanoLockSpray and NanoFlow Ion Sources • OptiMS Thermo MS Adapter for Nanospray Flex and Nanospray Flex NG Ion Sources • OptiMS Bruker MS Adapter for Bruker Mass Spectrometers
The principles for using these adapters are similar to what is shown in this manual, but for specific details refer to the Installation Guide for the mass spectrometer adapter in use.
Optional Detectors The CESI 8000 Plus High Performance Separation-ESI Module also supports traditional capillary electrophoresis experiments, using UV detection. The UV detector allows for 6 different bandpass filters. Two additional detectors are available from SCIEX:
• A photo diode array (PDA) detector that is in the 190 nm to 600 nm range. • A laser-induced fluorescence (LIF) detector that permits high-sensitivity analysis of labeled molecular species.
For instructions on using the UV, PDA, or LIF detector, go to the SCIEX web site at www.sciex.com and then click Products > Capillary Electrophoresis Instruments > PA 800 Plus Pharmaceutical Analysis System. Click the Resources tab.
To learn... Click the link for... How to install, calibrate, and maintain the detectors PA 800 Plus Maintenance Guide About the different detectors PA 800 Plus System Overview Guide About data from a PDA detector PA 800 Plus Method Development Guide
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Introduction
CESI-MS is the integration of Capillary Electrophoresis (CE) with Electrospray Ionization (ESI) in a dynamic process within a single device. It is designed for mass spectrometry applications analyzing charged and polar molecules. CESI is a front-end separation-and-ionization technology which combines the high efficiency characteristics of CE with ESI. This integration finally brings together the high resolution power of CE in an ultra low-flow format that enhances the sensitivity of mass spectrometry while reducing ion suppression. Within the CESI 8000 Plus System, sample components are separated by an electrical field that is applied between the inlet vial and the OptiMS sprayer (Figure 4.1). Conductive fluid is automatically delivered from a system vial in order to complete the CE circuit. The OptiMS sprayer does not require any make-up (sheath) liquid. This ensures that the intrinsic advantages of CE are delivered by the sprayer to the mass spectrometry without dilution or disturbance, resulting in ultra-low flow. The OptiMS cartridge assembly consists of a separation capillary composed of the porous sprayer, conductive liquid capillary, circulating liquid cooling, and sprayer housing (Figure 4.1). The circulating coolant maintains the capillary temperature, maximizing reproducibility by controlling Joule heating. The capillary is housed in a protective cartridge that allows for easy transfer and routine use in a rugged and robust manner. The cartridge simply clicks into place within the CESI 8000 Plus System, creating an easy-to-use plug-and-spray set-up for the mass spectrometer.
Figure 4.1 CESI 8000 Plus System Conceptual Overview
1. OptiMS Cartridge 5. Mass Spectrometer Adapter 8. High Voltage Power 2. Separation Capillary 6. Mass Spectrometer Power Supply Supply 3. Conductive Liquid Capillary 7. CESI 8000 Plus Current Monitor 9. HV Input Cable 4. OptiMS Sprayer Housing 10.HV Output Cable
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This manual will describe the major components of the CESI 8000 Plus System with OptiMS technology, and common procedures such as installation, integration with mass specs, as well as the more commonly used software procedures.
Appendices describe care and maintenance of CESI 8000 Plus components, and corrective actions for specific problems that may occur.
Instrument
The main components of the CESI 8000 Plus High Performance Separation-ESI Module include a CE platform and OptiMS sprayer. The CE platform has trays that hold sample vials, buffers, and other solutions, an interface block, a high-voltage power supply and electrodes, temperature control hardware, and a sample injection mechanism (Figure 4.2 and Figure 4.3).
Figure 4.2 CESI 8000 Plus System
1. OptiMS Cartridge Installed 4. High Voltage Input Connection from Mass Spectrometer 2. OptiMS Access Panel 5. High Voltage Output Connection to OptiMS Adapter 3. OptiMS Cartridge Sprayer 6. Holster for OptiMS Cartridge Sprayer
The main power switch is on the lower-right side of the front of the instrument. Most of the connections for external system components are on the upper-left side panel of the instrument, except for the AC inlet and the fuse holder which are located on the back of the instrument. The capillary exits out of the right side. Three fans supply cooling air flow for internal system
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components. Air is exhausted through the vents at the side and back of the instrument. Keep at least four inches (10 cm.) of clearance at each vent to ensure adequate air flow.
WARNING Electrical Shock Hazard. Completely connect the High Voltage Input and Output connection cables at both ends before turning on and operating the CESI 8000 Plus System.
High voltage (HV) from the mass spectrometer connects to the current monitor circuit on the CESI 8000 Plus System through the input HV cable. An output cable delivers the voltage from the outlet side of the current monitor circuit to the OptiMS capillary, required for the electrospray.
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, buffers and rinse solutions). The trays are on two parallel tracks. The trays on the left are inlet trays for sample and buffer; the trays on the right are outlet trays for buffer and/or conductive fluid (Figure 4.3).
Figure 4.3 CESI 8000 Plus System Trays
1. Inlet Sample Tray (48 Vials) 3. Outlet Sample Tray (48 Vials) 2. Inlet Buffer Tray (36 Vials) 4. Outlet Buffer Tray (36 Vials)
Each buffer tray has slots for 36 CESI-MS vials. The sample tray holds 48 CESI-MS vials. The trays are 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 4.4).
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Figure 4.4 Sample and Buffer Trays
8
7
6
6 5
5 4
4 3 A B C DE F 2 3
1 2 ABCDEF 1
1 2
1. Buffer Tray 2. 48-Vial Sample Tray for CESI-MS Vials (can also be used as a holder for micro vials containing samples)
WARNING Wear protective eyewear, lab coat, and gloves when opening the sample cover. The CESI-MS vials are pressurized during rinse and separation-with-pressure events. To reduce the risk of breakage and expelled particles, use only SCIEX vials designed for the CESI 8000 Plus System (PN B11648). Inspect every vial for damage before use. Do not use any vial that appears cracked or damaged in any way.
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Syringe Pump, Power Supply, LEDs and Interlock
Syringe Pump The CESI 8000 Plus System can generate pressures with an internal pump mechanism. This pump can supply 0.1 psi to 25 psi to perform pressure injections or low-pressure mobilizations. The pump can apply a maximum of 100 psi to move the fluids through the capillary. The pressure can also be applied to both ends of the OptiMS cartridge (for example, both capillaries) at the same time, allowing for both background electrolyte and conductive fluid to be simultaneously pumped into the OptiMS cartridge.
High Voltage (HV) Power Supply The HV power supply can deliver a maximum of 30 kV with a maximum current of 300 μA. The voltage programming range is from 1 kV to 30 kV in 100 V increments. The polarity is configured in the software. The current programming range is from 3.0 μA to 300 μA in 0.1 μA increments. The software allows the user to select current, voltage, or power operation. During operation the system will ramp the current, voltage, or power up to the programmed value. Limits for current, voltage, and power can be entered to protect the capillary. For example, if the user programs a voltage setting for 30 kV, but the setting for maximum current is only 3.0 μA, the system can reach the limit set for the current before reaching the programmed voltage.
LED Indicators The front panel of the instrument contains LED indicators for power, ultraviolet (UV), high voltage (HV+ and HV-) (Figure 4.5).
Figure 4.5 LED Indicators on the CESI 8000 Plus System
• PWR — indicates instrument power on or off.
• UV — for service only.
• HV+ — indicates high voltage normal polarity.
• HV- — indicates high voltage is on reverse polarity.
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Cartridge and Sample Cover Interlocks The hinged doors of the CESI 8000 Plus System have interlock sensors that prevent unsafe access to the inside of the instrument. The outer door is called the sample cover; the second inner door is called the cartridge cover. Opening the sample cover (Figure 4.6) does the following:
CAUTION Wear protective eyewear, labcoat, and gloves before opening the sample cover.
Figure 4.6 Opening the Sample Cover (Outer Door)
• Stops any tray movement immediately. • Prevents the execution of any programmed events that require tray movement. • Aborts a method or sequence when a step that requires tray movement is encountered. • Causes the instrument to beep every 5 seconds if a method is running or if high voltage is applied while the tray is not moving. Opening the cartridge cover (Figure 4.7) does the following:
CAUTION Do not open the cartridge cover during a run. If opened during a run, the method will be aborted and separation will fail.
Figure 4.7 Opening the Cartridge Cover (Inner Door)
PWR
UV CAUTION
HV+
HV-
• Turns off the high voltage if it is on. • Turns off the pump that circulates the capillary coolant.
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OptiMS Cartridge
The OptiMS cartridge assembly consists of a separation capillary which terminates in an ESI sprayer tip and a conductive liquid capillary, both of which are encased in a liquid cooling tube (Figure 4.8). These capillaries are shrouded at the inlet ends by protective covers which automatically retract when the OptiMS cartridge is installed in the CESI 8000 Plus System.
The spray end (5) is made of PEEK, which is a high performance plastic material that has been tested and proven to be inert under CESI-MS conditions. The OptiMS cartridge is available with OptiMS Silica Surface capillaries (which are bare fused- silica capillaries). For instructions on cleaning and storage of the OptiMS cartridge, refer to the Capillary Cleaning Procedure (Short Term Storage) and Capillary Storage Procedure sections in APPENDIX F of this manual.
Figure 4.8 The OptiMS Cartridge
1. Cartridge Body (installed in the CESI instrument) 6. Sprayer Tip 2. Separation Capillary (within liquid cooling tube) 7. Protective Sheath (shown 3. Conductive Liquid Capillary (within liquid cooling tube) retracted; exposing capillary end) 4. Protective Sheaths (retract upon installation of OptiMS 8. Exposed Capillary (when sheath cartridge) is retracted) 5. OptiMS Sprayer Housing (installs into an adapter that 9. Inlet End of Separation Capillary fits a specific mass spectrometer ion source) 10. Outlet End of Cartridge Body
The separation capillary (2) and the conductive liquid capillary (3) are separate capillaries, and the content of the separation capillary never contacts or mixes with the content of the conductive liquid capillary (Figure 4.9). The ESI needle, located in the spray end, closes the circuit between the CESI 8000 Plus System and the mass spectrometer, thus allowing for electrospray to occur.
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Figure 4.9 Conceptual View of Inside the Sprayer End
1. ESI Needle 4. Etched Segment of Separation Capillary 2. Conductive Liquid 5. Plume 3. Separation Capillary
The protective sheaths shown in Figure 4.8 have a locking mechanism that only exposes the inlet ends of the separation and conductive liquid capillaries when the OptiMS cartridge is installed in the CESI 8000 Plus System. The locking mechanism will not retract if the OptiMS cartridge is not installed on the CESI 8000 Plus System.
The OptiMS sprayer locks into adapters specifically designed to fit the SCIEX Nanospray® III ion source and ion sources for Thermo Scientific, Bruker, and Waters mass spectrometers. Refer to the Installation Guide for the mass spectrometer adapter in use. The following OptiMS adapters are available:
WARNING Electrical Shock Hazard. To prevent a shock hazard, do not touch an installed mass spectrometer adapter that is connected to the CESI 8000 Plus System when the separation voltage is on.
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• OptiMS adapter for SCIEX mass spectrometer (Figure 4.10)
Figure 4.10 Adapter for SCIEX Nanospray® III Ion Source
1. HV Input Cable 2. HV Output Cable
• OptiMS adapter for Thermo Scientific mass spectrometer (Figure 4.11)
Figure 4.11 Adapter for Thermo Scientific NanoSpray II ion source
1. HV Output Cable
NOTE The HV input cable for the Thermo Scientific adapter is shipped separately in the kit box.
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CESI-MS Integration Procedures
This chapter discusses the integration of the CESI 8000 Plus System with the following mass spectrometers:
• Thermo Scientific LTQ mass spectrometer series • Thermo Scientific Q Exactive mass spectrometer • SCIEX TripleTOF® 5600 mass spectrometer
Included in this chapter are the procedures for the installation of the OptiMS cartridge and mass spectrometer adapters, and the alignment of the instruments. The following procedures are discussed:
• CESI 8000 Plus System: Installing OptiMS Cartridge • Thermo Scientific Mass Spectrometer: Integration with the CESI 8000 Plus System • SCIEX TripleTOF® 5600 Mass Spectrometer: Integration with the CESI 8000 Plus System
WARNING Electrical Shock Hazard. Disconnect the power before any instrument disassembly. Failure to do so can cause electrical shock or other injury.
WARNING Electrical Shock Hazard. Maintenance or repair procedures not specifically described in this manual present a risk of electrical shock or injury. Contact a SCIEX Field Service Employee (FSE) for maintenance service and support.
WARNING Electrical Shock Hazard. Do not attempt to defeat any of the instrument interlocks or safety mechanisms.
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WARNING Electrical Shock Hazard. Do not touch the mass spectrometer adapter when connected to the CESI 8000 Plus System and the separation voltage is on.
CESI 8000 Plus System: Installing OptiMS Cartridge
The OptiMS interface plate must be installed onto a CESI 8000 Plus System before an OptiMS cartridge can be installed. Refer to Removing and Re-installing OptiMS Interface Plate in APPENDIX F: Using Vials and Hardware Maintenance for instructions.
OptiMS Cartridge Installation To install the OptiMS cartridge, perform the following steps:
1 Open the cartridge cover, loosen and raise the lock down bar, and then slide open the CESI 8000 Plus Access Panel (Figure 5.1).
Figure 5.1 CESI 8000 Plus Access Panel
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2 Insert the OptiMS Sprayer housing through the side panel and rest the sprayer into the holster (Figure 5.2). Make sure the protective sleeve (Figure 5.16) remains over the tip of the sprayer.
NOTE To prevent damage, do not let the tip of the sprayer touch any surfaces.
Figure 5.2 OptiMS Sprayer in Holster
3 To install the cartridge body, sit the cartridge’s ends on the aligning blocks of the OptiMS interface plate at a 45 degree angle, and lightly push it in (Figure 5.3).
Figure 5.3 Positioning of Cartridge Body onto Interface Plate
1. Aligning Blocks 2. “T” Slider
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4 Pivot the cartridge up until parallel to the interface plate (Figure 5.4).
5 Align the “T” slider into the groove of the interface plate and slide the cartridge down (set upper handle positions as shown in Figure 5.4 and Figure 5.5). As the cartridge is slid down, the sheaths on the inlet and outlet sides retract and the capillaries become visible beneath the ejectors (Figure 5.5).
Figure 5.4 Pivot Cartridge Upwards and Align onto Interface Plate
1. Align “T” Slider into the Groove
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Figure 5.5 Slide Cartridge All the Way Down
1. Capillaries (appear beneath interface block and within the ejectors)
6 Route coolant and capillary tubing through the notched arm (Figure 5.6).
Figure 5.6 Routing Coolant and Capillary Tubing
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7 Bring the lock down bar to the front of the cartridge and secure cartridge in place by tightening the thumbscrews (Figure 5.7).
Figure 5.7 Tighten Screws to Secure Cartridge
Thermo Scientific Mass Spectrometer: Integration with the CESI 8000 Plus System
Integration of the CESI 8000 Plus System with a Thermo Scientific mass spectrometer is described in the following sections: • Physical Integration with the CESI 8000 Plus System • OptiMS Cartridge Sprayer Tip Installation into a Thermo Scientific NanoSpray II Ion Source • Establishing Communication Between the CESI 8000 Plus Controller and the Thermo Scientific Mass Spectrometer • Aligning the CESI 8000 Plus System with a Thermo Scientific Mass Spectrometer
Physical Integration with the CESI 8000 Plus System Perform the following steps to make the connections between the CESI 8000 Plus System and a Thermo Scientific mass spectrometer.
1 Make sure that the CESI 8000 Plus controller (computer, monitor, keyboard, and mouse) is connected. Ensure that both the CESI 8000 Plus System and controller are connected to electrical power.
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2 Connect the GPIB-USB cable to the connector labeled “Controller” on the CESI 8000 Plus System. Plug the other end of the GPIB-USB cable to a USB port on the CESI 8000 Plus controller (Figure 5.8).
IMPORTANT A spacer may be needed to allow the GPIB cable connector to fit on the CESI 8000 Plus System with the relay cable connector.
Figure 5.8 GPIB-USB Integration to Controller
1. Spacer Connector (may be needed for CESI 3. Other End of GPIB-USB Cable (connected to 8000 Plus Controller Port) the CESI 8000 Plus Controller USB Port) 2. GPIB-USB Cable to Spacer to Controller Port
3 Connect the relay cable to the relay input (labeled I/O) on the CESI 8000 Plus System. Connect the other end of the relay cable to the Thermo Scientific mass spectrometer relay input (labeled Aux I/O on the LTQ model) for contact closure (Figure 5.9).
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Figure 5.9 Relay Cable Integration for Contact Closure
1. Relay Cable Installed on the CESI 8000 2. Relay Cable installed on the Thermo Plus System (connect at far right of the Scientific LTQ Model Mass Spectrometer controller as shown)
There are two cables included in the Thermo Scientific Source Adapter Kit (PN B07366): • B13901 — for installation with mass spectrometer models LTQ, LXQ, and LCQ. • B13902 — for installation with mass spectrometer models TQS and Q Exactive.
Use the relay cable for the particular mass spectrometer model in use.
4 The RS-232 Service Only connector (Figure 5.10) should only be used by a SCIEX Field Service Employee (FSE).
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Figure 5.10 RS-232 Connector on the CESI 8000 Plus System
OptiMS Cartridge Sprayer Tip Installation into a Thermo Scientific NanoSpray II Ion Source
WARNING Electrical Shock Hazard. Ensure that the MS ESI voltage is set to zero and idle. The CESI 8000 Plus System separation voltage should be off.
Prior to installing the NanoSpray II ion source, ensure the 1-inch adapter ring is not installed (Figure 5.11). Follow the manufacturer's instructions to remove this part.
Figure 5.11 Remove 1-inch Adapter Ring
1. Socket Head Screws 2. Ion Max Adapter 3. 1-Inch (Deep) Adapter Ring
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Additionally, ensure the ion sweep cone has been removed from the mass spectrometer (Figure 5.12).
CAUTION The ion sweep cone may be hot. Allow sufficient cooling time before handling.
Figure 5.12 Ion Sweep Cone
1. Gas Inlet
To install the OptiMS sprayer tip into the Thermo Scientific NanoSpray II ion source, perform the following steps:
1 Install the Thermo Scientific NanoSpray II ion source onto the mass spectrometer system following the manufacturer’s instructions.
2 Use the knob shown in Figure 5.13 to retract the stage as far as possible from the mass spectrometer inlet.
Figure 5.13 Thermo Scientific NanoSpray II Alignment Knob
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WARNING It is critical to perform Step 2 prior to installation of the adapter. During the installation of the OptiMS spray assembly, the glass tip of the OptiMS sprayer will be exposed. If the ion source stage is too close to the mass spectrometer inlet, then damage to the tip may result.
3 Position the Thermo Scientific mass spectrometer adapter onto the ion source by sliding it into the holder (Figure 5.14 and Figure 5.15).
Figure 5.14 Orientation of the Adapter Prior to Installation onto the Ion Source
3
2
1
1. High Voltage (HV) Output Cable 3. Thermo Scientific Mass 2. Thermo Scientific Mass Spectrometer Ion Source Spectrometer Adapter
NOTE Some ion sources may come with a metal set screw on the right side of the holder part. If present, replace the metal set screw with the plastic thumbscrew supplied in the adapter kit (Figure 5.15).
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Figure 5.15 Adapter for Thermo Scientific Mass Spectrometer in Place
1. Thumbscrew 2. Adapter (in place)
4 Remove protective sleeve from OptiMS sprayer’s tip (Figure 5.16).
IMPORTANT Save the protective sleeve. It will be needed if the OptiMS cartridge has to be removed and/ or stored for later use.
Figure 5.16 Protective Sleeve Removal
CAUTION Once the protective sleeve is removed, the protective sheet can retract. During the installation of the OptiMS spray assembly, if the NanoSpray’s stage is too close to the mass spectrometer inlet damage to the tip may result.
5 Carefully insert the OptiMS sprayer assembly into the adapter by aligning the OptiMS arrow with the adapter unlock position (Figure 5.17).
6 Twist the OptiMS sprayer assembly counter clockwise (Figure 5.17) to lock it into place within the adapter.
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Figure 5.17 Locking the OptiMS Spray Assembly into the Adapter
1. Twist OptiMS Counter Clockwise from Unlock to Lock Position 2. OptiMS in Lock Position
7 Connect the adapter input cable to the input cable on the mass spectrometer. Push the connector together and lock by twisting the lock (Figure 5.18).
WARNING Electrical Shock Hazard. Ensure that the MS ESI voltage is set to zero and idle. The CESI 8000 Plus System separation voltage should be off.
Figure 5.18 HV Cable to Mass Spectrometer Input Cable (Cable-to-Cable) Connection
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8 To expose the HV connectors, slide open the door located on the lower-left corner of the right-side panel of the CESI 8000 Plus System (Figure 5.19).
9 Connect the black ground banana plug from the cable to the matching black receptacle on the panel. Connect the HV cable from the adapter to the output connector in the HV Connection Panel. Twist the HV cable 90 degrees clockwise to lock it in place (Figure 5.19 and Figure 5.20).
IMPORTANT If the black banana ground plug is not installed, there may be electromagnetic interference from the HV cables.
Figure 5.19 HV Connection Panel
1. HV Panel Input Connection 2. HV Panel Output Connection to from Mass Spectrometer OptiMS Adapter (black banana plug) Source (red banana plug)
10 Connect the red banana ground plug into the matching red receptacle on the panel. Connect the HV cable from the mass spectrometer source to the input connection on the HV connection panel, and twist 90 degrees clockwise to lock it in place (Figure 5.19 and Figure 5.20).
IMPORTANT If the red banana ground plug is not installed, there may be electromagnetic interference from the HV cables.
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WARNING Electrical Shock Hazard. Install the HV Input and Output cables and then check the connections at both ends before turning on the CESI 8000 Plus System.
Figure 5.20 High Voltage Panel Connections
3 4
INPUT OUTPUT 1
2
INPUT OUTPUT
5 6
1. Input Red 4. Black Banana Plug 2. Red Banana Plug 5. HV Cable Input (from Mass Spectrometer) to 3. Input Black Input Red 6. HV Cable Output (to Adapter) to Output Black
Establishing Communication Between the CESI 8000 Plus Controller and the Thermo Scientific Mass Spectrometer The CESI 8000 Plus controller (PC) communicates with the Thermo Scientific mass spectrometer with a relay cable (Figure 5.9). To configure the CESI 8000 Plus controller, perform the following procedure:
1 Turn on the CESI 8000 Plus System and the CESI 8000 Plus controller.
2 On the CESI 8000 Plus controller, click Start > CESI 8000 Software folder and then right-click Change MS Type (Figure 5.21).
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Figure 5.21 Access Change MS Type from the CESI 8000 Plus Software
The Change MS Type dialog will open (Figure 5.22).
Figure 5.22 Change MS Type Dialog
3 If the Connect to AB SCIEX MS check box is selected, then clear the check box (Figure 5.22).
4 Click Change (Figure 5.22).
If there will be a need to switch from the Thermo Scientific mass spectrometer to a SCIEX mass spectrometer, then additional network reconfigurations required (refer to APPENDIX B: Nominal Network Configuration).
Aligning the CESI 8000 Plus System with a Thermo Scientific Mass Spectrometer The level of the buffer vials in the CESI 8000 Plus System must be at the same level as the OptiMS cartridge sprayer tip. This will prevent hydrodynamic (siphoning) flow within the capillaries, which may affect CE-MS separation performance. This leveling is done by aligning the height indicator line on the CESI 8000 Plus System with the OptiMS sprayer tip placed at the mass spectrometer adapter. Also, by having the CESI 8000 Plus System properly leveled, the coolant will properly drain out of the OptiMS cartridge before removal of the OptiMS cartridge assembly.
Perform the following steps to align the CESI 8000 Plus System with a Thermo Scientific mass spectrometer:
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1 If necessary, use the knob shown in Figure 5.23 to retract the ion source stage all the way back so that the sprayer tip does not collide with the mass spectrometer when moving the stage.
Figure 5.23 Move Ion Source Stage All the Way Back
1. Alignment Knob 2. Screw (secures 3. Screw (secures 4. Holder holder screw) holder with stage) 5. Screw (tightens 6. Screw (tightens 7. Thumb screws stage) stage) (secures faceplate)
2 The stage and stage holder for the Thermo Scientific LTQ mass spectrometer has a tendency to loosen with use. Be sure to frequently tighten the screw: that secures the holder screw, that secures the holder with the stage, that tightens the stage (both locations), and that secures the faceplate (items 2, 3, 5, 6, and 7 as indicated in Figure 5.23).
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WARNING It is critical to perform Step 1 prior to moving the CESI 8000 Plus cart towards the stage. This will avoid any potential damage to the tip of the OptiMS sprayer.
3 Move the CESI 8000 Plus cart close (approximately 4 inches) to the stage of the mass spectrometer so that the OptiMS coolant tubes can be fully extended (Figure 5.24).
Figure 5.24 Instrument Approximately 4 inches (10 cm.) from Stage
1. CESI 8000 Plus System 2. Ion Source Stage 3. CESI 8000 Plus Cart
4 Align the OptiMS sprayer tip to the same height as the mass spectrometer inlet. Then, align the height indicator on the CESI 8000 Plus System with the OptiMS sprayer tip (Figure 5.25).
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IMPORTANT The OptiMS sprayer’s tip needs to be aligned to the height indicator on the CESI 8000 Plus System while simultaneously centered with the mass spectrometer inlet on the mass spectrometer (Figure 5.25).
Figure 5.25 Alignment of OptiMS Sprayer Tip, Height Indicator, and Ion Transfer Tube
1. Height indicator 3. Ion Transfer Tube (mass spectrometer 2. OptiMS Sprayer Tip inlet)
CAUTION Always remove consumable before adjusting the height of the CESI 8000 Plus cart.
5 Use the up and down height-adjustment buttons on the front of the cart to adjust the height of the cart until the height indicator on the CESI 8000 Plus System is aligned to the OptiMS sprayer tip (Figure 5.26).
IMPORTANT Always press the height adjustments buttons one at a time, never simultaneously.
IMPORTANT If the CESI 8000 Plus cart will not power to its lowest height, or if it becomes stuck, then the cart must be re-programmed. Refer to the CESI 8000 Plus Mobile Cart Homing Procedure section in APPENDIX G.
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Figure 5.26 Cart Height Adjustment Buttons
1. Cart Up Button 2. Cart Down Button
6 Use the mass spectrometer alignment knobs to make fine adjustments to align the OptiMS sprayer tip to the center of the source inlet on the mass spectrometer (Figure 5.27).
IMPORTANT The sprayer tip must always be at least 2 mm away from the inlet on the mass spectrometer when centered to the inlet.
WARNING If the sprayer tip is too close to the mass spectrometer transfer tube, then it is possible that a mass spectrometer incompatible capillary rinse solution might get aspirated into the mass spectrometer. This could cause damage to the mass spectrometer.
Figure 5.27 Alignment Knobs on the Thermo Scientific NanoSpray II Ion Source
1. Y-axis (up and down) 3. Z-axis (side to side) 2. X-axis (towards and away from ion transfer tube) 4. Height Indicator
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7 When the CESI 8000 Plus System is visually aligned to the mass spectrometer, lock the wheels on the CESI 8000 Plus cart by stepping on the locking lever at each caster (Figure 5.28).
Figure 5.28 Locking CESI 8000 Plus Mobile Cart Wheels
When properly adjusted, the tubing from the OptiMS Spray Assembly should not dip and form pockets. This will help ensure that gravity flow will not occur within the OptiMS cartridge in the CESI 8000 Plus System, and that the coolant drains before the cartridge is removed.
SCIEX TripleTOF® 5600 Mass Spectrometer: Integration with the CESI 8000 Plus System
Integration of the CESI 8000 Plus System with a SCIEX TripleTOF® 5600 mass spectrometer is described in the following sections:
• Physical Connections with the CESI 8000 Plus System • OptiMS Cartridge Sprayer Tip Installation into the SCIEX Nanospray® III Source • Establishing Communication Between the CESI 8000 Plus Controller and the SCIEX Mass Spectrometer • Aligning the CESI 8000 Plus System with the SCIEX TripleTOF® 5600 Mass Spectrometer
Physical Connections with the CESI 8000 Plus System
Perform the following steps to make the connections between the CESI 8000 Plus System and a SCIEX mass spectrometer.
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1 Make sure that the CESI 8000 Plus controller (computer, monitor, keyboard, and mouse) is connected. Ensure that both the CESI 8000 Plus System and controller are connected to electrical power.
2 Connect the GPIB-USB cable to the connector labeled “Controller” on the CESI 8000 Plus System. Use spacer if needed. Plug the other end of the GPIB-USB cable to a USB port on the CESI 8000 Plus controller (Figure 5.29).
Figure 5.29 GPIB-USB Integration to the Acquisition Computer
1. Spacer Connector (may be needed for CESI 3. Other End of GPIB-USB Cable (installed into 8000 Plus controller port) the acquisition computer USB port) 2. GPIB-USB Cable to Spacer to Controller Port
3 Connect the CESI 8000 Plus communication cable (Ethernet cable) to the CESI 8000 Plus controller and to the SCIEX mass spectrometer acquisition computer (Figure 5.30).
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Figure 5.30 System Communication Setup
1. One End of Ethernet Cable (installed 2. Other End of Ethernet Cable (installed on the on the CESI 8000 Plus controller) SCIEX mass spectrometer acquisition computer)
IMPORTANT For the SCIEX CE-MS configuration, a 14-ft Ethernet cable is provided. The maximum space between the CESI 8000 Plus System and a SCIEX TripleTOF® 5600 acquisition computer should not exceed 13 feet.
WARNING Personal Injury Hazard. Route the relay cable where users can not trip on it.
4 The RS-232 Service Only connector on the CESI 8000 Plus System (Figure 5.31) should only be used by a SCIEX Field Service Employee (FSE).
Figure 5.31 RS-232 Connector on the CESI 8000 Plus System
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OptiMS Cartridge Sprayer Tip Installation into the SCIEX Nanospray® III Source
WARNING Electrical Shock Hazard. Ensure that the MS ESI voltage is set to zero and idle. The CESI 8000 Plus separation voltage should be off.
To install the mass spectrometer spray end of the OptiMS cartridge into the SCIEX Nanospray® III source, perform the following steps:
1 Install the SCIEX Nanospray® III ion source onto the mass spectrometer system following the manufacturer’s instructions.
2 Pull the ion source stage away from the mass spectrometer inlet (Figure 5.32).
Figure 5.32 Pulling Back the SCIEX Assembly
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WARNING It is critical to perform Step 2 prior to installation of the adapter. During the installation of the OptiMS spray assembly, the glass tip of the OptiMS sprayer will be exposed. If the NanoSpray’s stage is too close to the mass spectrometer inlet, then damage to the tip may result.
3 Insert the adapter on the SCIEX Nanospray® III’s source mount and push it as far forward as possible such that the hook at the end of the adapter goes under the end of the rail. Make sure the adapter fits securely into place. Then tighten the thumbscrew to secure the adapter (Figure 5.33 and Figure 5.34).
Figure 5.33 Orientation of the Adapter Prior to Installation onto the Ion Source
1. High Voltage (HV) Output Cable 3. SCIEX Nanospray® III Adapter 2. Hook 4. SCIEX Nanospray® III Ion Source
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Figure 5.34 Tighten Thumbscrew for Secure Fit
1. Thumbscrew to Tighten
4 Remove protective sleeve from the OptiMS sprayer tip (Figure 5.35).
IMPORTANT Save the protective sleeve. It will be needed if the OptiMS cartridge has to be removed and/ or stored for later use.
Figure 5.35 Protective Sleeve Removal
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CAUTION Once the protective sleeve is removed, then the protective tip guard can retract. During the installation of the OptiMS spray assembly, if the NanoSpray’s stage is too close to the mass spectrometer inlet then damage to the tip may result.
5 Carefully insert the OptiMS sprayer assembly into the adapter by aligning the OptiMS arrow with the adapter unlock position (use the keying pattern depicted on the side of adapter). Do not allow the tip to touch any surfaces (Figure 5.36).
Figure 5.36 Inserting the OptiMS Spray Assembly into Adapter
1. OptiMS Spray Assembly 2. Insert OptiMS Spray Assembly in Unlock Position
6 The OptiMS has a locking mechanism which keeps the OptiMS Spray Assembly securely attached to the adapter. Twist the OptiMS sprayer assembly counter clockwise (Figure 5.37) to lock it into place within the adapter.
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Figure 5.37 Locking the Spray Assembly into the Adapter
1. Twist OptiMS from Unlock to Lock Position 2. OptiMS in Lock Position
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7 To expose the HV connectors, slide the door open, which is located on the lower-left corner of the right-side panel of the CESI 8000 Plus System (Figure 5.38).
Figure 5.38 CESI 8000 Plus System HV Panel
WARNING Electrical Shock Hazard. Ensure that the MS ESI voltage is set to zero and idle. The CESI 8000 Plus separation voltage should be off.
8 Connect the black ground banana plug from the cable to the matching black receptacle on the panel. Take the HV cable from the adapter and connect it to the output connector in the HV connections panel. Twist the HV cable 90 degrees clockwise to lock it in place (Figure 5.39 and Figure 5.40).
IMPORTANT If the black banana ground plug is not installed, there may be electromagnetic interference from the HV cables.
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Figure 5.39 HV Connection Panel
1. HV Panel Input Connection from Mass 2. HV Panel Output Connection to OptiMS Spectrometer Source (red banana plug) Adapter (black banana plug)
CAUTION
Ensure that the default ESI voltage setting in the Analyst® TF Software on the mass spectrometer acquisition computer is set to zero before connecting the CESI 8000 Plus System to the mass spectrometer.
9 Connect the red banana ground plug into the matching red receptacle on the panel. Connect the HV cable from the mass spectrometer ion source into the input connection on the CESI 8000 Plus HV panel, and twist 90 degrees clockwise to lock it in place (Figure 5.39 and Figure 5.40).
IMPORTANT If the red banana ground plug is not installed, there may be electromagnetic interference from the HV cables.
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WARNING Electrical Shock Hazard. Completely connect and check (at both ends) the HV Input and Output connection cables before turning on and operating the CESI 8000 Plus System.
Figure 5.40 High Voltage Panel Connections
3 4
INPUT OUTPUT 1
2
INPUT OUTPUT
5 6
1. Input Red 4. Black Banana Plug 2. Red Banana Plug 5. HV Cable Input (from mass spectrometer) to 3. Input Black Input Red 6. HV Cable Output (to adapter) to Output Black
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Establishing Communication Between the CESI 8000 Plus Controller and the SCIEX Mass Spectrometer The CESI 8000 Plus controller (PC) communicates with the SCIEX mass spectrometer using an Ethernet cable (Figure 5.30). To configure the CESI 8000 Plus controller, perform the following procedure:
1 Power on the CESI 8000 Plus System and the CESI 8000 Plus controller.
2 Click Start > CESI 8000 Software folder and then right-click Change MS Type (Figure 5.41).
Figure 5.41 Access Change MS Type from the CESI 8000 Plus Software
The Change MS Type dialog will open (Figure 5.42).
Figure 5.42 Change MS Type Dialog
3 If the Connect to AB SCIEX MS check box is selected, then click the check box to clear it (Figure 5.42).
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4 The IP address that becomes enabled within the dialog should be the one that was pre- configured for the SCIEX mass spectrometer.
IMPORTANT Do not change the IP address unless you know that the IP address shown is being used by another network connection. If the IP address displayed needs to be changed, contact SCIEX Technical Support for assistance.
5 Click Change (Figure 5.42).
If there will be a need to switch to another mass spectrometer, or to switch back from another mass spectrometer to the SCIEX mass spectrometer, then additional network reconfigurations required (refer to APPENDIX B: Nominal Network Configuration).
Aligning the CESI 8000 Plus System with the SCIEX TripleTOF® 5600 Mass Spectrometer The level of the buffer vials in the CESI 8000 Plus System must be at the same level as the OptiMS cartridge sprayer tip. This will prevent hydrodynamic (siphoning) flow within the capillaries, which may affect CE-MS separation performance. This leveling is done by aligning the height indicator line on the CESI 8000 Plus System with the OptiMS sprayer tip placed at the mass spectrometer adapter. Also, by having the CESI 8000 Plus System properly leveled, the coolant will properly drain out of the OptiMS cartridge before removal of the OptiMS cartridge assembly.
To align the CESI 8000 Plus System with the SCIEX TripleTOF® 5600 mass spectrometer:
1 If necessary, turn the coarse Z-axis adjustment knob (shown in Figure 5.43) to retract the stage and the adapter on the ion source rail all the way back from the curtain plate. (This is to keep the OptiMS capillary from colliding with the mass spectrometer when moving the stage forward.)
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Figure 5.43 Move Stage All the Way Back
1. Coarse Z-axis Adjustment Knob 2. Stage
IMPORTANT Make sure that the Source Drain assembly on the mass spectrometer is covered. The Source Drain assembly must be completely covered for proper operation (Figure 5.44).
Figure 5.44 Source Drain Assembly on the Mass Spectrometer Must be Covered
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WARNING It is critical to perform Step 1 prior to moving the CESI 8000 Plus cart towards the stage. If the ion source stage is too close to the mass spectrometer inlet, then damage to the tip may result.
2 Move the CESI 8000 Plus cart close, approximately 4 inches (10 cm.) to the ion source stage so that the OptiMS coolant tubes can be fully extended (Figure 5.45).
Figure 5.45 Instrument Approximately 4 Inches from Stage
1. Coolant Tubes 3. CESI 8000 Plus Cart 2. Ion Source Stage
3 Align the OptiMS sprayer tip to the same height as the mass spectrometer inlet. Then, align the height indicator on the CESI 8000 Plus System with the OptiMS sprayer tip (Figure 5.46).
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IMPORTANT The OptiMS sprayer tip needs to be aligned to the height indicator on the CESI 8000 Plus System while simultaneously centered with the mass spectrometer inlet on the mass spectrometer (Figure 5.46).
Figure 5.46 Alignment of OptiMS Tip, Height Indicator, and Mass Spectrometer Inlet
1. Height Indicator 3. Mass Spectrometer Inlet 2. OptiMS Sprayer Tip
4 Move the stage forward toward the source inlet of the mass spectrometer until the stage clicks into place on the guide rails.
CAUTION Always remove consumable before adjusting the CESI 8000 Plus cart height.
5 Use the up and down height-adjustment buttons on the front of the cart to adjust the height of the cart until the height indicator on the CESI 8000 Plus System is aligned to the OptiMS sprayer tip (Figure 5.47).
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IMPORTANT Always press the height adjustments buttons one at a time, never simultaneously.
IMPORTANT If the table top of the CESI 8000 Plus cart will not power to its lowest height, or if it becomes stuck, then the cart must be re-programmed. Refer to the CESI 8000 Plus Mobile Cart Homing Procedure section in APPENDIX G.
Figure 5.47 Cart Height Adjustment Buttons
1. Cart Up Button 2. Cart Down Button
6 Use the SCIEX TripleTOF® 5600 mass spectrometer's other adjustment knobs to fine tune the alignment of the OptiMS sprayer tip with the center of the inlet on the mass spectrometer (Figure 5.48).
IMPORTANT The sprayer tip must always be at least 2 mm away from the inlet on the mass spectrometer when centered to the inlet.
WARNING If the sprayer tip is too close to the mass spectrometer transfer tube, then it is possible that a mass spectrometer incompatible capillary rinse solution might get aspirated into the mass spectrometer. This could cause damage to the mass spectrometer.
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Figure 5.48 X-axis, Y-axis, and Z-axis Alignment Knobs
1. CESI 8000 Plus Height Indicator 5. X-axis Adjustment Knob 2. Fine Z-axis Adjustment Knob (movement toward the curtain plate) (horizontal movement) 3. Coarse Z-axis Adjustment Knob (movement toward the curtain plate) 6. Stage 4. Y-axis Adjustment Knob (vertical movement) 7. OptiMS Sprayer Tip 8. MS Inlet
7 When the CESI 8000 Plus System is in visual alignment to the mass spectrometer, lock the wheels on the CESI 8000 Plus cart. This is done by stepping on the locking lever at each caster to lock each wheel in place.
Figure 5.49 Locking CESI 8000 Plus Mobile Cart Wheels
When properly adjusted, the tubing from the OptiMS Spray Assembly should not dip and form pockets. This will help ensure that gravity flow will not occur within the OptiMS cartridge in the CESI 8000 Plus System, and that the coolant drains before the cartridge is removed.
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Overview of 32 Karat™ Software
The 32 Karat™ Software is a full-featured chromatography and capillary electrophoresis software package that provides control of the CESI 8000 Plus System. For the CESI 8000 Plus user, there is no electrophoretic data collected by the 32 Karat™ Software. Only voltage, electrical current, pressure, and power data traces can be collected. The software allows the user to develop methods (the means of creating or editing instrument runs), and view data traces such as voltage and current. The 32 Karat™ Software also provides the mechanism to develop sequences (a list of methods to be run), and the means to launch either a single method or sequences. In addition to running methods and sequences, the software provides direct control – a means to immediately access many of the instrument features and actions.
The CESI 8000 Plus System also provides software with a workflow-oriented approach to running methods and sequences that have already been developed by a 32 Karat™ Software user. Detailed information on using the user interface software can be found in CHAPTER 7, CESI 8000 Software.
System Administration
IMPORTANT By default, user login and other security features in the CESI 8000 Software are disabled. Refer to the System Administration Guide for information on enabling these features.
System administration is a feature used to control access to the software. CESI 8000 Plus and 32 Karat™ Software access can be restricted by user, instrument, or project. At installation, system administration is disabled. The default user name is cesi and the default password is 8000. This user name and password are needed for any system changes or access to any instrument. The administrator, can grant access to additional users.
Another advantage of using the system administration features is that it stores any customized settings for each instrument. This is convenient when multiple users run different applications on the same system. System administration enables a much greater degree of security on your system.
When a user is added as a system user, the access for this user is customized using the System Administration wizard. To access the wizard, select System Administration Wizard from the Tools menu (Figure 6.1 and Figure 6.2).
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Figure 6.1 System Administration Wizard (from Tools menu)
When System Administrator Wizard is selected, the dialog shown in Figure 6.2 will appear.
Figure 6.2 Wizard Selection Type
Additional information on system administration is found in the System Administration Guide.
Controller and Instrument Start Up
Controller/Network Login If the instrument is installed on a network, the user name and password will be supplied by the network administrator. 32 Karat™ Software functionality is different when run on a network
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versus as a stand-alone system. In particular, the system administration features can use network names and passwords automatically. The default network identification is 32 Karat and the work group name is WORKGROUP. If more than one CESI 8000 Plus System is going to be installed on the network, unique network names are required for each workstation. Normally, these changes are performed by the network administrator.
License Key The software requires a license key in order to perform methods. This key is a USB Flash drive that must be put in an available USB slot on the controller. For instrument control, the license key is required. Without the license key, the software will only operate in “Demo” mode.
CAUTION The flash drive license key must remain in place at all times while the software is running.
Demo Mode A license key is not required to operate in “Demo” mode. Demo mode means that a license is not installed.
Launching 32 Karat™ Software Launch the 32 Karat™ Software by double-clicking the 32 Karat™ Software icon on the side menu of the CESI 8000 Plus Ready Window (Figure 7.1). The Enterprise window opens. From the View menu, select Hierarchy Pane. In the left pane, the group CESI 8000 opens. Select this group to display the available instruments in the right pane (Figure 6.3).
Figure 6.3 Enterprise Window in 32 Karat™ Software
IMPORTANT The Service Support Group should only be used by SCIEX Field Services. A UV Detector is required to use the Service Support selection.
Instrument Start Up The software can only control the CESI 8000 Plus System after it has completed its startup initialization process. Before starting the CESI 8000 Plus System: • Make sure the cartridge is installed. • Make sure that the coolant level for the capillaries is not low.
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The coolant level can be checked by viewing the level indicator through the coolant sight glass (Figure F.15). If the coolant level is low, add coolant as described in the Refilling Coolant section in APPENDIX F.
After pushing in the power button on the front bezel of the instrument, allow the instrument up to five minutes to initialize. Shortly after the power is turned on, the instrument will register a short tone, indicating that the circuit boards have activated and started to initialize. Approximately two minutes later, you should hear additional sounds as the transport and pressure systems initialize.
Instruments and Projects A project is a set of folders for file types commonly used in the 32 Karat™ and CESI 8000 Software. For example, if you are doing methods development, it would be convenient to store your method in project (or instrument) folders with the appropriate names. A project can be created for each method or sequence.
When opening an instrument, the user is asked to specify a project. When opened, all folders default to this project. This simplifies locating method and data files. If System Administration is used, then when users have logged on to an instrument they can select any project they have access to from the File menu (Figure 6.1).
NOTE Additional information about System Administration can be found in the System Administration Guide.
Configuration The instrument configuration required to run the mass spectrometer detector is pre-defined.
Online Versus Offline 32 Karat™ Software has two main modes of operation:
• Online mode gives full instrument control and the ability to run methods. • Offline mode allows opening of existing data files, but does not allow instrument control. Any instrument can be opened offline.
If an instrument is running, it is possible to open a second window for the same instrument in offline mode. Similarly, if you need to open a data file on a remote computer, you should open the instrument offline. An instrument is opened online by double-clicking the instrument icon. To open an instrument offline, right-click the instrument icon and select Open Offline.
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When a newly configured instrument is launched for the first time, the instrument wizard dialog opens (Figure 6.4).
Figure 6.4 Instrument Wizard — CE MS Dialog
If the instrument is opened online, then the current project name should be CE-MS and the current project will be “Default”. (This is due to the user login being disabled.) If the instrument is open under a different project, then it is possible to select the project from the File menu on the instrument window.
Using the Direct Control Window to Load the Cartridge and Samples
Direct Control can be selected from the Control menu (Figure 6.5).
Figure 6.5 Control Menu for Direct Control
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Create a quick access button to Direct Control window by going to View menu and selecting Preferences. The Preferences dialog opens (Figure 6.6).
Figure 6.6 Preferences Dialog
Select Direct Control as a toolbar option. Make sure the check box for Show toolbar is checked. The icon will open on the toolbar as shown in Figure 6.7.
Figure 6.7 Icon for Direct Control Dialog on Toolbar
The Direct Control window allows manual instrument control and reports the status of the instrument functions. Icons that confirm the status of the sample cover and cartridge are located in the lower part of the window (Figure 6.8).
Figure 6.8 Direct Control Window
Select Load to move the trays to the front of the sample compartment.
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IMPORTANT Install the OptiMS cartridge, correct vials, and close the sample cover to enable the instrument to run the method.
TripleTOF® 5600 Mass Spectrometer Using AAO The BCI CE-MS Analyst Access Object (AAO) software interface provides a software layer to communicate with the Analyst® TF Software used by the controller of the SCIEX TripleTOF® 5600 mass spectrometer. If AAO is being used, the Direct Control window will display an icon that indicates the mass spectrometer status of the AAO interface (Figure 6.9). The following icons indicate the mass spectrometer status of the AAO interface:
Mass spectrometer interface is Offline.
Mass spectrometer is on Standby, Stopping, Paused, Unknown, or AAO driver connection failed.
Mass spectrometer is Ready.
Mass spectrometer is Pre Run / Ready to acquire.
Mass spectrometer is Running.
Figure 6.9 Direct Control Window with AAO
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Methods and Sequences
What is a Method? A method is a collection of serially executable steps; each step resulting in a specific CESI 8000 Plus System setting or action. A method contains complete information for running the instrument. When you set up a method in the CESI 8000 Software, you are combining several steps into a logical series of events that are executed automatically by the instrument.
Method development takes place in the Instrument Window and can be done while the instrument is either online or offline.
Method Development Online — When you create or edit a method online, you also have access to instrument control and viewing data such as voltage and current. Method Development Offline — When you create a method offline, you do not have access to instrument control (starting or stopping runs), however, you can open data files. Additionally, when creating or editing a method while the instrument is offline, you can set up a complete method, including instrument configuration, which can be opened at a later time and used to run the instrument.
Additional information can be found in the Online Versus Offline section above. Detailed information on using Methods and Method Development can be found in “Chapter 6: Method Development” within the CESI 8000 Software Online Help. Refer to the Launching the CESI 8000 Software section in CHAPTER 7 for information on accessing the CESI 8000 Software Online Help.
Methods and Relay Events In the CE-MS mode of the CESI 8000 Software, relay events in a method serve as the trigger mechanisms to start data acquisition by the mass spectrometer. For a non-AAO configuration (such as for the Thermo Scientific mass spectrometer), a relay event actually closes the electronic switch, and the relay cable (Figure 5.9) transfers the electronic signal to the mass spectrometer to start the data acquisition.
For AAO configurations (such as for the SCIEX TripleTOF® 5600 mass spectrometer), the relay event will echo back to the software to send the start acquisition command through the AAO interface. (There is no relay cable with AAO; therefore, the relay event serves an internal loop back signal to the software in order to start mass spectrometer data acquisition.)
What is Sequence? A sequence is a list of methods for the 32 Karat™ Software that defines the order for runs to be acquired and processed. A sequence (or sequences) can be used to run a batch of samples without user intervention. Sequences can be used for preconditioning of the OptiMS cartridge, shutting down the OptiMS cartridge for storage, and running multiple samples. You can also insert priority samples into a sequence, or queue another sequence to start.
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Creating and Editing a Method
To create or edit a method, you need to be viewing the Instrument wizard (Figure 6.4). To open the Instrument wizard:
1 Access the 32 Karat™ Software Enterprise window.
2 Right-click the icon that represents your instrument system (create one if necessary) and select Open Offline. The instrument window opens after a few seconds. When the window opens, the Instrument wizard opens. It is now possible to open the method editing dialog by selecting Create or Modify a Method.
To write a new method or to run an instrument test sample while in Online mode, you need to be at the Instrument Setup window (Figure 6.10). Do the following:
1 Click File > Method > New. The name of the method in the Instrument Window title bar changes to untitled.met.
2 To access the instrument control and data acquisition sections of the method, click Method > Instrument Setup.
3 Click the tab marked Initial Conditions to bring it to the front. The
Instrument Setup Window
This section describes the fields in the Initial Conditions tab and the Time Program tab in the Instrument Setup window. More advanced information on Methods and Method Development can be found in “Chapter 6: Method Development” within the CESI 8000 Software Online Help. Refer to the Launching the CESI 8000 Software in CHAPTER 7 for information on accessing the CESI 8000 Software Online Help.
Instrument Setup — Initial Conditions
The Initial Conditions tab is used to set instrument parameters at the start of a method, before the separation begins.
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Figure 6.10 Instrument Setup Window — Initial Conditions Tab
IMPORTANT Capillary coolant temperature below dew point at a given environmental condition produces condensation on surfaces of tubing and cartridge, which results in current leak. Moisture collects on tubing and cartridge body which drips down into the interface area. Changing the coolant temperature setting to above the dew point prevents condensation and thus current leak.
Auxiliary Data Channels The CESI 8000 Software gives you the option to collect any one or all instrument parameters: voltage, current, power, and pressure. Select the channels to record by selecting the appropriate boxes. The max kV and max μA boxes are used to set the allowable limits for these parameters. Voltage and current are interrelated through Ohm’s Law. The system limits both parameters whenever one limit is reached. For example, assume a voltage set at 30 kV and a current limit set at 10 μA. With some buffer systems, a voltage of 12 kV generates a current of 10 μA. In this case the voltage does not exceed 12 kV, as the current limit is the determining factor.
Temperature Sets the initial temperature of the cartridge coolant and the sample storage unit.
Trigger Settings The CESI 8000 Software can be forced to wait until certain conditions are met before beginning a run. These are selected by selecting the appropriate box. If Wait for external trigger is selected, the CESI 8000 Plus System becomes a slave device and does not start until an external signal is received. The two Wait for temperature options assure that the system has reached the correct operating temperature before beginning a run. These options delay only the start of the time program. Parameters set in initial conditions occur without a wait.
Inlet and Outlet Trays The types of trays used when a method is run must be specified here. When the method is run, this information is compared to the tray types that are configured in the instrument. If there is a tray type mismatch, the method does not run.
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Additional information on these and other parameters of the Instrument Setup window can be found in the CESI 8000 Software Online Help. Refer to the Launching the CESI 8000 Software in CHAPTER 7 for information on accessing the CESI 8000 Software Online Help.
Instrument Setup — Time Program
The Time program tab is used to set up and define the events that actually make up the method during and after the separation process. The Time program window is arranged like a spreadsheet. An event is entered into each line. Events are executed in order, top to bottom (Figure 6.11).
Figure 6.11 Instrument Setup Dialog — Time Program Tab
The following subcategories (shown as columns) can be found in the Time program tab:
Time — the point after time zero at which the event occurs.
Event — the action that occurs (see below).
Value — this varies depending on the action selected.
Duration — length of time the event lasts.
Inlet and Outlet Vial — where the capillary ends are during the event.
Summary — a system-generated description of the event.
Comments — a user-generated annotation of the event.
Time is not a required event. Events that have no time associated with them are run in the listed order, top to bottom, and each is finished before the next event begins. Timed events must be grouped together; a group of timed events cannot be interrupted by an untimed event. Untimed events can only occur before a group of timed events. Some events do not have a time option, others can be timed or untimed. Collection of voltage and current data begins with the first
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timed event (time 0.00) and it ends when the method ends, or when a STOP DATA event is reached.
To program a line, click in the EVENT box. Click the down arrow to open a menu of events. Select an event to open a dialog for that event (Figure 6.12).
Figure 6.12 Time Program Event List
The following dialogs and field definitions are the ones that should be more commonly used by the CESI 8000 Plus user. For information on more advanced event types, refer to the CESI 8000 Software Online Help.
Separate Dialog
The Separate dialog is used to control the conditions under which the separation process takes place (Figure 6.13).
Figure 6.13 Separate Dialog
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This dialog has the following parameters:
Separation Type Electrically driven separations can be done at controlled voltage, current, or power. When one of these is selected, the other two float to a value determined by the resistance of the capillary contents. Voltage and current cannot exceed the limits set in the Initial Conditions window. Separations can also be programmed to use pressure or vacuum to move the fluid in bulk through the capillary. Voltage, current, or power can be combined with Pressure or Vacuum so that two processes are at work simultaneously.
Polarity Determines the direction of the current. The charge on the electrodes is indicated by the graphic in this dialog.
Values Allows for entry of the set points for the Separation Type parameters. The available options change depending on the Separation Type selected. Ramp time is only valid for electrical separations. It determines the length of time for the voltage, current or power to change from the present to the programmed level.
Tray Positions Can be selected graphically by clicking on Trays. The type of trays shown are determined by the settings in the Initial Conditions dialog (Figure 6.14). When a method is used in a sequence, you might want to change the vial positions after a specified number of cycles. The inlet position, outlet position, or both can be incremented automatically by selecting the appropriate boxes and entering the desired number of cycles between changes.
Figure 6.14 Tray Selection Dialog
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Pressure Direction Specifies whether pressure is applied to the inlet end of the separation capillary. Pressure can also be applied to both ends of the cartridge at the same time.
At Time Specifies this is a timed event at the time specified. Separation is usually a timed event.
Rinse Dialog The rinse event is used to clean the capillary and to load fresh buffer or other separation media (Figure 6.15).
Figure 6.15 Rinse Dialog
This dialog has the following parameters:
Pressure Type Selects the mechanism to be used to move fluid through the capillaries.
Tray Positions Functions exactly as described in the Separation event.
Values Specifies the magnitude of pressure to be delivered and for how long.
Pressure Direction Specifies whether pressure is applied to the separation or conductive liquid capillary
At Time Functions exactly as described in the Separation event.
Inject Dialog The inject event is used to deliver a precisely measured amount of sample into the capillary (Figure 6.16). This step is always untimed, and usually precedes the first separation step.
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Figure 6.16 Inject Dialog
This dialog has the following parameters:
Injection Type Sample can be injected into the capillary by positive pressure, or by the application of voltage (electrokinetic injection).
Polarity Specifies the charge on the electrodes during a voltage injection.
Pressure Direction Specifies whether pressure is applied to the separation (forward direction) or conductive liquid capillary (reverse direction).
Values Specifies the magnitude of the pressure or voltage, and how long it is applied. Higher values inject more sample. Select the For Capillary Fill box when running experiments where the injection step consists of filling the entire length of the capillary with a sample mixture. Clear the option for low pressure – high precision injections.
Tray Positions Functions exactly as described in the Separation event.
Sequence Table When a method is used in a sequence table, certain parameters in the inject event can be overridden by values entered for the sequence. This parameter determines whether the method Inject event or the sequence table has priority. Multiple injection events are allowed. If multiple injections are used, only the first injection event can be overridden or incremented in the sequence table.
NOTE The Allow Override box must be checked in the Sequence Table area (in Figure 6.16) to give the sequence parameters priority over the method parameters.
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Stop Data Collection of voltage and current data starts at time = 0.00 and continues until the end of the method, unless a STOP DATA event is encountered (Figure 6.17). This event can be used to avoid voltage and current data collection during steps such as post-run capillary cleaning. STOP DATA is always a timed event.
Figure 6.17 Stop Data Dialog
End End is an optional event. The method does not continue beyond an end event. It is always a timed event (Figure 6.18).
Figure 6.18 End Dialog
Saving a Method
IMPORTANT Remember to save your method.
1 Click File > Method > Save As.
2 Give the method a name with a title and date. For example, type: TestMethod_111518.
3 Click Save. The default path at installation is: C:\32Karat\Projects\Default\Methods. Your system administrator may have assigned you to a different default path.
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Overview of Creating a Sequence
Once a method is optimized, the method can be run repeatedly on different samples or multiple times on a single sample via a sequence to calculate the performance. A sequence is displayed as a spreadsheet, with each row representing a method run. The CESI 8000 Software includes a Sequence wizard to simplify the sequence generation process. From the Instrument window, click File > Sequences > New. The Sequence wizard opens (Figure 6.19).
Figure 6.19 Sequence Wizard — Method Page
The Sequence wizard can be used to create a sequence, which is used to automate data acquisition. Detailed information on Sequences and using the Sequence wizard can be found in “Chapter 9: Sequences” within the CESI 8000 Software Online Help. Refer to the Launching the CESI 8000 Software in CHAPTER 7 for information on accessing the CESI 8000 Software Online Help.
Single Run and Sequence Runs In Single Run mode a method must be manually started prior to every run. Single run mode is useful in method development where the results of one run suggests modifications to the method, or other procedures. Sequence mode is used to append multiple methods together as a sequence.
The CESI 8000 Plus System must be online to run samples and acquire data. If you are not online, “offline” opens in the title bar of the instrument window. Close any offline windows before proceeding. From the main CESI 8000 Software window, double-click an instrument icon to go online.
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Programming a Sequence
A Sequence is a list of methods and data files that will be used to run a batch of samples without user intervention. Sequences can be used to acquire data (run the instrument). An example of how to create a sequence to acquire data from multiple runs of the test mix is given in the steps below.
1 To create a new sequence, click File > Sequence > Sequence Wizard from the Instrument window. The Sequence wizard opens (Figure 6.20). The wizard consists of 5 windows. Not all of them are used for every sequence, and not every feature of each window is used in the example.
Figure 6.20 Sequence Wizard — Method Page
The first window requires that you select a method.
2 Choose a method file.
3 Click the yellow folder icon to navigate to the method file.
4 Under Data File Type, select For Acquisition. Amount Values are not used in this example.
5 Click Next. The Sequence Wizard — Unknowns page opens (Figure 6.21).
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Figure 6.21 Sequence Wizard — Unknowns Page
This dialog is used to create the Sample ID and Data File ID.
6 Type a text string in the Sample ID field. The blue arrow to the right of the Sample ID field opens the following menu:
Figure 6.22 Sample ID Text Options Menu
Selecting an item from this menu causes a symbol to be inserted in the Sample ID field. The selected parameter is automatically incorporated into the Sample ID when the sequence is run. You can select any combination of the items shown in Figure 6.22. The Data Path field specifies the directory where the data files are stored.
7 Click the green folder icon, and select Data Path, then navigate to the folder.
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8 Type a text string into the Data file field. The blue arrow to the right of the text box opens this menu:
Figure 6.23 Data File Text Options Menu
As described above for the Sample ID, this menu is used to insert codes that are automatically inserted into the filename when the sequence is run. (The Open File choice is used to reprocess existing data files). Number of Unknown Runs in Sequence determines how many lines there are in the Sequence Table. Repetitions per Run determines the number of times each line in the Sequence is run. An identifier is automatically added to the file name for each repetition when this option is used.
9 For this example, enter 1 for both the Number of Unknown Runs in Sequence and Number of Unknown Runs in Sequence fields.
10 Click Next. The Sequence Wizard — Vials page opens (Figure 6.24).
Figure 6.24 Sequence Wizard — Vials Page
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11 If the method being used allows vial position override, the desired starting position can be entered here. If override is not allowed, or if auto-incrementing has been set, any entry here is ignored. A set of calibration vials can also be identified in this window. Refer to APPENDIX C for information on manual and auto-calibration methods and the strategy behind each approach. Refer to the CESI 8000 Software Online Help for more information on calibrating through the Sequence Table. The Advance direction field determines the advance direction for the sequence. When the Advance box is enabled for either or both inlet and outlet vials, the software automatically fills down vial position values in the Sample Inject Inlet/Outlet columns of the sequence table. This automatic fill down eliminates the need for manual entry, one vial position at a time in the sequence table. If Row Major is selected as the Advance Direction, the software automatically fills down vial positions, incrementing them by rows in the same tray (for example, A1, A2, A3, A4, A5, A6, B1, B2, B3, B4 for buffer tray configuration). If Column Major is selected as the Advance Direction, the software automatically fills down vial positions, incrementing them by columns in the same tray (for example, A1, B1, C1, D1, E1, F1, A2, B2, C2, D2 for buffer tray configuration). Do not confuse Advancing vial positions with the vial incrementing operation in instrument runs. Advancing vial positions here refers to simplifying sequence table planning and organizing. These vial position values can be changed in the sequence table, and they are not saved with any method. They are saved in the sequence.
12 Clicking Next will eventually cause the Reports dialog to open (Figure 6.25). Keep check boxes cleared as none of the settings are relevant for CESI-MS.
Figure 6.25 Sequence Wizard — Reports Page
This page (Figure 6.25) allows you to have reports generated automatically at the end of the sequence run.
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13 Clicking Finish will cause a Sequence Table window to open similar to that shown in Figure 6.26.
Figure 6.26 Sequence Table (Part A)
Sequence Table The Sequence Table window is used to place in sequential order all of the contents that have previously been defined for the sequence. The Sequence Table window contains many more columns than can be shown at one time. The user can edit by right-clicking in a blank area of the sequence table, and selecting properties. Figure 6.27 shows the options available for selection.
Figure 6.27 Properties Available for Sequence Table
The Sequence Table shown in Figure 6.26 shows 9 columns, each defined as follows:
Status — Indicates if that particular run is running, or is completed.
Run Type — This category designates a particular line in the sequence as calibration, a system suitability or a shutdown run for example. Click the Run Type field and a blue arrow will show. Clicking on the blue arrow will display all options available. The example in Figure 6.26 shows the run type as unknown since no settings were chosen.
Reps — Stands for repetition and shows how many times to repeat the same line. When a number other than one is selected, the data files name will automatically be incremented with a rep 1, rep 2, etc., appended to the file name.
Sample Inject Inlet — Indicates the vial the sample is being injected from if sample is located in the inlet tray.
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Sample Inject Outlet — Indicates the vial the sample is being injected from if sample is located in the outlet tray. In the CESI 8000 Plus System the sample is always located in the inlet sample tray.
Sample ID — Name of sample being run.
Method — Separation method used to run that particular line.
Filename — Based on the File Name Text you entered in the Sequence wizard. The filename still shows the Date and Time symbol
When you are finished editing the table, click File > Sequence > Save As. Use a Sequence name that has significance to you, for example, TestSequence_111518.
Sequence Validation
The software validates that the methods in the sequence are appropriate for the current instrument configuration. If any problems are detected, a message opens and the sequence does not run. Correct any problems noted and restart the sequence.
After a successful sequence validation, the method in the first line is downloaded to the CESI 8000 Plus System and the run will begin. Observe the operation of the instrument as a guide to future operations and troubleshooting. You can open the Direct Control window during the acquisition of voltage and current information to view real-time information on instrument status. During the run, the data opens in real time in the instrument windows. At the end of each run, the method for the next run is downloaded before the new run begins. This feature allows you to make changes to a method while a sequence is processing. The version of the method that is current at download (last saved) is the version that runs. You can also edit the Sequence Table while the sequence is running. Existing lines that have not been started can be edited or deleted, and additional lines can be added to the table. (Lines that have already completed or are currently executing cannot be edited).
NOTE When a sequence is built using a method that has auto-incrementing enabled, edits that change the auto-generated positions are not allowed. If allow override is not selected, relevant sequence table entries are not editable.
It is helpful to experiment with the Sequence Table before proceeding to the next section. Make changes, process the sequence, and observe the effects. You may want to combine these experiments with the writing of new methods. A series of different methods can be entered into the Sequence Table. When processed, the results demonstrate the effects of changes in parameters. For example, you might create a series of methods at different separation temperatures to examine the effect of that parameter on your separation. Another series of experiments might examine the effects of changing the volume injected. If your method allows override, this can be done with a single method by editing the Sample Inject Duration column in the Sequence Table.
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Overview of the CESI 8000 Software
The CESI 8000 Software operates “on top” of the 32 Karat™ Software (32 Karat™ Software is discussed in CHAPTER 6, 32 Karat™ Software Overview). The CESI 8000 Software provides the user with a workflow-oriented approach to running methods and sequences that have already been developed by a 32 Karat™ Software user. There is no direct way to create or modify sequences and methods within the CESI 8000 Software, but the queuing, execution, and monitoring of methods and sequences is graphically enhanced. This relieves the routine user of some of the complexities of the 32 Karat™ Software. While the CESI 8000 Software provides some windows into the 32 Karat™ Software, the CESI 8000 Software has been developed with the routine user in mind.
The CESI 8000 Software steps the instrument operator through the tasks of running methods and sequences on the CESI 8000 Plus System. Routine users often receive Standard Operating Procedures detailing separation methods, sequences, and report templates developed in the 32 Karat™ Software by method developers. The CESI 8000 Software enables simplified use of routine methods and sequences. Use the CESI 8000 Software to run sequences and methods, and access the 32 Karat™ Software, version 10.1 or higher. The CESI 8000 Software also allows for instrument control and inherits the necessary electronic controls for 21 CFR part 11 compliance from the 32 Karat™ Software. The Describe Sequence feature in the CESI 8000 Software enables the user to vary the number of samples used in a sequence, or modify certain aspects of the sequence, without the need to generate or edit existing sequences developed in the 32 Karat™ Software.
Use the CESI 8000 Software to:
• Access an application • Describe Sequence • Run Application • Monitor auxiliary data such as voltage and current • Access the 32 Karat™ Software functions
In order for a routine user to run methods and sequences in the CESI 8000 Software, they must be properly created and configured within the 32 Karat™ Enterprise Software.
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Launching the CESI 8000 Software
Launch the CESI 8000 Software by double-clicking the CESI 8000 Plus icon on the Windows desktop. The Ready window opens as shown in Figure 7.1. The upper right buttons activate the major functions. Hover the mouse over the buttons to obtain a balloon tip regarding function. CESI 8000 Software Online Help access is also available here. The 32 Karat™ Software can be launched from the side menu.
NOTE The same license key used for the 32 Karat™ Software is required to operate the CESI 8000 Software. Refer to License Key in CHAPTER 6 for additional information about the license key.
Figure 7.1 CESI 8000 Plus Ready Window
The Run button runs a method or sequence.
The Describe button is used to prepare sequences.
This button is used to access the desktop.
This button launches the 32 Karat™ Software (see Login and Logout).
This button locks the CESI 8000 Software (see Lock and Unlock).
This button is used to access the About dialog.
This button is used to Exit the CESI 8000 Software.
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IMPORTANT Users are able to access only the methods or sequences to which they have permission as set by the 32 Karat™ Software System Administrator.
IMPORTANT We recommend that you enable security in the 32 Karat™ Software and use assigned User Names and Passwords to ensure system security. The CESI 8000 Software is optimized for use with a user-login enabled system.
At any point the side menu can be hidden by selecting .
The CESI 8000 Software visually displays the current instrument status in color, as follows: • Ready — Blue • Idle — Blue • Run Complete — Blue • Running — Green • Scheduled Run — Green • Rinsing Capillary — Light Green • Run Aborted — Red • Instrument Offline — Red • Instrument Error — Red • Waiting Instrument Status — Yellow • Sample Cover Open — Yellow • No Cartridge — Yellow • MS Connection Lost — Yellow
Login and Logout The 32 Karat™ Software enables 21 CFR part 11 compliance. User names and passwords for the CESI 8000 Software are administered through the 32 Karat™ Software. Options to enable or disable security, or allow users to save passwords are controlled through 32 Karat™ Software security options.
If 32 Karat™ Software security is enabled and an application and sequence or method are selected, the system prompts for a user name and password (Figure 7.2).
Figure 7.2 Login Dialog
NOTE If 32 Karat™ Software security is configured to use the Domain controller, then the system will also prompt for the Domain name.
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Lock and Unlock The lock feature on CESI 8000 Software prevents other users from manipulating any controls in the software while the current user is away from the controller. You will still be able to view, print, or export windows.
To unlock the window, select and enter the User Name and Password for the user that locked the window.
NOTE When the CESI 8000 Software is locked, other users will still be able to access other programs (such as the 32 Karat™ Software) on the system.
About Dialog To view the software versions for the CESI 8000 Plus, 32 Karat, and the instrument firmware
versions, select . To copy the version numbers to the clipboard, select Copy Version Numbers. The firmware version is only available if the instrument is online.
Help
Access the help by selecting , Help, or F1 on any of the windows.
Users
There are two types of users in the CESI 8000 Software:
•Routine User — An analyst using the system to access the same methods and sequences daily. Typically the methods do not need to be altered and only the number of samples for the sequence is edited. The routine user only has permission to run methods and sequences as defined by the 32 Karat™ Software system administrator or method developer. • Method Developer — An analyst that has permission to create methods and sequences, as well as configure the 32 Karat instruments and projects. Method developers are responsible for managing the 32 Karat instrument configuration, the 32 Karat™ Software project, and assigning users and permissions. Methods, sequences, and reports are developed by the method developer in the 32 Karat™ Software. Method developers are responsible for transferring these methods and sequences to the CESI 8000 Software for routine use. A method developers’ access can be limited to certain instruments and projects as needed by the system administrator.
NOTE All system administration is performed by a user with System Administrator privileges in the 32 Karat™ Software.
The CESI 8000 Software is developed primarily for the routine user.
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IMPORTANT In order for users to access the CESI 8000 Software for routine use, they must have these four permissions set by the system administrator in the 32 Karat™ Software: • Open Method • Open Sequence • Save Sequence (for the Describe Sequence function) • Lock Instrument
Run Application
Run Application ( ) enables the routine user to run 32 Karat™ Software methods and sequences that are defined by the method developer. From the Run Application dialogs, the user can select a sequence or method, configure the sample setup, and initiate a run.
IMPORTANT Do not attempt to start a method or sequence from the 32 Karat™ Software and the CESI 8000 Software at the same time. The CESI 8000 Software will prevent a user from starting a method or sequence from the same connected instrument.
To run an application, select . Running an application includes three steps:
• Application Selection
• Samples and Vials
• Run Acquisition
Application Selection
The first step of Run Application is to select an application (Figure 7.3).
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Figure 7.3 Selecting the Application
Select an Application
1 Select the Application from the application list.
2 Using the menu, select a sequence or method ( ) (Figure 7.4 and Figure 7.5). If the sequence or method is not shown, select Browse to locate the sequence or method.
NOTE Available sequences are shown above available methods.
NOTE When selecting a sequence the application is highlighted in blue or yellow. If no application was previously selected the application is highlighted in blue. If an application is currently selected and the CESI 8000 Software is currently connected to the instrument, the application is highlighted in yellow.
CAUTION If the CESI 8000 Software is already open with a method or sequence, do not open CE-MS project in the 32 Karat™ Software. Doing so will not allow the properties of the method or sequence to be changed or saved. Instead of using the 32 Karat button in this instance, use the “Show 32 Karat” button at the center bottom of the dialog.
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Figure 7.4 Select a Sequence
Figure 7.5 Select a Method
3 If 32 Karat™ Software security features are enabled, enter the User Name and Password for the application, see Login and Logout.
4 Upon successful login, a connection is made with the instrument. The instrument status is updated in the top banner of the CESI 8000 Software. The software will check the 32 Karat™ Software configuration for the instrument and compare it with both the selected application requirements and the instrument status and report any discrepancies. Real-time voltage and electrical current data from the instrument opens in the Instrument Status and Direct Control panel.
NOTE. Several instrument checks are made when the CESI 8000 Software connects to an instrument. If
there is an instrument error, a flashing will open following a successful login. Select to show the error message.
5 From the Instrument Status and Direct Control section of the dialog, prepare the instrument to run the selected application (bring the trays into the Home or Load position).
6 Click Next or select to prepare the samples and vials for the selected application, see Samples and Vials.
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Samples and Vials
In the Samples/Vials dialog, the application is configured for the selected method (Samples/Vials Dialog) or sequence (Sequence Dialog). Three actions are performed from the Samples/Vials dialog: • Select the number of samples to run • Enter Sample Information • Load Instrument Trays
NOTE The current user, application, and method/sequence is shown in the top-right corner of the CESI 8000 Software (Figure 7.6).
Figure 7.6 Current User, Application, and Method/Sequence
Depending on whether a method or sequence is selected, the Method Samples/Vials dialog or Sequence Samples/Vials dialog will open, see Samples/Vials Dialog or Sequence Dialog.
Samples/Vials Dialog
The Method Samples/Vials dialog allows configuration of the sample ID, output data path, output data file name, and the number of repetitions. The tray detail for the method is shown. To set up a sequence, see Sequence Dialog.
Figure 7.7 Method Setup Window
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Set Up Samples and Vials for a Method
1 Enter the Sample ID or select Insert to create a Sample ID using the increment number, instrument name, user name, date and time, or method name. See Insert Option for more information.
NOTE You can modify the number of placeholders and start number for the increment number (for example, change the increment number to <00200> to begin the increment at 00200.
2 Select Browse to change the output data path.
NOTE The default output data folder is the data folder for the selected application.
3 To specify the data file name, select Insert or enter the data file name for this run. See Insert Option for more information.
NOTE If no data file is specified, the default data file name is
4 Select the number of repetitions for the method.
NOTE The CESI 8000 Software will check for duplicate data file names. If there is more than one repetition the data file names must use a unique identifier such as the date and time or increment.
5 To print the method report select Print Method Report. The method report is printed as configured in the method.
6 Select to bring the instrument trays to the load position.
7 Place the vials into the positions as depicted in the Tray Detail view. For display options, see Display Options.
8 Once the trays are loaded into the instrument and the sample door is closed, start the method run by selecting .
9 A dialog will open to confirm that the samples are loaded. Verify that they are loaded, and select Yes - run now to start the run, see Run Acquisition.
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Insert Option The Insert option allows the user to increment the Sample ID or Data File name. To access the insert option, select Insert (Figure 7.8). In the Insert dialog, select the option and it will be appended to the contents of the Sample ID box (Figure 7.9) or Data File name (Figure 7.10).
Figure 7.8 How to Access the Insert Option
Figure 7.9 Insert Sample ID Dialog
Figure 7.10 Insert Data File Dialog
Options for the Sample ID (Figure 7.9) or Data File (Figure 7.10) dialogs include:
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• Increment Number — A count starting from the specified number. Use 0 to add placeholders for the number of digits to use in the increment, for example enter 010 to use three digits and start the count at 10. • User Name — The current user logged into the application. • Method Name — The method name for the data file. • Instrument Name — The name of the instrument that the sample was run on. • Date and Time — The date and time that the sample run started. • Sample ID — (Data File only) The current Sample ID for the Data file.
Sequence Dialog
The Sequence Samples/Vials dialog allows configuration of the sample IDs, output data path, output sequence path, output data file names, and the number of repetitions. The tray detail for the sequence is shown. When one or more runs are selected in the sequence table, the associated vials are highlighted in the tray view. To set up a method, see Samples/Vials Dialog.
NOTE The Samples/Vials dialog checks for potential tray collisions. An error message will open if there is the possibility of a collision. Use the Show Vial Preview to see where the collisions can occur, and make whatever tray correction is necessary to avoid the collision. To display the vial preview, select Display Options > Show Vial Preview.
NOTE To select a row, select the left-most column for the row and then drag to select multiple rows. Associated vials in the tray detail view will be highlighted.
To undo changes made to the Sequence Table, select Reload Sequence which will restore the original state. From the Sequence Samples/Vials dialog, switch to the Describe Sequence dialog
by selecting . If the sequence is modified and saved in the Describe Sequence dialog, select Reload Sequence to reload the described sequence.
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Figure 7.11 Sequence Setup
The default output sequence path is {Pre-defined Project folder}\Data\Sequence. Enter the desirable path in the text box or select Browse. The path will be available in the current instrument until there is a switch to a different instrument, or until the application is exited.
How to Set Up Samples and Vials for a Sequence
1 Select the Number of Samples.
NOTE The default Number of Samples is the maximum number of samples for the sequence. If Number of Samples is not available the sequence must first be described, see Describe Sequence.
Rows in the sequence are removed as the sample quantity is varied. For more information see Sequence Table Rows.
2 To change the output data path, select Browse.
NOTE The default output data folder is the data folder for the selected application.
3 In the Sequence Table, specify the number of repetitions, sample ID, and output data file name for each run, see Fill Down Option.
NOTE Mandatory fields that are blank are highlighted in yellow. These fields are configured when the sequence is described, see Describe Sequence.
NOTE When a particular run is selected, the vials used in the run are highlighted in the tray view.
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4 To print the method report select Print Method Report. The method report is printed as configured in the method.
5 To print the sequence report select Print Sequence Report. The sequence report is printed as configured in the sequence.
6 Select to bring the instrument trays to the load position.
7 Place the vials into the positions as depicted in the Tray Detail view. For display options, see Display Options.
8 Once the trays are loaded into the instrument and the sample door is closed, start the sequence run by selecting .
NOTE After selecting , the sequence about to be run is automatically saved within a Sequence folder created in the output data path field (Figure 7.11).
9 A dialog will open to confirm that the samples are loaded. Verify that they are loaded, and select Yes - Run Now to start the sequence run, see Run Acquisition.
NOTE You can select Schedule Run to start a run at a later time. The maximum schedule delay time is 100 hours and 59 minutes.
Sequence Table Rows When a sequence is run, the number of samples can be varied. Rows in the sequence are hidden as the sample quantity is reduced. When a sequence is described, rows are assigned types, Always, Control, and Sample. When the number of samples is set, rows are hidden based upon the following types:
• Sample — The rows from one row beyond the number of samples are hidden (not including rows labeled Control or Always) (Figure 7.12, Figure 7.13, Figure 7.14, and Figure 7.15). • Control — The control row is hidden when the first Sample row following it is hidden (Figure 7.14, Figure 7.15, Figure 7.16, and Figure 7.17). • Always — Never hidden and always run, regardless of the number of samples (Figure 7.13).
NOTE When the number of samples is set to 0, all rows are hidden except for rows set to Always.
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Figure 7.12 Sequence with All Nine Samples
Figure 7.13 Sequence with No Samples (All Rows Hidden)
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Figure 7.14 Sequence with Five Samples
Figure 7.15 Sequence with Four Samples
NOTE Notice how Standard C (Row 13) is hidden when the number of samples changes from five (Figure 7.14) to four (Figure 7.15).
Hidden rows that are not run can be shown by right-clicking the sequence and selecting Show Hidden Rows (Figure 7.16 and Figure 7.17).
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Figure 7.16 Sequence with Five Samples and Hidden Rows
Figure 7.17 Sequence with Four Samples and Hidden Rows
For more information on setting row types, see Step 6 of Describe Sequence. For more information on the number of samples see Sequence Dialog.
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Fill Down Option The Fill Down option allows the user to increment the Sample ID (Figure 7.19) or Data File name (Figure 7.20). In a sequence this will automatically increment the selected row and all following rows. If a range of rows is selected, the Fill Down option will be applied to the selected cells.
To access the fill-down option, select the cell. Right-click the cell and select Fill Down (Figure 7.18). In the Fill Down dialog, select the option and it will be appended to the end of the Sample ID or Data File name (Figure 7.19 and Figure 7.20).
Figure 7.18 How to Access the Fill Down Option
NOTE When Fill Down is selected for Reps (repeats or repetition), the selected cell’s value is copied into the following rows.
Options for Fill Down include:
• Calibration Level — The calibration level as defined in the sequence table and method. • Calibration Set — The calibration set as defined in the sequence table and method. • Date and Time — The date and time that the sample run started. • Increment Number — A count starting from the specified number. Use 0 to add placeholders for the number of digits to use in the increment, for example enter 010 to use three digits and start the count at 10. • Instrument Name — The name of the instrument used for the sample run. • Line Number — The run number in the sequence. • Method Name — The method name for the data file. • Sample ID — (Data File only) The current Sample ID for the Data file. • User Name — The current user logged into the application.
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Figure 7.19 Fill Down Sample ID Dialog
Figure 7.20 Fill Down Data File Dialog
Display Options The Display Options provide additional options for viewing information about the trays:
• Program Event — Changes the view of the trays to show the action performed on the vial (for example, separation, rinse, sample inject, etc.). • Vial Contents — Changes the view of the trays to the contents of the vial.
NOTE The Vial Contents information is obtained from the method definition. The first five characters of the Vial Contents are shown in the tray view. It is recommended that each of the vial content types have unique identifiers and are consistent between methods.
• Show Legend — Displays the vial legend, see Vial Legend.
NOTE The vial legend can be shown or hidden from the Display Options menu or by clicking the button highlighted in Figure 7.21.
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Figure 7.21 Alternative Access to the Display Options Menu
• Hide Legend — Hides the vial legend, see Vial Legend. • Show Tray Detail — Opens a window showing the full tray detail based upon the current tray view (Program Event or Vial Contents), see Tray Detail View. • Show Vial Preview — Opens a table to preview the methods and vials used in the method/ sequence and show tray collisions, see Vial Preview.
Vial Legend The Vial Legend displays a list of events or contents based upon the view (Program Event or Vial Contents). To change the color for the vial type, select the color next to the description and select a new color. To change the tray view, select Program Event or Vial Contents or select from the Display Options. The number of vials for each event type or content type is shown next to each item in the Vial Legend.
Reset changes the colors in the vial legend back to the default settings.
Tray Detail View The tray detail view shows the full tray detail based upon the current tray view (Program Event or Vial Contents). From this dialog, the user can view, export, or print the current trays. The vials can be viewed as circles or rectangles.
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Figure 7.22 Tray Detail View
Vial Preview The Vial Preview shows the order, cycle, and vials accessed for each of the methods in the sequence. Cycle number is the number of times that the specific method has been run. This window is used to show tray collisions (marked in red).
Figure 7.23 Sequence Vial Preview
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Run Acquisition
The Run Acquisition window shows information about the sequence or method that is running. The left-hand panel has three tables to show different levels of detail about a method or sequence. The Run Queue table is used to verify that the run initiated in CESI 8000 Plus System is in the 32 Karat™ Software Run Queue. If a sequence is running, the Sequence Run table will show which row of the sequence is currently running. The Current Run table shows which event the method is currently running. The graph consists of two tabs, Current Run shows real-time data as it is being collected, and All Runs shows all data collected so far for the sequence or method being run. For more information see Graph Options.
During a run the user can view the settings for the first two steps, Application and Samples/Vials
by selecting , , Back, or Next. Once a run has started none of the display settings can be changed. Only one Sequence or Method at a time can be started from the CESI 8000 Software.
Once the run is complete, the settings for the current run can be viewed, printed, or exported by selecting one of the three views and Print or Export.
To begin a new run, when the run is complete, select to signify that you are finished with viewing, printing, and exporting the data from this run.
Figure 7.24 Sequence Run Acquisition Window
NOTE Select to expand the graph and show more data (Figure 7.25).
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Figure 7.25 Sequence Run Acquisition Graph
During the scheduled run, the Run Acquisition page will be empty. The system state is Scheduled Run with the scheduled run starting time countdown below the main state as shown in Figure 7.26.
Figure 7.26 Scheduled Run Acquisition Window
Click Start Now to cancel the waiting and start the run. Click Stop to abort the scheduled run, and the system state will switch to Run Aborted.
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How to Stop a Run
IMPORTANT Once a method or sequence is stopped it cannot be resumed.
1 To stop the run, select .
2 The following options will open: • Stop current run only • Stop after current run (sequence only) • Stop current run and sequence run (sequence only) • Stop all run queue items (sequence only)
Select the appropriate option and select OK to stop the run.
How to Finish a Run
1 After viewing, exporting, and printing information from each of the Run Application steps, select from Acquisition.
2 Select Finished to close the Run application or Run More to run the same sequence or method or start a new one.
NOTE If Run More is selected, all user-entered settings will be retained in the Application Configuration and can be modified. If a different application sequence or method is selected all previously entered settings from the previous run is discarded.
SCIEX TripleTOF® 5600 Mass Spectrometer Using AAO The Analyst Access Object (AAO) software interface provides a software layer to communicate with the Analyst® TF Software used by the SCIEX TripleTOF® 5600 mass spectrometer acquisition computer. If AAO is being used, the Direct Control window (Figure 6.9) will indicate the mass spectrometer status with one of the following mass spectrometer status icons:
Mass spectrometer interface is Offline or Disconnected.
Mass spectrometer is on Standby, Stopping, Paused, Unknown or AAO driver connection failed.
Mass spectrometer is Ready.
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Mass spectrometer is on Pre Run / Ready to acquire.
Mass spectrometer is Running.
Graph Options Some graph settings are independent for the Current Run and All Runs tab. Any changes made to the Graph Options dialog are applied automatically to the selected graph tab.
Figure 7.27 Graph Options Dialog
Graph options include:
• Absolute Value — Shows the absolute value of the Current and Voltage in the graph. This can be used for voltages or currents. • Label with Run Number — Adds the run number to the graph (the Current Run tab shows text on top, the All Runs tab shows in the key for the trace). • Label with Sample ID — Adds the Sample ID to the graph (current run on top, all runs shows in the key for the trace). • Multiple Colors — If selected, then each trace will open in a different color. • Scale Y Axis — If selected, the Y-axis labels are scaled to show a number and a multiplier is shown in the Y-axis legend. • Separate Panels — If selected, then each electropherogram (egram)/trace is shown in its own separate panel on the tab. Max Displayed allows you to select how many panels are viewable on the tab (a maximum of 5 panels can be shown). If not selected, then each egram/ trace is shown on the same panel (select Multiple Colors to help distinguish between the traces). • Thin Lines — Changes the line thickness of the trace.
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• Other options — Expands the Graph Options dialog to show additional graph settings.
NOTE The additional graph settings only apply if Separate Panels is not selected and there is more than one trace.
— Spacing Between Traces — Adjusts how much space is between each of the traces. If there is no spacing between the traces they will overlay each other. — Height of Display Region — Changes the Y-axis scale of the display region.
• Max Displayed — If Separate Panels is selected then this controls how many separate panels can be seen at one time in the window. A maximum of five panels can be shown at a time. • Show/Hide Traces — Allows you to select the traces to show in the graph.
Sequence Transfer Overview
To complete the sequence transfer, the methods and sequences must be transferred from the 32 Karat™ Software to the CESI 8000 Software. Sequence Transfer should be done by a method developer with administrative rights. Method developers will first develop methods and sequences using the 32 Karat™ Software. Once the methods and sequences are finalized they can be transferred from the 32 Karat™ Software to the CESI 8000 Software.
To transfer a sequence the following steps are required: • Define an Instrument for the Routine User • Define the Project for the Instrument • Add the Routine User and Assign Privileges (Data System) or Add the Routine User and Assign Privileges (Domain Controller) • Transfer the Methods • Transfer the Sequences • Review the Sequence Properties • Describe Sequence • Run Application
IMPORTANT We recommend that you enable 32 Karat™ Software security and use assigned User Names and Passwords to ensure system security. The CESI 8000 Software is optimized for use with a user-login enabled system.
Sequence Transfer
Define an Instrument for the Routine User
1 Start the 32 Karat™ Software and login as an Administrator.
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2 Click File > New > Instrument to create a new instrument.
3 Enter a name for the instrument.
IMPORTANT The instrument name must be a valid Windows folder name. In order for the routine user to access an application from the CESI 8000 Software, the 32 Karat™ Software project name must match the 32 Karat™ Software instrument name.
4 Configure the instrument by right-clicking the new instrument name and selecting Configure > Instrument.
5 Select the instrument in the Instrument type field.
6 Select Configure.
7 Select CE-MS from the module list in the left window and select .
8 Configure CE-MS in the right window by right-clicking CE-MS and selecting Open.
9 Select tray sizes, home positions, and any other options as required.
10 After CE-MS is configured, select OK.
11 Select Options.
12 Configure General Options and Instrument Options. Select OK.
13 Select OK to close the remaining dialogs.
Define the Project for the Instrument
1 Select Tools > System Administration Wizard.
2 In the System Administration wizard, select Project and then select Next.
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3 Select Create a new project and then select Next.
IMPORTANT The project name must be the same as the instrument name and must be a 32 Karat folder name.
4 Enter the name of the instrument.
5 (Optional) Enter a description and select Next.
6 Configure the general project settings and select Next.
7 Define the electronic signature roles and select Finish.
NOTE Users will be added later.
Add the Routine User and Assign Privileges (Data System)
1 Select Tools > Options.
2 Select the Enterprise tab.
3 Select Add User.
4 Enter the User Name and Password, select Save.
5 Select OK.
6 Select Tools > System Administration Wizard.
7 Select User and select Next.
8 Select the User Name and select Next.
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9 Assign System administration or Instrument administration privileges as appropriate. Select Next.
NOTE System or instrument administration are not required settings for routine users.
10 Add instruments for the current user then select Next.
11 Add projects for the current user then select Next.
12 Add the Open Method, Open Sequence, Save Sequence, and Lock Instrument privileges. Add any additional privileges and select Next.
IMPORTANT In order for the routine user to run methods and sequences they must have the following privileges in the 32 Karat™ Software: • Open Method • Open Sequence • Save Sequence • Lock Instrument
13 As appropriate, assign electronic signature roles.
14 Select Finish. The sequence can now be transferred, see Transfer the Methods.
Add the Routine User and Assign Privileges (Domain Controller)
1 Select Tools > System Administration Wizard.
2 Select User and select Next.
3 Select the Domain and then enter the User Name.
4 Select Check Names. Add the name to the Selected Users by selecting .
5 Select Next.
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6 Assign System administration or Instrument administration privileges as appropriate. Select Next.
NOTE System or instrument administration are not required settings for routine users.
7 Add instruments for the current user then select Next.
8 Add projects for the current user then select Next.
9 For each of the projects that the routine user needs access to, add the Open Method, Open Sequence, Save Sequence, and Lock Instrument privileges. Add any additional privileges and select Next.
IMPORTANT In order for the routine user to run methods and sequences they must have the following privileges in the 32 Karat™ Software: • Open Method • Open Sequence • Save Sequence • Lock Instrument
10 As appropriate, assign electronic signature roles.
11 Select Finish. The sequence can now be transferred, see Transfer the Methods.
Transfer the Methods
1 Copy the methods into the project’s Method folder.
IMPORTANT The approved methods for the application should be in the Method folder defined for the 32 Karat project.
NOTE Method developers may have already created the 32 Karat instrument configuration and 32 Karat project for routine use on existing systems. As a result, methods may already be in the project folder.
Transfer the Sequences
1 Copy the sequences into the project’s Sequence folder.
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IMPORTANT The approved sequences for the application should be in the Sequence folder defined for the 32 Karat project.
NOTE Method developers may have already created the 32 Karat instrument configuration and 32 Karat project for routine use on existing systems. As a result, methods and sequences may already be in the project folder.
Review the Sequence Properties
1 Right-click the instrument in the 32 Karat™ Software and select Open.
2 Enter the User Name and Password. Select the project and select Login.
3 Open the transferred sequence. Click File > Sequence > Open.
4 Select Sequence > Properties and set the Method and Data folders to match the instrument’s project folders for method and data (Figure 7.28).
IMPORTANT Ensure that the properties for the transferred sequence point to the appropriate Method and Data folders for the application.
Figure 7.28 Sequence Properties Dialog
5 Ensure that the methods are selected from the corresponding method folder for this project in the Sequence Run Table (Figure 7.29). Verify that any file paths in the Method column are correct.
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Figure 7.29 Method in the Sequence Run Table
6 Click File > Sequence > Save.
7 Repeat steps 1 through 6 for all transferred sequences.
8 Close the 32 Karat instrument and the Enterprise window in the 32 Karat™ Software.
Describe Sequence In order for a routine user to vary the number of samples used in a sequence, the sequence must
be described using the Describe Sequence ( ) feature in the CESI 8000 Software. When a sequence is described, the rows in the sequence can be defined as a method representing a control (Control), unknown sample (Sample), or a method always required for the sequence (Always) that is always run. This allows the routine user to select the number of unknown samples to run, define the number of replicates, edit the Sample ID, or edit the output data file name.
NOTE The user must have the 32 Karat™ Software sequence file write permission in order to describe a sequence.
NOTE If a sequence is modified in the 32 Karat™ Software after it was described in the CESI 8000 Software, then the changes will be detected and only the full sequence can be run. The sequence will need to be re-described (using Describe Sequence) in order to vary the sample quantity.
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Figure 7.30 Describe Sequence Dialog
1 From the CESI 8000 Software main window, select . The Describe Sequence Start dialog opens (Figure 7.31).
NOTE This example is for an instrument named QC001 and a 32 Karat project named QC001.
Figure 7.31 Describe Sequence Start Dialog
2 In the Application field, select the application.
3 In the Sequence field, select the sequence to describe.
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4 If prompted, enter the User Name and Password.
5 For sequences that have not been described: • All of the sequence runs are set to Sample. • The Sample ID and Data File Name columns are set to Optional. • The Reps column is set to Required.
IMPORTANT The Save button will be enabled the first time a sequence is described since the sequence is automatically modified to these default settings.
6 To describe the rows, select the rows and then select one of the following:
• Sample ( ) — Identifies the selected rows as being unknown samples. These are the rows that will be either included or removed when the routine user selects a different quantity of samples to run. • Control ( ) — Identifies the selected rows as being control methods. These are the methods that will typically bracket a group of unknown samples. • Always ( ) — Identifies the selected rows that must always be run, regardless of how many unknown samples are to be run. For example, designate a shutdown method that should always be run at the end of a sequence with Always.
NOTE This affects how as sequence is run when the number of samples is changed. For more information see Sequence Table Rows.
7 To change the column settings, select the column header for Reps, Sample ID, or Data File to toggle through the options: • Optional — Allows the routine user to either enter information in the column or leave this field blank. • Required — Requires the routine user to enter information into every field of this column. No fields can be blank. • Fixed — Prohibits the user from making any changes to any of the fields in this column. The current column fields as defined by the 32 Karat™ Software sequence author will be used when the application is run.
8 Once the rows and columns are described, it is important to verify that each of the possible sample quantities that a routine user could select while running the application are valid. Select the different Sample quantities in Verification to make sure that the appropriate sequence rows will be run for every possible sample quantity. To see which rows are removed as the number of samples is varied, right-click the sequence table and select Show hidden rows. For more information on how rows are hidden based upon the number of samples see Sequence Table Rows.
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IMPORTANT Rows defined as sample or control may be excluded when the routine user varies the number of samples that are run. Care must be taken when using special run types in the 32 Karat™ Software sequence. Review the Run Type column when testing the different sample quantities to ensure that the sequence functions as expected for all possible sample quantities.
9 Select Save. The changes made are saved back to the template sequence file that was opened. If audit trails are enabled for the sequence, a dialog opens. Enter the reason for the change.
NOTE The change made to the sequence is saved in the first row of the sequence. This information can be viewed from the 32 Karat™ Software sequence editor (Figure 7.32). Attempts to alter the text within the brackets will invalidate the described sequence. To fix this, describe the sequence again and save the described sequence.
Figure 7.32 32 Karat™ Software Sequence Editor
10 Select Finish.
Sequence Verification To verify that the sequence transfer is correct a test run should be performed, see Run Application. To verify that the sequence rows are hidden properly, see Sequence Table Rows.
Sequence Display Options
The Display Options provide additional options for viewing information about the trays:
• Program Event — Changes the view of the trays to show the action performed on the vial (for example, separation, rinse, sample inject, etc.).
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• Vial Contents — Changes the view of the trays to the contents of the vial.
NOTE The Vial Contents information is obtained from the method definition in the 32 Karat™ Software. Edit vial contents using the 32 Karat™ Software method editor. The first five characters of the Vial Contents are shown in the tray view. It is recommended that each of the vial content types have unique identifiers and are consistent between methods.
• Show Legend — Displays the vial legend, see Vial Legend.
NOTE The vial legend can be shown or hidden from the Display Options menu or by selecting the Show/Hide button (Figure 7.33).
Figure 7.33 Show/Hide Button Location
1. Show/Hide Button
• Hide Legend — Hides the vial legend, see Vial Legend. • Show Tray Detail — Opens a window showing the full tray detail based upon the current tray view (Program Event or Vial Contents), see Tray Detail View. • Show Vial Preview — Opens a table to preview the methods and vials used in the method/ sequence and shows tray collisions, see Vial Preview.
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148 / 372 RUO-IDV-05-3897-C | B11949AG CHAPTER 8 Using a SCIEX TripleTOF® 5600 Mass Spectrometer with the CESI 8000 Plus System
Introduction
This chapter includes procedures for the OptiMS Silica Surface Cartridge, from capillary conditioning and instrument tuning, to obtaining a separation of a test mixture for CESI-MS system suitability evaluation.
The user should be familiar with the procedures described in CHAPTER 5, Integrating the CESI 8000 Plus System with a Mass Spectrometer.
This chapter is divided into the following sections:
• Stock Reagents and Other Consumables Required • Preparation of Reagent Solutions and Test Sample • Procedure for Installation of Adapter and OptiMS Cartridge • Methods for the CESI 8000 Plus System • Capillary Conditioning • Establishing a Stable Spray and Determining Optimum ESI Voltage • Running CESI-MS
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The workflow is as follows:
Before you start, ensure you have all the required reagents properly prepared. Description and preparation protocols can be found in the next section.
Stock Reagents and Other Consumables Required
Table 8.1 Stock Reagents
Stock Reagent Vendor Part Number Acetic Acid, glacial (HAc) Sigma-Aldrich A6283 or equivalent 0.1 N Sodium Hydroxide (NaOH) SCIEX 338424 0.1 N Hydrochloric Acid (HCl) EMD Chemical HX0603A 1 N Sodium Hydroxide (NaOH) Sigma-Fluka 319511 Methanol (MeOH) Fisher Chemical A454 7.5 M Ammonium Acetate (AmAc) Sigma-Aldrich A2706 Beta-Galactosidase Digest SCIEX 4465938 DDI water N/A 18 megaohm or higher
Table 8.2 Materials
Consumables Vendor Part Number OptiMS silica surface cartridge SCIEX B07367 20 mL glass vial VWR VW74511-20 Micro vials SCIEX 144709 CESI-MS vial SCIEX B11648 Green CESI-MS vial cap SCIEX B24699 0.5 mL centrifuge tube
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Table 8.2 Materials (Continued)
Consumables Vendor Part Number 50 mL volumetric flask 20 mL glass bottle 100 mL glass bottle pH meter Table top mini centrifuge Assorted pipettes and tips
Preparation of Reagent Solutions and Test Sample
Table 8.3 Required Reagents
Reagents Concentration Use 10% Acetic Acid (v/v) 10% HAc (v/v) Background electrolyte and conductive liquid 20% Acetic Acid (v/v) 20% HAc (v/v) pH adjustment of Leading Electrolyte (LE) 200 mM ionic strength in 200 mM Leading Electrolyte (LE) Buffer Sample and system suitability preparation ammonium, pH 4.0 1 μM Beta-Galactosidase Digest 1 μM System test mix
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Preparation of 10% (v/v) HAc—Background Electrolyte (BGE)
NOTE This solution must be prepared fresh every day.
IMPORTANT Sonicate in a water bath for 30 minutes prior to use.
IMPORTANT When filling the CESI-MS vials, care should be taken not to introduce air bubbles. Check for any trapped air bubbles on the wall of vials, and replace vials as needed.
1 Add 18.0 mL of DDI water into a clean 20 mL glass vial.
2 Inside a fume hood, add 2.0 mL of HAc into the DDI water and mix it well. Mix the contents by inverting the vial 3 times and store at room temperature.
Preparation of 20% (v/v) Acetic Acid (HAc)—For LE pH Adjustment
NOTE Prepare fresh when preparing LE only. Do not stock this solution.
1 Add 80.0 mL of DDI water into a clean 100 mL glass bottle.
2 In a fume hood, add 20.0 mL of acetic acid into the DDI water and mix it well. Mix the contents by inverting the bottle 3 times and store at room temperature.
3 Record the preparation date.
Preparation of 200 mM LE (Leading Electrolyte) Buffer
1 Prepare 50 mL of 400 mM ammonium acetate (AmAc) solution: a. Add 20 mL of DDI water into a 50 mL clean glass volumetric flask. b. In a fume hood, add 2.7 mL of 7.5 M ammonium acetate into the same flask. c. Bring the volume to 50 mL with DDI water. d. Mix the solution by inverting the flask 3 times.
2 Pour the 50 mL of 400 mM AmAc solution into a clean 100 mL beaker.
3 Using a calibrated pH meter, measure and record the initial pH value of the solution.
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4 Adjust the pH of the solution to 4.0 by adding aliquots of freshly prepared 20% HAc.
5 Transfer the solution into a clean 100 mL volumetric flask.
6 Bring the volume to the 100 mL mark with DDI water and mix the solution by inverting the flask 3 times.
7 Record the preparation date, and store at 2 °C to 8 °C when not in use.
NOTE This solution expires in 24 months when stored properly.
Reconstitution of 1 μM Beta-Galactosidase Digest Solution (Beta-Galactosidase Stock Solution)
1 Add 625 μL of DDI water to beta-galactosidase vial and vortex the vial for 5 seconds.
2 Aliquot 50 μL each for storage.
3 Store at -35 °C to -15 °C (frozen) up to one year.
4 Record the preparation date.
Preparation of Beta-Galactosidase Test Sample
1 Prepare a 0.5 μM beta-galactosidase test sample by mixing 50 μL of 200 mM LE Buffer, pH 4.0, with 50 μL aliquot of 1 μM beta-galactosidase in a 0.5 mL centrifuge tube.
2 Mix the sample thoroughly.
3 Centrifuge the sample tube for 15 minutes at 13 000 rcf.
4 Use only the supernatant (90 μL) as the CESI-MS test sample.
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5 Record the preparation date.
Preparation of Beta-Galactosidase for Auto-calibration
1 Prepare a 0.5 μM beta-galactosidase sample by mixing 50 μL of 10% HAc solution with 50 μL of 1 μM beta-galactosidase in a 0.5 mL centrifuge tube.
2 Mix the sample thoroughly.
3 Centrifuge the sample tube for 15 minutes at 13 000 rcf.
4 Use only the supernatant (90 μL) as the auto-calibration sample.
5 Sample must be placed in the inlet sample tray A1 position. Discard after each batch.
Buffer Tray Setup The buffer tray set up is described in this section. It is important to follow the specific location for each reagent as they match the CESI 8000 Plus methods described in this chapter.
NOTE Use only CESI-MS vials and CESI-MS caps in the CESI 8000 Plus System buffer and sample trays.
Table 8.4 provides the vial content, quantity, and volume for capillary conditioning beta- galactosidase auto-calibration, manual calibration, and system test methods.
Table 8.4 Solution/Reagent Requirements
Vial Content Quantity Volume BGE 3 vials (refer to Note) 1.4 mL Methanol – Optima LC/MS grade 2 vials 1.5 mL DDI water 2 vials 1.5 mL 0.1 N NaOH 1 vial 1.5 mL 0.1 N HCl 1 vial 1.5 mL Methanol – LC/MS grade 1 Falcon tube 50 mL 5 mL DDI water 1 Falcon tube 50 mL 10 mL
NOTE Add 2 more vials for a total of 5 vials if auto-calibration is required. Refer to APPENDIX C, CE Method for Auto-Calibration.
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Figure 8.1 shows the position of each vial reagent in both inlet and outlet trays. This layout is common to all five CESI 8000 Plus methods. Figure 8.2 shows vial layout for use with the CE method for auto-calibration.
Figure 8.1 Vial Layout for Inlet and Outlet Trays
Figure 8.2 Vial Layout for Inlet and Outlet Trays (for Use with CE Method for Auto-Calibration)
Sample Tray Setup The sample tray setup is described in this section. It is important to follow the specific location for each sample as they match the CESI 8000 Plus methods described in this chapter.
The beta-galactosidase sample used for the auto-calibration and the beta-galactosidase sample used for system testing are located in vial positions A1 and A2 respectively, as shown Figure 8.3. Samples to be analyzed can be placed in other positions; however, these positions must be specified in the CESI 8000 Plus methods or manually specified in the sequence table.
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IMPORTANT Samples are likely to evaporate over time. It is recommended to use a minimum of 50 μL of sample for a sequence with a duration of approximately 24 hours. If the duration of the sequence is over 50 hours, a minimum volume of 80 μL is strongly recommended.
Figure 8.3 Sample Tray Setup
Procedure for Installation of Adapter and OptiMS Cartridge
For installation instructions for the adapter and the OptiMS cartridge, refer to CHAPTER 5, Integrating the CESI 8000 Plus System with a Mass Spectrometer within this manual.
Prior to inserting the OptiMS sprayer into the adapter, ensure the positioning stage is aligned as far away from the entrance of the mass spectrometer as possible (Figure 8.4).
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Figure 8.4 Pull back Positioning Stage from the SCIEX Mass Spectrometer
For additional information, refer to the OptiMS Cartridge Sprayer Tip Installation into the SCIEX Nanospray® III Source section in CHAPTER 5.
IMPORTANT Before proceeding to the next step it is very important the mass spectrometer is idle and the ESI voltage is set to zero.
Methods for the CESI 8000 Plus System
The five methods we will use can be found at: C:\32karat\projects\CEMS\Methods and include capillary conditioning, auto-calibration, system performance test with beta-galactosidase, and
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cleaning and storage methods. The mass spectrometer suggested methods are described later on in this section. The CESI-MS Conditioning method is used for conditioning of the new OptiMS cartridge. This method is named CESI-MS Conditioning.met.
The CESI-MS auto-calibration is used (only on the SCIEX TripleTOF® 5600 mass spectrometer) for voltage-infusing of the beta-galactosidase in 5% HAc for auto-calibration of the mass spectrometer system during CESI-MS batch runs. This method is named CESI-MS Auto- calibration_ABSciex. This auto-calibration is needed to achieve high mass accuracy on a TOF instrument. It is recommended to run an auto-calibration at a minimum of every five hours. It is however, at the discretion of the user how frequently to run the auto-calibration. The CESI-MS Separation (beta-galactosidase) is a method used to inject and separate a beta- galactosidase control sample by CESI-MS. This method is used to ensure both the CESI 8000 Plus System and the SCIEX TripleTOF® 5600 mass spectrometer are set up properly. This method is named CESI-MS Separation for AB SCIEX MS.met.
CESI-MS Cleaning is a method used to clean the capillary at the end of a batch. This method is named CESI-MS Cleaning.met.
CESI-MS Storage is a method used to condition and store the capillary if capillary won't be used for more than 3 days. These cleaning and storage methods will be discussed in detail within APPENDIX F.
The initial conditions for all five CESI 8000 Software methods are the same and the settings used are as shown in Figure 8.5. Auxiliary data channel is checked for voltage and current with maximum values set at 30 kV and 10 μA respectively. The cartridge temperature is set at 25 °C, and 10 °C for sample temperature control. The Threshold and Peak Width are set to 2 and 9 respectively. The trigger settings are checked for both. Wait until cartridge and sample storage temperatures are reached. The inlet trays are set for 36 vials for buffer and 48 vials for sample tray. Outlet trays are set as 36 vials for the buffer tray and no sample tray.
Figure 8.5 Initial Conditions for All CESI 8000 Software Methods
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The time program for all 5 methods differ in a variety of steps; Figure 8.6 through Figure 8.8 show how each time program is set. Figure 8.6 shows the time program settings for the CESI-MS Capillary Conditioning method.
Figure 8.6 CESI-MS Capillary Conditioning Method Settings
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Figure 8.7 shows the time program settings for the CESI-MS Auto-calibration method to be used in a batch run requiring auto-calibration.
Figure 8.7 CESI-MS Auto-calibration_ABSciex Method Settings
The CESI-MS separation method, shown in Figure 8.8, is used for the separation of the beta- galactosidase digest as a system performance test.
Figure 8.8 CESI-MS Separation for SCIEX Mass Spectrometer Method Settings
IMPORTANT For the beta-galactosidase system performance test, do not use a separation voltage above 20 kV.
Figure 8.9 Voltage Ramp Down for Step 10 in Figure 8.8
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The CESI-MS Cleaning method used at the end of every sequence is shown in Figure 8.10. Refer to APPENDIX F for detailed information.
Figure 8.10 CESI-MS Cleaning Method Settings
CESI-MS Beta-Galactosidase System Performance Test
The CESI-MS Separation method is used to perform a 45 minute-long CESI-MS separation of beta- galactosidase digest as a system performance test. Figure 8.11 to Figure 8.16 show the various parameters settings and properties.
Figure 8.11 General Acquisition Settings
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IMPORTANT If creating a method from scratch, the following applies: 1. Synchronization Mode is set to No Sync. 2. Ensure that the Beckman CE Driver is present on the left pane under Acquisition Method. 3. The duration of the method, Duration (min), must be at least 1.5 minutes shorter than the method set on the CESI 8000 Software.
1 Click TOF MS (+) in the left pane to enter the mass spectrometer settings (Figure 8.12).
Figure 8.12 Parameter Settings for Mass Spectrometer Tab
2 Click Edit Parameters, and the Parameter Settings dialog will open. Enter values in both the Source/Gas and Compound tabs (Figure 8.13).
Figure 8.13 Parameter Settings for Source/Gas and Compound Tabs
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3 Go to Advanced MS tab to enter other parameters (Figure 8.14).
Figure 8.14 Parameter Settings for Advanced Mass Spectrometer Tab
4 Click Product Ion (+) to enter the mass spectrometer parameters for Product Ion scan (Figure 8.15).
Figure 8.15 Parameter Settings for Product Ion Scan
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5 Click Edit Parameters, and a dialog for Source/Gas and Compound will open. Enter parameters in both tabs (Figure 8.16).
Figure 8.16 Parameter Settings for Source/Gas and Compound tabs of Product Ion Scan
6 Save the method as CESI_MS_AB_Sciex_Betagal_45min.dam.
Capillary Conditioning
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Capillary conditioning is a procedure that must be performed when using an OptiMS Silica Surface Cartridge for the first time, and when using a cartridge that has been stored for a period longer than 3 days. (See APPENDIX F: Using Vials and Hardware Maintenance).
Capillary conditioning consists of 9 rinse steps at high pressure of various durations. This method performs forward rinses to condition the separation capillary using methanol (MeOH), DDI water, 0.1 N NaOH, 0.1 N HCl, and 10% acetic acid. Note the time duration for all rinses is 10 minutes at 100 psi. This method also performs reverse rinses of the conductive liquid capillary (CLC) with MeOH, DDI water, and 10% Acetic Acid for 3 minutes at 100 psi each.
1 Place a Falcon tube containing 10 mL of methanol in the holster on the side of the instrument and rest the sprayer in the tube. The sprayer tip must be fully immersed in methanol (Figure 8.18).
IMPORTANT Place the protective sleeve on the sprayer before placing sprayer in the holster.
Figure 8.17 Protective Sleeve Placement
Figure 8.18 Sprayer Immersed in 10 mL of Methanol
2 Prepare inlet and outlet buffer trays as shown in Figure 8.1. Then set the trays aside.
3 Open the CESI 8000 Software.
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4 Click the icon to open the 32 Karat™ Software.
5 In the 32 Karat™ Software, click the Direct Control icon on the toolbar (Figure 8.19).
Figure 8.19 Direct Control Icon on the Toolbar
NOTE If the quick access Direct Control icon is not visible as shown in Figure 8.19, then the procedure for creating it has not been done. Refer to Using the Direct Control Window to Load the Cartridge and Samples in CHAPTER 6: 32 Karat™ Software Overview for instructions on navigating to Direct Control, and also for instructions on creating the quick access Direct Control icon.
6 Click Load.
7 Wait until the trays are in the Load positions, and then open the front cover and place the inlet tray in the inlet carrier (left) and the outlet tray in the outlet carrier (right).
8 Close the door.
9 Inside the 32 Karat™ Software continue to the method menu and open the CESI-MS Conditioning method in the method folder of the CE-MS project.
10 Start the CE-MS Conditioning method using a Single run.
11 In the toolbar, click the blue arrow (Figure 8.20) to submit a Single Run acquisition.
Figure 8.20 Single Run Acquisition Icon on Toolbar
IMPORTANT After the last methanol rinse is completed (Method line 2 — approximately 13 min into the Conditioning method), replace the methanol Falcon tube (Figure 8.18) with a DDI water tube and immerse the sprayer into DDI water. Wait for CESI-MS Conditioning method to be completed. After completion of the method, remove sprayer from the DDI water tube and remove as much of the water as possible by gently padding it dry with dry paper wipes.
12 Figure 8.21 shows the dialog for the single run. Under Run information field, confirm the method path and name are correct for capillary conditioning. It is not necessary to enter sample ID nor data file name as no data file will be generated.
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Figure 8.21 Single Run Acquisition Dialog
The CESI 8000 Software will start the series of rinses and at the completion of the initial capillary conditioning method the OptiMS capillary cartridge is ready for the next step, establishing a stable spray.
Establishing a Stable Spray and Determining Optimum ESI Voltage
Prior to establishing a stable spray, it is important the sprayer tip position is optimized relative to the mass spectrometer inlet. For detailed information on how to adjust and align the sprayer
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on the SCIEX TripleTOF® 5600 mass spectrometer, refer to the Aligning the CESI 8000 Plus System with the SCIEX TripleTOF® 5600 Mass Spectrometer section within CHAPTER 5 of this manual. Ensure that both separation capillary and conductive liquid capillary are filled with 10% acetic acid and the ESI voltage is set to zero on the mass spectrometer.
To fill the conductive liquid capillary with background electrolyte (10% Acetic acid):
1 Go to the Direct Control window. Select the box next to Pressure and use the settings shown in Figure 8.22.
Figure 8.22 Pressure Rinse Setting to Fill Conductive Liquid Capillary with 10% Acetic Acid
2 Click OK.
NOTE Always look at the rinse icon represented by a “blowing face” (Figure 8.23) to determine the direction where the pressure is being applied. The reverse rinse direction will always refer to rinsing the conductive liquid capillary and the “blowing face” should then be visible on the right- hand side of the dialog.
Figure 8.23 Direct Control Window Showing the Reverse Rinse to Fill the Conductive Liquid Capillary
NOTE The “blowing face” on the right-hand side of the dialog (pointed by arrow).
When the conductive liquid capillary is filled with background electrolyte, a droplet is observed at the end of the stainless steel needle (Figure 8.24). After the rinse step completes, proceed to the next step, filling the separation capillary.
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Figure 8.24 Droplet at the Stainless Steel Needle End
To fill the separation capillary with background electrolyte (10% Acetic acid) follow these two easy steps:
1 Go to the Direct Control window, click Rinse, and set 100 psi for 2 minutes in the forward direction BI: A1 (Figure 8.25).
Figure 8.25 Pressure Rinse Setting for a Forward Rinse of the Background Electrolyte to Fill the Separation Capillary
2 Click OK. When the separation capillary is filled with background electrolyte, a droplet becomes visible at the end of the sprayer tip (Figure 8.26).
NOTE If for some reason the separation capillary is empty, it may take up to 7 minutes for the first drop of BGE to form at the sprayer tip.
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Figure 8.26 Droplet at the Tip of the Sprayer
WARNING As droplets form at the end of the sprayer tip, ensure that are not aspirated into the mass spectrometer inlet. Do not position the sprayer tip closer than 2 mm from the curtain gas plate.
3 With both separation capillary and conductive liquid capillary properly filled with background electrolyte, go to the Direct Control window in the 32 Karat™ Software and apply 20 kV (normal polarity) for 30 minutes using a 1.0 min ramp, as illustrated in Figure 8.27.
Figure 8.27 Voltage Settings Dialog
The CESI 8000 electrical current should be around 2 μA to 3 μA for 10% HAc.
4 In the Analyst® TF Software, go to the toolbar, under Tune and Calibrate (Figure 8.28).
Figure 8.28 Tune and Calibrate Mode Menu
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5 In the navigation bar on the left pane, double-click Manual Tuning (Figure 8.29).
Figure 8.29 Navigation Bar
6 In the pop-up window, fill in the fields for the Source/Gas tab as shown in the left pane of Figure 8.30.
Figure 8.30 Source, Curtain Gas, and Mass Spectrometer Settings
NOTE The minimum curtain gas value that can be entered in the Analyst® TF Software is 10. However, CE-MS runs use a curtain gas value of 5. It has been set (factory setting) to 5 when the TripleTOF® 5600 mass spectrometer is interfaced with CESI 8000 Plus mass spectrometer.
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7 From the Scan type list, select TOF MS, set the Accumulation time to 500 ms, and set the TOF Masses (Da) to Min. 300 and Max. 1500. Under Period, set the duration to 5 min, as shown in right pane of Figure 8.30.
8 Set the Ion Spray Voltage Floating (ISVF) voltage to 1.0 kV (also named ESI) and click Start.
9 Increase the ESI (ISVF) voltage in 0.1 kV increments until a continuous signal is achieved in the mass spectrum window (minimum ESI voltage). A typical profile is shown in Figure 8.31.
Figure 8.31 Typical Spray Profile of an OptiMS Silica Surface Cartridge Using 10% HAc as Background Electrolyte
10 Click Stop, set the ESI voltage to zero, and press Enter to accept the change. No spray and no background mass spectrum should be present.
11 Enter the minimum ESI voltage value determined earlier and click Enter. The electrospray should start again.
NOTE If spray is still observed at 0.0 kV ISVF, then there may be a connection problem. Refer to troubleshooting in APPENDIX G for a solution.
12 Increase the minimum ESI voltage by 100 V (ion collection ESI voltage for mass spectrometer methods).
13 While applying ESI voltage, optimize the position of the sprayer in relation to the mass spectrometer inlet by manipulating the XYZ-position knobs to obtain a maximum TIC signal. The sprayer tip should always be no closer than 2 mm from the curtain gas plate.
14 To check on the stability of the spray, continue applying 20 kV (normal polarity) for 20 minutes.
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15 In the Analyst® TF Software, go to the Advance MS tab and check the MCA box. Go back to the MS tab and set the Accumulation time to 1 (sec), set Duration to 5.00 min, set Scan Type to TOF MS, and set TOF Masses (Da) to min of 70 and max of 2000.
16 Press Acquire, enter a file name (for example: Baseline), and record the file. After the baseline has been recorded, zoom in the y-axis to maximize the baseline Fluctuations (Figure 8.32). If the baseline fluctuation is less than 40% (within 2 to 5 min), record ESI voltage value for mass spectrometer methods (ion collection ESI voltage): Baseline Fluctuation (%) = [(highest value - average value)/average value] x 100.
IMPORTANT If baseline fluctuation is higher than 40%, repeat sprayer position optimization procedure until a good baseline is achieved. If you are having problems establishing a good baseline, then refer to the Fine Tuning Sprayer Tip Position for SCIEX Mass Spectrometer in APPENDIX G. For additional help determining baseline fluctuation, refer to the Neutral OptiMS Cartridge Instruction Guide.
Figure 8.32 Determining the Baseline Fluctuation
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Running CESI-MS
This section provides a tutorial on the use of the CESI 8000 Plus System with the SCIEX TripleTOF® 5600 mass spectrometer. The tutorial uses a tryptic digestion of beta-galactoside as the sample. The beta-galactosidase peptide digest is a standard mixture commonly used to evaluate mass spectrometer performance and it will be used here to evaluate the performance of the capillary separation as well.
The workflow used to run the CESI 8000 Plus System in tandem with the TripleTOF® 5600 mass spectrometer will require setting up a sequence using the Analyst® TF Software followed by setting up a sequence on the CESI 8000 Plus System to run. This sequence of events is very important because the mass spectrometer must be set up first, prior to the CESI 8000 Plus System sending the start signal so the mass spectrometer can start data acquisition.
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IMPORTANT Prior to attempting to run the beta-galactosidase sample, make sure the procedures for establishing a stable spray (Establishing a Stable Spray and Determining Optimum ESI Voltage) have been followed and successfully completed.
Create a Sequence in the Analyst® TF Software
This section provides step by step instructions on how to build a sequence of 10 runs of beta- galactosidase performance test on the TripleTOF® 5600 mass spectrometer with auto-calibration every 5 runs in tandem with the CESI 8000 Plus System.
1 In the navigation bar, under Acquire, double-click Build Acquisition batch.
2 In the Batch Editor window, click Add Set.
3 A new set called SET1 is added. Click Add Samples.
4 A new Add Sample dialog opens. Under the Sample name, type BetaGal for prefix and 3 for Number of digits in sample number. A Data file name and Sub Folder are given and the number of samples to be run is set to 10 (Figure 8.33).
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Figure 8.33 Add Sample Dialog
5 Click OK to close the Add Sample dialog and the Batch Editor opens again with all the information entered. The Vial Position field must be set to 1 for all runs (Figure 8.34). Under the Acquisition field, browse to the applicable method using the Method Editor. In this example, select CESI_MS_AB_Sciex_Betagal_45min. This method was created earlier in this chapter (Methods for the CESI 8000 Plus System).
Figure 8.34 Batch Editor Window — Sample Tab
6 Click File > Save as to save this acquisition batch.
7 After saving the batch, in the Batch Editor, click the Calibrate tab and check the Auto Calibration box. The Reference Table used for auto-calibration is the B-Gal CE-MS Calibration Ref. Select BgalAutocal Installation as the mass spectrometer method to be used for auto calibration. The auto calibration can be set to perform once every 5 sample runs (Figure 8.35).
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Figure 8.35 Batch Editor Window — Calibrate Tab
8 Click View to ensure that the correct reference table is selected. There are 8 reference ions used for mass spectrometer calibration and the peptide at 729.3 m/z is selected for MS/MS calibration. The Retention time for all the reference ions is 2.5 minutes, since unlike an LC experiment, in the CE infusion mode, all the reference ions are present at the same time. The Retention time tolerance is set to +/- 30 sec. Reference tables are shown (Figure 8.36).
Figure 8.36 Reference Tables
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Start a Sequence in the Analyst® TF Software
1 Click Submit to submit the batch. This will submit the 10 beta-galactosidase separation runs with auto-calibration (Figure 8.37).
Figure 8.37 Batch Editor Window — Submit Tab
2 To preview the queue, go to View > Sample Queue. The Queue Manager window opens and shows that there are 12 runs (10 beta-galactosidase separations and two auto-calibrations) in the queue. The queue server shows the queue on Standby with an hourglass symbol in column 1 (Figure 8.38).
Figure 8.38 Queue Manager Window
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3 To start the sequence batch, make sure the mass spectrometer acquisition computer is in “Standby” mode before clicking Ready on the toolbar.
NOTE If the mass spectrometer acquisition computer is already in the Ready state, change it to Standby and then reset it to Ready.
At this point the TripleTOF® 5600 mass spectrometer is waiting for the trigger signal from the CESI 8000 Software to start data acquisition.
IMPORTANT To put the mass spectrometer acquisition computer in Standby mode, from the Main Header menu, click Acquire > Standby.
Load Sample and Reagents on the CESI 8000 Plus System
Sample Tray Setup The sample tray setup is described in this section. It is important to follow the specific location for each sample as they match the CESI 8000 Plus methods described in this chapter. The beta- galactosidase sample used for the auto-calibration and the beta-galactosidase sample used for system testing are located in vial positions A1 and A2, respectively (Figure 8.39). Samples to be analyzed can be placed in other positions; however, these positions must be specified in the CESI 8000 Plus methods or manually specified in the sequence table.
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IMPORTANT Samples are likely to evaporate over time. It is recommended to use a minimum of 50 μL of sample for a sequence with a duration of approximately 24 hours. If the duration of the sequence is over 50 hours, a minimum volume of 80 μL is strongly recommended.
Figure 8.39 Sample Trays
1 Refer to the procedure for preparation of required background electrolyte solutions and sample (Preparation of 10% (v/v) HAc—Background Electrolyte (BGE)) section for procedures on how to reconstitute and prepare the beta-galactosidase test sample.
2 Dispense the beta-galactosidase test sample in a micro vial and cap the vial using a CESI-MS cap. Place the beta-galactosidase sample for auto-calibration in position A1 of the sample inlet tray and the beta-galactosidase sample for the system performance test in sample tray position A2.
3 Open the CESI 8000 Software and click the icon to open the 32 Karat™ Software. In the 32 Karat™ Software, click the Direct Control icon (Figure 8.40) and click Load (Figure 6.8). Wait for the carriers to come forward, open the front cover, and place both the buffer inlet and outlet trays on their respective carriers and the sample inlet tray in the back of inlet carrier (left).
Figure 8.40 Direct Control Icon on the Toolbar
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Create a Sequence in the CESI 8000 Software
1 In the CESI 8000 Software, go to Menu > Sequence > Sequence Wizard.
2 In the Sequence Wizard — Method page, click the yellow folder icon in Method to browse to the CESI Plus method for the separation of beta-galactosidase (Figure 8.8), as shown in Figure 8.41.
Figure 8.41 Sequence Wizard — Method Page
3 Click Next.
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4 Fill in the fields for the Sample ID, Data path, and Data file. By clicking on the blue arrow next to the Data file field, a series of options come up to increment the file name. In this example, the Increment Number is chosen as the increment feature to be appended to the Data file name (Figure 8.42).
Figure 8.42 Sequence Wizard — Unknowns Page
5 Under the Number of unknown runs in sequence field, type the number of beta-galactosidase runs set to run on the mass spectrometer. This number excludes the number of auto- calibration runs.
6 Click Next.
7 In the First unknown vials of sequence dialog, click the Trays button. A dialog showing a 6x8 position tray opens. Click position A2 and clear the check box for Advance. The outlet vial position defaults to BO:A1 (buffer outlet position A1) which is the correct position for the sequence.
NOTE When running more than one sample, the check box for Inlet vial advance must be checked.
8 Click Finish. The sequence table will be shown (Figure 8.43).
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Figure 8.43 Sequence Table
IMPORTANT The autocal mix is always on sample inlet tray position A1, and in this example the sample is on position A2. Ensure that the sample position is designated properly in the sample table before submitting the sequence to run.
9 Both the CESI 8000 Software and the mass spectrometer sequences must match in order to run the sample and auto-calibration runs. To edit the sequence table in the CESI 8000 Software: highlight the first run, right-click the highlighted run, and click Insert Line (Figure 8.44).
Figure 8.44 Editing the Sequence Table
10 Go to the Method field and browse to the CESI-MS Auto-calibration_AB Sciex.met calibration method by clicking on the green side button. Type the Sample ID and File name.
11 Add another line for auto-calibration by highlighting the run on line number 7 and right- clicking it. Click Insert line and a line will be added to the sequence. Repeat steps 9 and 10. The final sequence will open as shown in Figure 8.45.
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Figure 8.45 Sample Sequence Example
NOTE Auto-calibration data file names must be different.
12 In the toolbar, go to Sequence > Properties. In the Sequence Properties dialog (Figure 8.46) ensure that for the File paths, the data is saved in the desired folder. A folder can also be created.
Figure 8.46 Sequence Properties Dialog
13 Go to File > Sequence > Save As to save the sequence. In this example, the sequence name is BGal_Installation.
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Start a Sequence in the CESI 8000 Software
1 To run the sequence, click the Run Sequence icon and the Run Sequence dialog will open (Figure 8.47). Click Start to initiate the sequence.
Figure 8.47 Run Sequence Dialog
2 The status of the first auto-calibration run can be checked by monitoring the Bgal_Installation sequence (Figure 8.48). The first auto-calibration run shows that it is being acquired.
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Figure 8.48 Monitoring the Sequence Run
3 Once the CE method starts, the status in the mass spectrometer Queue Server will change from Ready to PreRun.
NOTE The moment the trigger signal is sent from the CESI 8000 Software to the TripleTOF® 5600 mass spectrometer, the Queue Server status will change from PreRun to Acquiring.
4 As the batch run progresses, for each run that is acquired successfully, a green check mark will open and the run status will change to Acquired.
NOTE If setting up a method from scratch, make sure the duration of the Relay On step is at least 0.05 minutes.
IMPORTANT For the beta-galactosidase system performance test, do not use a separation voltage above 20 kV.
Automatic Rinse Feature The CESI 8000 Plus System makes use of an “Automatic Rinse” feature. In the event of a hardware failure during the rinse steps as well as the injection step, either during a method run or sequence run, the instrument will (without user intervention) rinse the separation capillary for 3 minutes at 50 psi from the first separation event vial position.
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Beta-Galactosidase Analysis
As an example, from all 150 peptide fragments of beta-galactosidase detected, the 729 and 714 m/z ions are used to evaluate the system. Table 8.5 shows the mass assignment for the two fragments of interest*.
Table 8.5 Beta-Galactosidase 729 and 714 m/z Mass Assignment
m/z Peptide Sequence Monoisotopic Molecular Weight (Da) Charge State 714 DWENPGVTQLNR 1427.6793 2 729 APLDNDIGVSEATR 1456.7158 2
1 Go to peak view and open the beta-galactosidase file with the “.wiff” extension.
2 A typical total ion chromatogram (TIC) for a beta-galactosidase run of a beta-galactosidase system performance test separation (in 100 mM LE separated using 10% HAc as background electrolyte), is shown in Figure 8.49.
* Ref. LC/MS Peptide/Protein Mass Standards Kit for Tuning of the AB SCIEX ESI Instrument Protocol.
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Figure 8.49 Total Ion Chromatogram (TIC)
3 Create the extracted ion chromatogram (XIC) for peaks 729.3652 m/z and 714.8469, both with a width of 0.05.
IMPORTANT The peak range 729.3652 m/z and 714.8469 m/z may need to be adjusted based on the mass spectrometer mass calibration.
4 Efficient system performance can be gauged by the identification and clear separation of the beta-galactosidase 714.8469 m/z and 729.3652 m/z peaks. Calculate the difference in the migration time between the two peaks. This difference must be greater than 0.05 minutes.
Figure 8.50 shows both peaks of interest for XIC and mass spectra with 729.3652 m/z and 714.8469 m/z for the beta-galactosidase tryptic digest. There is a migration time difference of 0.2 minutes.
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Figure 8.50 Extracted Ion Chromatogram (XIC) and Mass Spectra for a Beta-Galactosidase Run
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Introduction
This chapter includes procedures for the OptiMS Silica Surface Cartridge, from capillary conditioning and instrument tuning, to obtaining a separation of a test mixture for CESI-MS system suitability evaluation.
The user should be familiar with the procedures described in CHAPTER 5, Integrating the CESI 8000 Plus System with a Mass Spectrometer.
This chapter is divided into the following sections:
• Stock Reagents and Other Consumables Required • Preparation of Reagent Solutions and Test Sample • Procedure for Installation of Adapter and OptiMS Cartridge • Capillary Conditioning • Establishing a Stable Spray and Determining Optimum ESI Voltage • Tuning • Running CESI-MS The workflow is as follows:
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Before you start, ensure you have all the required reagents properly prepared. Description and preparation protocols can be found in the next section.
Stock Reagents and Other Consumables Required
Table 9.1 Stock Reagents
Stock Reagent Vendor Part Number Acetic Acid, Glacial Sigma-Aldrich A6283 or equivalent 0.1 N Sodium Hydroxide (NaOH) SCIEX 338424 0.1 N Hydrochloric Acid (HCl) EMD Chemical HX0603A 1 N Sodium Hydroxide (NaOH) Sigma-Fluka 319511 Methanol (MeOH) Fisher Chemical A454 7.5 M Ammonium Acetate (AmAc) Sigma-Aldrich A2706 Beta-Galactosidase Digest SCIEX 4333606 DDI water N/A 18 megaohm or higher
Table 9.2 Materials
Consumables Vendor Part Number OptiMS silica surface cartridge SCIEX B07367 20 mL glass vial VWR VW74511-20 Micro vials SCIEX 144709 CESI-MS vial SCIEX B11648 Green CESI-MS vial cap SCIEX B24699 0.5 mL centrifuge tube 50 mL volumetric flask 20 mL glass bottle 100 mL glass bottle pH meter Table top mini centrifuge Assorted pipettes and tips
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Preparation of Reagent Solutions and Test Sample
Table 9.3 Required Reagents
Reagents Concentration Use 10% Acetic Acid (v/v) 10% HAc (v/v) Background electrolyte and conductive liquid 20% Acetic Acid (v/v) 20% HAc (v/v) pH adjustment of Leading Electrolyte (LE) 200 mM ionic strength in 200 mM Leading Electrolyte (LE) Buffer Sample and system suitability preparation ammonium, pH 4.0 1 μM Beta-galactosidase digest (beta- 1 μM System stock solution galactosidase performance test)
Preparation of 10% (v/v) Acetic Acid (HAc)—Background Electrolyte (BGE)
NOTE This solution must be prepared fresh every day.
IMPORTANT When filling the CESI-MS vials, care should be taken not to introduce air bubbles. Check for trapped air bubbles on the wall of vials, and replace vials as needed.
1 Add 18.0 mL of DDI water into a clean 20 mL glass vial.
2 In a fume hood, add 2.0 mL of HAc into the DDI water and mix it well. Mix the contents by inverting the vial 3 times.
3 Sonicate the vial contents for 30 minutes.
4 Store at room temperature.
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Preparation of 20% (v/v) Acetic Acid (HAc) — For LE Buffer pH Adjustment
NOTE Prepare fresh and discard after use. Do not stock this solution.
1 Add 80.0 mL of DDI water into a clean 100 mL glass bottle.
2 Inside a fume hood, add 20.0 mL of acetic acid into the DDI water and mix it well. Mix the contents by inverting the bottle 3 times and store at room temperature.
3 Record the preparation date.
Preparation of 200 mM LE (Leading Electrolyte) Buffer
1 Prepare 50 mL of 400 mM ammonium acetate (AmAc) solution: a. Add 20 mL of DDI water into a 50 mL clean glass volumetric flask. b. In a fume hood, add 2.7 mL of 7.5 M ammonium acetate into the same flask. c. Bring the volume to 50 mL with DDI water. d. Mix the solution by inverting the flask 3 times.
2 Pour the 50 mL of 400 mM AmAc solution into a clean 100 mL beaker.
3 Using a calibrated pH meter, measure and record the initial pH value of the solution.
4 Adjust the pH of the solution to 4.0 by adding aliquots of freshly prepared 20% HAc.
5 Transfer the solution into a clean 100 mL volumetric flask.
6 Bring the volume to the 100 mL mark with DDI water and mix the solution by inverting the flask 3 times.
7 Record the preparation date, and store at 2 °C to 8 °C when not in use.
NOTE This solution expires in 24 months when stored properly.
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Reconstitution of 1 μM Beta-Galactosidase Digest Solution (Beta-Galactosidase Stock Solution)
1 Add 625 μL of DDI water to beta-galactosidase vial and vortex the vial for 5 seconds.
2 Dispense 50 μL aliquot in a 0.5 mL centrifuge tube for storage.
3 Store at -35 °C to -15 °C (frozen) up to one year.
4 Record the preparation date.
Preparation of Beta-Galactosidase Test Sample
1 Prepare a 0.5 μM beta-galactosidase test sample by mixing 50 μL of 200 mM LE Buffer, pH 4.0, with 50 μL aliquot of 1 μM beta-galactosidase in a 0.5 mL centrifuge tube.
2 Mix the sample thoroughly.
3 Centrifuge the sample tube for 15 minutes at 13 000 rcf.
4 Use only the supernatant (90 μL) as the test sample.
5 Record the preparation date, and store at 2 °C to 8 °C for no more than 3 days before use.
Sample Tray Setup for System Performance Test The sample tray setup is described in this section. It is important to follow the specific location for each sample as they match the CESI 8000 Plus methods described in this chapter.
The beta-galactosidase sample used for the system performance test is located in vial positions A1, as shown Figure 9.1. Samples to be analyzed can be placed in other positions; however, these positions must be specified in the CESI 8000 Plus methods or manually specified in the test sequence.
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IMPORTANT Samples are likely to evaporate over time. It is recommended to use a minimum of 50 μL of sample for a sequence with a duration of approximately 24 hours. If the duration of the sequence is over 50 hours, a minimum volume of 80 μL is strongly recommended.
Figure 9.1 Sample Tray Setup
Procedure for Installation of Adapter and OptiMS Cartridge
For installation instructions for the adapter and the OptiMS cartridge, refer to CHAPTER 5, Integrating the CESI 8000 Plus System with a Mass Spectrometer within this manual.
Prior to inserting the OptiMS sprayer into the adapter, ensure the positioning stage is aligned as far away as possible from the mass spectrometer inlet.
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For additional information, refer to the OptiMS Cartridge Sprayer Tip Installation into a Thermo Scientific NanoSpray II Ion Source section in CHAPTER 5.
IMPORTANT Before proceeding to the next step it is very important the mass spectrometer is idle and the ESI voltage is set to zero.
Capillary Conditioning
Capillary conditioning is a procedure that must be performed when using an OptiMS Silica Surface Cartridge for the first time.
Capillary conditioning consists of 9 rinse steps at high pressure at various durations. This method performs forward rinses to condition the separation capillary using methanol (MeOH), DDI water, 0.1 N NaOH, 0.1 N HCl, and 10% acetic acid. Note the time duration for all rinses are 10 minutes at 100 psi. This method also performs reverse rinses of the conductive liquid capillary (CLC) with MeOH, DDI water, and 10% Acetic Acid for 3 minutes at 100 psi each.
Buffer Tray Setup The buffer tray setup is described in this section. It is important to follow the specific location for each reagent as they match the CESI 8000 Plus methods described in this chapter.
NOTE Use only CESI-MS vials and CESI-MS caps in the CESI 8000 Plus System buffer and sample trays.
Table 9.4 provides the vial content, quantity and volume needed for capillary conditioning, and system performance test methods.
Table 9.4 Solution/Reagent Requirements
Vial Content Quantity Volume BGE 4 vials 1.5 mL Methanol - Optima LC/MS grade 2 vials 1.5 mL
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Table 9.4 Solution/Reagent Requirements
Vial Content Quantity Volume DDI water 2 vials 1.5 mL 0.1 N NaOH 1 vial 1.5 mL 0.1 N HCl 1 vial 1.5 mL Methanol - LC/MS grade 1 Falcon tube 50 mL 10 mL DDI water 1 Falcon tube 50 mL 10 mL
Figure 9.1 shows the position of each vial reagent in both inlet and outlet trays. This layout is common to all five CESI 8000 Plus methods.
NOTE While dispensing BGE, avoid introducing air bubbles into the vial.
Figure 9.2 Vial Layout for Inlet and Outlet Trays
Capillary Conditioning Procedure
1 Place a Falcon tube containing 10 mL of methanol in the holster on the side of the instrument and rest the sprayer in the tube. The sprayer tip must be fully immersed in methanol (Figure 9.3).
Figure 9.3 Sprayer Immersed in 10 mL of Methanol
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2 Prepare inlet and outlet buffer trays as shown in Figure 9.1. Then set the trays aside.
3 Open the CESI 8000 Software.
4 Click the icon to open the 32 Karat™ Software.
5 In the 32 Karat™ Software, click the Direct Control icon on the toolbar (Figure 9.4).
Figure 9.4 Direct Control Icon on the Toolbar
NOTE If the Direct Control icon is not visible as shown in Figure 9.4, refer to Using the Direct Control Window to Load the Cartridge and Samples in CHAPTER 6: 32 Karat™ Software Overview for instructions on opening the Direct Control window and how to create a Direct Control icon on the toolbar.
6 Click Load.
7 Wait until the trays are in the Load positions, and then open the front cover and place the inlet tray in the inlet carrier (left) and the outlet tray in the outlet carrier (right).
8 Close the door.
9 In the 32 Karat™ Software, open the CESI-MS Conditioning method, located in the Method folder for the CE-MS project.
10 Start the CESI-MS Conditioning method using a Single run.
11 In the toolbar, click the blue arrow (Figure 9.5) to submit a Single Run acquisition.
Figure 9.5 Single Run Acquisition Icon on Toolbar
IMPORTANT After the last methanol rinse is completed (Method line 2 — approximately 13 minutes into the Conditioning method), replace the methanol Falcon tube (Figure 9.3) with a DDI water tube and immerse the sprayer into DDI water. Wait for CESI-MS Conditioning method to be completed. After completion of the method, remove sprayer from the DDI water tube and remove as much of the water as possible by gently padding it dry with dry paper wipes.
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12 Figure 9.6 shows the dialog for the single run. Under Run information field, confirm the method path and name are correct for capillary conditioning. It is not necessary to enter the sample ID nor data file name as no data file will be generated.
Figure 9.6 Single Run Acquisition Dialog
The CESI 8000 Software will start the series of rinses and at the completion of the capillary conditioning method, the OptiMS capillary cartridge is ready for the next step which is establishing a stable spray.
Establishing a Stable Spray and Determining Optimum ESI Voltage
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Prior to establishing a stable spray, it is important the sprayer tip position is optimized relative to the mass spectrometer inlet. For detailed information on how to adjust and align the sprayer on the Thermo Scientific mass spectrometer, refer to the Aligning the CESI 8000 Plus System with a Thermo Scientific Mass Spectrometer section within CHAPTER 5 of this manual.
Ensure that both separation and conductive liquid capillaries are filled with 10% acetic acid and the ESI voltage is set to zero on the mass spectrometer.
To fill the conductive capillary with background electrolyte (10% Acetic acid), follow these two easy steps:
1 Go to the Direct Control window, click Rinse, and set it to 100 psi for 2 minutes in the reverse direction (conductive liquid capillary) BO: A1, as illustrated in Figure 9.7.
Figure 9.7 Pressure Rinse Setting to Fill Conductive Liquid Capillary with 10% Acetic Acid
2 Click OK.
NOTE Always look at the rinse icon represented by a “blowing face” (Figure 9.8) to determine the direction where the pressure is being applied. The reverse rinse direction will always refer to rinsing the conductive liquid capillary and the “blowing face” should then be visible on the right- hand side of the dialog.
Figure 9.8 Direct Control Window Showing the Reverse Rinse to Fill the Conductive Liquid Capillary
NOTE The “blowing face” on the right-hand side of the dialog (pointed by arrow).
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As the conductive liquid capillary fills with background electrolyte, a droplet is observed at the end of the stainless steel needle. After the rinse step completes, proceed to the next step to fill the separation capillary.
To fill the separation capillary with background electrolyte (10% Acetic acid) follow these two easy steps:
1 Go to the Direct Control window. Click the box next to Pressure and use the settings as shown in Figure 9.9.
Figure 9.9 Pressure Rinse Setting for a Forward Rinse of the Background Electrolyte to Fill the Separation Capillary
2 Click OK. As the separation capillary fills, a droplet becomes visible at the end of the sprayer tip.
NOTE With an empty separation capillary, it may take up to 7 minutes for a droplet to become visible.
WARNING As droplets form at the end of the sprayer tip, ensure that are not aspirated into the mass spectrometer inlet. Do not position the sprayer tip closer than 2 mm from the mass spectrometer inlet.
3 With both separation and the conductive liquid capillaries properly filled with background electrolyte, go to the Direct Control window in the 32 Karat™ Software and apply 20 kV (normal polarity) for 30 minutes using a 1.0 min ramp, as illustrated in Figure 9.10.
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Figure 9.10 Voltage Settings
The CESI 8000 Software electrical current should be around 2 mA to 3 mA.
4 While the CESI 8000 Plus separation voltage is on, apply ESI voltage in increments of 100 V starting at 500 V.
5 Monitor the Total Ion Count in the mass spectrometer software until ionization is detected. This is your minimum ESI voltage. Figure 9.11 shows a typical TIC profile of a 10% HAc as a background electrolyte.
Figure 9.11 Typical Spray Profile Using 10% HAc as Background Electrolyte
6 Monitor the baseline and fine tune the position of the sprayer tip at the mass spectrometer inlet by slowly adjusting the knobs in the xyz positioning stage. The better the sprayer is aligned, the more stable the TIC will be.
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7 Once the OptiMS sprayer position has been optimized based on the stability of the TIC, the minimum ESI voltage is determined. Increase the ESI spray voltage by approximately 200 V. This is your optimal ESI voltage.
NOTE Ion spray voltage should not exceed 1800 volts.
A stable Total Ion Count baseline is indicative of a stable ion spray. Spray stability can be optimized by adjusting the sprayer tip position in relation to the mass spectrometer inlet and by adjusting the ion spray voltage.
IMPORTANT Fine tuning the position of the sprayer tip may become necessary. Refer to the Fine Tuning Sprayer Tip Position for Thermo Scientific Mass Spectrometer in APPENDIX G.
8 If stable spray cannot be achieved, refer to troubleshooting in APPENDIX G.
Tuning
IMPORTANT Ensure the mass spectrometer system is properly calibrated before using CESI-MS. As a general guideline, any known peptide can be used as a tuning mix. For metabolomic applications, the tuning should be generated by using a metabolite type compound. The tune mix buffer must be in 10% acetic acid.
It is necessary to create two tune files. These tune files differ only in ESI voltage. The first tune file is set with 0 kV spray voltage, and the second tune file is set with a spray voltage that has been found to produce a stable spray. See Establishing a Stable Spray and Determining Optimum ESI Voltage.
Both tune files will be used to run the beta-galactosidase performance test separation. The tune file with zero ESI voltage is used for 1 minute, which corresponds to the time period where the
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CE voltage is ramping to 20 kV. Once the separation voltage reaches the desired magnitude of 20 kV, the second tune file with the optimal spray voltage will be applied. This will ensure that the correct voltage is being applied during the separation of the sample.
NOTE It is the responsibility of the customer to calibrate the mass spectrometer. Refer to the manufacturer’s documentation for calibration procedures and recommended frequency.
Once the tune files have been successfully acquired and saved, proceed to the section Running CESI-MS for a description on running a beta-galactosidase digest as the system performance test sample.
Running CESI-MS
This section provides a tutorial on the use of the CESI 8000 Plus System with the Thermo Scientific mass spectrometer. The tutorial uses a tryptic digestion of beta-galactoside as the sample. The beta-galactosidase tryptic digest is a standard mixture commonly used to evaluate mass spectrometer performance, and it will be used here to evaluate the system performance.
The workflow used to run the CESI 8000 Plus System in tandem with the Thermo Scientific mass spectrometer will initially require setting up a method and sequence using the Xcalibur software, and then setting up a separation sequence on the CESI 8000 Software. This sequence of events is very important because the mass spectrometer must be set up first so that it is waiting for the CESI 8000 Plus System to send the start signal, which will cause the mass spectrometer to
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start data acquisition.
IMPORTANT Prior to attempting to run the beta-galactosidase sample, make sure the procedures for establishing a stable spray (refer to Establishing a Stable Spray and Determining Optimum ESI Voltage) and tuning (refer to Tuning) have been followed and successfully completed.
Create a Method with the Xcalibur Software
Perform the following procedure:
1 Create one method with two segments. The first segment with duration of 1 minute, will use the tune file with zero ESI voltage created during tuning of the mass spectrometer. The second segment for 44 minutes duration will use the tune file with the optimum ESI voltage for a stable spray. Figure 9.12 and Figure 9.13 illustrates one method with two segments. The tune method for the segment shown in Figure 9.12 is set with zero ESI voltage.
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Figure 9.12 First Segment of a Method Used to Run Beta-Galactosidase Digest Test Sample
The tune method for the segment shown in Figure 9.13 is set with optimized ESI voltage for a stable spray.
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Figure 9.13 Second Segment of a Method Used to Run Beta-Galactosidase Digest Test Sample
2 In the scan event settings, as illustrated in Figure 9.14, set the Data type to Profile and the Scan Ranges to 300 m/z to 1500 m/z.
Figure 9.14 Scan Event Settings
3 Save the method.
IMPORTANT It is recommended to set the temperature of the ion transfer capillary to 200 °C for the analysis of beta-galactosidase. This temperature may change depending on the sample.
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Create a Sequence with the Xcalibur Software
The newly created beta-galactosidase mass spectrometer method should be programmed into a sequence. The sequence can be queued to start. The mass spectrometer will not begin to run the method until it receives a relay start trigger from the CESI 8000 Software.
IMPORTANT Ensure that the system will go into standby mode after the sequence completes.
1 In the Xcalibur software, open the New Sequence Template window, shown in Figure 9.15, to create a sequence.
Figure 9.15 New Sequence Template
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2 Click OK, and the sequence table dialog will open as shown in Figure 9.16.
Start a Sequence with the Xcalibur Software
Figure 9.16 Sequence Setup-Home Page
1 Highlight row #1 corresponding to the sample to run, and then click the toolbar icon.
2 The run sequence dialog will open. Set the instrument to standby after the sequence has finished. Otherwise, the spray voltage will remain on. Click OK. The Acquisition Queue tab on the left pane will show the sequence currently running, and the sequence row being acquired.
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The Status tab, which is also on the left pane, will show the status of the sequence. As the sequence is downloaded and queued, the status will change from ready to downloading and finally to waiting for contact closure.
3 Next load the sample and reagents onto the CESI 8000 Plus System.
IMPORTANT Before starting a sequence on the CESI 8000 Plus System, ensure that the status on the Xcalibur software is waiting for contact closure.
Load Sample and Reagents on the CESI 8000 System
1 To perform this method, transfer the following solutions and volume into CESI-MS vials according to the table below.
Table 9.5 Solution/Reagent Requirements
Vial Content Quantity Volume BGE 3 vials 1.5 mL DDI water 1 vial 1.5 mL 0.1 N NaOH 1 vial 1.5 mL 0.1 N HCl 1 vial 1.5 mL
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2 Take the two buffer trays and place the vials with the reagents in the location as shown on Figure 9.17.
Figure 9.17 Buffer Trays Inlet and Outlet for Beta-Galactosidase Separation Method
3 Dispense the beta-galactosidase test sample into a micro vial, and place it inside of a CESI- MS vial.
4 Cap the vial using a CESI-MS cap.
5 Place the beta-galactosidase sample vial on position A1 of sample inlet tray.
6 Open the CESI 8000 Software and click the icon to open the 32 Karat™ Software.
7 In the 32 Karat™ Software, click Direct Control icon, and then click Load (Figure 9.18).
Figure 9.18 Direct Control Icon on the Toolbar
8 Wait for the carriers to come forward, and then open the front cover and place both the buffer inlet and outlet tray on the respective carriers.
9 Place the sample inlet tray in the back of inlet carrier (left).
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Create a Sequence in the CESI 8000 Software
In the CESI 8000 Plus System, the sequence to run beta-galactosidase will be performed using a method with initial conditions and time program as illustrated in Figure 9.19 and Figure 9.20, respectively.
Figure 9.19 Initial Conditions for CESI-MS Separation for Thermo Scientific Mass Spectrometer
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Figure 9.20 Time Program for CESI-MS Separation for Thermo Scientific Mass Spectrometer
NOTE If setting up a method from scratch, make sure the duration of the Relay On step is at least 0.05 minutes.
Figure 9.21 Voltage Ramp Down for Step 10 in Figure 9.20
To create a sequence, follow the procedure as described in CHAPTER 6, Programming a Sequence.
IMPORTANT For the beta-galactosidase system performance test, do not use a separation voltage above 20 kV.
Automatic Rinse Feature The CESI 8000 Plus System makes use of an “Automatic Rinse” feature. If there is a hardware failure during the rinse steps and also the injection step, either during a method run or sequence run, the instrument will (without user intervention) rinse the separation capillary for 3 minutes at 50 psi from the first separation event vial position.
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Start a Sequence in the CESI 8000 Software
Prior to starting a sequence, make sure that all reagents and sample required to run the experiment are in their appropriate place.
1 Go to the toolbar and click the Run Sequence Table icon as shown on Figure 9.22.
Figure 9.22 Run Sequence Table Icon
The Run Sequence dialog will open as illustrated in Figure 9.23.
Figure 9.23 Run Sequence Dialog
2 Ensure the name of the sequence created in the prior section is present in the sequence name field. If not, click the yellow folder icon to browse to the proper location.
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3 Click Start.
Beta-Galactosidase Analysis
This section provides an example of beta-galactosidase analysis. From all 150 peptide fragments of beta-galactosidase detected, the 729 and 714 m/z will be used to evaluate the system performance. Table 9.6 shows the mass assignment for the two fragments of interest.
Table 9.6 Beta-Galactosidase 729 and 714 m/z Mass Assignment
m/z Peptide Sequence Monoisotopic Molecular Weight (Da) Charge State 714 DWENPGVTQLNR 1427.6793 2 729 APLDNDIGVSEATR 1456.7158 2
1 Go to the Qual Browser software and click File > Open, and then browse to the location where the beta-galactosidase data file was saved.
2 Click Open. A TIC (Total Ion Chromatogram) opens. Figure 9.24 shows an example of a typical beta- galactosidase Total Ion Chromatogram.
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Figure 9.24 Example of a Typical Beta-Galactosidase Total Ion Chromatogram
3 Change the view from TIC (total ion chromatogram) to Base Peak Chromatogram.
4 Configure the Base Peak Chromatogram in order to view the following two peaks: 714 and 729.
5 Configure the Base Peak Chromatogram to the following settings: a. Peak range 714.5 to 714.7, 729.2 to 729.4. b. Set automatic processing to include smoothing with a smoothing value of five. Your Base Chromatogram should look similar to what is shown in Figure 9.25.
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IMPORTANT The peak range 714.5 to 714.7 and 729.2 to 729.4 may need to be adjusted based on the mass spectrometer mass calibration.
Figure 9.25 Identification and Clear Separation of the Beta-Galactosidase 714 and 729 m/z Fragments
Efficient system performance can be gauged by the identification and clear separation of the beta-galactosidase 714 and 729 peaks in the Base Peak Chromatogram.
6 Calculate the migration time of each peak and ensure that there is at least 0.05 minute separation time between the two peaks.
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Shutdown and Decoupling Overview
This section describes the shutdown procedures and decoupling of the following hardware and instruments within a CESI 8000 Plus System:
• Cartridge Removal and the CESI 8000 Plus System Shutdown • Decoupling the CESI 8000 Plus System • Decoupling from the Thermo Scientific Mass Spectrometer • Decoupling from the SCIEX Mass Spectrometer
Cartridge Removal and the CESI 8000 Plus System Shutdown
The OptiMS cartridge should be removed and properly stored anytime the CESI 8000 Plus System is shutdown and completely powered off.
Cartridge Removal and Instrument Shutdown To remove the OptiMS cartridge from the CESI 8000 Plus System, make sure that the MS ESI voltage is off and then perform the following steps:
1 If the cartridge is not going to be used for a short period of time (up to three days), then run the capillary cleaning method for short term storage. Refer to Capillary Cleaning Procedure (Short Term Storage) in the Hardware Maintenance section of APPENDIX F. If the cartridge is not going to be used for a longer period of time (three days or longer), then run the Capillary Storage Procedure. Refer to Capillary Storage Procedure in the Hardware Maintenance section of APPENDIX F.
2 Place the CESI 8000 Plus System into the Load position. To do this use, open the Direct Control window, and then enable the Load Command by clicking Load (Figure 10.1); or from a method dialog use the following path: Control menu > Direct Control > Load.
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Figure 10.1 Load Button on Direct Control Window
3 Open the cartridge cover (Figure 4.7). The CESI 8000 Plus coolant pump will start up and expel the coolant from the cartridge coolant lines (Figure 10.2). This will take about 30 seconds. Wait for the pump to turn off before removing the cartridge.
Figure 10.2 Coolant Lines on Cartridge
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4 Loosen the thumbscrews from the lock down bar (Figure 10.3).
Figure 10.3 Loosen Thumbscrews from the Lock Down Bar
5 Lift the lock down bar all the way up.
6 Retract the positioning stage as far away as possible from the mass spectrometer inlet. This is done so that the sprayer tip is not bumped against the mass spectrometer, which could damage the tip.
CAUTION Let the ion source cool for at least 30 minutes before removing the sprayer from the adapter. Surfaces of the ion source become hot during operation.
To retract the stage for a Thermo Scientific mass spectrometer, refer to Aligning the CESI 8000 Plus System with a Thermo Scientific Mass Spectrometer in CHAPTER 5. To retract the stage for a SCIEX mass spectrometer, refer to Aligning the CESI 8000 Plus System with the SCIEX TripleTOF® 5600 Mass Spectrometer in CHAPTER 5.
7 Loosen the sprayer end from the adapter by turning the arrow on the sprayer to the unlock position (Figure 10.4).
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Figure 10.4 Unlock Sprayer from Cartridge
1. Sprayer in Unlocked Position 2. Unlocking Sprayer from Adapter
8 Remove sprayer from adapter (Figure 10.5).
Figure 10.5 OptiMS Capillary Sprayer Removal
9 Insert the protective sleeve onto the OptiMS sprayer to protect it from breakage (Figure 10.6).
Figure 10.6 Placing OptiMS Plastic Sleeve on Sprayer Tip
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10 Pass the tubing with sprayer end through the access panel, and then remove coolant tubing from the notched arm (Figure 10.7 and Figure 10.8).
Figure 10.7 Putting Tubing/Sprayer through Access Panel
Figure 10.8 Removing Coolant Tubing from Notch
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11 While holding the cartridge sprayer with one hand, grab the OptiMS cartridge by its mid-section and remove by lifting cartridge up and pulling it outwards (Figure 10.9).
Figure 10.9 Removing the OptiMS Capillary Cartridge
NOTE A small amount of liquid coolant may drip from cartridge ends. That is normal and does not harm the hardware.
IMPORTANT As the cartridge is slid upwards, the sheaths on the inlet and outlet sides retract downwards and cover the capillary ends.
12 Store the OptiMS cartridge at room temperature.
Decoupling the CESI 8000 Plus System
If the mass spectrometer is to be used for a period of time without the CESI 8000 Plus System, or if one or both instruments are to be moved, then it is best to decouple the CESI 8000 Plus System from the mass spectrometer. The following decoupling instructions are provided:
• Decoupling from the Thermo Scientific Mass Spectrometer • Decoupling from the SCIEX Mass Spectrometer
Decoupling from the Thermo Scientific Mass Spectrometer
1 If you have not yet done so, prepare the OptiMS cartridge for storage and then remove the cartridge. If necessary, refer to the Cartridge Removal and Instrument Shutdown section earlier in this chapter.
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2 Shut down the CESI 8000 Plus System, and then unplug the power cord for the CESI 8000 Plus System and CESI 8000 Plus cart.
3 Decouple both the mass spectrometer adapter’s high-voltage cables from the output and input connectors in the CESI 8000 Plus high voltage connection panel (Figure 10.10).
Figure 10.10 High Voltage Connection Panel
1. High Voltage Panel Input Connection 2. High Voltage Panel Output Connection to from Mass Spectrometer Source (red OptiMS Adapter (black banana plug) banana plug)
4 Disconnect the black and red ground banana plugs from the CESI 8000 Plus panel.
5 Unlock the CESI 8000 Plus cart wheels by lifting the lever at each caster upwards with your foot (Figure 10.11). Lower the cart to its lowest setting. Then slowly move the CE instrument away from the mass spectrometer.
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IMPORTANT Always lower the CESI 8000 Plus cart to its lowest setting before moving the CESI 8000 Plus System.
Figure 10.11 Unlocking CESI 8000 Plus Mobile Cart Wheels
6 Use the thumbscrew on the Thermo Scientific mass spectrometer to loosen the adapter. Then remove the mass spectrometer adapter from the stage (Figure 10.12). Put the adapter in a safe place.
Figure 10.12 Loosen Thumbscrew to Remove Adapter
1. Thumbscrew 2. Adapter
7 Disconnect the relay cable from the mass spectrometer and keep it with the CESI 8000 Plus System (Figure 10.13).
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Figure 10.13 Remove Relay Cable
1. USB_GPIB Cable 2. Relay Cable
Decoupling from the SCIEX Mass Spectrometer
1 If you have not yet done so, prepare the OptiMS cartridge for storage and then remove the cartridge. If necessary, refer to the Cartridge Removal and Instrument Shutdown section earlier in this chapter.
2 Shut down the CESI 8000 Plus System, and then unplug the power cord for the CESI 8000 Plus System and CESI 8000 Plus cart.
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3 Decouple both mass spectrometer adapter’s high-voltage cables from the output and input connectors in the CESI 8000 Plus high voltage connection panel (Figure 10.14). Remove the black and red ground banana plugs as well.
Figure 10.14 High Voltage Connection Panel
1. High Voltage Panel Input Connection 2. High Voltage Panel Output from Mass Spectrometer Source (red Connection to OptiMS Adapter banana plug) (black banana plug)
4 Decouple both mass spectrometer adapter’s high-voltage cables from the output and input connectors in the CESI 8000 Plus high voltage connection panel (Figure 10.14). Remove the black and red ground banana plugs as well.
5 Disconnect the black and red ground banana plugs from the CESI 8000 Plus System panel.
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6 Unlock the CESI 8000 Plus cart wheels by lifting the lever at each caster upwards with your foot (Figure 10.15). Lower the cart to its lowest setting. Then slowly move the CE instrument away from the mass spectrometer.
Figure 10.15 Unlocking CESI 8000 Plus Mobile Cart Wheels
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7 Loosen the mass spectrometer adapter locking screw and then slide the adapter all the way off of the stage (Figure 10.16). Put the mass spectrometer adapter in a safe place.
Figure 10.16 Loosen Thumbscrew to Remove Adapter
1. Loosen Thumbscrew to Remove the SCIEX Adapter
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CESI 8000 Plus System
Table A.1 System Specifications
Item Description Dimensions (H x W x D) 99.1 cm (cover open), 73.7 cm (cover closed) × 63.5 cm × 72.4 cm (39 inches (cover open), 29 inches (cover closed) × 25 inches × 28.5 inches) Weight 85.3 kg (188 lbs) Electrical Power requirement: 100 VAC to 240 VAC, 5.0 A, 50 Hz or 60 Hz Power consumption: supply voltage must not exceed 10% of nominal Fuses (depending on supply voltage in use): 8.0 A slow blow; 1/4 inch (2 ea.): 100 VAC to 120 VAC 6.3 A time delay; 20 mm (2 ea.): 200 VAC to 240 VAC Installation (overvoltage) category: Category II Working Environment Altitude: up to 2000 m (6562 feet) Humidity: <80% (non-condensing) at 15 °C to 30 °C Humidity: <60% (non-condensing) at 30 °C to 40 °C Temperature: 15 °C to 40 °C (15 °C to 30° C recommended) Maximum Heat Dissipation 400 W (1024 BTU/hour) Pollution Degree 2 I/O TTL: 2 Contact closures: 2
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Validated Controller Configuration
The system includes a computer and a monitor (also referred to as a “controller”). The software has been validated on a controller with the following specifications:
• Lenovo M720s workstation • 8th Generation Intel Core i7-8700 3.2 GHz processor • 16 GB RAM • 22 inch wide-screen monitor with True Color and 1680 x 1050 resolution • Windows 10 Enterprise LTSB 2016 (Windows 10 IoT) with Cybersecurity • Operating system language set to English (United States) • 500 GB hard drive • DVD-RW drive • 2 serial ports • 2 Ethernet ports • 8 USB ports
NOTE SCIEX fully validates and supports the controllers supplied with the system. Only limited support is available for customer-supplied computers.
NOTE Specifications are subject to change without notice.
Cart Specifications
Table A.2 Cart Specifications
Item Description Dimensions (W × D) 91.4 cm × 73.7 cm (36 inches × 29 inches) Height Adjustable from 68.6 cm to 111.8 cm (27 inches to 44 inches) Weight 69.0 kg (152 lbs) Electrical 120 VAC at 60 Hz, optional 230 VAC at 50 Hz Supports 136.0 kg (300 lbs)
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Sample Temperature Control
Table A.3 Sample Temperature Control Specifications
Specification Type Description Temperature Range 20 °C below ambient to 60 °C (140 °F) Minimum setting: 4 °C (39 °F) Temperature Stability ±1 °C at 25 °C (77 °F) ±3 °C at 4 °C (37 °F) and 60 °C (140 °F) Temperature Accuracy ±2 °C within a range of ±15 °C from ambient temperature ±3 °C outside a range of ±15 °C from ambient temperature
Capillary Temperature Control
Table A.4 Capillary Temperature Control Specifications
Specification Type Description Temperature Range 10 °C below ambient to 60 °C (140 °F) Minimum setting: 15 °C (59 °F) Temperature Stability ±1 °C at 25 °C (77 °F) Temperature Accuracy ±1 °C within a range of ±1 °C from ambient ±2 °C outside a range of ±5 °C from ambient
Pressure and Vacuum System
Table A.5 Pressure and Vacuum System Specifications
Specification Type Description Pressure Range Injection: 0.1 psig to 25 psig (pressure) or 0.1 psig to 5.0 psig (vacuum) Rinse: 0.1 psig to 100 psig (pressure) or 0.1 psig to 5.0 psig (vacuum) Pressure Stability ±0.3 psi at 25 psi ±1.0 psi at 100 psi Pressure Direction Applied at inlet or outlet for all pressure functions, rinses, and injections User-settable in software
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Detector Specifications
UV Detector
Table A.6 UV Detector Specifications
Specification Type Description Wavelength Range 190 nm to 600 nm Wavelength Accuracy ±2 nm UV Source 30 W pre-aligned deuterium lamp Filter Selection 214 nm (standard) with 7 open positions for additional filters Filter dimensions must be: Diameter: ½ inch (127 mm) Thickness: 0.20 inch (5 mm) Analog Output Output 1 is data Full scale output is 1.0 AU/V. Multipliers of 1.0, 0.5, 0.2, 0.05, 0.02, and 0.01 to provide lower AU/V values can be set in the software. Output 2 not used Output 3 depends on what is programmed: Current signal when voltage is programmed Voltage signal when current or power is programmed
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Laser Induced Fluorescence (LIF) Detector (Optional)
Table A.7 LIF Detector Specifications
Specification Type Description Wavelength Range (for Excitation: 300 nm to 700 nm optics) Emission: 350 nm to 750 nm Solid State Laser Laser output delivered to the capillary: 2.5 ±0.5 mW Laser wavelength 488 nm nominal Sensitivity Minimum signal/peak-peak noise ratio of 10,000:1 for 50 nM sodium fluorescein in a 75 μm i.d. capillary Relative Fluorescence Units 0 RFU to 1000 RFU Range Filters (optional) For 488 nm laser: 488 notch filter and 520 nm band-pass filter For user-supplied lasers, two filters are required: a filter to block stray laser light and an emission filter to select the wavelength of the emitted light. Filter dimensions must be: • Outer diameter: 0.500 inch (+0.000 inch, –0.010 inch); 12.7 mm (+0.000 mm, –0.25 mm) • Thickness: ≤ 0.350 inches (0.889 mm) • For multiple filters used in a single channel, total thickness: ≤ 0.350 inches (0.889 mm) Dynamic Range > 104 Baseline Noise < 0.005 RFU peak to peak Baseline Drift < 0.2 RFU/hour Analog Outputs Output 2 is Data Channel 2 Full scale output is 1.0 AU/V. Multipliers of 1.0, 0.5, 0.2, 0.05, 0.02, and 0.01 to provide lower AU/V values can be set in the software. Output 3 depends on what is programmed: Output 1 is Data Channel 1 Current signal when voltage is programmed Voltage signal when current or power is programmed
Photo Diode Array (PDA) Detector (Optional)
Table A.8 PDA Detector Specifications
Specification Type Description Wavelength Range 190 nm to 600 nm Wavelength Accuracy 2 nm UV Source Lifetime 1000 hours UV Source 30 W pre-aligned deuterium lamp Scan Collection Frequency 0.5 Hz to 32 Hz Detector 256 element diode array Bandwith 6 nm minimum (absorbance averaging)
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Table A.8 PDA Detector Specifications (Continued)
Specification Type Description Analog Output Output 1 is Data Channel 1 Output 2 is Data Channel 2 Full scale output is 1.0 AU/V. Multipliers of 1.0, 0.5, 0.2, 0.05, 0.02, and 0.01 to provide lower AU/V values can be set in the software. Output 3 depends on what is programmed: Current signal when voltage is programmed Voltage signal when current or power is programmed
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Overview
This appendix describes the nominal network configuration for connecting a CESI 8000 Plus controller (PC) to or from a SCIEX TripleTOF® 5600 mass spectrometer. In the nominal configuration, there are three network connections available on the SCIEX TripleTOF® 5600 mass spectrometer acquisition computer. One port can be used for another system (such as a SCIEX LC system), and one port can be dedicated to a CESI 8000 Plus System, and the third port is connected to the Internet.
In the CESI 8000 Plus controller (PC), at least one network connection is available. One CESI 8000 Plus System network port should be used for connection to the mass spectrometer acquisition computer, and this connection will not change. That is, once connected, the network cable should never be removed from the CESI 8000 Plus controller. There are four different reconfiguration procedures discussed in this appendix:
• Switching from the CESI 8000 Plus System to Different Mass Spectrometer Systems • Switching to the CESI 8000 Plus System from Different Mass Spectrometer Systems • Switching from a SCIEX Mass Spectrometer to Another Mass Spectrometer • Switching to a SCIEX Mass Spectrometer from Another Mass Spectrometer
A more advanced networking and computer configuration for the Analyst® TF Software is also possible. Refer to “Advanced Networking and Computer Configuration for the Analyst® TF Software 1.7”.
If a reconfiguration is done on the CESI 8000 Plus System, then the corresponding procedure must be done for the SCIEX mass spectrometer in the 32 Karat™ Software. For instance, if “Switching from the CESI 8000 Plus System to Different Mass Spectrometer Systems” is done, then the corresponding procedure “Switching to a SCIEX Mass Spectrometer from Another Mass Spectrometer” must also be done.
IMPORTANT If your system does not match the nominal configuration described in this appendix, then it might not be possible to apply these instructions to your network configuration.
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Switching from the CESI 8000 Plus System to Different Mass Spectrometer Systems
When switching the mass spectrometer interface from the CESI 8000 Plus System to some other system, such as a SCIEX LC system, closely note the network connection on the mass spectrometer acquisition computer (PC) used by the CESI 8000 Plus System. When reconnecting back to the CESI 8000 Plus System, you will want to reconnect to exactly the same network connector on the mass spectrometer acquisition computer that was used before switching.
NOTE If the mass spectrometer acquisition computer is using nominal configuration, then it is not necessary to disconnect the CESI 8000 Plus System.
Switching to the CESI 8000 Plus System from Different Mass Spectrometer Systems
When switching the mass spectrometer back to the CESI 8000 Plus System from some other system, such as a SCIEX LC system, reconnect the CESI 8000 Plus System to the same network connection from which it was originally switched. If the crossover network cable was disconnected, then also connect it as it was originally connected.
Verify that the network settings for the CESI 8000 Plus System connection by performing the following steps:
NOTE These are the network settings on the mass spectrometer acquisition computer for the port/ adapter connected to the CESI 8000 Plus System.
1 The required settings were recorded at installation in a text file called MSNetworkSettings.txt. Click C:\Program Files\BCI CE-MS Driver\MSNetworkSettings.txt, open it and write down the following settings: • IP address • Subnet mask
2 Navigate to the network configuration window (Start > Settings > Network Connections) and select the appropriate Local Area Connection for the CESI 8000 Plus System (or CE-MS).
3 Right-click the selected Local Area Connection and select Properties. A window titled Internet Protocol (TCP/IP) Properties similar to that shown in Figure B.1 opens.
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Figure B.1 Example Internet Protocol (TCP/IP) Properties Window
4 The settings shown in your Internet Protocol (TCP/IP) Properties window should match the MSNetworkSettings.txt text file. If not, correct the settings in the Internet Protocol (TCP/IP) Properties window and restart the mass spectrometer acquisition computer (PC).
5 If a restart was necessary, verify the settings in the Internet Protocol (TCP/IP) Properties window again after the controller has restarted.
Switching from a SCIEX Mass Spectrometer to Another Mass Spectrometer
1 Close the 32 Karat™ Software on the CESI 8000 Plus controller.
2 Disconnect the crossover network cable connecting the CESI 8000 Plus System to the mass spectrometer acquisition computer.
3 Connect the relay/trigger cable from the CESI 8000 Plus System to the target mass spectrometer.
4 On the CESI 8000 Plus controller, click Start > CESI 8000 Software folder and then right-click Change MS Type (Figure B.2).
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Figure B.2 Change MS Type within CESI 8000 Plus Software
The Change MS Type dialog will open (Figure B.3).
Figure B.3 Change MS Type Dialog
5 Clear the check box: Connect to AB SCIEX MS (Figure B.4).
Figure B.4 Change MS Type Dialog
6 Click Change.
The CESI 8000 Plus System and the 32 Karat™ Software are now properly configured for a non- SCIEX mass spectrometer.
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Switching to a SCIEX Mass Spectrometer from Another Mass Spectrometer
1 Close the 32 Karat™ Software on the CESI 8000 Plus controller.
2 Disconnect the relay/trigger cable from the CESI 8000 Plus System.
3 Reconnect the crossover network cable from the CESI 8000 Plus System to the SCIEX mass spectrometer acquisition computer.
4 Verify the correct network settings by performing the steps in the Switching to the CESI 8000 Plus System from Different Mass Spectrometer Systems section.
5 On the CESI 8000 Plus controller, click Start > CESI 8000 Software Folder and then right-click Change MS Type (Figure B.5).
Figure B.5 Change MS Type within CESI 8000 Plus Software
The Change MS Type dialog will open (Figure B.6).
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Figure B.6 Change MS Type Dialog
6 Click the Connect to AB SCIEX MS check box (Figure B.7).
Figure B.7 Change MS Type Dialog
7 Verify that the IP address shown is correct. If not, close the dialog and perform the steps in the Switching to the CESI 8000 Plus System from Different Mass Spectrometer Systems section above within this appendix.
8 Click Change.
The CESI 8000 Plus System and the 32 Karat™ Software are now properly configured for a SCIEX mass spectrometer.
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Advanced Networking and Computer Configuration for the Analyst® TF Software 1.7
CE-MS Networking
1 Connect the crossover Ethernet cable from the CESI 8000 Plus controller to the mass spectrometer acquisition computer (Figure B.8).
Figure B.8 Connection for the CESI 8000 Plus System to the Mass Spectrometer Acquisition Computer Ethernet
1. Ethernet Cable Installed on the CESI 8000 Plus Controller 2. Ethernet Cable Installed on the SCIEX Mass Spectrometer Acquisition Computer
The “standard” installation/configuration is to connect the CESI 8000 Plus controller to the mass spectrometer acquisition computer via a Crossover Network cable. The following section of this document refers to this configuration.
PC Configurations This procedure describes the nominal network configuration for connecting a CESI 8000 Plus controller (PC) to a SCIEX TripleTOF® 5600 mass spectrometer. In the nominal configuration, there are three network connections available on the SCIEX TripleTOF® 5600 mass spectrometer computer. One port should be dedicated to another system (such as a SCIEX LC system), and one port should be dedicated to CESI 8000 Plus System. The third port connection is dedicated to the Internet. In the CESI 8000 Plus controller (PC), there are one (or more) network connections available. One CESI 8000 Plus network port to be used for connection to the mass spectrometer
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acquisition computer, and this connection will not change. That is, once connected, the network cable need never be removed from the CESI 8000 Plus controller. • The mass spectrometer acquisition computer has three network ports. • The CESI 8000 Plus controller has two available network ports.
Configuring Network Settings on the Mass Spectrometer Acquisition Computer 1. If the TripleTOF® 5600 mass spectrometer is using Analyst® TF Software 1.7 on a Windows 7 acquisition computer, then refer to the “Configuring Network Settings on the Analyst 1.7 Windows 7 Mass Spectrometer Acquisition Computer” section. If the TripleTOF® 5600 mass spectrometer is not using Analyst® TF Software on a Windows 7 controller, then continue with step 2 below. 2. Click Start > Network Connections (Figure B.9).
Figure B.9 Network Connections
3. Right-click an available network connection and rename it Internet (Figure B.10).
NOTE To ensure that you are configuring the correct network port, unplug the Ethernet cable from the back of the computer and watch the connection status change in the window from Connected to Network cable unplugged.
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Figure B.10 Available Network Connection
4. Right-click the newly renamed network connection (Internet), and select Properties.
IMPORTANT The nominal three ports system will configure Local Area Connection for the “built-in” network port. The two “add-on” network ports on will be configured as Local Area Connection 2 and Local Area Connection 3. 5. Highlight the Internet Protocol (TCP/IP) and click Properties (Figure B.11).
Figure B.11 Internet Properties
IMPORTANT Leave the Internet Protocol TCP/IP Properties as shown (default) for normal Internet connection. Different IT environment can have different settings. Work with the local IT group to define those settings.
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6. Select Advanced (Figure B.12).
Figure B.12 Internet Protocol (TCP/IP) Properties
7. Clear the Automatic metric check box and enter 1 in Interface metric (Figure B.13).
Figure B.13 Advanced TCP/IP Settings
8. Close all windows and go back to the Network Connections page.
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9. Right-click the second available network connection (usually Local Area Connection 2) and rename it CESI 8000 (Figure B.14).
Figure B.14 Local Area Connection 2
10. Right-click the renamed network connection (CESI 8000) and select Properties (Figure B.15).
Figure B.15 CESI 8000 Plus Network Connection Properties
11. Highlight the Internet Protocol (TCP/IP) and click Properties (Figure B.16).
Figure B.16 CESI 8000 Properties Dialog
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12. Click Use the following IP address. 13. Enter the following settings (Figure B.17): • IP address: 192.168.1.1 • Subnet mask: 255.255.255.0 • Default gateway: Leave it blank. • Preferred DNS server: Do not change; leave it blank.
Figure B.17 Internet Protocol (TCP/IP) Properties
14. Select Advanced.
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15. Clear the Automatic metric check box and enter 2 in Interface metric (Figure B.18).
Figure B.18 Advanced TCP/IP Settings
16. Click OK and exit to the Network Connections window. 17. Right-click the CESI 8000 network connection again and select Properties (Figure B.19).
Figure B.19 Network Connections
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18. Click Configure (just to the right of Connect using; Figure B.20).
Figure B.20 CESI 8000 Properties Dialog
19. Select the Advanced tab and highlight Link Speed & Duplex property. 20. Change Value from Auto Negotiation to 100Mbps/Full Duplex (Figure B.21).
Figure B.21 Link Speed and Duplex Settings
21. Click OK and exit all windows.
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Setting Mass Spectrometer Acquisition Computer Firewall Parameters 1. Click Start > Control Panel (Figure B.22).
Figure B.22 Control Panel
2. Switch the Windows view to Classic View, if not already set (Figure B.23).
Figure B.23 Selecting Classic View
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3. Double-click Windows Firewall (Figure B.24).
Figure B.24 Windows Firewall
4. Click the Advanced tab, and clear all the enabled firewall check boxes (Figure B.25).
Figure B.25 Windows Firewall Settings
5. Exit out of all network settings. 6. Restart the mass spectrometer acquisition computer. 7. After the restart, navigate back to Network Connections and verify that all TCP/IP configurations set in this procedure are still set. 8. Following the steps above, modify the network interface metric to 3 for Network Connection 3 (or rename to other device interface name).
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Configuring Network Settings on the CESI 8000 Plus Controller 1. Navigate to Start > Control Panel > Network and Sharing Center (Figure B.26).
Figure B.26 Network and Sharing Center
NOTE There will be more than one connection on the above window if the system has more than one network port installed. Locate an available network connection to set up the CESI 8000 Plus connection.
2. Click Change advanced sharing settings in the left pane (Figure B.27).
Figure B.27 Change Advanced Sharing Settings
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3. Select Turn on network discovery and Turn off file and printer sharing (Figure B.28).
Figure B.28 Update Advanced Sharing Settings
4. Click Change adapter settings in the left pane (Figure B.29).
Figure B.29 Change Adapter Settings
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5. Right-click Ethernet and select Properties (Figure B.30).
Figure B.30 Ethernet Properties
6. Highlight Internet Protocol Version 4 (TCP/IPv4) and click Properties (Figure B.31).
Figure B.31 Internet Protocol Version 4 (TCP/IPv4) Properties
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7. Click Use the following IP address (Figure B.32).
Figure B.32 Internet Protocol Version 4 (TCP/IPv4) Properties Dialog
8. Enter the following IP settings (Figure B.33): • IP address: 192.168.1.2 • Subnet mask: 255.255.255.0 • Default gateway: Do not change; leave it blank. • Preferred DNS server: Do not change; leave it blank.
Figure B.33 DNS Server Address
9. Click Advanced.
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10. Clear the Automatic metric check box and enter 2 in Interface metric (Figure B.34).
Figure B.34 Advanced TCP/IP Settings Dialog
NOTE Follow the above method to change the existing Internet network connection Interface metric to 1 (for multiple network connections configurations only).
11. Click OK and exit the network connection windows. 12. Right-click Ethernet and select Properties. 13. Click Configure, which is located just below and to the right of Connect using (Figure B.35):
Figure B.35 Configuring the Network
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14. Select the Advanced tab, and then select the Speed & Duplex property. Set the value to 100 Mbps Full Duplex from the Value menu (Figure B.36).
Figure B.36 Value Menu
15. Exit all network configuration windows.
16. Turn off Windows Firewall in Customize Settings. Navigate to Start > Control Panel > All Panel Control Items > Windows Firewall > Customize Settings (Figure B.37). Select Turn off Windows Firewall in both Private and Public network settings. Click OK.
Figure B.37 Firewall Settings
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Configuring Network Settings on the Analyst 1.7 Windows 7 Mass Spectrometer Acquisition Computer 1. Navigate to Start > Control Panel > User Accounts > Change User Account Control settings (Figure B.38).
Figure B.38 User Account Control Settings Window
NOTE You may need to restart the system for the change to take effect.
2. Navigate to Start > Control Panel > Network and Sharing Center (Figure B.39).
Figure B.39 Network and Sharing Center
NOTE There will be more than one connection at the above window if the system has more than one network port installed. Locate an available network connection to set up the CESI 8000 Plus connection.
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3. Click Change adapter settings in the left pane (Figure B.40).
Figure B.40 Change Adapter Settings
4. Right-click Local Area Connection and select Rename. 5. Type in CESI 8000 as the new name. 6. Right-click Local Area Connection and select Properties (Figure B.41).
Figure B.41 Local Area Connection Properties
7. Highlight Internet Protocol Version 4 (TCP/IPv4) and click Properties (Figure B.42).
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Figure B.42 CESI 8000 (Local Area Connection) Properties Dialog
8. Click Use the following IP address (Figure B.43).
Figure B.43 Internet Protocol Version 4 (TCP/IPv4) Properties
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9. Enter the following IP settings (Figure B.43): • IP address: 192.168.1.1 • Subnet mask: 255.255.255.0 • Default gateway: Do not change; leave it blank. • Preferred DNS server: Do not change; leave it blank.
10. Click Advanced. 11. Clear the Automatic metric check box and enter 2 in Interface metric (Figure B.44).
Figure B.44 Advanced TCP/IP Settings
NOTE Follow the above method to change the existing Internet network connection Interface metric to 1 (for multiple network connections configurations only).
12. Click OK and exit the network connection windows. 13. Right-click CESI 8000 and select Properties. 14. Click Configure, which is located just below and to the right of Connect using (Figure B.45).
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Figure B.45 Configuring the Network
15. Select the Advanced tab, and then select the Link Speed & Duplex property. Set the value to 100 Mbps Full Duplex from the Value menu (Figure B.46).
Figure B.46 Value List
16. Exit all network configuration windows. 17. Perform the steps in the “Network Sharing Setting for the Crossover Connection” section.
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Network Sharing Setting for the Crossover Connection
1 Navigate to the Network and Sharing Center and select Choose homegroup and sharing options (Figure B.47).
Figure B.47 Network and Sharing Center
2 Select Change advanced sharing settings (Figure B.48).
Figure B.48 Homegroup Options
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3 Under Network discovery, select Turn on network discovery, and under File and printer sharing, select Turn on file and printer sharing (Figure B.49). Leave everything else as the default selection.
Figure B.49 Advanced Sharing Settings
4 Select Save changes to save all changes (Figure B.50).
Figure B.50 Saving Changes
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5 Restart the computer.
6 After the restart, navigate back to Network Connections and verify the TCP/IP configurations set in the steps above are still set.
Setting the Mass Spectrometer Type
1 From the Start menu, select All Programs > CESI 8000 Software > Change MS Type.
Figure B.51 Changing the MS Type
2 Select the Connect to AB SCIEX MS check box, and verify/set the IP address to: 192.168.1.1.
3 Select Change (Figure B.52).
Figure B.52 Changing the MS Type
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Curtain Gas Patch Installation
NOTE Before installing the patch, open the hardware manager and deactivate all profiles. Make sure the Analyst® TF Software is closed before installing the patch.
1 Locate the patch download file on the CESI 8000 Plus Sharepoint site.
2 Unzip the file onto the desktop.
3 Double-click the .msi file to launch the patch (Figure B.53).
Figure B.53 MSI File
4 Follow the installation wizard for the patch by clicking Next on the wizard shown in Figure B.54, Figure B.55, and Figure B.56, and then clicking Finish in the wizard shown in Figure B.57.
Figure B.54 Welcome Wizard
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Figure B.55 License Agreement
Figure B.56 Program Installation
Figure B.57 Completion Wizard
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CE-MS Driver Installation
1 Load the BCI interface driver tool.
2 Double-click CESI-MS AOO Driver.exe file (Figure B.58).
Figure B.58 BCI Interface Driver Tool
3 Click on Setup. Follow the wizard for driver installation by selecting Next/OK in the wizard shown in Figure B.59, Figure B.60, waiting for the wizard shown in Figure B.62 to complete, and then selecting Finish in Figure B.64.
Figure B.59 BCI CE-MS Driver — InstallShield Wizard
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Figure B.60 Install Folder
Figure B.61 BCI CE-MS Driver Setup
Figure B.62 Completion Wizard
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Saving Network Configurations
NOTE The CE-MS driver must be installed on the mass spectrometer acquisition computer prior to saving the network configuration file.
Prior to setting up the hardware profile, it might be beneficial to save the networking parameters for future reference. Once saved, these text files can be used for network troubleshooting and will serve as a reminder in the event that the customer changes the settings inadvertently.
To save a network configuration profile:
1 Browse the computer for the NetworkConfig.exe file.
2 Double-click the file to run the executable. The program generates a network configuration summary file in the following locations: • CESI Computer: — File name: CENetworkSettings.txt — File location: C:\32Karat\Debugging\Diagnostics
• MS Computer: — File name: MSNetworkSettings.txt — File location: C:\Program Files\BCI CE-MS Driver
NOTE Refer to Figure B.63 for an example of a network configuration file.
Figure B.63 Network Configuration File — Example
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Analyst® TF Software Hardware Profile Configuration
A hardware profile in the Analyst® TF Software is very similar to an instrument configuration in the 32 Karat™ Software. In the 32 Karat™ Software, you can build an instrument configuration that uses certain tray positions, detectors, and algorithms. Similarly, in the Analyst® TF Software, a hardware profile that specifically recognizes the CE instrument needs to be built. A CE-MS hardware profile has the following 3 parts:
1. TripleTOF® 5600 mass spectrometer 2. Agilent Autosampler 3. Software Application (Curtain Gas Patch)
To configure the Mass Spectrometer Hardware Profile
1 Open the Hardware Configuration editor in the Analyst® TF Software (Figure B.64).
Figure B.64 Hardware Configuration Editor
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2 Create a new profile by selecting New Profile (Figure B.65).
Figure B.65 Creating a New Profile
3 Name the profile by entering a name, such as “CESI 8000 Plus profile”, into the Profile Name field (Figure B.66), and then selecting OK.
Figure B.66 Naming the Profile
4 Select Add Device.
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5 Select Mass Spectrometer TripleTOF 5600 (Figure B.67).
Figure B.67 Available Devices Dialog
6 Click OK.
7 Click Add Device again.
8 Select Software Application from the Device Type menu (Figure B.68).
Figure B.68 Device Type Menu in the Available Devices Dialog
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9 Select Add Device again.
10 Add the Autosampler Agilent from the Device Type menu (Figure B.69).
Figure B.69 Device Type Menu in the Available Devices Dialog
11 Set up the Mass Spec device: a. From the Create New Hardware Profile window, click the mass spectrometer and select Setup Device (Figure B.70).
Figure B.70 Added Devices
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b. Click the Communication tab, and make sure the defaults are selected (Figure B.71), and press OK.
Figure B.71 Mass Spectrometer Device Setup
12 From the Create New Hardware Profile window, select the Software Application, and then click Setup Device. The Software Application Settings window opens (Figure B.72).
Figure B.72 Software Application Settings
13 Make sure the BCI CE Driver is selected, and click OK.
14 From the Create New Hardware Profile window, click the Autosampler. a. Select Setup Device.
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b. Select the Communication tab > Advanced. Then, check the Simulation mode check box (Figure B.73).
Figure B.73 Agilent Autosampler Window
15 Close all settings windows and navigate back to the Create New Hardware Profile dialog.
16 Verify that the final hardware configuration profile opens (Figure B.74).
Figure B.74 Final Hardware Configuration Profile
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17 Activate the profile in the Hardware Configuration Editor by clicking Activate Profile. Once the profile is active, a green check mark opens next to it (Figure B.75).
Figure B.75 Hardware Configuration Editor Dialog
18 Restart the mass spectrometer acquisition computer. Once the profile has been activated, the right corner of the Analyst window should open as shown in Figure B.76.
NOTE If the icons are not yellow, deactivate the Hardware profile and re-activate it.
Figure B.76 Status Icons in Analyst® TF Software Window
Curtain Gas Setting and Verification
Curtain Gas Setting
1 In the mass spectrometer acquisition computer, click Start > All Programs > BCI CE-MS Driver > Adjust Curtain Gas (Figure B.77).
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Figure B.77 Adjust Curtain Gas
2 Enter the following password to access the Curtain Gas Adjustment dialog: beckmanMMDDYY (Figure B.78).
Figure B.78 Enter Password Dialog
3 Set the curtain gas to 5 in the Adjust Curtain Gas Settings window and click OK (Figure B.79).
Figure B.79 Adjust Curtain Gas Setting Window
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4 Adjust the curtain gas value by entering the read-back value into the Current Curtain Gas Reading field and clicking on Suggest Set Value (Figure B.79). Refer to Curtain Gas Verification for how to obtain the read-back value.
5 Enter the suggested value into the Set curtain gas to field.
6 Repeat the process until the read back value (see Curtain Gas Verification) is between 4 to 4.5.
Curtain Gas Verification To verify and adjust the curtain gas:
1 Power on the CESI 8000 Plus System.
2 In the Analyst® TF Software, double-click Manual Tuning on the left side of the window (Figure B.80).
Figure B.80 Manual Tuning
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3 At the bottom of the window, click the icon to the left of Run (Figure B.81).
Figure B.81 Status Icons in Analyst® TF Software Window
NOTE If the icons at the bottom of the window are yellow, deactivate and reactivate the Hardware profile.
4 Verify that the value in the Curtain Gas (psi) field is approximately 4 to 4.5 (Figure B.82).
NOTE The Curtain Gas CUR field cannot be changed while the CESI 8000 Plus hardware profile is active.
Figure B.82 Checking the Curtain Gas Readback Value
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NOTE As long as the user is running the CESI 8000 Plus hardware profile, the instrument pulls its curtain gas value from the curtain gas patch, rather than the SW input. Therefore, the Curtain Gas(psi) field in the Analyst® TF Software will not reflect the curtain gas setting of 5, nor can it be changed to 5 while the CESI 8000 Plus hardware profile is active (Figure B.83).
Figure B.83 Source/Gas Tab
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Overview
This appendix outlines the methods for manual and automatic calibration of the SCIEX TripleTOF® 5600 mass spectrometer while performing CE-MS runs. When performing CESI-MS runs, the mass spectrometer can be calibrated periodically by infusing a calibrant solution through the OptiMS capillary and using it to automatically calibrate the mass spectrometer. This ensures that the deviation in the mass accuracy is minimal in the sample runs that follow the calibration.
NOTE Although the frequency in which the calibration is performed is at the user’s discretion, it is strongly recommended an auto-calibration to be performed every 5 hours of data acquisition.
Required Reagents
Table C.1 Stock Reagents
Chemical Vendor Part Number Acetic Acid, glacial Sigma-Aldrich A6283 or equivalent 0.1 N Sodium Hydroxide (NaOH) SCIEX 338424 0.1 N Hydrochloric Acid (HCl) SCIEX 391646 Methanol (MeOH) Fisher Chemical A454 7.5 M Ammonium Acetate (AmAc) Sigma-Aldrich A2706 Beta-galactosidase digest SCIEX 4465938
Reagent and Sample Preparation
Refer to CHAPTER 8: Using a SCIEX TripleTOF® 5600 Mass Spectrometer with the CESI 8000 Plus System for the preparation of reagents and sample.
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Manual Calibration Before Auto-calibration It is important to manually calibrate the instrument before setting up for auto-calibration. This ensures that the calibration coefficients a and t0 are at good values to begin with. The auto- calibration will not be successful if the reference ions are outside of the 100 ppm tolerance for peak finding. In such situations, manual calibration has to be performed to get the mass accuracies within 100 ppm. Both manual and auto-calibration require a reference table containing precursor and product ion values. Create the reference table before proceeding as described below.
Create a Reference Table for CE-MS Calibration
1 The reference table used for both manual and auto-calibration is B-Gal CE-MS Calibration Ref. To create the reference file, from the navigation bar, click Acquire and go to Tools > Settings > Tuning Options (Figure C.1). The Tuning Options dialog will open (Figure C.2).
Figure C.1 Create Reference File
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2 Click Reference (Figure C.2) to open the Reference Table Editor dialog for the reference table that is currently being used (in this case, PPGs Pos. Calibration Ref. is currently loaded in the instrument; Figure C.3).
Figure C.2 Tuning Options Dialog
1. Reference Button
Figure C.3 Reference Table Editor Dialog
3 Click New to open a new empty reference table (Figure C.4).
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Figure C.4 Empty Reference Table in the Reference Table Editor Dialog
4 For Name, type B-Gal CE-MS Calibration Ref and enter the values in all the fields as shown in Figure C.5. Ensure that the radio button for Positive (“Positive Ion” mode) is checked and the Retention time tolerance +/- is set to 30 000 seconds.
Figure C.5 Reference Tables
5 Ensure that the peptide at m/z 729.36250 is selected for MS/MS, and the Retention Time Tolerance has been set to +/- 30 seconds.
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6 Click OK to open the Tuning Options window containing the newly created reference table (Figure C.6).
Figure C.6 Tuning Options Dialog
7 Ensure that the B-Gal CE-MS Calibration Ref is selected as the Positive Reference. Click OK.
Manual Calibration of the Mass Spectrometer The following instructions are for manually calibrating the mass spectrometer by infusing 0.5 μM beta-galactosidase digest solution in 5% HAc (see Preparation of Beta-Galactosidase for Auto-calibration) through the OptiMS capillary.
1 Dispense 1.5 mL of 10% HAc in two CESI-MS vials, and place them in position A1 on both inlet and outlet trays.
2 Dispense 90 μL of 0.5 μM beta-galactosidase digest supernatant solution in 5% HAc in a sample vial and place in vial in the sample tray at the A1 (SI:A1) position.
3 In the Direct Control window, click Inject to open the Inject Parameters dialog. Select the injection parameters (Figure C.7).
NOTE Ensure the For Capillary Fill check box is checked in order to inject at 100 psi for 180 seconds.
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Figure C.7 Direct Control Window and Inject Parameters Dialog
4 After the capillary is filled with the 0.5 μM beta-galactosidase digest solution, apply 20 kV for 20 minutes by clicking Voltage. Ensure that the capillary inlet is in the BI:A1 position and the outlet is in the BO:A1 position (Figure C.8).
Figure C.8 Adjust Settings in the Inject Parameters Dialog
5 For manual calibration of the mass spectrometer, the beta-galactosidase digest solution has been infused from the OptiMS capillary and the CE voltage has been applied.
Manual Calibration in TOF Mass Spectrometer Mode In this section, the mass spectrometer parameters will be set up.
1 In the navigation bar, under Tune and Calibrate, double-click Manual Tuning.
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2 In the Source/Gas tab, set GS1 and GS2 to 0, Curtain Gas (CUR) to 5, and Interface Heater Temperature to 50.
3 In the right pane, under the MS tab, for the Scan type, select TOF MS.
4 Set the Accumulation time to 0.999 (secs). For TOF Masses (Da), set Min to 350, and Max to 1000.
5 Under Period, set the Duration to 1 minute.
6 Figure C.9 shows the MS tab in the Tune Method Editor dialog after setting up all the parameters.
Figure C.9 Tune Method Editor Dialog: Source/Gas Tab
7 In the Advanced MS tab, ensure that the MCA check box is clear (Figure C.10).
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Figure C.10 Tune Method Editor Dialog — Advanced MS Tab
8 Set the ISVF (spray voltage) and adjust the sprayer position until the spray is stable.
9 Click Start, a TIC window will be acquired in approximately 1 minute. The TIC and Mass Spectrum windows are shown in Figure C.11.
Figure C.11 TIC and Mass Spectrum Windows
10 Right-click the TIC window and click Open File as shown in Figure C.12.
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Figure C.12 Opening a File from the TIC Window Pane
11 The TIC and MS of the data file that was acquired will open as shown in Figure C.13.
Figure C.13 TIC and MS Spectrum Windows from the Acquired Data File
12 Click the MS pane to highlight it, and delete it by clicking on the Delete icon shown highlighted in red (Figure C.14).
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Figure C.14 Deleting MS Data Pane
13 After deleting the MS pane, the TIC will look as shown in Figure C.15.
Figure C.15 TIC Pane (Only)
14 Click the TIC pane to select, double-click it to obtain an “average mass spectrum” (Figure C.16).
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Figure C.16 Highlighted TIC Panel
15 Right-click the mass spectrum and click Re-calibrate TOF, a TOF Calibration dialog will open. In this dialog, from the Reference Table list, select reference file B-Gal CE-MS Calibration Ref. Set the Tolerance to 0.2 Da (Figure C.17).
NOTE If the reference file B-Gal CE-MS Calibration Ref is not found, then refer to Create a Reference Table for CE-MS Calibration for the procedure on how to create this file.
Figure C.17 TOF Calibration Dialog with Reference File Selected in TOC Calibration Window
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16 Click the Calculate new calibrations to calculate the Average error for this new calibration. Check the average error to make sure it is within the routine operating standards for the mass spectrometer instrument being calibrated.
17 Click Calibrate spectrum (Figure C.18). New calibration values will open. Ensure the check box for Set as Instrument Default is checked.
18 In the SAVE CURRENT CALIBRATION area, click the Entire file icon to apply this calibration to all samples in the file. Click Save.
Figure C.18 TOC Calibration Toolbar
Figure C.19 TOC Calibration Dialog with Saved Calibration Spectrum Values
NOTE If an ion is not found during calibration, highlight the missing ion in the table and delete it. Re- calculate new calibration values.
19 The calibration is now saved and a dialog opens, which shows where the recalibrated data is saved. Click OK.
20 Next, the Only TOF MS scans, positive polarity, high resolution were recalibrated dialog will open. Click OK.
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21 Click Close to close the TOF calibration window. The recalibrated mass spectrum opens. Close the TIC and Mass Spectrum windows to return to the Tune window. The TOF MS calibration is complete.
Manual Calibration in Product Ion Mode
1 In the right pane, under the MS tab, set Scan type to Product Ion, set Product Of to 729.3, and set TOF Masses (Da) to 100 for Min and 1500 for Max. In the left pane, under the Compound tab, set Collision Energy to 48 and Collision Energy Spread to 5. Ensure that High Sensitivity mode is selected. Figure C.20 shows the Tune window after setting up all the parameters.
Figure C.20 Tune Window
2 From the top right corner, click Start to acquire the spectrum (Figure C.20). The TIC and Mass Spectrum windows generated during the Product Ion calibration are shown in the Tune window (Figure C.21).
Figure C.21 TIC and Mass Spectrum Windows
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3 Right-click the TIC pane and click Open File (Figure C.22).
Figure C.22 Opening a File from the TIC Window
4 This will open both the TIC and MS of the Product Ion calibration data file that was acquired. The spectrum displayed is not the average MS and cannot be used for calibration. Highlight this pane and click the Delete icon (shown by arrow pointer in Figure C.23).
Figure C.23 Deleting the TIC Pane
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5 To open the average MS, click and drag to highlight the TIC pane. Double-click it to obtain an average mass spectrum. Right-click the mass spectrum and click Re-calibrate TOF (Figure C.24).
Figure C.24 Re-Calibrate TOF
6 This opens up the TOF Calibration dialog for the Product Ion mode calibration. Select the reference table B-Gal CE-MS Calibration Ref, which is the same as the one used before for TOF mass spectrometer calibration. Set the Tolerance to 0.2 Da. The Product ion masses will open in the table (Figure C.25).
Figure C.25 TOF Calibration Window
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7 Click the Calculate new calibrations to calculate the Average Error for this new calibration. Then click Calibrate spectrum. Ensure that Set As Instrument Default box is checked. Click the Entire File icon to apply this calibration to all samples in the file. Click Save.
8 The calibration is now saved and a dialog opens which shows location of the recalibrated data. Click OK.
9 Next, another dialog that reads Only TOF MSMS scans, positive polarity, high resolution were recalibrated will open. Click OK.
10 Close the TOF Calibration window by clicking Close. The recalibrated spectrum will open. Close the TIC and Mass Spectrum windows to return to the Tune window.
11 The CESI instrument has been manually calibrated in the TOF MS and Product Ion modes.
Methods for Auto-Calibration
CE Method for Auto-Calibration The CE auto-calibration method is used to infuse the beta-galactosidase in 5% HAc (sample kept in S1:A1 position) for automatic calibration of the MS during CE-MS batches. Figure C.26 shows the Time Program of the CE auto-calibration method. The settings in the Initial conditions tab are the same as the “Beta-gal separation” method. This method is saved as CESI-MS Auto- calibration_ABSciex.
NOTE This method uses BGE vials that are to be placed in the A4 position of both the inlet and the outlet buffer trays.
Figure C.26 Time Program
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MS Method for Auto-Calibration The MS method is used for performing auto-calibration. In order for auto-calibration to be performed, the Agilent 1100 Autosampler has to be configured in the hardware profile to be present as an available device (Figure C.27). The different tabs available in this method are shown.
Figure C.27 Acquisition Method and Configuration of the Agilent 1100 Autosampler
1. Acquisition Method Pane 2. Agilent Autosampler Available
Figure C.28 TOF MS (+) — MS Tab and Edit Parameters
1. TOF MS (+) 2. Edit Parameters
1 In the Acquisition Method pane, click TOF MS (+).
2 In the MS tab, click Edit Parameters (Figure C.28). This will open a new window (Figure C.29) containing 2 tabs: Source/Gas and Compound.
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3 Under the Source/Gas tab, the ISVF (determined in step 8 of Manual Calibration in TOF Mass Spectrometer Mode) must be entered, and the values for all the other parameters set up as shown in the steps below.
Figure C.29 TOF MS (+) — Source/Gas Tab and ISVF
1. Ion Spray Voltage Floating (ISVF)
Perform the following steps:
1 After setting the parameters in the Source/Gas tab, click the Compound tab in the same window to set up the parameters (Figure C.30).
Figure C.30 TOF MS (+) — Compound Tab
2 In the Acquisition Method pane, click Product Ion (+) 729.4. In the MS tab, click Edit Parameters to set up the parameters under Source/Gas and Compound for the MS/MS experiment (Figure C.31).
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Figure C.31 Product Ion (+) 729.4 — MS Tab and Parameters Settings Dialog
1. Product Ion (+) 729.4
Figure C.32 MS Tab and Parameters Settings Dialog
Figure C.33 Acquisition Method and the Beckman CE Driver
1. Beckman CE Driver
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Figure C.34 Acquisition Method and the Agilent 1100 Autosampler
1. Agilent 1100 Autosampler
Executing Auto-Calibration
The following is the step-by-step instructions for setting up the CE-MS batch with 3 beta- galactosidase separations along with auto-calibration. The auto-calibration run is performed while executing this batch.
Create a Sequence in the Analyst® TF Software
This section provides step-by-step instructions on how to build a sequence of 10 runs of beta- galactosidase performance test on theTripleTOF® 5600 mass spectrometer with auto-calibration every 5 runs in tandem using the CESI 8000 Plus System.
1 In the navigation bar, under Acquire, double-click Build Acquisition batch.
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2 In the Batch Editor window, click Add Set.
3 A new set called SET1 has been added. Click Add Samples. A new Add Sample dialog opens.
4 For Prefix, type BetaGal and for Number of digits, type 3. A Data file name and Sub Folder are given and the number of samples to be run is set to 10 (Figure C.35).
Figure C.35 Add Sample Dialog
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5 Click OK to close the Add Sample dialog and the Batch Editor re-opens with all the information shown. The Vial Position field must be set to 1 for all runs (Figure C.36). Under Acquisition, browse to the applicable method. In this example choose CESI_MS_AB_Sciex_Beta-Gal_45min. Refer to CHAPTER 8: Methods for the CESI 8000 Plus System for steps to create this method.
Figure C.36 Batch Editor Window and Sample Tab
6 Click File > Save as to save this acquisition batch.
7 After saving the batch, in the Batch Editor, click the Calibration tab and check the Auto Calibration check box. The Reference Table used for auto-calibration is the B-Gal CE-MS Calibration Ref. Select the Bgal_Autocal Installation as the MS method for auto-calibration. The auto-calibration can be set to perform once every 5 sample runs (Figure C.37).
Figure C.37 Calibrate Tab
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8 Click View to ensure that the correct reference table is selected. There are 8 reference ions used for MS calibration and the peptide at 729.3 m/z is selected for MS/MS calibration. The Retention time for all the reference ions is 2.5 minutes, since unlike an LC experiment, in the “CE infusion” mode, all the reference ions are present at the same time. The Retention time tolerance is set to +/- 30 seconds. Reference tables are shown in Figure C.38.
Figure C.38 Reference Tables
Start a Sequence in the Analyst® TF Software
1 Click Submit on the right to submit the batch. This will submit the 10 beta-galactosidase separation runs with auto-calibration (Figure C.39).
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Figure C.39 Submit Batch Runs using Auto-Calibration
2 To preview the queue, go to View > Sample Queue. The Queue Manager window opens and shows that there are 12 runs (10 beta-galactosidase separations and two auto-calibration) in the queue. The queue server shows the queue as Standby with an hourglass symbol (Figure C.40).
Figure C.40 Queue Manager Window
3 To start the sequence batch, click Ready on the toolbar. At this point the TripleTOF® 5600 mass spectrometer is waiting for the trigger signal from the CESI 8000 Software to start data acquisition.
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Load Sample and Reagents on the CESI 8000 System
Sample Tray Setup It is important for each sample to be placed in the specific location shown in Figure C.41 as they match the CESI 8000 Plus methods described in this chapter. The beta-galactosidase sample used for the auto-calibration and the beta-galactosidase sample used for system testing are located in vial positions A1 and A2 respectively. Samples to be analyzed can be placed in other positions; however, these positions must be specified in the CESI 8000 Plus methods or manually specified in the sequence table.
IMPORTANT Samples are likely to evaporate over time. It is recommended to use a minimum of 50 μL of sample for a sequence with a duration of approximately 24 hours. If the duration of the sequence is over 50 hours, a minimum volume of 80 μL is strongly recommended.
Figure C.41 Sample Trays
1 Refer to the procedure for preparation of required background electrolyte solutions and sample in CHAPTER 8. Preparation of 10% (v/v) HAc—Background Electrolyte (BGE) and for procedures on how to reconstitute and prepare the beta-galactosidase test sample.
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2 Dispense the beta-galactosidase test sample in a micro vial and cap the vial using a CESI-MS cap. Place the beta-galactosidase sample for auto-calibration on position A1 of the sample inlet tray and the beta-galactosidase sample for system performance test on the sample tray position A2.
3 Open the CESI 8000 Software and click the 32 Karat™ Software icon. In the 32 Karat™ Software, click the Direct Control icon (Figure C.42) and click Load (Figure 6.8). Wait for the carriers to come forward, open the front cover, and place both the buffer inlet and outlet tray on the respective carriers and the sample inlet tray in the back of inlet carrier (left).
Figure C.42 Direct Control Icon on the Toolbar
Create a Sequence in the CESI 8000 Software
1 In the CESI 8000 Software, click Menu > Sequence > Sequence Wizard.
2 In the Method field, click the yellow folder to browse to the CESI method for the separation of beta-galactosidase (Figure 8.8), as shown in Figure C.43.
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Figure C.43 Sequence Wizard — Method Page
3 Click Next.
4 Fill in the fields for Sample ID, Data path, and Data file. By clicking on the blue arrow next to the Data file field, a series of options come up to increment the file name. In this example, the increment number is chosen as the increment feature to be appended to the Data file name (Figure C.44).
Figure C.44 Setting up the Sequence Wizard — Unknowns Page
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5 In the Number of unknown runs in the sequence field, type the number of beta-galactosidase runs to run on the mass spectrometer. This number excludes the number of auto-calibration runs.
6 Click Next.
7 In the First unknown vials of sequence dialog, click Trays. A window showing a 6x8 position tray should open. Click on position A2 and clear the check box for Advance. The outlet vial position defaults to BO:A1 (buffer outlet position A1), which is the correct position for the sequence.
NOTE When running more than one sample, the check box for the Inlet vial advance, must be cleared.
8 Click Finish. The Sequence table will open as shown in Figure C.45.
Figure C.45 Sequence Table
IMPORTANT The autocal sample is always on sample inlet tray position A1, and in this example the sample is on position A2. Ensure that the sample position is designated properly in the sample table before submitting the sequence to run.
9 Both the CESI 8000 Software and the mass spectrometer sequences must match in order to run the sample and auto-calibration runs. To edit the sequence table in the CESI 8000 Software: highlight the first run, right-click the highlighted run, and click Insert Line (Figure C.46).
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Figure C.46 Editing the Sequence Table
10 Go to the Method field and browse to CESI-MS Auto-calibration_AB Sciex.met as the calibration method by clicking the green side button. Type the Sample ID and File name.
11 Add another line for auto-calibration by highlighting the run on line number 7 and right- clicking it. Click Insert line and a line will be added to the sequence. Repeat steps 9 and 10. The final sequence will open as shown in Figure C.47.
Figure C.47 Sample Sequence Example
NOTE Auto-calibration data file names must be different.
12 In the toolbar, click Sequence > Properties. In the Sequence Properties dialog (Figure C.48), ensure that under File paths, the data is being saved on a desired folder. A folder can also be created.
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Figure C.48 Sequence Properties Dialog
13 Go to File > Sequence > Save As to save the sequence. In this example, the sequence name is BGal_Installation.
Start a Sequence in the CESI 8000 Software
1 To run this sequence, click the Run Sequence icon and the Run Sequence dialog opens. Click Start to initiate the sequence.
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Figure C.49 Run Sequence Dialog
2 The status of the first auto-calibration run can be checked by monitoring the Bgal_Installation sequence (Figure C.50). The first auto-calibration run shows that it is being acquired.
Figure C.50 Monitoring the Sequence Run
3 Once the CE method starts, the status in the MS Queue Server will change from Ready to PreRun.
NOTE When the trigger signal is sent from the CESI 8000 Plus System to the TripleTOF® 5600 mass spectrometer, the Queue Server will change from PreRun to Acquiring.
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4 As the run progresses, for each run that is acquired successfully, a green check box will open and the run status will change to Acquired.
NOTE If setting up a method from scratch, make sure that the duration of the Relay On step is at least 0.05 minutes.
IMPORTANT For the beta-galactosidase system performance test, do not use a separation voltage above 20 kV.
Automatic Rinse Feature The CESI 8000 Plus System makes use of an “Automatic Rinse” feature. If there is a hardware failure during the rinse steps and also the injection step, either during a method run or sequence run, the instrument will (without user intervention) rinse the separation capillary for 3 minutes at 50 psi from the first separation event vial position.
Auto-Calibration Data Analysis
The following is the step-by-step instructions for analyzing the auto-calibration data.
1 To open the auto-calibration data, select Explore in the navigation bar.
2 Each auto-calibration performed is saved as a separate data file. The auto-calibration data file is saved in the Sub Folder Cal Data.
NOTE The auto-calibration data is saved if the Keep calibration data file check box in the Queue Options dialog was selected.
3 The auto-calibration data files are named with Cal plus the time stamp and calibration sample index; for example, Cal20130910162907040.wiff.
4 The TIC of the auto-calibration data is opened in the Analyst Peak Explorer. The mass spectra can be extracted from the TIC by highlighting the region and double-clicking it. To evaluate the mass accuracy before and after calibration, extract mass spectra from a region before and after calibration. This is done by extracting one mass spectrum between 2 minutes to 2.5 minutes (this spectrum was obtained before the instrument was calibrated) and another mass spectrum between 4 minutes to 4.5 minutes (this spectrum was obtained after calibration). This will open one set of mass spectrometer and MS/MS spectra before calibration and one after calibration (Figure C.51).
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Figure C.51 Data Analysis
1. MS Before Calibration 2. MS/MS Before Calibration 3. MS After Calibration 4. MS/MS After Calibration
5 To get the list of ions present in the mass spectrum, right-click the mass spectrum and select List data. This is shown for the MS before calibration in Figure C.52.
Figure C.52 Obtaining List Data
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1. MS Before Calibration 2. MS/MS Before Calibration 3. MS After Calibration 4. MS/MS After Calibration
6 This will open a table with a list of all the ions present in that mass spectrum. Repeat the above step for the MS after calibration too. Each mass spectrum will now have a data table. Click the Calibration Peak List tab in each data table and it will show all the reference ions used for calibration (Figure C.53).
Figure C.53 Reference Ions
1. Mass Spectrometer Before Calibration 2. Data Table From Mass Spectrometer Before Calibration 3. Mass Spectrometer After Calibration 4. Data Table From Mass Spectrometer After Calibration
7 Check to see if the right reference table has been selected. Right-click the table and click the Beta Galactosidase Digest CESI reference table and click Use as reference. This is the same reference table previously used for auto-calibration. This is shown in Figure C.54 for the MS before calibration.
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Figure C.54 Reference Table Verification for MS Before Calibration
8 Repeat the above step for MS after calibration. Now, both the mass spectra have data tables showing the Calibrant Peak List using the MS Beta Galactosidase Digest CESI as the reference table. Shown are the Target Mass or the theoretical mass and the Found At mass or the experimental mass for all the 8 reference ions chosen for auto-calibration. The mass shift between the Target mass and the Found at mass is given as Mass Shift (ppm) in the table (Figure C.55).
Figure C.55 MS After Calibration Analysis
1. Mass Spectrometer Before Calibration 2. Data Table From Mass Spectrometer Before Calibration 3. Mass Spectrometer After Calibration 4. Data Table From Mass Spectrometer After Calibration
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9 The mass shift is higher in the MS before calibration table and lower in the MS after calibration data table for all ions except the ion at 949.8 m/z (indicated by red square in the above figure). For this ion, the mass shift is much higher both before and after calibration. This is an artifact and does not affect the auto-calibration. For the ion at 949.8 m/z, the second isotopic peak in the envelope was chosen instead of the monoisotopic peak (Figure C.56); the red arrow denotes the second isotopic peak, and the blue arrow indicates the monoisotopic peak. The second isotopic peak was chosen since it is higher in abundance than the monoisotopic peak. Therefore, the mass shift calculated with the second isotopic peak is showing up as being very high both before and after calibration. When the acquisition software does the auto-calibration, the peaks are selected with a tolerance of +/- 100 ppm and only the monoisotopic peak will be selected.
Figure C.56 Evaluation of Peak Data
1. Monoisotopic Peak 2. Second Isotopic Peak
10 For evaluating the MS/MS mass accuracy, repeat steps 6 to 8 for MS/MS before calibration and MS/MS after calibration.
11 For the MS/MS experiment, the mass shift after calibration is lower than it was before calibration (Figure C.57).
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Figure C.57 Evaluation of Peak Data in MS/MS Experiment
1. MS Before Calibration 2. Data Table From MS Before Calibration 3. MS After Calibration 4. Data Table From MS After Calibration
Troubleshooting Auto-Calibration Failure
In order for the auto-calibration to pass, the following criteria have to be met:
• Intensity of the reference ions must be at least 10 counts per second (cps) on the TripleTOF® 5600 mass spectrometer and 3.3 cps on MS/MS. • Reference ions have to be within a mass tolerance of 100 ppm. • Greater than or equal to 80% of the selected ions in the reference table must be present.
IMPORTANT Refer to the AB SCIEX Mass Calibration Tutorial found under Start Menu > All Programs > AB SCIEX > Analyst TF 1.7 > Hardware and Software Guides for more information.
Successful auto-calibration runs have a green check mark. An open red circle with a diagonal line through it indicates that the sample was acquired but the calibration failed due to one or more of the reference ions failing to meet the calibration criteria. For more information, double-click the icon and the error message will open as shown in Figure C.58 below.
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Figure C.58 Calibration Failed Error Message
1 Start the troubleshooting: a. Open the corresponding cal data file that failed. b. Select a region in the TIC after 3 min (after the mass calibration has been updated) and extract a spectrum from this region. c. From the spectrum, obtain the data table that shows the calibration peak list (make sure the right reference table has been selected). These steps have been outlined in the Auto-Calibration Data Analysis section above.
2 Check to make sure the intensity of the reference ions is greater than 10 cps in the TripleTOF® 5600 MS data, and over 3.3 counts in the MS/MS data. The auto-calibration will fail in the TripleTOF® 5600 mass spectrometer or the MS/MS experiment if the intensity of the 80% of the selected ions in the reference table doesn’t meet the criteria.
3 Check that the mass accuracy of the reference ions is within 100 ppm. To do this, follow the steps outline in the Auto-Calibration Data Analysis section and check the Mass shift (ppm) in the Calibration peak list table.
IMPORTANT Make sure the threshold for peak detection is set to 1% in the spectrum. This can be done in the Tune and Calibrate mode by going to Tools > Settings > Appearance Options > Other Graph options and then changing the Default Threshold for the Spectrum to 1%.
If the mass shift is greater than or equal to 80% of the reference ions is over 100 ppm, manual calibration has to be performed.
4 Additional troubleshooting can be found in Auto-calibration Troubleshooting Table.
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Auto-calibration Troubleshooting Table
Error Message Possible Cause Solutions Auto-calibration Check the auto-calibration If the intensity of the reference ions is less than 10 cps: failed on the TOF data file to see if the intensity 1. Check ISVF in the method for the TOF mass spectrometer MS experiment. of the reference ions on the experiment to make sure if the voltage is correct and the spray TOF mass spectrometer is at is stable. least 10 cps for ≥ 80% of the 2. Prepare a fresh beta-galactosidase sample for auto- reference ions. calibration. 3. Check the TOF MS calibration data to find the missing ion, and then clear that ion from the Reference Table. Re-run the sequence. Auto-calibration Check the auto-calibration If the mass shift is more than 100 ppm: failed on the TOF data file to see if the mass 1. Do a manual calibration to correct for the mass accuracy. MS experiment. shift of the reference ions on 2. If manual calibration doesn’t help, instrument optimization has the TOF mass spectrometer to be performed. is less than 100 ppm for 80% or more of the reference ions. Auto-calibration Check the auto-calibration If the intensity of the reference ions is less than 10 cps: failed on the MS/MS data file to see if the intensity 1. Check ISVF in the method for the Product ion experiment to High Sensitivity of the reference ions on the make sure the voltage is correct and the spray is stable. experiment. MS/MS is at least 10 cps for ≥ 2. Check if the collision energy (CE) is optimum for the 729.3 m/ 80% of the reference ions. z Product ion experiment. If not, the quality of the 729.3 MS/MS spectra has to be checked, and CE has to be adjusted. 3. Prepare a fresh beta-galactosidase sample for auto- calibration. Auto-calibration Check the auto-calibration If the mass shift is more than 100 ppm: failed on the MS/MS data file to see if the mass 1. Do a manual calibration to correct for the mass accuracy. High Sensitivity shift of the reference ions on 2. If manual calibration doesn’t help, instrument optimization has experiment. the MS/MS is less than to be performed. 100 ppm for ≥ 80% of the reference ions.
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322 / 372 RUO-IDV-05-3897-C | B11949AG APPENDIX D Auto Vial Increment Feature
Overview
The auto vial increment feature is useful when repeating the same method for an extended period of time. This feature will automatically change to the next row of buffer vials after a specified number of replicates has been achieved with the same method. In this manner, there is a lower risk of running out of reagent volume and depleting the buffers when performing multiple analyses. The auto vial increment feature is available on the method rinse, injection and separation steps of the time program.
Setting Auto Vial Increment on a Rinse Step To set a rinse step with auto vial increment, open the method in the 32 Karat™ Software and then open the rinse step. The auto vial increment section is indicated on the figure below by a red square.
Figure D.1 Rinse Dialog: Auto Vial Increment
• Check Inlet to increment only the rinse vial at the inlet buffer tray. • Check Outlet to increment only the rinse vial at the outlet buffer tray. • Check both Inlet and Outlet to increment the rinse vials at inlet and outlet buffer trays.
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Once an increment has been checked, the number of cycles (repetitions) can be specified.
Figure D.2 Rinse Dialog: Specifying the Number of Cycles
The figure above shows that both inlet and outlet buffer trays will be incremented every 6 cycles, which is equivalent to 6 repetitions of the same method. The table below summarizes which buffer vial positions will be used at each cycle during the above rinse step.
Table D.1 Vial Positions Used During Rinse
Cycles Inlet Buffer Position Outlet Buffer Position (BI) (BO) 1 through 6 E1 A1 7 through 12 E2 A2 13 through 18 E3 A3 19 through 24 E4 A4 25 through 30 E5 A5 31 through 36 E6 A6
NOTE There are six rows in the buffer trays. Vials will not be incremented beyond row 6. Start auto vial increment with vial positions at Row 1 to maximize the number of cycles.
Setting Auto Vial Increment on an Injection To set an injection step with auto vial increment, open the method on the 32 Karat™ Software and then open the injection step. The auto vial increment section is indicated on the figure below by a red square.
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Figure D.3 Inject Dialog: Auto Vial Increment
• Check Inlet to increment only the injection vial at the inlet buffer tray or at the inlet sample tray. • Check Outlet to increment only the vial at the outlet buffer tray. • Check both Inlet and Outlet to increment the vials at inlet and outlet trays.
After an increment has been checked, the number of cycles (repetitions) can be specified.
Figure D.4 Inject Dialog: Specifying the Number of Cycles
The figure above shows that only the inlet buffer tray will be incremented every 12 cycles, which is equivalent to 12 repetitions of the same method. The table below summarizes which vial position will be used at each cycle during the above injection step.
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Table D.2 Vial Positions Used During Injection Step
Cycles Inlet Buffer Position Outlet Buffer Position (BI) (BO) 1 through 12 A1 B1 13 through 24 A2 B1 25 through 36 A3 B1 37 through 48 A4 B1 49 through 60 A5 B1 61 through 72 A6 B1
NOTE There are six rows in the buffer trays. Vials will not be incremented beyond row 6. Start auto vial increment with vial positions at Row 1 to maximize the number of cycles.
IMPORTANT A better alternative for changing the sample injection position during repetitions of the same method is to use the Allow Override option on the injection step (indicated on figure below by red square). Saving the method with checked Allow Override permits the change of injection vials on any sequence that utilizes the method. This will ensure that the sample injection vial is the one indicated on the sequence, and not by a cycle count.
Figure D.5 Inject Dialog: Allow Override
Setting Auto Vial Increment on a Separation Step To set a separation step with use auto vial increment, open the method on the 32 Karat™ Software and then open the separation step. The auto vial increment section is indicated on the figure below by a red square.
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Figure D.6 Separate Dialog: Auto Vial Increment
• Check Inlet to increment only the separation vial at the inlet buffer tray. • Check Outlet to increment only the separation vial at the outlet buffer tray. • Check both Inlet and Outlet to increment the separation vials at inlet and outlet buffer trays.
Once an increment has been checked, the number of cycles (repetitions) can be specified.
Figure D.7 Separate Dialog: Specifying the Number of Cycles
The figure above shows that both inlet and outlet buffer trays will be incremented every 12 cycles, which is equivalent to 12 repetitions of the same method. The table below summarizes which buffer vial positions will be used at each cycle during the above separation step.
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Table D.3 Vial Positions Used During Separation Step
Cycles Inlet Buffer Position Outlet Buffer Position (BI) (BO) 1 to 12 A1 A1 13 to 24 A2 A2 25 to 36 A3 A3 37 to 48 A4 A4 49 to 60 A5 A5 61 to 72 A6 A6
NOTE There are six rows in the buffer trays. Vials will not be incremented beyond row 6. Start auto vial increment with vial positions at Row 1 to maximize the number of cycles.
Auto Vial Increment in a Method If the auto vial increment is used in a method, it will be listed in the Summary column of the time program. In the time program shown below, auto vial increment has been used on steps 4, 5, 7, and 8. The term In / Out vial inc 12 means that both inlet and outlet vials will be incremented after 12 cycles. For example, In vial inc 6 means that only the inlet vial will be incremented after 6 cycles. In another example, Out vial inc 3 means that only the outlet vial will be incremented after 3 cycles.
Figure D.8 Time Program
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Reviewing Vial Positions before Running a Sequence
Utilize Sequence Vials Preview to ensure that the trays contain all necessary vials to run a specified sequence on its entirety. To do this, open the 32 Karat™ Software and then open the sequence to be previewed. Next, go to the main header and select Sequence followed by Sequence Vials Preview. This will open the preview window (example shown below). The Sequence Vial Preview is also helpful in identifying vial collisions. To solve a vial collision, change the identified vial position in the method and save the method.
Figure D.9 32 Karat™ Software Preview Window
NOTE The CESI 8000 Software provides a preview of all trays before the start of a sequence.
IMPORTANT • If the sequence is stopped and re-started, the next running method will revert to the initial set of vials. In other words, the cycle count will re-start at 1. • If the sequence has different methods, the CESI 8000 Plus System will revert to the initial set of specified at the start of each method. Therefore, do not combine different methods if the auto vial increment feature is been used.
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330 / 372 RUO-IDV-05-3897-C | B11949AG APPENDIX E Conductive Liquid Capillary Conditioning
Conductive Liquid Capillary (CLC) Conditioning Procedure
If the baseline mass spectrum shows a series of peaks with an m/z difference of 98 (Figure E.1), it is likely due to a phosphate contamination. Perform the procedure below to condition the conductive liquid capillary in the OptiMS cartridge.
The CLC conditioning method is a series of reverse rinses with 1.0 N and 0.1 N sodium hydroxide, 0.1N HCl, DDI water, and 10% HAc and one last forward rinse at the end of the method to fill the separation capillary with 10% HAc.
Figure E.1 Profile of Phosphate Contamination
NOTE During this method, the sprayer must rest in the holster and remain immersed in no more than 10 mL of DDI water.
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1 To clean up phosphate contamination, set up the CESI 8000 Plus method (Figure E.2).
Figure E.2 CLC Conditioning Method
IMPORTANT This method uses reverse rinses. Only one vial is required in the buffer inlet tray, at position A1.
2 Set up the buffer inlet and outlet tray for the Conductive Liquid Capillary conditioning method, as shown in Figure E.3.
Figure E.3 CLC Conditioning Buffer Tray Layout
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Overview
This appendix describes how to properly use CESI-MS vials, CESI-MS vial caps, and micro vials. Also shown are the following maintenance procedures:
• Capillary Cleaning Procedure (Short Term Storage) • Capillary Storage Procedure • Removing and Re-installing OptiMS Interface Plate • Opening Levers, Electrodes, and the Interface Block • Clean Electrodes, Opening Levers, and the Interface Block • Refilling Coolant • Replacing Quad Rings • Replacing Fuses
IMPORTANT To achieve optimal instrument performance, consistent user maintenance must be performed regularly on the CESI 8000 Plus System. A yearly planned maintenance by a SCIEX FSE is recommended.
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Using CESI-MS Vials and Caps
NOTE When you fill the CESI-MS vials, some important items to remember are: • Never reuse vials and vial caps. • Do not overfill vials. An overfilled vial will allow liquid to enter the pressure system and it has the potential of causing system damage. • Do not under-fill the vial or let the liquid level become too low. Low liquid level in separation vials can cause poor separations, or cause the separation capillary to be filled with air which could lead to its breakage if voltage is applied.
CESI-MS Vials and Micro Vials Items required:
• CESI-MS vials • CESI-MS vial caps
1 Fill the CESI-MS vials as shown in Figure F.1. Do not put more than 1.4 mL in a vial.
Figure F.1 CESI-MS Vials
1. CESI-MS Vial Cap 2. Maximum Fill Level 3. CESI-MS Vials
2 Put the cap on the vial and press it into position.
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Filling the Micro Vials Items required:
• Micro vials • CESI-MS vials • CESI-MS vial caps
1 Using care as not to introduce any bubbles, fill the micro vial with at least 50 μL of sample (Figure F.2).
NOTE If there are air bubbles present, centrifuge the vial for a few seconds to remove the bubbles.
Figure F.2 Micro Vials
1. CESI-MS Cap 3. CESI-MS Vial 2. Micro Vial 4. Micro Vial inside CESI-MS Vial
2 Place the micro vial in the CESI-MS vial.
3 Put the cap on the CESI-MS vial.
4 Put the capped vial in the tray.
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Hardware Maintenance Procedures
Specifically covered are storage procedures, the removal and replacement of electrodes and opening levers, and the cleaning of the electrodes, opening levers, and the interface block. Also covered is how to refill coolant, replace quad rings, and replace fuses.
WARNING Electrical Shock Hazard. Disconnect the power before any instrument disassembly. Failure to do so can cause electrical shock, and/or damage the CESI 8000 Plus System.
WARNING Electrical Shock Hazard. Maintenance or repair procedures not specifically described in this manual present a risk of electrical shock or injury. Refer additional servicing to SCIEX Technical Support.
WARNING Electrical Shock Hazard. Do not attempt to defeat any of the instrument interlocks or safety mechanisms.
Capillary Cleaning Procedure (Short Term Storage) The method shown in Figure F.4 cleans the capillaries at the end of a sequence. It is used for short-term storage (up to 3 days) of the OptiMS Silica Surface Cartridge. The reagents for the capillary cleaning method are: • 0.1 N NaOH • 0.1 N HCI • DDI water • 10% HAc
Prepare the reagents for the capillary shutdown by placing the reagent vials on the inlet and outlet buffer trays as indicated in Figure F.3.
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Figure F.3 Capillary Cleaning Buffer Tray Configuration
The time program for the capillary cleaning method (Figure F.4):
Figure F.4 Capillary Cleaning Method
Capillary Storage Procedure This method is used to prepare the OptiMS cartridge for storage. This method cleans and dries the OptiMS capillaries. This method should always be performed if the OptiMS cartridge will not be used for 3 or more days. The reagents for capillary cleaning:
• 0.1 N NaOH • 0.1 N HCI • DDI water • MeOH
CAUTION Do not blow air through the capillary using the pressure rinse function of this method. Doing so will contaminate the internal surface of the OptiMS separation capillary. The vacuum function should be used instead.
Prepare the reagents listed above for the capillary shutdown by placing the reagent vials on the inlet and outlet buffer trays as indicated in Figure F.5.
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Figure F.5 Capillary Shutdown Buffer Tray Configuration
NOTE An empty vial with a cap should be placed where Empty is indicated.
The method used to run the capillary shutdown method (Figure F.6):
Figure F.6 Capillary Shutdown Method
NOTE This CESI-MS storage method cannot be left running automatically in the sequence because MeOH is very volatile. Thus, MeOH filled vials must be used immediately or refilled just before use. The ESI needle can safely remain in air but not in liquid so that it can be dried by vacuum.
Removing and Re-installing OptiMS Interface Plate If there is a spill, then it may become necessay to remove the OptiMS interface plate to clean the CESI 8000 Plus System. To remove and then re-install the OptiMS interface plate into a CESI 8000 Plus System, perform the following steps:
Removing OptiMS Interface Plate
1 Remove the OptiMS cartridge. Refer to Cartridge Removal and Instrument Shutdown.
2 Power off the CESI 8000 Plus System by clicking ON/OFF on the front of the instrument.
3 Loosen the thumbscrews on either side of the OptiMS interface plate (Figure F.7).
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Figure F.7 OptiMS Interface Plate Thumbscrews
1. Thumbscrews (for holding OptiMS interface plate)
4 Remove plate by holding the handle on the top of the device and pull it outwards. Store the plate in a safe place.
Re-installing OptiMS Interface Plate
1 Hold the handle of the OptiMS interface plate and insert it as shown in Figure F.8.
Figure F.8 OptiMS Interface Plate Insertion
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IMPORTANT Make sure that the OptiMS interface plate is sitting flush with the mounting surface of the CESI 8000 Plus System.
2 Tighten the plate thumbscrews (on both sides of plate; Figure F.9).
Figure F.9 Tighten Thumbscrews
Opening Levers, Electrodes, and the Interface Block
Remove and Replace Opening Levers
1 Use Direct Control to move the trays to the load position.
2 Lift the cartridge cover and then wait for the coolant to drain from the cartridge.
3 Power off the instrument.
4 Loosen the two thumbscrews and lift the cartridge lock down bar.
5 Remove the OptiMS cartridge from the interface block.
6 If the trays are still in the way, remove them for access to both opening levers.
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7 Remove an opening lever by grasping it with both hands and pulling down firmly (Figure F.10). Repeat for second lever.
Figure F.10 Interface Block, Electrodes, and Opening Levers
1. Interface Block 2. Electrodes 3. Opening Levers
CAUTION Puncture Hazard. Do not place fingers directly below the electrodes when installing opening levers. Electrode ends are sharp; handle them with care.
8 Install an opening lever. a. Align the o-ring and electrode hole in the lever directly under the electrode. The short cylinder side of the lever should be under the spring. b. With your fingers on the sides of the lever, press the lever up into the interface block until it snaps into place. Repeat for the second lever.
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Remove and Replace Electrodes
1 The vial opening levers must be removed prior to removing the electrodes. Repeat steps 1 to 6 of Remove and Replace Opening Levers.
2 Remove the electrodes one at a time as shown in Figure F.11.
Figure F.11 Remove Electrodes from Interface Block
1. Electrode Tool 2. Electrode 3. Nub at End of Electrode Tool Handle
a. Align the electrode tool flush with the bottom of the interface block. b. Push straight forward under the interface block to capture the electrode in the tool. c. Make sure the nub at the end of the electrode tool handle fits into the notch on the interface block as shown in the circled area of Figure F.11. d. Pry gently with the electrode tool to remove the electrode from the interface block. e. Remove the electrode from the electrode tool. Repeat for second electrode.
3 Install an electrode into the interface block.
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Figure F.12 Install an Electrode into the Interface Block
1. Electrode Tool 2. Electrode Key
a. Place an electrode in the tool with the electrode key facing you (Figure F.12). b. Align the tool below the notch and parallel to the bottom of the interface block. c. Press upward to snap the electrode into the interface block. d. Remove the tool by pulling straight back. Repeat for second electrode.
Clean Electrodes, Opening Levers, and the Interface Block The items required for cleaning are:
• Dry paper wipes • Mirror • Pen Light • Cotton swabs • 150 mL of DDI water • Methanol • Beaker
1 Repeat steps 1 to 6 of Remove and Replace Opening Levers.
2 Immerse both levers in a beaker containing at least 150 mL of DDI water.
3 Perform steps under Remove the electrodes one at a time as shown in Figure F.11.
4 Immerse both electrodes in the DDI water with the opening levers.
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5 Sonicate these four parts for five minutes.
6 Retrieve the parts and dry them thoroughly with a dry paper wipe.
7 Use cotton swabs to clean the interface block with water followed by methanol. Allow the interface block surface to dry.
8 Inspect the interface block using a mirror and pen light. Repeat this process until the interface block is clean.
9 Perform the steps under Install an electrode into the interface block.
CAUTION Puncture Hazard. Do not place fingers directly below the electrodes when installing opening levers. Electrode ends are sharp; handle them with care.
10 Perform the steps under Install an opening lever.
11 Reinstall the OptiMS cartridge in the interface block.
12 Lower the clamp bar and tighten the two thumbscrews.
13 Close the cartridge cover.
IMPORTANT To avoid corrosion of the ion source, frequently clean the dry chemical waste that accumulates beneath the sprayer.
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Refilling Coolant
Items required:
• Capillary Cartridge Coolant (PN 359976) • Coolant Fill Adapter (PN 144647)
1 Open the sample cover (Figure F.13).
Figure F.13 Opening the Sample Door (Outer Door)
2 Attach the coolant fill adapter to the coolant fill port (Figure F.14).
Figure F.14 Attaching Coolant Fill Adapter
3 The instrument power should be on with the OptiMS cartridge installed.
4 Slowly fill the coolant supply until the fill indicator is within the yellow lines in the coolant sight glass (Figure F.15).
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Figure F.15 Coolant Sight Glass Location on the CESI 8000 Plus System
5 Remove the coolant fill adapter and close the sample cover.
6 The coolant fill adapter comes with a plunger. When adding coolant, the plunger should not be used. Gravity supplies enough force to pull the coolant into the system. Using the plunger can cause damage.
Replacing Quad Rings
The interface block quad rings provide a coolant seal between the interface block and the OptiMS cartridge. If coolant is leaking between the interface block and OptiMS cartridge, the quad rings might require replacement. The following procedure describes how to change the quad rings. You must have new quad rings to do the replacement.
How to Replace the Quad Rings
1 Use the Direct Control window to move the trays to the load position.
2 Lift the cartridge cover.
3 Loosen the two thumbscrews and lift the clamp bar.
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4 Turn off instrument power. Remove the OptiMS interface plate from the instrument.
5 Find the location of the quad rings (Figure F.16). Use the tweezers that were supplied with the instrument (or your thumbnail and forefinger) to remove the quad rings.
Figure F.16 Location of the Quad Rings on the CESI 8000 Plus System
6 Install the new quad ring in the quad ring recess of the interface block.
7 Reinstall the OptiMS interface plate.
8 Lower the clamp bar and tighten the two thumbscrews.
9 Close the cartridge cover.
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Replacing Fuses
CAUTION Make sure to replace the fuses with the correct type and rating for continued protection against risk of fire and/or improper instrument operation.
CAUTION If the fuses continue to blow after being replaced, contact a SCIEX FSE for additional assistance.
Items required: • Flat-tip screwdriver, #2 • Replacement fuses (as required)
How to Replace the Fuses
1 Turn off instrument power and disconnect the power plug from the AC power outlet.
2 Using the flat-tip screwdriver, remove the fuse block (Figure F.17).
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Figure F.17 Replacing Fuses
1. Fuse Block 2. Air Conditioning Vents 3. External Connections Panel
3 Replace the fuses: Table F.1 Fuse Type and Rating
Line Voltage 100 VAC to 120 VAC 200 VAC to 240 VAC Fuse Type and Rating 8.0 A Slow Blow; ¼ inch (2 ea.) 6.3 A Time Delay; 20 mm (2 ea.)
4 Reinstall the fuse block and reconnect the power cable.
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350 / 372 RUO-IDV-05-3897-C | B11949AG APPENDIX G Troubleshooting and Mobile Cart Homing Procedure
Overview
This appendix discusses problems that might occur when using the CESI 8000 Plus System with a mass spectrometer, and provides corrective actions for each problem. Troubleshooting and Flow Diagrams are provided.
Steps for fine tuning the OptiMS sprayer tip position for a Thermo Scientific mass spectrometer or a SCIEX mass spectrometer are provided in the Fine Tuning Sprayer Tip Position for Thermo Scientific Mass Spectrometer and Fine Tuning Sprayer Tip Position for SCIEX Mass Spectrometer sections. The homing procedure for the CESI 8000 Plus cart is given in the CESI 8000 Plus Mobile Cart Homing Procedure section.
Maintenance procedures are described in APPENDIX F: Using Vials and Hardware Maintenance.
IMPORTANT For any maintenance not covered in this manual, contact SCIEX Technical Support.
Troubleshooting and Flow Diagrams
The following is a list of possible problems when using the CESI 8000 Plus System with a mass spectrometer:
NOTE Whenever the problem involves positioning the sprayer tip, make certain that the droplets fall freely from the tip and the sprayer tip does not touch the mass spectrometer inlet.
• Sample Preparation Considerations for Analysis with CESI-MS • Properties of a Method or Sequence Cannot be Modified or Saved • Blocked Sprayer Tip • Electrospray Cannot Be Established Between the CESI 8000 Plus System and Mass Spectrometer • Electrospray is Detected at Zero Ion Spray (IS) Voltage while Applying CE Voltage • Unstable Electrospray while Applying CE Voltage and IS Voltage
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• Droplet at Capillary Tip Disappeared Indicating that Electrospray has Started, but No Mass Spectrometer Signal is Detected • Signal of Total Ion Current (TIC) is Lower Than Usual and/or Background Signal Intensity Fluctuates More Than 40% • Unstable CE Current • No Analyte Signals Detected by Mass Spectrometer During a CE-MS Separation • Some Analyte Peaks are Missing or Smaller Than Expected • CE Current Gradually Decreases During CE-MS Separation • The Iron-Acetate Cluster m/z 537 is Suppressing the Sample Signals • High Voltage System Unable to Deliver the Required Current
Sample Preparation Considerations for Analysis with CESI-MS Surfactants and chaotropic agents (such as SDS, urea and guanidine hydrochloride) are commonly used in sample preparation protocols. These reagents however have deleterious effect at high concentrations when using CESI-MS. Problems such as irreproducible migration time, non-specific adsorption onto capillary inner surface, and clogging of the separation capillary may occur.
To ensure optimum performance of CESI-MS analysis, it is recommended to use ultrafiltration, or a desalting step after lysis and/or protein extraction, prior to enzymatic digestion.
Properties of a Method or Sequence Cannot be Modified or Saved This problem occurs if the CESI 8000 Software is in the process of loading (or has just loaded) a method or sequence, and you attempt to reopen the 32 Karat™ Software with the 32 Karat™ Software button. The 32 Karat™ Software will reopen, but the effect on the method or sequence is that the properties of the method or sequence cannot be saved. Instead of using the 32 Karat™ Software button in this instance, use the Show 32 Karat button at the center bottom of the user interface dialog.
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Blocked Sprayer Tip During capillary rinse, there is no droplet observed at the sprayer tip.
Possible Cause Sprayer tip has become clogged with debris.
Possible Solution Perform the following procedure:
1 Fill a 50 mL Falcon tube with approximately 10 mL of DDI water.
CAUTION Do not add more than 10 mL of DDI water. More than that may cause it to splash up into the metal components of the sprayer end.
2 Screw the tube into the side of the instrument (Figure G.1).
Figure G.1 Falcon Tube Screwed into Holster
3 Very gently submerge the sprayer end of the cartridge in the 50 mL tube (Figure G.2).
NOTE When inserting sprayer end into tube, try to not touch the sides of the tube.
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Figure G.2 Sprayer Submerged in 50 mL Falcon Tube
4 Using the CESI 8000 Software Direct Control window, apply 100 psi in forward direction for 5 minutes. Pump 10% acetic acid through capillary to dislodge the clog. If necessary, refer to Using the Direct Control Window to Load the Cartridge and Samples in CHAPTER 6, and Capillary Cleaning Procedure (Short Term Storage) in APPENDIX F.
5 After 5 minutes, gently remove the capillary from the Falcon tube and wipe sprayer end with a dry paper wipe to remove excess liquid (Figure G.3).
CAUTION Gently wipe dry the sprayer end. Be careful not to move the retractive tip protector while wiping. The sprayer tip can break if the retractive tip protector moves while wiping.
Figure G.3 Wipe Clean Sprayer End with Dry Paper Wipe
6 Reinsert the sprayer back into the mass spectrometer adapter.
7 Remove the 50 mL tube from holster and empty contents.
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Electrospray Cannot Be Established Between the CESI 8000 Plus System and Mass Spectrometer
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Electrospray is Detected at Zero Ion Spray (IS) Voltage while Applying CE Voltage
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Unstable Electrospray while Applying CE Voltage and IS Voltage
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Droplet at Capillary Tip Disappeared Indicating that Electrospray has Started, but No Mass Spectrometer Signal is Detected
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Signal of Total Ion Current (TIC) is Lower Than Usual and/or Background Signal Intensity Fluctuates More Than 40%
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Unstable CE Current
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No Analyte Signals Detected by Mass Spectrometer During a CE-MS Separation
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Some Analyte Peaks are Missing or Smaller Than Expected
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CE Current Gradually Decreases During CE-MS Separation
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The Iron-Acetate Cluster m/z 537 is Suppressing the Sample Signals
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High Voltage System Unable to Deliver the Required Current
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Fine Tuning Sprayer Tip Position for Thermo Scientific Mass Spectrometer
Before performing a CESI-MS separation, it is critical that the OptiMS sprayer tip position in front of the mass spectrometer inlet is optimized for proper ESI voltage. Having the sprayer tip too close to the mass spectrometer inlet (less than 2 mm) may cause accidental suction of rinsing solutions (such as 0.1 N NaOH and 0.1 N HCl) into the mass spectrometer system. Having the sprayer too far from the mass spectrometer inlet may require the use of high ESI voltage which may lead to undesired peptide fragmentation. Figure G.4 depicts in green the sprayer tip distance and ESI voltage values recommended when using Thermo Scientific mass spectrometer. The red area should be avoided.
Figure G.4 Thermo Scientific Mass Spectrometer Sprayer Tip Distance and ESI Voltage Correlation
Perform the following steps to optimize the sprayer tip position after placing OptiMS cartridge on CESI 8000 Plus System, the OptiMS sprayer on mass spectrometer adapter, and before performing a CESI-MS separation:
1 Fill both OptiMS separation capillary and CLC with background electrolyte.
2 Align sprayer tip at 3 mm from mass spectrometer inlet by adjusting xyz-knobs.
3 Apply CESI separation voltage (for example, 20 kV) that will be used in CESI-MS separation.
4 Ensure that CESI electrical current is stable.
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5 Ensure OptiMS cartridge does not spray at 0 ESI voltage during mass spectrometer scanning.
6 Set ESI voltage to 0.5 kV and then increase in 0.1 kV increments until electrospray is detected.
7 Increase ESI voltage by 0.2 kV.
8 Adjust the xyz position of OptiMS sprayer tip to maximize mass spectrometer signal intensity while minimizing ion trap time, and then ensure sprayer tip is approximately 3 mm away from the mass spectrometer inlet.
9 Once the sprayer tip position has been optimized, set ESI voltage to zero.
10 Set ESI voltage to 0.5 kV and then increase in 0.1 kV increments until electrospray is detected.
NOTE This ESI voltage value is the minimum and it is not high enough to maintain an effective spray during CESI-MS separation.
11 Increase minimum ESI voltage by 0.2 kV.
NOTE This ESI voltage value should be used with the mass spectrometer method to provide a stable electrospray during CESI-MS separation.
12 Set ESI voltage to zero.
13 Turn off CESI separation voltage.
Repeat this procedure:
• After installing an OptiMS cartridge. • If signal CESI separation voltage is changed. • If a different background electrolyte is used.
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Fine Tuning Sprayer Tip Position for SCIEX Mass Spectrometer
Before performing a CESI-MS separation, it is critical that the OptiMS sprayer tip position in front of the curtain gas plate is optimized for proper ESI voltage. The sprayer tip should be positioned outside the curtain gas plate to avoid rinsing solutions dripping into the curtain gas plate. Having the sprayer too far from the curtain gas plate may require the use of high ESI voltage which may lead to undesired peptide fragmentation. Figure G.5 depicts in green the sprayer tip distance and ESI voltage values recommended when using a SCIEX mass spectrometer. The red area should be avoided.
Figure G.5 SCIEX Mass Spectrometer Sprayer Tip Distance and ESI Voltage Correlation
Perform the following steps to optimize the sprayer tip position after placing OptiMS cartridge on the CESI 8000 Plus System, the OptiMS sprayer on mass spectrometer adapter, and before performing a CESI-MS separation:
Repeat this procedure:
1 Fill both OptiMS separation capillary and CLC with background electrolyte.
2 Align sprayer tip at 3 mm from curtain gas plate by adjusting xyz-knobs.
3 Apply CESI separation voltage (for example, 20 kV) that will be used in CESI-MS separation.
4 Ensure that CESI electrical current is stable.
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5 Ensure OptiMS cartridge does not spray at 0 ESI voltage while performing mass spectrometer scan.
6 Increase ESI voltage by 200 V.
7 Set ESI voltage to 1000 V, and then increase in 100 V increments until electrospray is detected.
8 Adjust the xyz position of OptiMS sprayer tip to maximize mass spectrometer signal intensity, and then ensure sprayer tip is approximately 3 mm from curtain gas plate.
9 Once the sprayer tip position has been optimized, set the ESI voltage to zero.
10 Set ESI voltage to 1000 V, and then increase in 100 V increments until electrospray is detected.
NOTE This ESI voltage value is the minimum and it is not high enough to maintain an effective spray during CESI-MS separation.
11 Increase minimum ESI voltage by 200 V.
NOTE This ESI voltage value should be used with the mass spectrometer method to provide a stable electrospray during CESI-MS separation.
12 Set ESI voltage to zero.
13 Turn off CESI separation voltage.
Repeat this procedure:
• After installing an OptiMS cartridge. • If signal CESI separation voltage is changed. • If a different background electrolyte is used.
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CESI 8000 Plus Mobile Cart Homing Procedure
The electric cart is pre-programmed to its home position which is the lowest height that the internal firmware allows. This firmware home is slightly above the physical lower limit and is nominally at 27 inches from the floor. Also pre-programmed is an upper limit which is relative to the home position and is 44 inches from the floor. Each power module has a firmware commanded bottom position that must be taught specific to that table. Replacing this module, such as converting from 110 V to 230 V will cause that taught position to be lost. If that happens the table top may not reach to lowest height, so it must be re-programmed by the procedure below. The upper limit, being relative to the lower limit, is not lost when switching modules.
IMPORTANT Always press the height adjustments buttons one at a time, never simultaneously.
1 Press Down until downward travel stops.
Figure G.6 Cart Height Adjustment Buttons
1. Cart Up Button 2. Cart Down Button
2 Click Down on the keypad three times. On the third time, hold it down for 5 seconds to 10 seconds until you hear a slight click and the table bumps up-down-up.
3 If the legs fail to function properly, unplug the power, wait 10 seconds, plug the power back in and repeat the above step.
4 The above procedure must be performed anytime the power module is replaced.
If this does not resolve the problem, then contact SCIEX Technical Support.
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Customer Training
• In North America: [email protected] • In Europe: [email protected] • Outside the EU and North America, visit sciex.com/education for contact information.
Online Learning Center
• SCIEX University™
Purchase Consumables
Reorder SCIEX consumables online at store.sciex.com. To set up an order, use the account number, found on the quote, order confirmation, or shipping documents. The SCIEX online store is currently limited to the US, UK, and Germany but will be expanding to other countries in the future. For customers in other countries, contact the local SCIEX representative.
SCIEX Support
SCIEX and its representatives maintain a staff of fully-trained service and technical specialists located throughout the world. They can answer questions about the system or any technical issues that might arise. For more information, visit the SCIEX website at sciex.com or contact us in one of the following ways.
• sciex.com/contact-us • sciex.com/request-support
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CyberSecurity
For the latest guidance on cybersecurity for SCIEX products, visit sciex.com/productsecurity.
Documentation
This version of the document supercedes all previous versions of this document. To view this document electronically, Adobe Acrobat Reader is required. To download the latest version, go to https://get.adobe.com/reader.
To find software product documentation, refer to the release notes or software installation guide that comes with the software.
To find hardware product documentation, refer to the Customer Reference DVD that comes with the system or component. The latest versions of the documentation are available on the SCIEX website, at sciex.com/customer-documents.
Note: To request a free, printed version of this document, contact sciex.com/contact-us.
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