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Coulochem III Analyst Driver

1. Coulochem Ill Analyst Driver Version 1 0 59 User s Manual Appendix B Parameter Ranges and Default Values B 1 Configuration and Method Parameter Ranges and Defaults Table B1 below lists all configuration and method parameters along with each parameter s range units and default value Table B1 Parameter Ranges and Default Values Parameter Range Units Default System Configuration Mode DC or Pulse DC Communications port COM1 COM4 USB COMI Enable channel 2 yes or no no Enable guard channel yes or no no Enable thermal organizer yes or no no Enable event marker on AZ yes or no no Event marker height 20 to 20 5 Lock keypad yes or no yes DC Mode Ch 1 Potential 2000 to 2000 mV 50 mV Ch 1 Filter Constant 0 2 0 5 1 0 2 0 5 0 10 0 sec 5 0 sec Ch 1 Gain Range 10 pA to 1 mA in steps of 1 2 100 uA 5 10 20 50 100 200 500 Ch 1 Output Voltage 1 0 0 1 0 1 or 1 0 V 1 V Ch 1 Baseline Offset 50 to 50 0 Ch 2 Potential 2000 to 2000 mV 50 mV Ch 2 Filter Constant 0 2 0 5 1 0 2 0 5 0 10 0 sec 5 0 sec Ch 2 Gain Range 10 pA to 1 mA in steps of 1 2 100 uA 5 10 20 50 100 200 500 Ch 2 Output Voltage 1 0 0 1 0 1 or 1 0 V 1 V Ch 2 Baseline Offset 50 to 50 0 Guard Potential 2000 to 2000 mV 0 mV Post Run Ch 1 Enable Clean Cell yes or no no Ch 1 Clean Cell Potential 2000 to 2000 mV 0 mV Ch 1 Clean Cell Duration 1 0 to 120 0 sec 1 sec Ch 2 Enable Clean Cell
2. Coulochem Ill Analyst Driver Version 1 0 vii User s Manual License Agreement COMMUNICATIONS BETWEEN YOU AND ESA RELATING TO THE SUBJECT MATTER OF THIS EULA This software is copyright ESA Biosciences Inc 1986 2006 Rev 3a viii Coulochem III Analyst Driver Version 1 0 User s Manual Table of Contents 1 TOG MUCH toit I 1 ld USB TSW RV eee uu EM E 1 1 2 Contents of the Coulochem III Analyst Driver Software Application Manual l 1 37 Getting Helpauns censeo e ate Uso ER Sree AL am nt O 2 1 3 1 Help Buttons and the Coulochem III Help File eee 2 102 Help Navigator TOOL d onset ihm cellos meted uno eR ten caer ted alsa R 2 13730 Help Men ars e ere ART e Nid Gas ee ee pe eG o EPI Isa qs 3 1 3 4 Entire Manual in Adobe PDF Format 4 2 System Requirements and Installation eee e eee creen eene 5 217 System Requirements oscinina naaa es nto e beat aa 5 2 2 Additional Hardware Requirements ui e db act lai deno ace Pat eque qai d 5 2 3 Installing the Coulochem III Analyst Driver eere 6 2 44 Hardware Configuration ocak oe tuna eter Di e bie e uten rota bns iipa tabes uas talos 7 2 5 Connecting the Communications Cables doe erras dba uaep erra oA be oU ua do uiid 8 2 6 Verifyimg the Systemic ene eaedem e erede pee ire eI eA 10 3 Starting and Configuring the Driver ecce esee e reete e ee
3. Connected Found Coulochem III Lunam Serial i ES5400 B Software version 3 04g Sent Lines Transferred EN NN Update Mode Failed Version 1 00 Figure A 1 Firmware Update Utility for Coulochem III 58 Coulochem IIl Analyst Driver Version 1 0 User s Manual Updating the Coulochem Ill Firmware When properly connected through the USB cable the message Connected appears near the top of the utility window and the utility shows both the serial number and the current firmware software version number 4 Click the Update button When the Open dialog appears navigate to the Program Files folder normally on Drive C then to the ESA Biosciences subfolder and finally to the Corona Coulochem Firmware Update Utility folder i e to the folder C Program Files ESA Biosciences Corona Coulochem Firmware Update Utility 5 Select the file coulochem run where is the version number of the new firmware update for example coulochem 3 07 run If more than one such file is found select the one with the highest latest version number Then click Open 6 The update utility will replace all of the existing firmware with the version on the update CD The process should take no more than a few minutes When finished the message Download complete Coulochem III rebooting will appear near the top of the window and the Coulochem III will reboot automatically 7 Click Exit to close the update utility
4. Version 1 0 41 User s Manual 6 Acquiring Data with the Coulochem Ill Driver 6 1 Overview The basic steps in running samples with the Coulochem III driver are 1 Start Analyst 2 Activate a hardware profile See Section 4 for more on hardware profiles involving the Coulochem III detector Note Once a profile has been activated it is normally not necessary to deactivate the profile when closing the Analyst program unless other components require deactivation the Coulochem III driver does not If Analyst is closed without deactivating the active profile restarting Analyst will automatically restart the Coulochem III driver 3 Build acquisition methods as appropriate for the samples The same method may be used for all samples or different methods may be used for different samples Remember to attach Coulochem III parameters to the method See Section 4 for more on building acquisition methods 4 Build an acquisition batch 5 Submit batch samples to the batch queue 6 Run the samples in the queue Figure 6 1 illustrates a simple 5 sample batch using method DC 2ch with Guard 4 Acquire Tools Explore Window Script Help E x A Acquire Mode v A B Default x 83 X nHB xx Durati 5 AJ Acquiring Sample of Period o of o pane 00 00 00 e Bose ced a a io 0 100 Elapsed Stand By Normal u i ia onm Data File Project plate x Waiting DC 2ch with Guard New Batch DataSET1 Default x 2 2 2006 11 09
5. 57 A 2 Preparation and Installation of the Utility eed ress ette etnia donee Rd resp ene 57 A 3 Updating the Firmware with the Update Utility eere 58 Appendix B Parameter Ranges and Default Values 61 B 1 Configuration and Method Parameter Ranges and Defaults sss 61 x Coulochem III Analyst Driver Version 1 0 User s Manual 1 Introduction 1 4 Overview This manual describes the basic setup and operation of ESA Biosciences Coulochem III Electrochemical Detector for use as an integrated device within Applied Biosystems MDS Sciex Analyst Software The Analyst program enables you to control ABI s mass spectrometers and other devices such as autosamplers pumps column ovens and valves Once installed the Coulochem III plug in driver is provided as a companion Software Application in the Available Devices list within Analyst This allows you to configure and control the Coulochem III detector within the Analyst interface in a regulated environment using electronic records and the associated features in Analyst software The Coulochem III driver application supports control of the Coulochem III device by serial RS232 or USB communications and uses an analog to digital converter ADC supported by Analyst for data acquisition Documentation for the Coulochem III detector includes e Coulochem III User s Guide Part Number 70 4789 desig
6. and editing is done in exactly the same way as described in the sections above To view or modify the configuration parameters select Coulochem III Configuration from the dialog s Instrument menu To view or modify the Thermal Organizer parameters select Thermal Organizer from the dialog s Instrument menu In either case a new sub dialog will appear with all of the same parameters as are found on the main driver interface Remember to click the OK button when closing the sub dialog if you wish to save any changes made Coulochem III Analyst Driver Version 1 0 37 User s Manual Chapter 4 To attach the modified method data to the original Analyst file click the Attach Changes to Analyst button at the bottom of the editing dialog or select File gt Attach Changes to Analyst File from the dialog s menu Click the Cancel button if you decide not to change the original Analyst file A WARNING It is possible to modify the actively running acquisition method using this dialog However when a sample is submitted to the sample queue the then current values of the method parameters are stored in the queue s sample parameters Modifying a method after samples have been added to the queue will not change the method parameters in the existing queue samples from their original values but samples added to the queue after the method change will show the changes Further once the acquisition method parameters have been loaded into the Co
7. provides a description of driver functions that can be accessed while the Coulochem III detector is not actively running an acquisition method Acquiring Data with the Coulochem III Driver Chapter 6 describes the procedure for performing a sample data acquisition run with the Analyst program and Coulochem III driver Application Examples Chapter 7 provides some useful examples of how the Coulochem III detector can be combined with a mass spectrometer to detect and identify various compounds of interest Updating the Coulochem III Firmware Appendix A describes the procedure for updating the Coulochem III firmware Parameter Ranges and Default Values Appendix B Tabulates all of the program parameters used in the Coulochem III driver along with their range of values and default values 1 3 Getting Help The Coulochem III driver provides many ways to obtain help about a particular parameter topic or feature while using the program These include Help buttons a Help Navigator tool Help menu items and access to the entire manual as an Adobe PDF file 1 3 1 1 3 2 Help Buttons and the Coulochem Ill Help File Most windows and dialog boxes in the driver program provide a Help button for obtaining help Clicking the Help button automatically opens the Coulochem III Analyst Driver Help file to a topic that is relevant to the window or dialog box For example clicking the Help button on the main driver window while
8. Channel value is selected by drop list but may be grayed out if not applicable to the line s event Value entries may be made by drop list or manually depending on the event and may be grayed out if not applicable to the line s event as ESA Coulochem III EC Detector File Instrument Help Detector Setup Pre Run Timed Events Auto Zero Time Event Channel Value 1 00 Gain Range 1 10 p Apply Gain H 1 2 200 Potential Guard 400 Q Cells On 3 3 00 Auto Zero Cells Off 4 5 400 Cells Off Apply Settings 5 00 Event Mark 5 00 Select to add event Thermal Organizer v Activate Actual Temp 251 4E Set Temp 2 m Current ST 270 4E sT 2 Apply Lemp Sort Events Clear All Heb Acquisition Status Ide Logged onto Analyst Connected to Cclll on USB Idle time 2080 min Figure 4 8 The Timed Events Tab with Event Examples The total duration of Coulochem III data acquisition is determined by the greater of the time of the last event after sorting and the MS acquisition time 32 Coulochem III Analyst Driver Version 1 0 User s Manual Creating and Modifying Acquisition Methods Each timed event consists of a time point and an event type Some events may also include an event channel as well as an event value Timed events are tabulated in the Timed Events grid The default grid has no events To add an event simply click in the cell that reads Select to add event the
9. EC signal using the ADC Set this value above the highest current expected for the analyte of interest over the course of the experiment Default is 100 uA Signal output voltage This is the maximum analog signal the detector will output to a strip chart recorder or ADC unit Select from values of 1 V 100 mV 100 mV and 1 V Default is 1 V Baseline offset This is the ratio by which a flat signal baseline is offset on a chart recorder or ADC unit expressed as a of full scale Offset values from 50 to 50 are allowed Default is 0 Post Run Clean Cell Check this box if the selected channel is to be used in post run cleaning A clean cell period is used to help desorb products that may have a high affinity for the electrodes and is typically used with potentials that are higher than those normally used in detection Clean cell is normally used only when indicated typically when the signal for a certain compound begins to decline from its normally observed value after repetitive injections Coulochem III Analyst Driver Version 1 0 User s Manual Creating and Modifying Acquisition Methods Cell potential clean cell Select the post run potential for cleaning the selected channel electrode The range for clean cell potentials is 2000 mV to 2000 mV When using a clean cell cycle use a potential that is higher than that normally used for the compound s being detected Clean time Clean times may be selected from a
10. Manual Chapter 7 Oxidation at 1000 mV produces 3 additional oxidation products Figure 7 3 with peaks at m z 299 8 D 356 8 A and 388 8 B EC Potential 1000mV TIC ESI Time min 120 120 120 100 A XIC m z 356 8 100 B XIC m z 388 8 10 C XIC m z 358 8 80 80 80 ON CB e P 60 3 J 60 Y e0 D o Anp du 40 f Y 40 ba 40 20 20 20 O 0 0 0 100 200 300 400 100 200 300 400 100 200 300 400 mz mz m z 120 120 1 D XIC m z 299 8 100 E XIC m z 372 8 80 80 60 60 OC O ud d ig i S in 20 20 3 0 0 100 200 300 400 100 200 300 400 m z m z Figure 7 3 Tamoxifen at a Cell Potential of 1000 mV This example demonstrates that the EC oxidation products for a given compound often coincide with metabolites observed in biological assays The Coulochem III Analyst driver allowed for automated analysis of tamoxifen using various EC cell potential The simplicity and speed of on line EC LC MS often provides an effective means of characterizing some of the many unknown metabolites encountered in multivariate profiling studies 54 Coulochem III Analyst Driver Version 1 0 User s Manual Application Examples 7 4 Carbohydrate Detection Pulsed Mode The following example illustrates the use of the Coulochem HI Analyst Driver for automated analysis in which a dynamic pulsed potential EC waveform is used In this case the pulsed waveform is used with a gold
11. ionization for mass tagging and protein cleavage and to study the susceptibility of compounds to oxidation or reduction the nature of products and short lived intermediates and the associated reaction pathways These applications are of interest for generating and characterizing products that often correspond to biological metabolites degradants or reactive species of interest The use of EC in parallel with MS provides complementary orthogonal detection with high sensitivity and selectivity for redox active species Typical applications are DC detection of antioxidants and markers of oxidative stress and pulsed detection of carbohydrates Each example tabulates the conditions for the liquid chromatography LC setup Coulochem III EC setup and mass spectrometer MS setup Coulochem III Analyst Driver Version 1 0 49 User s Manual Chapter 7 7 2 Assisted lonization DC Mode Many neutral or non polar compounds do not ionize very readily However oxidative derivatives of electrochemically active compounds may be more readily ionizable In this example the Coulochem III is used to control an EC reactor cell placed in series upstream with an electrospray ion ESI source The EC cell is used to convert solution phase neutral species of butylated hydroxyanisole BHA to those that are more readily ionized for ESI MS analysis LC Conditions e Mobile phase 50 MeOH 20 mM NHzAcetate e Flow rate 0 1 ml min e Injection 10 ul B
12. on the Coulochem III and the wide end to an available USB port on the PC 4 Close all running software applications Then insert the Corona Coulochem Update Utility CD into your CD ROM drive The installation program should start automatically unless CD auto run is disabled on the PC If it does not start automatically click Start gt Run then type drive setup where drive is the drive letter of your CD ROM drive e g D setup then click OK 5 When the installation program starts click the Install button This will automatically install the Corona Coulochem Firmware Update Utility program files in the Coulochem III Analyst Driver Version 1 0 57 User s Manual Appendix A C Program Files ESA Biosciences folder It will also ensure that the ESA USB driver is properly installed 6 When finished click the Finish button 7 Remove the installation CD and store it in a safe place A 3 Updating the Firmware with the Update Utility 1 Ensure that the USB cable is properly connected as described in the previous section 2 Turn on the Coulochem III and allow it to complete its power up tests 3 Start the update utility by selecting Start gt All Programs gt Corona Coulochem Firmware Update Utility gt Corona Coulochem Firmware Update Utility from the Windows Start menu When operating on the Coulochem III the update utility will appear as in Figure A 1 below i Corona Coulochem Firmware Update Utility m m Message
13. set to 10 0V to 10 0V and the Mode must be set to Referenced Single Ended ADC Configuration A D Settings Alias Cell A D National Instruments 6032E PCI Board Polarity Range 10 0V to 10 0 wy Mode Referenced Single Ended Y ADC Channel Channel No 2 z V Channel Connected Channel Name Channel 2 Voltage 1 0 Simulation Simulation Mode T Figure 4 3 ADC Configuration for the NI 6032E PCI Board 22 Coulochem III Analyst Driver Version 1 0 User s Manual Creating and Modifying Acquisition Methods 11 Click OK to close the Create New Hardware Profile dialog 12 Enter a profile name in the Profile Name edit box then click OK A profile s instrument components can be examined by clicking the small box to the left of the profile name Figure 4 4 shows the profile MS with CcHI and ADC configured with the MS Coulochem III Detector and A D Converter Once configured the same profile can be used over and over again and profiles may be modified to accommodate changes in devices connected within a profile Multiple profiles can be created stored and reused but only one profile may be active at a time 13 Prior to building or using an acquisition method a profile must be activated Activate the newly created profile by selecting highlighting it and then clicking the Activate Profile button Since the Coulochem III device is a part of the new profile the Coulochem III drive
14. the following steps 1 il iii iv v Exit the Coulochem III Analyst driver by selecting File gt Exit from the menu From the main menu of the Coulochem III detector COULOCHEM MAIN MENU visible on the display press the SYSTEM button This should activate the SYSTEM SETUP MENU on the display If the SYSTEM button is inactive it may be necessary to turn the Coulochem III off and back on again Then repeat and continue from this step Press the NEXT button Press the DEFAULT button Press the NEXT button NOTE When properly configured both the Logged onto Analyst and Connected to CcIII indicators should appear on a dark green background as described in Section 3 2 An improper connection is indicated by a bright red background 18 Coulochem III Analyst Driver Version 1 0 User s Manual 4 Creating and Modifying Acquisition Methods 4 1 Overview An acquisition method consists of a set of parameters that describe the detector environment in which data acquisition is to occur as well as the duration and sequence of events that are to occur during the acquisition In a setting where both mass spectrometry and electrochemical detection are utilized acquisition method parameters must be assembled for both instrument environments The Analyst program manages all aspects of the mass spectrometry while the Coulochem III driver manages all aspects of electrochemical detection receiving status updates on the executi
15. the original shipping point transportation charges prepaid within the applicable warranty period set forth above and upon examination ESA determines to its satisfaction that such product was defective in material or workmanship ESA will at its option repair or replace the product or the defective part thereof or refund the original purchase price of the product The foregoing notwithstanding ESA will not be responsible for damage to any product resulting from misuse negligence or accident or resulting from repairs alterations or installation made by any person or firm not duly authorized by ESA in writing or for any damage to any cell assembly resulting from the flow being impeded If any cell assembly is used with control modules or potentiostats other than those manufactured by ESA this warranty shall be void B If at any time during the period ending ninety 90 days after delivery of any product to Buyer Buyer reports and documents any error in any software provided with such product and developed by ESA or any failure of any such software substantially to conform to ESA s software description that limits or prevents use of the software by Buyer ESA at its option will use reasonable efforts to correct any such error or failure will replace such software or will terminate Buyer s license to use the software and refund the price of the related product In connection with any such termination and refund Buyer will return the related pro
16. 52 AM Sample002 waiting DC 2ch with Guard New Batch DataSET1 Default x 2 2 2006 11 11 33 AM Sample003 0 0 Waiting DC 2ch with Guard New Batch DataSET1 Default Jplir x 2 2 2006 11 13 14 AM Sample004 0 0 Waiting DC 2ch with Guard New Batch DataSET1 Default Opl x 2 2 2006 11 14 55 AM Sample005 0 0 Waiting DC 2ch with Guard New Batch DataSET1 Default ing i Figure 6 1 A Simple Batch with 5 Samples Coulochem Ill Analyst Driver Version 1 0 43 User s Manual Chapter 6 Instructions for building acquisition batches and running the queue samples can be found in the Analyst manual and are no different when involving the Coulochem III detector and driver Refer to the manual for details 6 2 Viewing the Progress of a Coulochem Ill Acquisition As the Analyst program manages the acquisition of each sample in a batch it dispatches information at various times to each of the instruments or drivers in the active hardware profile including the Coulochem III driver Just prior to the start of the pre run phase Analyst provides the Coulochem III driver with all of the Coulochem III detector specific configuration and method data for the sample that is about to be run Analyst obtains this data from the acquisition method file and its attached Coulochem HI method data The Coulochem III then updates its interface with these values As the run begins its pre run phase the Coulochem III driver automatically switches to the Pre Run tab and begin
17. Acquisition Methods 4 4 1 1 DC Mode DC mode method parameters are illustrated in Figure 4 5 a ESA Coulochem III EC Detector File Instrument Help Detector Setup Pre Run Timed Events Channeli 325 2 0 10 p 100 mv n Baseline offset 10 DC Mode Cell potential Filter time constant Full scale gain range Signal output voltage Iv Post Run Clean Cell Channel 1 Cell potential 300 z n Clean time 0 0 Post clean cell delay f 00 Acquisition Status Idle 12 67 min Logged onto Analyst Idle time AutoZero Apply Gain Q Cells Qn Cells Off Apply Settings Thermal Organizer IV Activate Actual Temp 251 nt SetTemp 92 C Current ST 27 0 T sT 2 Apply Temp Help Connected to Celll on USB Figure 4 5 Detector Tab Parameters for DC Mode Note that when channel 2 and or the guard channel are enabled the same fields are used to enter parameters for channel 1 channel 2 and the guard cell Use the drop list control to indicate which channel s parameters you wish to view or modify Channel Drop List Select either Channel 1 Channel 2 or Guard Selections allowed depend on the system configuration parameters so for example Channel 2 will not appear in the list if the checkbox Enable channel 2 was not checked in the ESA Coulochem III Detector Configuration dialog see section 3 Coulochem Ill Analyst D
18. Analyst by selecting File gt Open from the Analyst menu then selecting Acquisition Method dam from the drop list Select the file from the file list 2 Click on Coulochem III Detector in the left panel of the Acquisition method display The Software Application Properties tab will display all of the Coulochem III configuration data pre run conditions thermal organizer Coulochem III Analyst Driver Version 1 0 35 User s Manual Chapter 4 settings mode parameters DC or Pulse and timed events for the method as illustrated in Figure 4 10 To print the Coulochem III method data right click anywhere in the tabulated data and select Print from the context menu Analyst Acquisition Method C Analyst Data Projects Wefault Acquisition Methods CC3 Verification DC 2 ek El Edit View Acquire Tools Explore Window Script Help x OE A IESE Acquire Mode x 8 Default 8m X ft xx GA Configure Acquisition method 3 A Security Configuration 25 Method 9 R 5 Mass Spec 1 675 min Ta GH Hardware Configuratio af amp Period 1 675 min X Report Template Editc 8 01 ESA Biosciences Coulochem Ill Configuration ap T Tr AJD Converter 0 0 mins Mode DC une l l E Coulochem III Detector Enable channel 2 yes Resolution Optimizatio Enable quard yes Quantitative Optimizati Enable thermal organizer yes E Manual Tuning Mark on autozero yes Acquire 1 NADA Melhod WA Pre run condi
19. Coulochem IIl Analyst Driver Version 1 0 User s Manual Starting and Configuring the Driver 3 3 Accessing the Coulochem Ill Driver Window The Coulochem III driver works alongside the Analyst program so normally both programs are open at the same time This will often cause the driver window to be hidden behind the Analyst program There are many simple ways to make the driver visible when this happens including any of the following e Click the ESA Coulochem III button on the Windows task bar at the bottom of the display e Double click the Coulochem III Detector item in Analyst s Navigation Bar This will not open another instance of the driver e Press Alt Tab from the keyboard and select the driver NOTE Although the normal procedure is to start Analyst first and then start the Coulochem ITI driver it is possible to run the Coulochem III Analyst driver without running Analyst first by double clicking the Coulochem III Analyst Driver icon on the desktop or selecting Coulochem III Analyst Driver from the Start gt All Programs menu under ESA Biosciences When the driver appears to be running without Analyst it is actually still connected to Analyst through a Windows service This service allows changes to be made to the driver configuration without having to start the Analyst program It is also possible to make changes to acquisition methods this way without having to open the Analyst program It is not possible howe
20. Full scale gain range RETE 32 Zt Signal output voltage Current ST 27 0 t Baseline offset Qo ST 2 Apply Temp Filter setting a m Acquisition Status Idle Idle time Figure 4 6 Detector Tab Parameters for Pulse Mode Acquisition Delay Select a value from 50 to 495 msec default 300 The value of this parameter is primarily relevant when monitoring the EC signal using the ADC 28 Coulochem III Analyst Driver Version 1 0 User s Manual Creating and Modifying Acquisition Methods E1 E4 Potentials Values from 2000 to 2000 mV are allowed Default values for potentials E1 thru E4 are El 200 mV E2 1000 mV E3 600 mV E4 100 mV E1 EA Pulse Widths The pulse width for E1 must be at least 5 msec greater than the acquisition delay Maximum value for all pulse widths is 1000 msec Minimum and default values are El AD 5 msec minimum 500 msec default E2 4 msec minimum 10 msec default E3 0 msec minimum 1 msec default E4 0 msec minimum 10 msec default Full scale gain range The full scale gain range runs from 10 pC to 10 mC in steps of 1 2 5 10 20 50 100 200 and 500 Set this value above the highest signal expected over the course of the experiment Signal output voltage Select from values of 1 V 100 mV 100 mV and 1 V Default is 1 V Baseline offset Offset values from 50 to 50 are allowed Default is 0 95 Filter setting Select None Low Med or High Use
21. HA 20 pg ml EC Conditions ESA Model 5021A Conditioning Reactor Cell Ground Decoupling Kit ESA P N 70 5971 connected between EC cell and MS Cell potential 600 mV Filter time constant 0 5 sec Full scale gain range 10 pA Signal output voltage 1V Baseline offset 0 e Pre equilibration 2 min autozero prior to run MS Setup e Electrospray positive ion mode e QI Scan 100 to 500 m z 50 Coulochem III Analyst Driver Version 1 0 User s Manual Application Examples Results 120 120 EC Potential 200mV EC Potential 600mV 100 Max intensity 10057 199 Max intensity 417280 80 m z OH 80 m z O 181 5 179 5 60 60 C CH3 X C CH3 40 40 20 OCH 20 OCH 0 0 1 fi hoc wl la 100 200 300 400 500 100 200 300 400 500 m z m z Figure 7 1 Results of Assisted Ionization Run with BHA The left panel in Figure 7 1 above shows an injection of 200ng of BHA with the EC cell at 200mV a potential at which no EC reaction is expected The spectrum is very low intensity with little evidence of protonated BHA m z 181 5 The right panel shows a similar injection with the EC cell potential at 600mV There is an intense peak at m z 179 5 representing an oxidized species derived from BHA This is a simple demonstration of the ability of EC to convert a solution phase neutral species to a more readily ionized species The ability to automate control of the EC cell potenti
22. I front panel display while the detector is running its self test sequence just after powering on Improvements to the software logic may make it advantageous to update the detector s firmware ESA provides a utility for updating the firmware logic on your Coulochem III The utility updates only the executable firmware It does not update system variables such as the serial number and baud rate and it will not affect your stored methods This chapter describes how to update the firmware using the Corona Coulochem Firmware Update Utility CD ESA P N 70 6968 which may be obtained from your ESA representative The CD uses the same basic utility for updating both the Coulochem III and the Corona CAD instruments The steps below detail both the installation and use of the utility A 2 Preparation and Installation of the Utility Ensure that the Coulochem III detector is turned off 2 The Corona Coulochem Firmware Update Utility cannot operate over the serial port it must use a USB port If the Coulochem III has already been connected and operated successfully through the USB port the required USB driver has already been installed If not it may be necessary to install the USB driver The utility will detect either condition for you 3 If not already connected through the USB port a USB cable will be required standard type A to B ESA P N 70 5713 is a 3 meter cable of this type Connect the small end of the cable to the USB Remote port
23. LVIGN S use Used pe gut eine edv i pro d RR VISTA eaten ti o X vdd tins 39 5 2 Startup and Shutdown Sequences oet nt rase ei isa d REIR REV piod 39 21241 Startup SCCM CMCC usc ceo ves quest te dea M e OU ed R E DAC 39 Coulochem III Analyst Driver Version 1 0 ix User s Manual Table of Contents 5 2 2 Shutdown SECON CE eo Acosta mtn n stood tus dedu citu dt eMe 39 5 3 Direct Couloehem III Detector CODBUOD sa ee dle rete t ac eal spe qudd tn 40 54 Thermal Organizer Controls ues ero ru rh aw neta ia T pe o vea ones 40 6 Acquiring Data with the Coulochem III Driver 43 OM CDVOPVIO oas edna ctu watt itae noie ue earns aa M EQ UNE 43 6 2 Viewing the Progress of a Coulochem III Acquisition eene 44 6 3 Aborting a Batch from the Driver Program sse 46 6 4 X Viewing the Coulochem III Electrochemical Data Profile and Method Properties 46 7 Application Examples 1er etate ner eet hoo een enint eee ueun o ha nua cde 49 W AXOYSLVIGW eee cates cated tete Mta edet diee ues tad e m EA Rep e 49 7 2 Assisted Tonization DC Modes CR RHEIN FARINE DURAN REN iaat 50 7 3 Metabolite Identification DC Mode eese tenete 51 7 4 Carbohydrate Detection Pulsed Mode sese 55 Appendix A Updating the Coulochem III Firmware 57 WM P
24. Manual
25. RANTY SHALL NOT BE ENLARGED OR OTHERWISE AFFECTED BY ESA S RENDERING OF TECHNICAL OR OTHER ADVICE OR SERVICE IN CONNECTION WITH THE SYSTEM OR THE LICENSED PROGRAM ESA SHALL NOT BE HELD RESPONSIBLE FOR THE PERFORMANCE OF OR OUTPUT OBTAINED FROM THE LICENSED PROGRAM AND OR THE SYSTEM NOR FOR ANY LIABILITY TO ANY PARTY ARISING OUT OF USE THEREOF OR USE OF ITEMS DESIGNED WITH THE SYSTEM OR THE LICENSED PROGRAM 7 LIMITATIONS ON REMEDIES ESA s liability in contract tort or otherwise arising out of or in connection with use of the Licensed Program and or the System any output thereof and or this EULA shall not exceed the amounts that Licensee has paid ESA or its authorized distributor hereunder An essential purpose of the limitation on remedies provided in this paragraph is the allocation of risks between Licensee and ESA IN NO EVENT SHALL ESA BE LIABLE FOR SPECIAL INCIDENTAL TORT CONSEQUENTIAL OR ANALOGOUS DAMAGES INCLUDING WITHOUT LIMITATION ANY DAMAGES RESULTING FROM LOSS OF USE LOSS OF DATA LOSS OF PROFITS LOSS OF GOODWILL OR LOSS OF BUSINESS ARISING OUT OF OR IN CONNECTION WITH THE PERFORMANCE OF THE LICENSED PROGRAM AND OR THE PERFORMANCE OF THE SYSTEM AND OR ESA S PERFORMANCE OF SERVICES EVEN IF ESA HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES 8 U S GOVERNMENT RESTRICTED RIGHTS If you are a government agency you acknowledge that the Licensed Program was developed at private expense and that the computer software compo
26. Sample 11 Mix 20ng FINAL of DataSET 1 wiff Max 100 0 18 52 Intensity 96 a Companion Software WB cul 440 Coulochem IIl Detector For Help press F1 User Name NBA1lnorm c Analyst Data ra Idle s Idle T Idle Time min Figure 6 5 Example Electrochemical Data Profiles in Analyst Bottom 2 Panels Coulochem Ill Analyst Driver Version 1 0 47 User s Manual Chapter 6 To view the method properties associated with the file highlight any plot and select Explore gt Show gt Show File Information or press Ctrl I on the keyboard Analyst displays a considerable amount of property data for the file including a section for Software Application Properties This section displays the Coulochem III method properties as shown in Figure 6 6 Analyst File Information for Sample 38 Sample005 of DataSET1 wiff He Edit view Tools Explore Window Script Help eus E explore mode Bloat Jex zx GH Configure 2 File Info Software Application Properties l EX Log Info T Display Name Coulochem Ill Detector Security Configuration ACQUERIOQ INO Identifier Key AASB68C0 8214 4BEF 8D5E 8E 1636427BAA Analog Digital Converte 5 GE Hardware Configuration Method Filename None Report Template E ditor Quant Info Period 1 Method Data Tune Resolution tables eR e Resolution Optimization Calibration tables ESA Biosciences Coulochem Ill Configuration Instrument Parameters Mode Key
27. al and change potential within a run via timeline and or between runs via different methods in an Analyst batch provides a significant amount of experimental flexibility 7 3 Metabolite Identification DC Mode Typical applications of serial EC MS involve on line generation and analysis of EC products using LC MS conditions that are typical of metabolic studies e g in vitro microsomal analysis Serial EC LC MS can thus be used with neat parent compound solutions for preliminary optimization of LC and MS conditions for subsequent metabolite analysis in biological samples By using identical conditions EC data may then be used as input to automated metabolite identification software i e Analyst Metabolite ID to aid in finding metabolites present in more complex biological matrices Furthermore when the data from an EC generated product corresponds to that of a biological metabolite the EC technique may then be viewed as a selective and rapid synthetic route to small quantities of this metabolite This can then be used to facilitate structural elucidation or more generally to produce additional chemical entities The following example shows the utility of the Coulochem III Analyst Driver for automated analysis of tamoxifen with an EC reactor cell controlled at different oxidative potentials for each injection Coulochem IIl Analyst Driver Version 1 0 51 User s Manual Chapter 7 LC Conditions e Column Shiseido C18 MG 3u
28. atus states in green as in Figure 3 1 Coulochem III Analyst Driver Version 1 0 13 User s Manual Chapter 3 Ru Acquisition Status Idle Logged onto Analyst Connected to Cclll on USB Idle time 0 30 min Coutlachem III Figure 3 1 Status Bar Showing Successful Connections During the self test sequence the Coulochem III connection status appears in a yellow field as in Figure 3 2 e Acquisition Status Idle Logged onto Analyst Idle time 0 36 min Cautechem III Figure 3 2 Status Bar During Coulochem III Self Test The appearance of either of the above status conditions indicates a successful connection with the Analyst program and the Coulochem III If either the Analyst or Coulochem III connection fails for any reason the failure is indicated by a red background as in Figure 3 3 14 eS Acquisition Status Idle Logged onto Analyst Not connected to Celll Idle time 1 04 min Cautechem III Figure 3 3 Status Bar Indicating a Failed Coulochem III Connection P WARNING After the communications port has been properly configured the Communications Error message box Figure 2 4 should no longer appear when starting up the driver If it should appear at a later time the warning should not be disregarded since it indicates a possible problem with communications between the detector and the PC Note that the problem could be as simple as forgetting to turn on the Coulochem III detector
29. button to change the Coulochem III s stored setpoint temperature to the value provided in the Set Temp field ST 2 Indicator lamp This indicator turns green whenever the thermal organizer s actual temperature is within 2 C of its setpoint temperature 4 4 5 Attaching the Method to an Analyst File The Coulochem III constituents of the data acquisition method consist of all of the parameters found on the Detector Setup tab Pre Run tab Timed Events tab and Thermal Organizer control group if active In order for these parameters to become part of an Analyst method they must be attached to an existing Analyst acquisition method dam file To attach the Coulochem III method parameters select File gt Attach Method to Analyst File and select the Analyst acquisition file from among the dam files listed Click Save to attach and save the modified method Note that an error message will appear if you enter a new file name and click the Save button The Coulochem III method data must be saved to an existing Analyst acquisition method Remember that without this step the Coulochem III method data will not be known by Analyst and will not be available to it 4 4 6 Viewing the Coulochem Ill Method from within Analyst Once the Coulochem III method data has been attached to an Analyst acquisition method file its parameters can be viewed from within Analyst To view the method follow these steps 1 Select and open the dam file in
30. cator Coulochem Ill Analyst Driver Version 1 0 User s Manual 45 Chapter 6 RECEN LLL Acquisition Status Clean cell Logged onto Analyst Connected to Cclll on USB Post run time 0 11 min Coulachem III Figure 6 4 Acquisition Status Bar During Post Run Clean Cell Phase After all 3 phases of the run have been completed the driver reverts to the idle state and displays idle time in the Status indicator in preparation for the next sample The 3 phase acquisition repeats for each remaining sample in the Analyst queue until all samples have been run 6 3 Aborting a Batch from the Driver Program A batch can be aborted from within the Coulochem III driver during any phase of the acquisition To stop the batch select Instrument Abort Batch from the driver menu Aborting a batch causes the driver to shift immediately to the post run clean cell phase of the currently executing run The driver also sends a signal to Analyst to stop any further execution of the batch 6 4 Viewing the Coulochem Ill Electrochemical Data Profile and Method Properties During the acquisition phase of a run the analog electrochemical data signal is sent to the A D Converter box converted to a digital stream of data and fed into the Analyst program for storage inside an Analyst To view the electrochemical profile s select File Open Data File from the Analyst main menu Then select the data file from the files listed and then th
31. detector display panel will show the current default mode settings This startup behavior occurs whether the Coulochem III driver is initiated from the icon on the desktop from the Companion Software items in Analyst or as the result of activating an Analyst hardware profile 5 2 2 Shutdown Sequence After the user attempts to close the Coulochem III driver by File Exit or other standard means the driver stops the Coulochem III detector unlocks the keypad and restores the detector display to the main menu It then stores the current configuration data before finally closing Coulochem III Analyst Driver Version 1 0 39 User s Manual Chapter 5 5 3 Direct Coulochem Ill Detector Control The main display surface of the Coulochem III driver provides a set of 5 buttons for directly controlling the Coulochem III detector These buttons are shown in Figure 5 1 below Auto Zero Apply Gain Qo Cells On Cells Off Apply Settings i Figure 5 1 The Driver s Detector Control Buttons and Indicators Auto Zero Click this button to autozero the detector Autozeroing adjusts the gain to produce a zero signal In a 2 channel setup in DC mode the autozero is applied across both channels The Auto Zero button is disabled during the pre run acquisition and post run clean cell phases of a run In DC mode post autozero stabilization can last up to 15 seconds or more while in Pulse mode it is nearly instantaneous During auto
32. duct to ESA forthwith upon request This warranty shall apply only to those portions of the software that were developed by ESA and that incorporate all program corrections and modifications if any delivered to Buyer It shall not apply to any error or failure due to machine error or to the misuse by or negligence of any person or entity other than ESA or to any software which is modified by any person or entity other than ESA C If Buyer shall fail to pay when due any portion of the purchase price of any product or any other payment required from Buyer to ESA whether under the contract or otherwise all warranties and remedies granted in this Section and all of ESA s warranties and obligations to service repair replace correct or otherwise remedy defects errors or failures under any other contract between Buyer and ESA may at ESA s option be terminated D THE WARRANTIES STATED HEREIN ARE THE ONLY WARRANTIES GIVEN BY ESA WITH RESPECT TO THE PRODUCTS AND THE SOFTWARE PROVIDED WITH THE PRODUCTS AND ARE GIVEN IN LIEU OF ALL OTHER WARRANTIES EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION ALL WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE BUYER S Coulochem Ill Analyst Driver Version 1 0 iii User s Manual Warranty EXCLUSIVE REMEDIES AND ESA S SOLE LIABILITY FOR ANY NON CONFORMITY OR DEFECT IN THE PRODUCTS OR SUCH SOFTWARE WILL BE THOSE EXPRESSED HEREIN E With respect to products sold to Buyer but n
33. e Value field at the specified time into the run Auto Zero This event implements an autozero at the specified time An autozero event must not occur at the end of the timed events Furthermore the next event following the autozero event must not occur before 15 sec 0 25 min have elapsed since the autozero These conditions are checked prior to attaching the method properties to an Analyst file a warning is provided if the conditions are not met Cells Off This event turns the cells off at the specified time Once turned off cells cannot be turned back on by another timed event There is no Cells On Coulochem III Analyst Driver Version 1 0 33 User s Manual Chapter 4 34 4 4 4 event However cells can be turned back on manually by clicking the Cells On button on the driver window Event Mark This event sets a marker spike in the raw analog data at the specified time into the run In DC mode the event can indicate which channel or channels are to receive the marker Marker height depends on the value provided in the Coulochem III Configuration dialog Section 3 4 Potential This DC mode only event changes the cell potential to a new value given by the Value field in mV on the channel selected from the list box in the Channel field at the specified time into the run Click the Sort Events button to sort the events by time Click the Clear All button to clear all events from the Timed Events grid Thermal Orga
34. e available If unsure start with COMI If this causes problems later return to the configuration and try COM2 COM3 16 Coulochem III Analyst Driver Version 1 0 User s Manual Starting and Configuring the Driver and COMA in order e Event marker height During the course of a data acquisition run the user may specify that a marker be placed in the acquisition data to designate a particular event such as an autozero The event marker height is the height of this marker designated as a percent of the full scale data output range Select a value between 20 and 20 full scale e Enable channel 2 Check this box if the Coulochem III detector has been fitted with a second channel cell and you wish to use the cell in your next first acquisition Most applications that use an EC cell primarily as a reactor typically only require a single channel channel 1 Many applications of EC for detection and quantitation use dual EC cells to obtain higher selectivity for example through oxidation reduction or oxidative screening e Enable guard channel Check this box if the Coulochem III detector has been fitted with a guard cell and you wish to use the guard channel in your next first acquisition The signal from the guard channel cannot be monitored using an ADC e Enable thermal organizer Check this box if the Coulochem III detector has been fitted with an optional thermal organizer ESA P N 70 5499T and you wish to display the the
35. e sample name and click OK Analyst opens the file and displays the total ion chromatogram TIC data by default To view the electrochemical profile s right click in the TIC plot and select Show ADC Data from the context menu Select A D Converter Channel 1 and click OK then repeat to display channel 2 if using 2 channels Figure 6 5 shows an example of electrochemical data profiles for 2 channels 46 Coulochem IIl Analyst Driver Version 1 0 User s Manual Acquiring Data with the Coulochem lll Driver Analyst Channel 2 from Sample 11 Mix 20ng FINAL of DataSET1 wiff BAE Hte Edit view Tools Explore Window Script Help EEA amp a p Bs K 2 C Explore Mode E E Defaut ES ja X I8 7 C1 ER EE Tic of FEMS from Sample 11 Mix 20ng FINAL of DataSET4 witf Turbo Spray Max 5 2e7 ops GH Configure amp Security Configuration 52e7 GH Hardware Configuration 4 07 amp Report Template Editor ill Tune 2 0e7 088459 272 Intensity cps v Resolution Optimization Quantitative Optimization Time min Manual Tuning Wl Channel 1 from Sample 11 Mix 20ng FINAL of DataSET 1 wiff M J Acquire 1 zol IDA Method Wizard i TE Build Acquisition Method Build Acquisition Batch Intensity 96 ZZ Express View Explore 1 Bae 11 12 13 14 16 Open Data File G Open Compound Database EZ Quantitate 100 E Build Quantitation Method N amp Quantitation Wizard Review Results Table Channel 2 from
36. ed Text Enable channel 2 Manual Tuning Enable guard 2 Acquire 1 Enable thermal organizer IDA Method Wizard Mark on autozero JA Quantitative Optimization TE Build Acquisition Method Pre run conditions Build Acquisition Batch Enable pre equilibration Pre equilibration time Autozero prior to run Explore 1 Wait for thermal organizer ig Open Data File amp Open Compound Database Thermal organizer M Organizer set temp E Quaniitale Activate thermal organizer E Build Quantitation Method X Quantitation Wizard DC Mode Parameters Channel 1 c Potential 123 mV Havia Mente Lebe Filter constant 0 5 sec amp Companion Software Gain range 2uA 5B ccu aao Output voltage 1 lt i gt ZZ Express View Coulochem Ill Detector For Help press F1 User Name NBA1 norm C l nalyst Data ra Idle Eg Idle TT Idle Figure 6 6 Coulochem III Method Properties Displayed in a Data File s Software Application Properties 48 Coulochem IIl Analyst Driver Version 1 0 User s Manual 7 Application Examples T 1 Overview This chapter provides examples of the use of the Coulochem III detector to control electrochemical EC cells with fixed DC or dynamic pulsed potential waveforms Cells can be used as reactors in series upstream of the MS ion source and or as detectors in parallel via flow split with MS Common applications are for on line derivatization e g to assist MS
37. esa MAGELLAN BIOSCIENCES BIOSCIENCES Coulochem III Analyst Driver Version 1 0 SOFTWARE APPLICATION MANUAL ESA Biosciences Inc 22 Alpha Road Chelmsford MA 01824 4171 U S A Telephone 978 250 7000 Fax 978 250 7090 www esainc com P N 70 6989 Rev A Coulochem is a registered trademark of ESA Biosciences Inc ANALYST is a registered trademark owned by Applera Corporation or its subsidiaries in the United States and certain other countries Microsoft and Windows are registered trademarks of Microsoft Corporation 2006 ESA Biosciences Inc All rights reserved No part of this manual may be reproduced or transmitted in any form or by any means without the written permission of ESA Biosciences Inc Warranty A ESA Biosciences Inc ESA warrants to Buyer that each product manufactured by ESA will be free from defects in material and workmanship in normal use from the date of delivery to Buyer as the original purchaser for the following periods instruments and equipment one year expendable items such as cells sensors and electrodes 90 days This warranty does not cover and no warranty is provided for consumables and parts that by their nature are normally required to be replaced periodically consistent with normal maintenance including without limitation fuses tubing pump piston seals injector rotors check valves and filters If any product covered by this warranty is returned to
38. eturn to ESA or destroy all copies of the Licensed Program 5 LICENSEE S RESPONSIBILITIES FOR SELECTION AND USE OF THE LICENSED PROGRAM AND THE SYSTEM The System and the Licensed Program when used together are a tool that ESA hopes you will find useful in your efforts to test and analyze chemicals HOWEVER ESA DOES NOT WARRANT THAT THE SYSTEM AND OR THE LICENSED PROGRAM IS SUITABLE FOR ANY SPECIFIC TASK OR IS ACCURATE TO ANY SPECIFIC DEGREE You are responsible for the supervision management and control of the use of the System and the Licensed Program and output thereof including but not limited to a selection of the System and the Licensed Program to achieve your intended results b determining the appropriate uses of the System and the Licensed Program in your business c establishing adequate independent procedures for testing the accuracy of the System and the Licensed Program and d establishing adequate backup to obtain accurate data and prevent the loss of data in the event of a malfunction of the System or the Licensed Program 6 LIMITED WARRANTY Subject to the other provisions in this EULA ESA warrants that for a period of 90 days from receipt by you the Licensed Program will conform in all material respects to the user documentation furnished to you ESA s sole responsibility under this warranty will be at its option 1 to use reasonable efforts to correct documented errors in said Licensed Program that are reported to
39. ew minutes is normally used when transitioning from high to low or low to high potentials between samples and methods Larger differences in potentials would indicate longer stabilization times Coulochem III Analyst Driver Version 1 0 User s Manual Creating and Modifying Acquisition Methods Autozero prior to run If this option is checked the Coulochem III detector will perform an autozero at the end of the pre run interval The pre run interval will automatically be extended as necessary in order to ensure that at least 15 seconds expire for proper detector stabilization after the autozero Wait for Thermal Organizer Checking this option ensures that the pre run period will not end until the thermal organizer temperature has reached within 2 C of its setpoint If Autozero prior to run is also checked the autozero is performed only after the thermal organizer has reached its setpoint Pre run options are available for both DC and Pulse mode operation of the Coulochem III detector Coulochem Ill Analyst Driver Version 1 0 31 User s Manual Chapter 4 4 4 3 Timed Events Tab The Timed Events tab Figure 4 8 lets the user specify various actions to be performed during data acquisition These actions include adjusting the gain range adjusting the cell potentials DC mode only performing an autozero turning cells off placing an event marker in the data Events are entered by selection from a drop list
40. ion file from among the dam files listed It does not have to be the same file that was previously read Click Save to attach and save the modified method data 36 Coulochem III Analyst Driver Version 1 0 User s Manual Creating and Modifying Acquisition Methods 4 6 Modifying a Coulochem Ill Method While Acquiring Data During active data acquisition the parameters of the actively running Coulochem III method are loaded and displayed in the Analyst driver window and tabs All fields are disabled in order to prevent modification during a run However it is still possible to modify any acquisition method during acquisition a separate menu item and dialog are available for this purpose To modify an existing attached method select File gt Edit Method from Analyst File from the driver menu select the file from the list and click Open The method editing dialog shown in Figure 4 11 will appear Edit Method CC3 Verification DC 2 dam File Instrument Detector Setup Pre Run Timed Events DC Mode Channeli Cell potential 123 E Filter time constant 0 5 v Full scale gain range 2 p X Signal output voltage 1 Baseline offset 5 Iv Post Run Clean Cell Channel 1 Cell potential 1111 Clean time 10 0 Post clean cell delay 9 20 H Attach Changes to Analyst Cancel Help Figure 4 11 Method Editing Dialog This dialog has all of the same tabs as the main driver window
41. it within the foregoing warranty period or 2 to refund the fees paid for the LICENSE to the Licensed Program ESA does not warrant that the Licensed Program will be error free or that all program errors will be corrected AII requests for warranty assistance shall be directed to Customer Service ESA Biosciences Inc 22 Alpha Road Chelmsford MA 01824 4171 Asserted errors must be documented by you to ESA detailing the error describing how to recreate the error and including if available sample output EXCEPTIONS ESA s warranty does not apply insofar as a any Licensed Program is subjected to misuse neglect accident or exposure to environmental conditions beyond those specified by the documentation b claims result from acts or omissions caused by persons other than ESA or from products material or software not provided by ESA or c you use a version of the Licensed Program that does not include all required updates provided by ESA NO OTHER WARRANTIES THE EXPRESS WARRANTY SET FORTH IN THIS ARTICLE 6 IS THE ONLY WARRANTY GIVEN BY ESA WITH RESPECT TO THE LICENSED PROGRAM EITHER BY ITSELF OR IN CONJUNCTION WITH THE SYSTEM ESA MAKES NO OTHER WARRANTIES EXPRESS IMPLIED OR ARISING vi Coulochem III Analyst Driver Version 1 0 User s Manual License Agreement BY CUSTOM OR TRADE USAGE AND SPECIFICALLY DISCLAIMS THE IMPLIED WARRANTIES OF NONINFRINGEMENT MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE SAID EXPRESS WAR
42. l ensure that the Coulochem III Analyst driver is configured to match the detector configuration This chapter discusses how to set up the initial configuration of the driver including the port connection and hardware cell options Once configured changes to the configuration should seldom be needed 3 2 Starting the Coulochem Ill Analyst Driver 1 Turn on the Coulochem III detector and allow ample time for it to complete its startup routine 2 Start the Analyst program If required enter a login name and password required when running in Mixed mode 3 Ensure that the Navigation Bar is visible Select View gt Navigation Bar if not 4 Double click on the Coulochem III Detector item in the Navigation Bar This should start the Coulochem III Analyst driver which will display its splash screen as shown in the previous chapter If running Analyst in Mixed mode it will be necessary to log on again prior to seeing the splash screen 5 After a few seconds the main driver window will appear 6 Ifa message box like the one shown in Figure 2 4 above appears click OK It may be possible to correct the problem in Section 3 3 but observe the warning note at the end of this section As soon as successful connections are made with both the Analyst program and the Coulochem III detector and the Coulochem III detector has completed its self test sequence the Status area at the bottom of the driver window displays the connection st
43. m 4 6 mm ID x 75 mm e Mobile phase gradient o A 1 ACN 20 mM NH Acetate o B 80 ACN 20 mM NH Acetate Flow Time B ml min 0 00 20 1 10 00 100 1 12 00 100 1 12 01 20 1 15 00 20 1 e Flow rate 1 ml min e Injection 10 ul Tamoxifen 20 ug ml 200 ng e After diode array flow splitter ESA P N 70 6228 adjusted to allow 200 ul into the MS EC Conditions ESA Model 5021A Conditioning Reactor Cell Ground Decoupling Kit ESA P N 70 5971 connected between EC cell and MS Cell potential 500 mV repeat at 1000 mV Filter time constant 0 5 sec Full scale gain range 10 pA Signal output voltage 1V Baseline offset 0 Pre equilibration 2 min autozero prior to run MS Setup e Electrospray positive ion mode e Enhanced MS Scan 100 to 500 m z 52 Coulochem III Analyst Driver Version 1 0 User s Manual Application Examples Results Figure 7 2 shows results for 200 ng Tamoxifen with the EC cell at a potential of 500 mV Two forms of Tamoxifen are primarily observed at m z 358 8 A and 372 8 B 4e 6 EC Potential 500mV 3e 6 TIC 2e 6 cps 1e 6 0 2 4 6 8 10 12 14 Time min 120 120 1001 A XIC m z 358 8 100 B XIC m z 372 80 CI m 80 Ca e l0 7 60 AI A 40 40 O 20 v 20 io0 200 300 400 0 i00 200 300 400 m z m z Figure 7 2 Tamoxifen at a Cell Potential of 500 mV Coulochem Ill Analyst Driver Version 1 0 53 User s
44. minimum of 1 sec to a maximum of 120 sec Typical clean cell times are 30 60 sec Post clean cell delay This is the amount of time given to allow the detector to stabilize after the clean cell period Delay times may be selected in the range 0 1 to 10 0 min Typical clean cell delay time is 0 5 min or less when using the cell only as a reactor A longer delay typically 2 minutes is required when using EC for sensitive detection and depends largely on the gain range setting 4 4 1 2 Pulse Mode Pulse mode method parameters are illustrated in Figure 4 6 Pulse mode is typically used for EC detection of specific analyte classes such as carbohydrates that are not readily measured using DC techniques In this case the parameter settings are typically based on literature values e g ESA Biosciences Application Notes This mode however provides a great deal of flexibility to experiment with dynamic waveforms to effect reactions not readily observed with DC for MS study Coulochem Ill Analyst Driver Version 1 0 27 User s Manual Chapter 4 as ESA Coulochem III EC Detector File Instrument Help Detector Setup Pre Run Timed Events Pulse Mode Auto Zero Acquisition delay 300 Apply Gain E1 potential 200 E Pulse width 500 E2 potential 1 000 Pulse width Apply Settings E3 potential Pulse width Thermal Organizer ica IV Activate potential Actual Temp 251 3E Pulse width
45. n choose an event from the drop list Click in either adjacent cell to activate the event Edit the event s time in minutes and if a cell has either a channel option or an associated value select or edit the appropriate channel or value cell Cells with no expected entries are displayed in light gray Depending on personal preference timed events can be entered using primarily the mouse or the keyboard When primarily using the keyboard each time the Tab key is pressed the focus moves to the next cell in the row Pressing the Tab key at the end of a row advances the focus to the Time column of the next row Shift Tab reverses the flow When a cell has the focus its contents can be edited by using the up and down arrow for drop lists Event Channel and Gain Range Value or by editing directly Time and Potential Value Time Specifies the time of the event in minutes from the beginning of EC data acquisition Event Specifies the event to occur at the specified time Channel Specifies the channel or channels on which the event is to apply The channel is irrelevant in Pulse mode In DC mode the channel can be specified for the Gain Range Potential and Event Mark events only Value Specifies the value for the event This applies only to the Gain Range and Potential DC mode only events The following events can be designated Gain Range This event changes the gain range to a new value selected from the drop list in th
46. n the Ready to Install dialog appears click Install to start the driver installation process 6 The installer will add the Coulochem III Analyst driver files to the folder C Program Files ESA Biosciences Coulochem III Analyst Driver It will then automatically install the USB driver for the Coulochem III detector 7 When finished click the Finish button 8 Remove the installation CD and store it in a safe place After installation an icon will appear on the desktop for starting the Coulochem III driver with or without first starting Analyst and a menu entry will appear in the Start gt All Programs menu under ESA Biosciences 2 4 Hardware Configuration The Coulochem III detector can be customized to accommodate a wide variety of analytical needs The Coulochem III for MS is the standard configuration for use with ABI s mass spectrometers This platform allows the use of the Coulochem III module to control electrochemical EC cells with fixed DC or dynamic pulsed potential waveforms Cells can be used as reactors in series upstream of the MS ion source and or as detectors in parallel via flow split with MS Serial EC LC MS configurations allow MS study of EC reactions and products while parallel EC with MS provides complementary and highly sensitive detection of redox active species Some examples of these applications are provided in Chapter 7 of this manual For additional detail please contact your ESA Biosciences re
47. ned to provide sufficient information to allow the analyst to collect analytical data in DC mode DC Mode requires the use of the DC Potentiostat Board o Coulochem III Reference Manual Part Number 70 4779 provides a detailed discussion on the care and use of the detector including method development installation maintenance and advanced topics such as the use of Pulse Scan and Redox Modes Pulse and Scan Modes require the Pulse Scan Potentiostat Board 1 2 Contents of the Coulochem Ill Analyst Driver Software Application Manual This manual includes the following material e System Requirements and Installation Chapter 2 describes the minimum computer system requirements for operation of the driver software as well as the procedure for installing the driver and verifying the installation e Starting and Configuring the Driver Chapter 3 describes the procedure for starting the driver software and adjusting the configuration parameters to meet the system arrangement e Creating and Modifying Acquisition Methods Chapter 4 describes the procedure for creating and modifying the method files describing the set of Coulochem III Analyst Driver Version 1 0 1 User s Manual Chapter 1 parameters and series of events that make up the acquisition method This chapter also includes a description of how to add the Coulochem III driver to an Analyst Hardware Profile Basic Coulochem III Driver Functions Chapter 5
48. nent is provided to you subject to RESTRICTED RIGHTS Notwithstanding any other lease or license agreement that may pertain to or accompany the delivery of this restricted computer software the rights of the government regarding its use duplication reproduction or disclosure by the Government is subject to the restrictions set forth in subparagraph c 1 and 2 of the Commercial Computer Software Restricted Rights clause at FAR 52 227 19 Contractor manufacturer is ESA Biosciences Inc 22 Alpha Road Chelmsford MA 01824 4171 9 GENERAL The terms of this LICENSE shall be construed in accordance with the substantive laws of the Commonwealth of Massachusetts Without limiting the generality of the foregoing this EULA and such relations shall not be governed by the United Nations Convention on Contracts for the International Sale of Goods even if such Convention would otherwise be applicable in some respect to this license of software The English language text of this EULA shall be the authorized text for all purposes If you have any questions about this EULA you may contact Customer Service ESA Biosciences Inc at 978 250 7000 YOU ACKNOWLEDGE THAT YOU HAVE READ THIS EULA UNDERSTAND IT AND AGREE TO BE BOUND BY ITS TERMS AND CONDITIONS YOU FURTHER AGREE THAT IT IS THE COMPLETE AND EXCLUSIVE STATEMENT OF THE LICENSE AGREEMENT BETWEEN YOU AND ESA INC WHICH SUPERSEDES ANY PROPOSAL OR PRIOR AGREEMENT ORAL OR WRITTEN AND ANY OTHER
49. nge 100 p v Cells Off Pree eee T Click the Apply Settings button to Baseline offset 0 x Z change the Coulochem Ill detector settings to those currently set in the Detector Post Run Clean Cell Channel 1 Setup tab Pell nntential mit P Figure 1 2 Help Navigator Text for the Apply Settings button 1 3 8 Help Menu The Help menu provides direct access to the entire Coulochem III Analyst Driver Help file Select Help Contents to open the help file to its table of contents Select Help gt Index to open the help file to its index of terms Coulochem III Analyst Driver Version 1 0 3 User s Manual Chapter 1 1 3 4 The Coulochem III Analyst Driver Help file provides all of the text of this manual in an easy to navigate hypertext form You can use the Search feature to search for all information about a particular term or terms e g guard cell Entire Manual in Adobe PDF Format Select Help Open Manual in PDF Format to open the Adobe PDF version of this Software Manual Adobe Reader or Adobe Acrobat is required Coulochem III Analyst Driver Version 1 0 User s Manual 2 System Requirements and Installation 2 1 System Requirements The Coulochem III driver application has been designed and tested to operate under the minimum hardware and software specifications listed below Microsoft Windows based PC with Intel Pentium 4 processor operating at 1 5 GHz Microsoft Window
50. nizer Controls If the thermal organizer option is included in the Coulochem III system and this has been indicated in the Coulochem III Configuration dialog Section 3 4 controls for the thermal organizer will appear in the driver window Figure 4 9 Thermal Organizer IV Activate Actual Temp 25 1 ME Set Temp 32 4 te Curent ST 270 C ST 2 Apply Temp Figure 4 9 The Thermal Organizer Group Controls To activate the thermal organizer check the Activate checkbox When active the group of controls in the Thermal Organizer group box indicate actual and setpoint temperatures in Celsius The display is updated about every second The following describes each of the controls in the Thermal Organizer group Activate Check this checkbox to activate the thermal organizer Uncheck to disengage the organizer When disengaged the organizer has no effect on temperature control Actual Temp This is the actual temperature in the thermal organizer chamber updated every second Coulochem III Analyst Driver Version 1 0 User s Manual Creating and Modifying Acquisition Methods Current ST This is the current active setpoint temperature stored within the Coulochem III detector Set Temp This edit field allows the user to change the Coulochem III s stored setpoint temperature The Apply Temp button see below must be clicked in order for the new Set Temp to be applied to the Coulochem III Apply Temp button Click this
51. ommunications Error Communications with the Coulochem III were lost or could not be established Ensure that the Coulochem is on and that the serial or USB cable is properly connected Select Instrument Coulochem III Configuration From the main menu and check that the port type COM or USB matches the connecting cable Figure 2 4 Coulochem III Driver Communications Warning If this occurs click OK The message appears because the initial default configuration assumes that the detector is connected through the serial port This situation will be corrected during configuration of the driver in a later section 8 Confirm that the term Logged onto Analyst appears in a dark green background in the Acquisition section at the bottom of the driver window 9 Confirm that the term Connected to CcIII appears also in a dark green background If the term Not connected to CcIII appears in a bright red background check the serial cable connection If connected through a USB port ignore the warning the situation will be corrected in a later section Coulochem Ill Analyst Driver Version 1 0 11 User s Manual 3 Starting and Configuring the Driver 3 4 Overview Prior to using the Coulochem III Analyst driver to collect data the driver needs to be configured to match the actual Coulochem III detector configuration including the connection port The detector connections were considered in Chapter 2 In this chapter we wil
52. on of the mass spectrometer MS for synchronization Despite this separation of instrument environments all acquisition parameters are consolidated into a single method file an Analyst dam which is managed entirely by the Analyst program This necessitates a somewhat strict protocol by which the MS and electrochemical detection method details are to be entered in a new method file specifically a Create a Hardware Configuration Profile a profile in Analyst This profile specifies all of the MS instrument parameters as well as basic properties of connected devices like pumps and autosamplers The profile also provides the ability to specify that a Coulochem III detector is connected to the system Once configured the same profile can be used over and over again until it is necessary to change basic instrument or device parameters Multiple profiles can be created stored and reused but only one profile may be active at a time b Build an acquisition method within Analyst This method initially will contain all parameters to be used for MS control The method is saved as a dam file by Analyst c Build a Coulochem III method This method details the parameters to be used and activities to be run on the Coulochem III detector Coulochem III method parameters can only be set within the Coulochem III Analyst driver d Attach the Coulochem III method data to the Analyst method data file Without this step the Coulochem III me
53. onfiguration item in the Navigation Bar This opens Analyst s Hardware Configuration Editor dialog 2 Click the New Profile button to open Analyst s Create New Hardware Profile dialog 3 Click the Add Device button 4 Adda Mass Spectrometer device to the profile as well as any pumps ovens autosamplers and other devices that you may require by selecting from the Device Type drop list then the Devices list and clicking OK Refer to the Analyst program manual for specific details on adding and setting up these devices The Coulochem III module and ADC card additions are described in the next steps 5 Add the Coulochem III detector to the profile by clicking the Add Device button and then selecting Software Application from the Device Type drop list Highlight Software Application from the Devices list and click OK Figure 4 1 shows the Create New Hardware Profile dialog for a simple profile just after adding the Coulochem III driver as a Software Application 20 Coulochem III Analyst Driver Version 1 0 User s Manual Creating and Modifying Acquisition Methods Create New Hardware Profile Profile Name MS with Celll Devices in current profile Mass Spectrometer 0 on GPIB Board 1 Add Device Software Application not configured 0 Delete Device Setup Device ccn Heb Figure 4 1 Hardware Profile with Coulochem III Detector Unconfigured 6 Highlight the Software Application text then click
54. ot install or use the Licensed Program ESA provides the Licensed Program to you the end user hereinafter referred to as Licensee and you Licensee accept the Licensed Program on the following terms and conditions 1 DEFINITIONS As used herein Licensed Program means the computer software associated media printed materials and online or electric documentation provided with the System and System means a Coulochem HI Detector and accompanying equipment supplied by ESA to you Licensee 2 GRANT OF LICENSE ESA grants to you Licensee a nonexclusive non transferable LICENSE to use a copy of the Licensed Program on a System in accordance with this EULA 3 OWNERSHIP OF THE LICENSED PROGRAM ESA owns and will retain all title copyright trademark and other proprietary rights in and to the Licensed Program This LICENSE is not a sale of the Licensed Program or any copy of it You the Licensee obtain only such rights as are provided in this EULA You understand and agree as follows You may load and use the Licensed Program on a System and one 1 additional independent computer for processing data produced with the System in accordance with the documentation provided therewith You may make one backup copy of the Licensed Program for archival purposes as permitted by the United States Copyright Act You must reproduce and include the copyright notice with the backup copy You may not reverse compile reverse assemble reverse enginee
55. ot manufactured or in the case of software not developed by ESA ESA MAKES NO WARRANTY OF ANY KIND EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION ANY WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE but ESA will make available to Buyer to the extent permitted by law and relevant contracts the warranties of the manufacturer of the relevant product or the developer of the software F This warranty shall be governed by and construed and enforced in accordance with the substantive laws of the Commonwealth of Massachusetts G This warranty shall be non transferable and shall run to the benefit of the original purchaser only iv Coulochem III Analyst Driver Version 1 0 User s Manual License Agreement ESA BIOSCIENCES INC Coulochem III Analyst Driver Software END USER LICENSE AGREEMENT IMPORTANT READ CAREFULLY THE FOLLOWING TERMS AND CONDITIONS BEFORE INSTALLING THIS SOFTWARE INSTALLING AND OPERATING THE SOFTWARE ON THE EQUIPMENT INDICATES YOUR ACCEPTANCE OF THESE TERMS AND CONDITIONS This ESA Biosciences Inc End User License Agreement EULA is a legal agreement between you either an individual or a single entity and ESA Biosciences Inc ESA for the ESA software product s identified in Section 1 below By installing copying or otherwise using the Licensed Program as hereinafter defined you agree to be bound by the terms of this EULA If you do not agree to the terms of this EULA do n
56. presentative The Coulochem III for MS ESA P N 70 6045A includes the Controller with DC and Pulse Mode capability the Model 5021A EC cell reactor cell with ca 16uL swept volume Model 5030 EC cell reactor cell with ca 3uL swept volume cables and Ground Decoupling Kit Cells and optional accessories include Model 5021A Conditioning Reactor Cell ESA P N 70 6068 Model 5030 Low Volume Reactor Cell ESA P N 70 6069 Model 5010A Dual Analytical Cell sss ESA P N 70 5560 Model 5040 Analytical Cell with Au Target ESA P N 55 0185 Adjustable Analytical Flow Splitter ESA P N 70 6337 Thermally Controlled Cell Organizer Module ESA P N 70 5499TA Coulochem III Analyst Driver Version 1 0 7 User s Manual Chapter 2 To ensure proper grounding it is essential that a Ground Decoupler Kit ESA P N 70 5971 be used for connections between the mass spectrometer and the electrochemical cell s and modules 2 5 Connecting the Communications Cables The Coulochem III detector can be connected to the PC by either an RS232 serial cable or a USB cable USB is preferred and a USB connection is required in order to perform firmware updates see Appendix A If using a serial cable connect one end of the cable to the RS232 Remote port on the Coulochem III detector Fig
57. r modify or incorporate the Licensed Program in whole or in part in any other product or create derivative works based on all or any part of the Licensed Program You may not remove any copyright trademark proprietary rights disclaimer or warning notice included on or embedded in any part of the Licensed Program You may not sell license sublicense assign rent or otherwise transfer the Licensed Program without the written permission of ESA ESA will not withhold permission to transfer the LICENSE as long as Coulochem Ill Analyst Driver Version 1 0 V User s Manual License Agreement you permanently transfer the Licensed Program including all components and archival copies if any in conjunction with the System with which it was acquired and the person who wishes to obtain the System and the LICENSE agrees to the terms and conditions of this EULA You may not use the Licensed Program in any country other than that in which it was obtained or otherwise export the Licensed Program 4 TERM The term of this LICENSE will continue until ESA provides written consent to assignment or transfer of this LICENSE or until ESA terminates this LICENSE as provided herein ESA may terminate this LICENSE by giving written notice to you specifying any failure or default in the performance of any provisions of this LICENSE if you fail to cure said failure or default within 30 days after such notice Upon termination of this License you will promptly r
58. r should open soon after clicking the Activate Profile button Allow the driver to finish its splash screen sequence then click its Minimize button upper right corner to minimize its window to the Windows task bar 14 Click Close to close the Hardware Configuration Editor dialog Hardware Configuration Editor Hardware Profiles CC3 without MS New Profile A MassSpecOnly Edit Profile Mass Spectrometer 0 on GPIB Board 1 Software Application Coulochem II Detector 0 Deis Prue A D Converter NIDAQ16 0 Activate Profile Available Devices Help Figure 4 4 Example Configuration MS with CcIII and ADC Coulochem IIl Analyst Driver Version 1 0 23 User s Manual Chapter 4 4 3 Building an Analyst Acquisition Method All acquisition methods must be initiated in Analyst Begin building a method by double clicking the Build Acquisition Method item in the Acquire section of the Analyst Navigation Bar Set the parameters for the mass spectrometer according to the requirements of the experimental run Refer to the Analyst and MS manuals To set the A D Converter properties click on the A D Converter item in the left panel 1 In the Analog Digital Converter Properties tab adjust the Rate value to the desired sampling rate 2 Ensure that a check appears in the checkbox corresponding to each channel for which you wish to have Analyst collect and store data 3 Adjust the Interp
59. r s Manual Acquiring Data with the Coulochem lll Driver Note that during the entire run the Cells On and Cells Off buttons are the only controls that are accessible to the user All other user input is disabled a ESA Coulochem III EC Detector File Instrument Help Detector Setup Pre Run Timed Events H Time Event 1 1 00 2 125 3 150 4 1 75 2 00 2 25 2 50 2 75 3 00 325 Acquisition Gain Range Gain Range Gain Range Potential Potential Potential Event Mark Event Mark Event Mark Auto Zero Channel Value 1 Status Aemuing 1 58 Run time 100 pA 500 pA 500 pA 400 500 600 Cells On Cells Off Thermal Organizer 7 Actual Temp 25 1 C SetTemp 7 ec Cunentst 270 occ E Que i Heb Logged onto Analyst Connected to Celll on USB Figure 6 3 Driver Display During the Acquisition Phase of a Run After the last timed event has been processed the post run phase begins This phase involves cleaning the cells so the driver switches to the Detector Setup tab where the clean cell properties are displayed The phase lasts for as long as the longest combination of Clean time and Post clean cell delay for either channel or the guard cell that is enabled During the post run phase the Coulochem III driver displays the post run time in the Acquisition status region at the bottom of the driver window Figure 6 4 The post run phase is designated in blue in the Status indi
60. required for connecting to the detector 2 3 Installing the Coulochem Ill Analyst Driver To install the Coulochem III Analyst driver on your PC perform the following steps 1 Ensure that the Coulochem III detector is off or disconnected If on the detector could interfere with the installation of the USB driver 2 Close all running software applications Then insert the Coulochem III Analyst Driver CD into your CD ROM drive The installation program should start automatically unless CD auto run is disabled on the PC If it does not start automatically click Start gt Run then type drive setup where drive is the drive letter of your CD ROM drive e g D setup then click OK The InstallShield Wizard should appear as in Figure 2 1 below Coulochem III Analyst Driver InstallShield Wizard 7 Welcome to the InstallShield Wizard for y e S a Coulochem Ill Analyst Driver MAGELLAN BIOSCIENCES The InstallShield Wizard will install Coulochem III Analyst Driver on your computer To continue click Next Cancel Figure 2 1 Installation Introductory Page 3 Click the Next button This will display the License Agreement 6 Coulochem III Analyst Driver Version 1 0 User s Manual System Requirements and Installation 4 Read the License Agreement and print a copy if desired by clicking the Print button If you agree to the terms of the License Agreement select the I Accept button and click Next 5 Whe
61. reted Value Full Scale and Interpreted Unit values for each channel as required Note that clicking on the Coulochem III Detector item in the left panel displays an empty Software Application Properties tab After the Coulochem III method has been configured in the next section this tab will display all Coulochem III method related values Save and close the method using a file name that will make it easy to remember the method s purpose or content 4 4 Building a Coulochem Ill Method and Attaching to Analyst Display the Coulochem III driver by clicking the ESA Coulochem III task button on the Windows task bar If the driver does not appear on the task bar restart it by double clicking Coulochem III Detector in Analyst s Navigation Bar The Coulochem III driver window displays its method parameters in three different tabs Detector Setup Pre Run and Timed Events In addition if a thermal organizer is attached to the Coulochem III module and indicated in the configuration setup controls for activating the organizer and for setting its setpoint temperature are displayed on the window Note All of the method parameters and their ranges and default values are tabulated in Appendix B 4 4 1 Detector Setup Tab The Detector Setup tab displays the parameter settings for either DC mode or Pulse mode depending on the configuration set up earlier 24 Coulochem III Analyst Driver Version 1 0 User s Manual Creating and Modifying
62. river Version 1 0 User s Manual 25 Chapter 4 EN WARNING Remember to set potentials filter constants etc for all channels in the configuration Forgetting to set a channel s parameters may lead to unexpected experimental results 26 Cell potential Select the cell potential for the designated channel in mV The spin buttons in this field change the potential 10 mV at a time The up and down arrows on the keyboard have the same effect For more exacting control enter the required potential from the keyboard The range is 2000 to 2000 mV The choice of potentials depends on the oxidation reduction properties of the compounds being investigated Typically the choice of potentials is made after running a series of stepped potentials over multiple injections of the compounds of interest Filter time constant Values of 0 2 0 5 1 0 2 0 5 0 and 10 0 sec are allowed The value of this parameter is only relevant when monitoring the EC signal using the ADC A small filter constant provides the least smoothing of data Optimizing this parameter is primarily relevant when using the EC cell for highly sensitive detection In that case select the lowest filter constant needed to improve the signal to noise ratio Default is 5 0 sec Full scale gain range The full scale gain range runs from 10 pA to 1 mA in steps of 1 2 5 10 20 50 100 200 and 500 The value of this parameter is primarily relevant when monitoring the
63. rmal organizer properties in the main driver window Activation of the organizer is controlled in the main driver window e Enable event marker on autozero Check this box if you would like to have the driver automatically create a marker in the acquisition data whenever an autozero event occurs The autozero marker height is determined by the Event marker height set above Lock keypad This box is normally checked and is not accessible to the user The Set Defaults button can be clicked at any time to set all values to their hard default values See Appendix B for a table of all user accessible values along with default and range values 3 Click OK to accept the changes made to the configuration 4 Ifa change was made to the Comm port value ensure that the connection status in the Acquisition section of the driver window now indicates Connected to CcIII on Port where Port is either USB or one of the COM ports If the connection status appears as Coulochem III Analyst Driver Version 1 0 17 User s Manual Chapter 3 Not connected to CcIII in a bright red background try one of the following to fix the problem Ensure that the Coulochem III detector is on a b C d Ensure that the connection USB or serial cable is secure If using a serial connection try another COM port in the configuration Ifusing a serial connection check the serial port settings on the Coulochem III detector To do this perform
64. s XP Professional with Service Pack 2 SP2 installed Installation of the Microsoft NET runtime added automatically during program installation if not already present 1 GB RAM 40 GB hard drive less than 1 GB available space required for program installation Available serial port or USB port for Coulochem III detector connection Monitor with 1024 x 768 minimum pixel resolution Two button mouse Analyst software version 1 4 1 or later including April and June 2005 hotfixes If using Analyst version 1 4 1 the Analyst hotfixes for April and June 2005 must be installed in order for Analyst to work with the Coulochem III driver If necessary check with your Applied Biosystems MDS Sciex representative for information on how to obtain and implement these hotfixes 2 2 Additional Hardware Requirements RS232 serial cable ESA P N 70 1743 This cable is not required if connecting through the USB port Coulochem III firmware version 3 07 or later See Appendix A for instructions on updating the firmware Standard type A to B USB cable ESA P N 70 5713 Not required if connecting through the RS232 port but required if updating the firmware becomes necessary Coulochem III Analyst Driver Version 1 0 5 User s Manual Chapter 2 e Optional Analyst supported ADC for analog signal output to Analyst Obtained from National Instruments see your ABI representative Either an RS232 cable or a USB cable is
65. s to display the pre run time in the Acquisition status region at the bottom of the driver window Figure 6 2 The Pre run phase is also designated in yellow in the Status indicator in this region ORI Acquisition Status Penn Logged onto Analyst Connected to Celll on USB Pre run time 0 38 min Coutachem III Figure 6 2 Acquisition Status Bar During Pre Run Phase The duration of the pre run phase depends on the parameters activated and assigned in the Pre Run tab After the pre equilibration time designated in the Pre equilibrate for field the driver will extend the pre run phase by as much time as needed for the thermal organizer to reach within 2 C of its setpoint if activated and checked If checked the autozero is performed only after the Pre equilibrate for time has elapsed and the setpoint has been approached An additional 15 seconds is added post autozero to ensure adequate stabilization from the autozero event The acquisition phase begins immediately after the pre run phase The Coulochem III driver automatically switches to the Timed Events tab and begins to display the run time in the Acquisition status region at the bottom of the driver window Figure 6 3 The acquisition phase is designated in green in the Status indicator As the run progresses and as each timed event is processed in the timed events grid the last executed event is highlighted 44 Coulochem IIl Analyst Driver Version 1 0 Use
66. se eran 13 Sul OV CT VIG si osi ose past DN cou ies ER nd f tal D puel eR aesti s OAS 13 3 2 Starting the Coulochem III Analyst Driver eeeseeeeenenenee 13 3 3 Accessing the Coulochem III Driver Window sese 15 3 4 Configuring the Coulochem III Analyst Driver esee 16 4 Creating and Modifying Acquisition Methods 19 d CTV CRN Sees aedi D M a e aA ala UN A CE 19 4 2 Creating and Activating Profiles sio eee cutis ERI Ut dec ver ea oce eae 20 4 3 Building an Analyst Acquisition Method ssssssseeeeennees 24 4 4 Building a Coulochem III Method and Attaching to Analyst ssssss 24 44 1 Detector Setup FaDa osten at ce ee Depas cete oci Lec ue 24 442 Pre Run Eab i ossotoscteetoi ie mU ueneno e a led Aaa res AE E Ua RE AT eda 30 AAS Timed Events Tabeio gnre peta osansa anena aga eo i ast testas A A EGT EEEo 32 4 4 4 Thermal Organizer Controls eur endet iet eie tito eid in Enter ids 34 4 4 5 Attaching the Method to an Analyst File sssseeen 35 4 4 6 Viewing the Coulochem III Method from within Analyst sus 35 4 5 Modifying an Attached Coulochem III Method sense 36 4 6 Modifying a Coulochem III Method While Acquiring Data sessss 37 5 Basic Coulochem III Driver Functions eere eee e eene tenen 39 Sd o C VO
67. the Pre Run tab is visible opens the Help file to the Pre Run Tab topic Help Navigator Tool Each window or dialog box in the driver program also provides a Help Navigator tool for obtaining help The tool is enabled by clicking the amp button in the upper right corner of the window or dialog box When activated the cursor changes to a question mark Clicking on any control 1 e any edit box check box selection box or icon produces a brief help statement describing the function of the control Figure 1 1 shows both the Help Navigator button upper right corner and the help Coulochem IIl Analyst Driver Version 1 0 User s Manual Introduction cursor over the Auto Zero button and Figure 1 2 shows the help statement that appears when the Help Navigator cursor is clicked over the Apply Settings button as ESA Coulochem III EC Detector File Instrument Help Detector Setup Pre Run Timed Events M DC Mode Channel 1 O site Zero Cell potential 50 mv Apply Gain Filter time constant 5 0 sec Q Cells On Full scale gain range 100 p Y Cells Off Signal output voltage 1 V Apply Settings Figure 1 1 Help Navigator Icon upper right and Cursor inm a ESA Coulochem III EC Detector File Instrument Help Detector Setup Pre Run Timed Events DC Mode Channel 1 Q AEN Cell potential 50 2 mV Apply Gain Filter time constant 5 0 sec 3 Cells On Full scale gain ra
68. the Setup Device button and select highlight Coulochem III Detector from the list of software applications in the Software Application Settings dialog Figure 4 2 Software Application Settings Software applications smf Coulochem III Detector Simulate device Alias name Cell Enable debug messages Cancel Help Figure 4 2 Software Applications Settings Dialog Coulochem III Analyst Driver Version 1 0 21 User s Manual Chapter 4 7 Enter CcIIP or something similar as the alias name then click OK 8 If an ADC connection has been implemented between the Coulochem III detector and the PC for collecting analog data from the Coulochem III detector click the Add Device button again to configure the ADC If an analog connection will not be used continue at step 12 below 9 Select A D Converter from the Device Type drop list then highlight A D Converter NIDAQI6 or the particular ADC to be used and click OK 10 Highlight the A D Converter chosen and click Setup Device The correct settings for the NIDAQI6 converter NI 6032E PCI board are illustrated in Figure 4 3 below for a 2 channel system Note that each channel is configured separately If 2 channels are to be used be sure to configure the second channel by selecting 2 as the Channel No and ensuring that the Channel Connected checkbox is checked for channel 2 as in the figure Note that for the NI 6032E PCI board the Polarity Range must be
69. the lowest filter needed to reduce output noise Default is None Coulochem Ill Analyst Driver Version 1 0 29 User s Manual Chapter 4 4 4 2 30 Pre Run Tab Just prior to actual data collection Analyst provides a pre run period during which any instrument attached to the active profile may delay the start of data acquisition The Coulochem III driver see Figure 4 7 provides the user with three options for the pre run interval These include a simple pre equilibration period of up to 2 hrs autozero prior to run and waiting to reach the thermal organizer setpoint temperature as ESA Coulochem III EC Detector File Instrument Help Detector Setup Pre Run Timed E vents Pre run Conditions Q Auto Zero Iv Pre equilibrate for f D min REUS Q Cells On v Autozero prior to run Cells Off Apply Settings Thermal Organizer IV Activate Actual Temp 51 XB SetTemp 92 C Current ST 27 0 T sST 2 Apply Temp Help Acquisition Status Idle Logged onto Analyst Connected to Cclll on USB Idle time 16 29 min Figure 4 7 Pre Run Tab Parameters DC and Pulse Modes Pre equilibrate for n minutes Checking this option and setting a non zero equilibration time tells the Analyst program to delay data acquisition by at least the period indicated Other options may extend it This allows the Coulochem II detector to stabilize any new cell potentials between runs A pre equilibration period of a f
70. thod would not be known by Analyst or available toit Attaching the Coulochem III method data to the Analyst dam file also allows the parameter data to be managed under the control of the Analyst program s secure filing system Other configuration and method data such as data for pumps and autosamplers may be communicated to Analyst through their respective drivers Coulochem III Analyst Driver Version 1 0 19 User s Manual Chapter 4 The sections below describe each of these steps in detail 4 2 Creating and Activating a Profile A profile specifies all mass spectrometer instrument parameters as well as basic properties of connected devices like pumps autosamplers the Coulochem III detector and the A D converter The instrument profile for a typical metabolite identification study see section 7 may include a binary pump autosampler Coulochem III module and mass spectrometer The Coulochem III module may be used to control a pre column EC reactor cell For a given parent compound a series of injections is made each with the cell set at a different applied potential EC oxidation or reduction products are then separated chromatographically and characterized by mass spectrometry The signal current or charge produced from the EC reactions may also be simultaneously monitored using the ADC The basic steps for creating a profile that includes the Coulochem III detector and ADC are 1 From Analyst double click the Hardware C
71. tions E Build Acquisition Meth Enable pre equilibration yes Build Acquisition Batct Pre equilibration time 3 z7 Express View Autozero prior to run yes Wait for thermal organizer yes Explore g y i Open Data File T gj Open Compound Date Thermal organizer amp LZ Quantitate Organizer set temp 27 Activate thermal organizer yes E Build Quantitation Met 3 y N amp Quantitation Wizard Review Results Table DC Mode Parameters Channel 1 amp Companion Software Potential 123 ilter constant D BB cci aao F h i 2 Gain range 2 uA R Caianham Il Datart lt gt Tu For Help press F1 User Name NBAl norm C yAnalyst Data a Idle Ex Idle ive Idle UU ous Figure 4 10 Analyst Program Showing Coulochem III Method Properties 4 5 Modifying an Attached Coulochem Ill Method Once Coulochem III method data has been attached to an Analyst acquisition it is possible to modify the file data at any time However method data that is in use in an actively acquiring method cannot be modified To modify the Coulochem III method data that is attached to an Analyst file select File gt Read Method from Analyst File from the Coulochem III driver menu select the dam file to be read and click Open This loads the method data into the driver window The data can then be modified as required To save and reattach the data to an Analyst file select File gt Attach Method to Analyst File and select the Analyst acquisit
72. ulochem III driver just prior to the run for each sample they remain fixed throughout the run Keep this in mind when modifying Coulochem III method parameters The modifications will not affect samples already submitted to the queue but they will affect new samples added to the queue after the modification 38 Coulochem IIl Analyst Driver Version 1 0 User s Manual 5 Basic Coulochem Ill Driver Functions 5 1 Overview The Coulochem III driver provides some basic detector functionality even when not actively involved in data acquisition such as the ability to apply settings to the Coulochem II detector perform an autozero and turn cells on and off This section examines these independent functions and begins with a discussion of the behavior of the driver during startup and shutdown as it connects or disconnects with the Coulochem III detector 5 2 Startup and Shutdown Sequences 5 2 4 Startup Sequence Immediately after confirming the connections to both Analyst and the Coulochem III detector the Coulochem III driver recalls the last configuration it was in DC vs Pulse mode Enable channel 2 Enable guard cell etc prior to shutdown and attempts to reestablish the same configuration It then disables most of the keys on the Coulochem III keypad sends default method parameters to the Coulochem III for the recalled mode and starts the Coulochem III in DC or Pulse mode After completing this sequence the Coulochem III
73. ure 2 2 and the other on an available COM port on the PC If using a USB cable connect the small end of the cable to the USB Remote port on the Coulochem III detector Figure 2 2 and the wide end to an available USB port on the PC 8 Coulochem III Analyst Driver Version 1 0 User s Manual System Requirements and Installation THERMAL ORGANIZER CONTROL RS2352 SIGNAL OUT 2 REMOTE USB REMOTE DC PSTAT MODULE COULOCHEM III ESA INC PULSE SCAN MODULE COULOCHEM IN ESA INC LOGIC MODULE COULOCHEM III ESA INC WARNING FOR CONTINUED PROTECTION AGAINST RISK OF FIRE REPLACE ONLY WITH FUSE THERMAL ORGANIZER POWER po LISTED TO UL61010 EH us Y T Ty un ETL TESTING LABORATORIES INC CORTLAND NEWYORK 13046 T2 0A 250VAC T1 0A 250VAC 100 120VAC 230 240VAC d s t d i id A i Figure 2 2 Coulochem III Back Panel Coulochem Ill Analyst Driver Version 1 0 User s Manual Chapter 2 2 6 Verifying the System To operate properly the Analyst program must be able to locate and communicate with the Coulochem III driver companion software Proper installation of both the Analyst program with any required hotfixes and the Coulochem III driver is essential in order for this communication to occur If necessary check with your Applied Biosystems MDS Sciex representative to ensure that you have the required hotfixes To verify the installation perform the follo
74. ver to create new methods without Analyst All acquisition methods must be initiated in Analyst as explained in subsequent sections Coulochem Ill Analyst Driver Version 1 0 15 User s Manual Chapter 3 3 4 Configuring the Coulochem Ill Analyst Driver The Coulochem III can be configured in either DC or pulsed mode In DC mode 1 or 2 channels can be utilized along with an optional guard cell and thermal organizer Operation in pulsed mode uses a single channel and does not use the guard cell The driver must be configured to reflect actual hardware as well as experimental preferences prior to use with the Analyst program for data acquisition 1 From the driver menu select Instrument gt Coulochem III Configuration ESA Coulochem III Configuration Instrument CC III operating mode Set Defaults Comm port COM1 Event marker height 5 5 x full scale Options Enable channel 2 Enable guard channel Enable thermal organizer Enable event marker on autozero r DK Cancel Help Figure 3 4 The Coulochem III Configuration Dialog 2 From the configuration dialog Figure 3 4 select the appropriate options for each of the following parameters e CCIII operating mode Select either DC or Pulse mode e Comm port Select USB if connected to the Coulochem III through an available USB port If connected though a serial port select either COMI COM2 COMG or COMA depending on which ports ar
75. wing steps 10 1 Turn the Coulochem III detector on and allow ample time for it to complete its startup routine Start the Analyst program If required enter a login name and password required when running in Mixed mode Ensure that the Analyst Navigation Bar is visible Select View gt Navigation Bar if not Verify that the item Coulochem III Detector appears in the Companion Software section at the bottom of the Navigation Bar Double click on the Coulochem III Detector item in the Navigation Bar This should start the Coulochem III driver which will display its introductory splash screen as shown in Figure 2 3 below If running Analyst in Mixed mode it will be necessary to log on again prior to seeing the splash screen ESA Biosciences Inc Coulochem III Electrochemical Detector Analyst driver Version 1 0 0 Build 23 Beta For Research Use Only Terms of use of this software are specified in the Software End User License Agreement EULA which was accepted during installation 4 Initializing Please wait LLLLLLELLLLLLLLLLL MAGELLAN BIOSCIENCES Figure 2 3 Coulochem III Driver Introductory Splash Screen Coulochem III Analyst Driver Version 1 0 User s Manual System Requirements and Installation 6 After a few seconds the main driver window will appear 7 Ifthe Coulochem III detector is connected to the PC using a USB cable a message box like the following may appear C
76. working electrode EC cell A typical application is for sensitive EC detection of carbohydrate species in parallel with MS Serial EC MS with pulsed waveforms is also of interest in effecting reactions that may not be observed with fixed DC conditions LC Conditions e Column ESA Carbohydrate RCX 10 e Mobile phase 100 mM ammonium hydroxide e Flow rate 1 5 ml min EC Setup e Analytical cell ESA 5040 Amperometric cell gold working electrode e Potentials and pulse widths Potential Pulse width mV msec El 200 500 E2 1000 10 E3 600 1 E4 100 10 Acquisition delay 300 msec Full scale gain range 1 uC e Signal output voltage 1V e Baseline offset 0 9o e Filter setting None e Cleaning T2 E2 El Acquisition Measurement Delay E3 T3 Conditioning Coulochem III Analyst Driver Version 1 0 55 User s Manual Chapter 7 Results Figure 7 4 shows the oxidation profile for a simple mixture of carbohydrates 20 pmoles total 1 1 Glucose 2 Fructose 3 Lactose 4 Sucrose Time minutes 0 7 Figure 7 4 EC Detection of a Simple Carbohydrate Mixture 56 Coulochem III Analyst Driver Version 1 0 User s Manual Appendix A Updating the Coulochem Ill Firmware A 1 Overview The Coulochem III detector is shipped with the latest version of its firmware the internal software that is responsible for its operation The firmware version is shown on the Coulochem II
77. yes or no no Ch 2 Clean Cell Potential 2000 to 2000 mV 0mV Coulochem III Analyst Driver Version 1 0 User s Manual 61 Ch 2 Clean Cell Duration Guard Enable Clean Cell Guard Clean Cell Potential Guard Clean Cell Duration Post Clean Cell Delay 1 0 to 120 0 yes or no 2000 to 2000 1 00 to 120 0 0 00 to 10 00 but gt longest Duration above sec mV sec min Provides post clean cell delay values for both channels and guard Pulse Mode Acquisition Delay AD El Potential El Pulse Width E2 Potential E2 Pulse Width E3 Potential E3 Pulse Width E4 Potential E4 Pulse Width Full Scale Gain Range Signal Output Voltage Baseline Offset Filter Constant Pre run Parameters Pre equilibrate Pre equilibrate time Auto zero prior to run Wait for thermal organizer 50 to 495 2000 to 2000 AD 5 to 1000 2000 to 2000 4 to 1000 2000 to 2000 0 to 1000 2000 to 2000 0 to 1000 10 pC to 10 mC in steps of 1 2 5 10 20 50 100 200 500 l or 1 50 to 50 none low medium high yes or no 0 00 to 120 00 yes or no yes or no Thermal Organizer Parameters Activate Set Temp 62 yes or no 15 to 60 msec mV msec mV msec mV msec mV msec min 1 sec no 0 mV 1 sec 1 00 300 msec 200 mV 500 msec 1000 mV 10 msec 600 mV msec 100 mV 10 msec 1 uC 1 V 0 none no 1 00 no no yes 32 Coulochem III Analyst Driver Version 1 0 User s
78. zero the indicator next to the Auto Zero button lights green Apply Gain Click this button in DC mode to apply the current full scale gain range to each enabled channel The Apply Gain button is disabled in Pulse mode The button is also disabled during the pre run acquisition and post run clean cell phases of a run Cells On Off Click either of these buttons to turn the cells on or off When on the indicator next to the Cells On button lights green These buttons always remain enabled even during a run Apply Settings Clicking this button sends all of the current settings DC or pulse to the Coulochem III detector and resets the detector to activate the new values The Apply Settings button 1s disabled during the pre run acquisition and post run clean cell phases of a run 5 4 Thermal Organizer Control If the system is equipped with a thermal organizer and the organizer is enabled the Apply Temp button see Figure 4 10 is used to set the setpoint temperature from the Set Temp field in the Coulochem III The Apply Temp button is disabled during the pre run acquisition and post run clean cell phases of a run 40 Coulochem III Analyst Driver Version 1 0 User s Manual Basic Coulochem lll Driver Functions The ST 2 indicator turns green whenever the thermal organizer s actual temperature is within 2 C of its setpoint temperature For more on the thermal organizer see Section 4 4 4 Coulochem Ill Analyst Driver

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