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LCi Operating Instructions

1. Exclusive Access Time 0 905 min z Back Cancel Help FIGURE 4 16 Program wizard autosampler options Set the Inject Mode to Normal For the Connected Pump Device the correct pump must be selected For Timebase 1 PumpLeft has to be selected If no pump or the wrong pump is selected the Exclusive Access Time cannot be calculated because the pump flow rate is unknown The Synchronize injection with pump box is recommended to be used This feature synchronizes the pump stroke with the autosampler injection and increases reproducibility If activated the Bypass checkbox will allow the bypass of the sampler loop The Flush Out Factor is the time after which the injection valve switches to the Load position Bypass Time The solvent flow bypasses the needle and sample loop to reduce the gradient delay times For Parallel LC it is always set to 1 0 to reduce the time to switch from one analytical column to the other analytical column The Maximum Inject Volume is the injection maximum injection volume that will be used in the sequence The Flow value is taken over from the flow rate as set in the pump options step of the wizard The Bypass Time is calculated from the injection volume capillary seat volume and the flow rate The Exclusive Access Time is the time that the autosampler can be solely used in one timebase The other timebase will be put on hold if it attempts to use the autosampler during Operating Instructions
2. 3 26 Other query criteria can also be used for the sequences Query Wizard Sequences Data Field Operator Value Sequence Directory starts with TEMPA QUERY EXAMPLE Data Field Data Field Operator Data Field Operator lt Back Cancel Help FIGURE 3 26 Sequence criteria settings in the Query Wizard 3 The sample type can be specified in FIGURE 3 27 depending on how the samples of interest are specified in the sequence Other sample parameters e g sample injection time can also be used as a query criteria Query Wizard Samples e hd Data Field Value Data Field p Data Field lt Back Cancel Help FIGURE 3 27 Sample criteria in the Query Wizard 30 Operating Instructions Automated Method Scouting 4 Result restrictions can be set in FIGURE 3 28 For Automated Method Scouting the SST result of the quantification method qnt sst_result is used to restrict the results This will limit the results to samples that have passed the SST as defined above Query Wizard Result Restrictions Formula _ Operator Value gnt sst_result ial kd Fassedl Ez Formula T Operator Value Formula E Operator Value Formula a Operator Value lt Back Cancel Help FIGURE 3 28 Results restrictions in the Query Wizard 5 Apply and save the query The results will be displayed in the directory where the query is performed General Nativ
3. As soon as the exclusive access is released by Timebase_ 1 Timebase 2 can take over exclusive access to the autosampler and inject the sample Both timebases are now running their analysis simultaneously on both detectors Start Batch on timebase 1 Batch List Reporting Eror Handling Transfer Name Operator Parallel LC Template 1_Parallel_ Template ViKarsten Remove Move Up Shutdown Delayed St Add Move Down Cancel Ready Check Help FIGURE 4 18 Start of the batch on timebase_ 1 Start Batch on timebase 2 Batch List Reporting Eror Handling Transfer Name Operator Delayed St Add E Farallel_LC_Template 2_Parallel_Template MKarsten Mowe Up Move Down Shutdown re e Cancel Ready Check Help FIGURE 4 19 Start of the batch on timebase_ 2 52 Operating Instructions Tandem LC 5 Tandem LC Tandem LC is an LCi solution for increasing the throughput with 50 up to 100 for gradient separations With Tandem LC two different flow paths are used allowing off line equilibration of one column while the second column is used for the analysis Tandem LC offers an increase in throughput without the need for new method development The system includes e One DGP 3600 pump that can make two ternary gradients independently and simultaneously The left pump can be used off line for the wash step and equilibration while the right pum
4. List of capillaries and accessories supplied with the Parallel Operation Kit a a cwis recons rome i o e id o epi Tcr n osm e D f cwm recon wesa i o F fowmrazosmron ooo o o oo a Column Clips Kit 4 Quick Installation Guide U3000 Application Kits Operating Instructions 37 Parallel LC 4 2 Configuring Software 4 2 1 Configuring Hardware in the Server Configuration The UltiMate 3000 Parallel LC system is configured in the Server Configuration of Chromeleon To configure the system first create two timebases e g Timebase 1 and Timebase 2 in the Server Configuration FIGURE 4 3 The DGP 3600 is a dual gradient pump it is essentially two separate pumps in one housing Therefore a DGP can also be shared in a Parallel LC system To share the pump column compartment and autosampler between the two timebases add those devices to one timebase and set the options for sharing as described below When the fluidic connections are made as described in the Quick Installation Guide apply the same settings as described below for the sharing of the devices between the two timebases fe Server Configuration Chromeleon Server Configuration File Edit View Server Help tay CP TREA DP aA FF i Sharable Devices Timebase_1 fey DGP 3600 Pump Virtual Right TCC 3x00 Column Compartment Virtual Oven Column A fq WwPS 3000 Autosampler Virtual fq VW0 3x00 Detector Timebase 2 fq DGP 3600 Pump Virt
5. Lower Pressure Limit 0 O 400 bar Upper Pressure Limit 400 O 400 bar Ripple Limit 3 0 1 0 5 0 4 W Purge Pump Purge via Sampler C Purge via Valve Screw i Flush System Solvent Composition Durations 5 000 miri B 72 0 0 0 100 0 2B 72 0 0 0 100 0 E 0 0 0 10 100 0 4 ol m 0 0 100 0 Cancel Help FIGURE 8 7 SmartStartup wizard left pump equilibration settings 6 FIGURE 8 8 shows the autosampler settings for equilibration with the option to prime the syringe wash the buffer loop and set the desired temperature settings Sampler Equilibration Conditions W Prime Syringe Mumber i 1 100 W Wash Buffer Loop Wolume 300 000 O 000 500 000 pl W Use Temperature Control Temperature nominal 1 0 0 4 0 45 0 C Lower Lirit 40 4 0 45 0 C Upper Limit 45 0 4 0 45 0 C Ready Temp Delta i 0 0 Mone 10 0 C Cancel Help FIGURE 8 8 SmartStartup wizard sampler equilibration conditions Operating Instructions 91 Automated Application Switching 7 The ColumnOven Equilibration Conditions screen allows the user to set the temperature valve positions and active column FIGURE 8 9 Choose the correct active column and valve positions for the application FIGURE 8 9 depicts the settings for the first application For the second application Valve Left is set to 1_2 and Valve Right is set to 6 1 ColumnOven Equilibration Cond
6. Sampler Temperature Nominal 10 0 c Sampler Temperature LowerLimit 4 0 C Sampler Temperature UpperLimit 45 0 c Sampler ReadyTempDelta 10 0 c ColumnOven TempCtrl on ColumnOven Temperature Nominal 50 0 c ColumnOven Temperature LowerLimit 5 0 c ColumnOven Temperature UpperLimit 85 0 c EquilibrationTime 0 5 min ColumnOven ReadyTempDelta 1 0 c ValveLeft 5 Dionex Acclaim ValveRight 5 Dionex Acclaim FIGURE 3 7 Program File created with the Chromeleon Wizard The temperature and the valve positions analytical column are fixed values An additional program has to be created for different temperatures or columns The semi colon after ValveRight 5 is used to comment the column name The previously created User Defined Columns Columnoventemp ColumnSelector and Column Name Section 3 2 3 1 are used as a variable in the program files Chromeleon will read the value entered in the UDC and use that value for the variable in the program file Chromeleon will read the UDC value per line in a sequence making it possible to adjust values while the sequence is running The variable in the program file is specified by the name used for the UDC preceded with Sample separated by a dot In the example shown in FIGURE 3 8 the column oven temperature specified in the program file will be changed to a variable as defined in the UDC Chromeleon reads the variable Sample ColumnOvenTemp from the pro
7. a split loop well plate autosampler and a UV or a PDA detector that is fully controllable by Chromeleon Chromatography Management Software Operating Instructions 5 LCi Solutions 2 6 Automated Application Switching Automated Application Switching 1s an LCi solution for increased efficiency by using two applications on one UltiMate 3000 Automated Application Switching eliminates the manual equilibration that has to precede any application change The system will equilibrate and perform a set of runs with one method After this first application the system will wash and prepare for the second application The second application will be started automatically when the equilibration 1s complete Columns solvents and samples can be completely different for the two applications The HPLC system that is used with the Automated Application Switching program includes a pump with the ability to perform two ternary gradients a thermostatted column compartment with two 2 position 6 port valves to allow easy switching a split loop well plate autosampler and a UV or a PDA detector that is fully controllable by Chromeleon Chromatography Management Software 6 Operating Instructions Automated Method Scouting 3 Automated Method Scouting The development of a new HPLC method can be a time consuming step that dramatically reduces the efficiency and productivity of an analytical laboratory When an analyst starts to develop a method the
8. 1s installed Table continues on next page 10 Operating Instructions Automated Method Scouting TABLE 3 1 continued A s 1 4 TCC port 6r H ValveRight Port 6 to Column 6 Inlet for 5 cm columns 5 cm Col 6 in gt l 4 TCC port 6r H ValveRight Port 6 to Column 6 Inlet for 15 cm columns l 15 cm Col 6 in gt 4 Col 1 out TCC port 1 gt 4 Col 2 out Column 2 Outlet to ValveLeft Port 2 for 5 and 15 cm columns l TCC port 2 gt 4 Col 3 out K Column 3 Outlet to ValveLeft Port 3 for 5 and 15 cm columns l TCC port 3 gt 4 Col 4 out L Column 4 Outlet to ValveLeft Port 4 for 5 and 15 cm columns l TCC port 4 gt 4 Col 5 out Column 5 Outlet to ValveLeft Port 5 for 5 and 15 cm columns l TCC port 5 gt 4 Col 6 out Column 6 Outlet to ValveLeft Port 6 for 5 and 15 cm columns TCC port 6 gt 4 TCC center 1 ValveLeft Center Port to VWD 3x00 Detector Det VWD gt 4 TCC center I ValveLeft Center Port to PDA 3000 Detector Det PDA gt Incl with Detector I Column 1 Outlet to ValveLeft Port 1 for 5 and 15 cm columns l Waste line from Detector es 4 TCC valve 1 Bypass tubing Multi Position Valve l TCC valve r gt Operating Instructions 11 Automated Method Scouting 3 2 Configuring Software 3 2 1 Configuring Hardware in the Server Configuration The UltiMate 3000 system is configured in the server configuration of Chromeleon To configure the system create a
9. 49 Parallel LC this time This time depends on the injection volume capillary void volume the capillary seat volume and the flow rate The calculation is 3 x injection vol ml 3 x capillary seat volum eni mn capillary void volume imed na flow rate ml mi n Now the program for Timebase_ 1 is created Continue by starting over the wizard to create the program file for Timebase 2 50 Operating Instructions Parallel LC 4 2 3 2 Program fine tuning When performing the same gradient separation on both columns it is necessary to adapt the gradient to Parallel LC Compared to a single gradient system the Parallel LC gradient requires an isocratic part before the start of the gradient FIGURE 4 17 This isocratic part should be at least equal to the duration of the Exclusive Access Time The wizard will give a warning when this isocratic part is not long enough Lol ae I Lee eS I Peale a rUPr To I I akats aa a as a I I gaia aa a ma leita TENSA A I VARSI SA ee S FIGURE 4 17 Gradient profile Top Original gradient for a single gradient system Bottom Gradient adjusted for Parallel LC Parallel LC is intended to run the same application on both columns to increase the throughput It is however possible to run two different applications simultaneous with Parallel LC The mobile phases must be fully miscible and the start of the injection on the second timebase should be delayed sufficiently to equilibr
10. Automated Method Scouting a 4 3 3 Finding the Best Chromatographic conditions with the SST and Query Combined with the Method File for Automated Method Scouting The steps to take when using the combination of the SST query and method file are l a2 Define parameters in the SST Section 3 2 4 3 2 The SST 1s first defined to narrow down the results before proceeding with the method file The SST is created as described in Section 3 2 4 3 2 where different parameters can be used It is recommended to at least use the parameter for the number of peaks in the SST eliminating all runs in which the sample is separated incompletely For example setting the number of peaks parameter to a value equal to the number of compounds in the sample will eliminate results with un separated compounds Perform a query Section 3 2 4 4 The query will limit the amount of results depending on the SST parameters Use the method file SCOUTING gqnt Section 3 2 4 3 1 Use the method on the results of the query to find the best results 4 4 Query Search for the Results The query will extract relevant data from the sequence as determined by the user A Report Column is an additional column in the sequence that can display the results of the SST The report column will be used later in the query to filter the results 1 Create a Report Column by right clicking in the sequence and select Report Columns New Report Column 2 Click the do
11. Hardware in the Server Configuration ccccccccccccccccsccceeeeeeeeeeeeeeeeeeeeeeeseeeeeeeeeeeseeeeeees 38 4 22 Direct Instrument Control from Chromeleomsniasierecneari hnn eine 42 A 23 Programs and s COMEMCCS c3cir atte ee aetna aie sec ea cel eam nae T AA T 43 5 Tandem EC rencanana a 53 ST Preparation ofthe SysteM cnsissicie denora a eea ae aia 54 52 Conhourne SOLQW ALTE essenin aE a 56 5 2 1 Configuring Hardware in the Server Configuration ccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 56 5 2 2 Direct Instrument Control from Chromeleon cccccccceeseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 56 52 Prosrams and Seguent CS sesh foc Sahar a haat La i aa a Ha i a Aa ata fe a 58 6 Automated On Line SPE LC cavescctscccatacetcssdstaccecs secu seedetasdeatosiusdieiaddets tase uenietaidettocieee 65 61 Preparation OL UNE SY SCCM xi ciiced cesectsccccedeheceecssbeeccediccesecdehenccedecececessteenesdecesccedosesessbct 66 02 COMMBUPING SOLOW ALE saei aa aeee a a eee 68 6 2 1 Configuring Hardware in the Server Configuration ccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 68 6 2 2 Direct Instrument Control from Chromeleon cccccccccceseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 68 Operating Instructions EA LCi Solutions 02 PFO Oral and SCOUCI CES welt ccieeteakeasheliast a a a lias a nase a N a a aa 69 7 Automated 2D LC ssrin ara a
12. OTF Autogenerated j No 3 ScouTING 3 3 934 min 5 000 AG Least Square O 4 950 Off Off Autogenerated No E SCOUTING 4 5 234 min 5 000 AG Least Square O d 950 Off Off Autogenerated No 5 SCOUTING 5 5 642 min 5 000 AG Least Square O fe 950 Off OFF Autogen rated No 6l SCOUTING 6 367 min 5 000 AG Least Square O 4s 950 Off OTF Autogenerated No 7 SCOUTING T 6 492 min 5 000 AG Least Square O 4 950 Off Off Autogenerated No 3 SCOUTING 6 975 min 5 000 AG Least Square O Ae 950 Off Off Autogenerated Yes 3 SCOUTING 9 15 933 min 5 000 AG Least Square O d 950 Off Off Autogenerated Yes FIGURE 3 14 Method file SCOUTING qnt example presenting an autogenerated peak table in the Peak Tracking tab of the method file The last column in the peak table labeled Check_Rs enables the user to select the peaks of interest If an entry is set to Yes the resolution will be evaluated in the Find Best_Method rdf report 3 2 4 3 2 Finding the Best Chromatographic conditions with the SST and Query 22 Operating Instructions Automated Method Scouting The System Suitability Test SST verifies if the desired criteria are reached Each defined parameter will be checked separately and has to meet the specified criteria to pass the SST The overall result of the SST can be displayed as a column with the result failed or passed in the sequence and or the results of the specified parameters The SST para
13. Which kind of Program do you want to create C Regular Program Program for On Line SPE LC Solid Phase Extraction Liquid Chromatography Program forx2 Tandem Operation FIGURE 5 5 Program wizard elect the type of program to create 58 Operating Instructions Tandem LC 3 After selecting Program for x2 Tandem Operation click Next The Tandem System Schematic will appear FIGURE 5 6 to assist you further in making the program Set the autosampler and column compartment temperature parameters in the following wizard screens not shown here Tandem System Schematic Column 2 from Analytical m Pump Autosampler _ 5 js from Reconditioning Pump Position 10_1 Columnt Column from Analytical A eee Autosampler from Reconditioning Position 1_2 FIGURE 5 6 Program wizard schematic overview of the fluidic connections Operating Instructions 59 Tandem LC 4 In the Tandem Pump Options of the wizard the solvents names the pressure limits and maximum flow acceleration deceleration can be specified If the system is setup according to the Quick Installation Guide the analytical pump must be set to PumpRight This pump is connected to the autosampler PumpLeft will be automatically set as the reconditioning pump Program Wizard Tandem Pump Options Solvents Pump Selection Name Analytical Pump Pump Right Reconditioning Pump Pump Left Pressure Limits Maximum Flow Accel
14. a PDA detector that is fully controllable by Chromeleon Chromatography Management Software 2 4 Automated On Line SPE LC Automated on line solid phase extraction 1s an LCi solution allowing easy and automated isolation of analytes of interest from a complex matrix The automated on line SPE reduces time labor and cost thus increases productivity After injection of an untreated sample the On Line SPE LC allows automated sample cleanup and or analyte enrichment Samples can run unattended increasing the workload per system The HPLC system that is used with the Automated On Line SPE LC program includes a pump with the ability to perform two ternary gradients a thermostatted column compartment with one 2 position 6 port valve to allow easy switching a split loop well plate autosampler and a UV or a PDA detector that is fully controllable by Chromeleon Chromatography Management Software 2 5 Automated 2D LC 2D LC is an LCi solution for separating complex samples It enables the combination of two orthogonal chromatographic techniques for example IEX and RP The sample is injected on the first dimension column Fractions of sample eluting from the first dimension are transferred to the second dimension for further analysis The HPLC system that is used with the 2D LC program includes a pump with the ability to perform two ternary gradients a thermostatted column compartment with one 2 position 10 port valve to allow easy switching
15. and WPS 3000SL analytical autosampler 1 6 mL delay volume approx 530 uL up to the valve UltiMate 3000 micro LPG 3400M and WPS 3000SL micro autosampler 0 9 mL delay volume approx 300 uL up to the valve UltiMate 3000 micro HPG 3x00M and WPS 3000SL micro autosampler 0 3 mL delay volume approx 100 uL up to the valve Program Wizard Tandem Operation Options Analytical Pump Pump Right fata a Void Volume Purge Flow 1 000 ml min Volume 2 500 ml Time 2 500 min 1 00 200 100 Reconditioning Pump FumpLeft 202 Reconditioning Flow Flow ml min Cancel Help FIGURE 5 9 Program wizard Tandem Gradient Options continued Operating Instructions Tandem LC 7 The next two steps in the wizard not shown will allow the user to set the injections parameters in the Sampler Options 8 In the Acquisition Options step of the wizard the acquisition channels can be selected and the acquisition time can be specified The default data collection time is the time entered in the Off Line Reconditioning Start Time in the Tandem Gradient Options 9 The next steps in the wizard depend on the acquisition channels selected The steps in the wizard include options for the acquisition parameters e g pressure column compartment temperature and UV detector settings including wavelength selection 10 After specifying the detection settings the valve settings are determined in the last step of the w
16. at 6 different temperatures the user would be required to prepare 6 different programs with the only difference being the column oven temperature With a UDC it is possible to create only one program and define the column oven temperature in the UDC Li Note It is not possible to use UDC commands in the program during the acquisition time commands that need to be known in advance e g gradient profile Anything that can be set in the program before or after the acquisition can be changed e g the flow rate in isocratic runs column oven temperature mobile phase composition in isocratic runs and the valve positions to switch between analytical columns A UDC is datasource dependent and is created by right clicking the server s standard datasource the green Chromeleon icon in the browser and selecting properties A new a Operating Instructions Automated Method Scouting UDC can be created or an existing one can be modified on the User defined Columns tab FIGURE 3 6 Properties of Datasource ULT 2_local General Access Control Statistics User defined Columns Availabilty Columns Properties Name column name Value type String Dimension or comment leample iti s i Empty values are possible Maximum string length 25 Default string fl mein Import Columns Append Column FIGURE 3 6 User Defined Columns tab from the Datasource Properties In the window in FIGURE 3 6 two colu
17. elution profile of the analyte WL 260 nm FIGURE 6 5 Analyte Break Through Time t A is the time just before the elution profile of the analyte Flow 2 0 m Lmin 10 Minutes The Transfer Time t T is the time it takes at a given flow rate to completely elute the analyte from the SPE column on to the analytical column To determine the value for t T the system should be setup as depicted in FIGURE 6 1 but without analytical column With the valve in position 1 2 the analyte is injected on the SPE column After the sample is trapped on the SPE column the valve is switched to position 6 1 The analyte will elute from the SPE column with the mobile phase from PumpLeft The Transfer Time is the time from the valve switch observed in the baseline to the time at which the analyte is eluted from the SPE column VivL BO nm FIGURE 6 6 Transfer Time t T is the time from the valve switch observed in the baseline to the time at which the analyte is eluted from the SPE column Minutes Operating Instructions Automated On Line SPE LC 6 2 3 2 Program settings A new Program File can be created by clicking File New Program File in Chromeleon A wizard will appear allowing the user to set the various program settings such as column compartment temperature flow rate etc The steps of the wizard are described below Program settings that are specific for Automated On Line SPE LC are explained in detail Program settings th
18. in the manual of each device and in the Chromeleon online help Use the default device names in the server configuration Enable column A and column B in the TCC 3x00 configuration allowing easy selection between the two columns It is recommended to use column identification chips P N 6710 1505 set of 5 The column identification chips allow storing column specific data More information regarding the column identification chips can be found in the Chromeleon online help or the manual of the TCC 3100 8 2 2 Direct Instrument Control from Chromeleon The panel tabset offers the interface to control the system It is automatically generated based on the hardware configuration and can be used by clicking the panel tabset button FIGURE 8 3 A panel tab for each device is automatically added to the tabset each tab will allow direct control via the default panel for that device For information about direct instrument control of each device refer to the user manual or the Chromeleon online help for that device FIGURE 8 3 Panel Tabset Button Operating Instructions 87 Automated Application Switching 8 2 3 Programs and Sequences A new Program File can be created by clicking File New Program File in Chromeleon A wizard will appear allowing the user to set the various program settings such as column compartment temperature flow rate etc The program wizard has no special options for Automated Application Switching Th
19. loop autosampler e One PDA 3000 or one VWD 3x00 detector Automated Method Scouting experiment stages e Analysis of sample on one column with various mobile phases e Reduce flow prepare for the next column e Repeat the above steps for all available columns e Change temperature and repeat all the above steps Operating Instructions 7 Automated Method Scouting 3 1 Preparation of the system 3 1 1 Considerations for Sample Analysis The number of parameters in HPLC method development to determine the optimum separation can be infinite therefore some basic information about the sample 1s required to preselect the conditions and hence limit the amount of scouting to be performed Some examples of this point include e The choice of organic modifier has an influence on the selectivity of most compounds on a given column e For ionizable compounds the pH can have a very large influence on the selectivity e Cig column selectivity for very polar compounds can be very different per column manufacturer For polar samples a selection of different Cig columns can help to determine the best separation of the desired compounds e If no information is available about the sample a broad range of different stationary phases and basic and acidic conditions with different organic modifiers can help to determine preliminary conditions The found conditions can then be further optimized 3 1 2 System Hardware The schematic of the HPLC set up f
20. on Press Finish to show the batch list where you can start the equilibration fou can also add sequences to be run afterwards Press Finish to prepare the equilibration start Equilibration Start Log z Back Cancel Help FIGURE 8 11 SmartStartup wizard warning before starting the equilibration of the system After clicking Finish in the SmartStartup wizard the SmartStartup program can be saved Select Yes to save the SmartStartup program to be used for the application for which the startup program is intended Specify a name for the SmartStartup program It is recommended to choose a name that identifies the program with the application The program file will be saved in the Equilibration sequence in the folder with the timebase name under the default Chromeleon datasource After saving the program file an equilibration panel will appear and the Start Batch on lt Timebase name gt window will appear Remove the Equilibration sequence from the batch The panel and the batch window can be closed Repeat the above steps to create a startup program for the second application Copy the SmartStartup program file for the application to the sequence of the application Set the sequence up to first run the SmartStartup program before starting with the analysis of samples Set the sample type for this run to Blank If it is required to automatically turn on the devices of the system and the UV and or VIS lamp the Equilibra
21. port valve allows easy switching between the first and second dimension e One WPS 3000SL split loop autosampler e One PDA 3000 or one VWD 3x00 detector 2D LC experiment stages The sample is injected on the IMAC column Part of the sample is not retained by the IMAC column and enters the loop The flow rate on the IMAC column is stopped and the valve is switched The fraction in the loop is analyzed on the RP column The valve is switched back after the RP separation is finished The gradient and flow rate on the IMAC column are resumed to elute the trapped peptides in to the loop 7 Steps 3 6 are repeated for the eluted phosphopeptides oe For a more elaborate explanation of the example setup please refer to Technical Note 705 Automated Enrichment and Determination of Phosphopeptides Using Immobilized Metal Affinity and Reversed Phase Chromatography with Column Switching The technical note can be found on the Dionex website and on the Intelligent LC Solutions Reference Library CD 78 Operating Instructions Automated 2D LC 7 1 Preparation of the System The schematic of the HPLC set up for Automated 2D LC is shown in FIGURE 7 1 FIGURE 7 1 Schematic representation for Automated 2D LC All components except the columns that are required for a fully operable system are provided with the system modules or the Automated 2D LC kit For a list of supplied capillaries and accessories in the Auto
22. the application This startup program is created with the SmartStartup wizard of Chromeleon For each application a separate startup program has to be created The startup programs for 88 Operating Instructions Automated Application Switching the applications are saved in the sequence that is used for the application This startup program has to be applied in the first line of the sequence For the startup programs to work properly it is necessary to turn on the devices of the system and UV and or VIS lamp manually before starting the batch If it 1s required that the devices of the system and UV and or VIS lamp are turned on automatically the SmartStartup wizard can be used To accomplish this the Equilibration sequence in the batch created with the SmartStartup wizard has to run To create a SmartStartup program 1 Click Batch and SmartStartup in Chromeleon A wizard will appear that allows to define limits and device settings for the equilibration 2 First the timebase is selected After selecting the timebase the settings for the equilibration are specified FIGURE 8 5 A program file can be used for initial equilibration settings or the equilibration settings can be specified manually SmartStartup Wizard Extract Equilibration Conditions for Timebase DGP3600A Choose Equilibration Conditions Select the startup conditions Select Choose Program gt to read startup conditions Choose Program gt from a p
23. the left pump This step will flush the shared flow path of both applications e The right pump flow rate is stopped to bring the column for the second application online e Valve Right is switched from position 2 to position 6 1 e The flow rate of the right pump can be resumed for the equilibration Operating Instructions 95 Automated Application Switching 8 2 3 2 Creating a batch Click Batch Edit while in the Chromeleon browser A window with the title Batch lt Timebase Name gt appears If there are sequences in the batch remove them by clicking Remove Add the sequences to the batch by clicking Add and selecting the sequence for the first application flush wash sequence and the second application Start Batch on timebase 1 Batch List Reporting Error Handling Transfer Name Operator Delayed Start Add EE AAS IINSULIN mkarsten iM AAS 2SWITCH TO APPLICATION mkarsten ee fa AAS SMPA mkarsten Move Up Move Down Shutdown Cancel Ready Check Help FIGURE 8 12 Example batch with application 1 sequence flush wash sequence application 2 sequence and shutdown program Create a shutdown program by clicking the Shutdown button in the batch window Use a previously created program or create a new shutdown program Guided by a wizard the user can create a shutdown program to stop the pumps or to reduce the flow rate in the case buffers are used The wizard also p
24. timebase in the server configuration and add the various devices e g pump detector sampler and column compartment to the timebase Details about adding a device to the timebase are presented in the manual of each device and in the Chromeleon online help Use the default device names in the server configuration Li Tip Make sure that the correct valves are selected for the TCC 3200 FIGURE 3 3 the valves are identified as either 6 ports 6 positions or 7 ports 6 positions in the Chromeleon server configuration depending on the Chromeleon version TCC 3x00 Column Compartment General aaas Configuration Relays Inputs Eror Levels Installed components Left Valve Right Valve Column A Column E Column C Column D FIGURE 3 3 Configuration of the TCC 3200 in the Server Configuration 3 2 2 Direct Instrument Control from Chromeleon The panel tabset offers the interface to control the system It is automatically generated based on the hardware configuration and can be used by clicking the panel tabset button FIGURE 3 4 A panel tab for each device is automatically added to the tabset each tab will allow direct control via the default panel for that device For information about direct instrument control of each device refer to the user manual or the Chromeleon online help for that device FIGURE 3 4 Panel Tabset Button 12 Operating Instructions Automated Method Scouting The p
25. 5 Now the Matrix Depletion Time t M Analyte Break Through Time t A and Transfer Time t T are required These values have been determined in previous experiments and are used by Chromeleon to calculate the Calculated Switching Times marked as Begin Transfer t V1 and End Transfer t V2 The Begin Transfer time is in the middle after elution of the matrix from the SPE column and before the analyte breaks through The valve switches at that time to elute the analytes from the SPE column to the analytical column The End Transfer time is the time after the Begin Transfer time plus the Transfer Time plus one additional minute The analyte is transferred from the SPE column to the analytical column The valve switches back and the SPE column can be reconditioned while the analytes are separated on the analytical column Program Wizard Column Switching Options Please enter the evaluated column switching parameters SPE Extraction Parameters Matrix Depletion Time t M i 000 min Anahte Break Through Time t A 4 000 min Transter Time t T 2 000 min Calculated Switching Times Begin Transfer t V 1 min End Transfer t V2 min FIGURE 6 11 Program wizard SPE Extraction parameters for Column Switching time 74 Operating Instructions Automated On Line SPE LC A separate window FIGURE 6 12 will appear to show the separation steps graphically On Line PE LC Column Switching On Line SPE LC C
26. Automated Application Switching FIGURE 8 2 Stack of the Automated Method Scouting system A complete description of the installation and configuration of the hardware is presented in the Quick Installation Guide The application kits P N 6037 0009 Application Switching Kit Dual Ternary Analytical or P N 6037 0010 Application Switching Kit Dual Ternary Micro contain all the tubing to make all fluidic connections for a micro or analytical Automated Application Switching system TABLE 8 1 List of capillaries and accessories supplied with the Automated Application Switching kit ea O Capillary TCC 6p port 4 r or 10p port 5 Col 2 5 cm Capillary TCC 6p port 4 r or 10p port 5 Col 2 15 cm Capillary TCC 6p port 4 r or 10p port 5 Col 2 25 cm Capillary TCC 6p port 2 r or 10p port 1 Det G Capillary TCC 6p port 2r or 10p port 1 Det o T id T Sd M enormem o Capillary Right Pump TCC port 6 1 D conan Tocos rT O ooo P omowe ooo PEEK capillary for VWD waste connection B MEA Tei nsanaion Gude U000appicion ke OOOO ooo 86 Operating Instructions Automated Application Switching 8 2 Configuring Software 8 2 1 Configuring Hardware in the Server Configuration The UltiMate 3000 Automated Application Switching system is configured in the server configuration of Chromeleon Create a timebase in the server configuration and add the devices Details about adding a device to the timebase are presented
27. EJ DIONEX LCi Solutions for UltiMate 3000 Operating Instructions Now sold under the Thermo Scientific brand Version 2 0 Date January 2008 Thermo SCIENTIFIC 2008 DIONEX M164338 LCi Operating InstructionsLCi Solutions All information in this manual is subject to change without notice and does not represent a commitment on the part of Dionex Corp CHROMELEON LCi and UltiMate are registered trademarks of Dionex Corp Any other mentioned trade or company names are subject to the copyright and the property and trademark rights of the respective companies All rights reserved including those for photomechanical reproduction and storage on electronic media Without the written permission of Dionex no part of this publication may be reproduced in any form by means of photocopy microfilm or any other process for any purpose or processed copied transmitted or distributed in any other form independent from the means electronic or mechanical that is used Page II Operating Instructions LCi Solutions Warnings The Warning sign and the Important sign shown below are included in various locations in this manual or in the manuals provided with the instruments and software described herein These signs provide the following information OP Warning Indicates that failure to take note of the accompanying information may result in personal injury A Important Indicates that failure to take not
28. IGURE 3 11 in the Autogenerate Peak Table options No Peak Name Ret Time Window Standard Int Type Cal Type Peak Type Group Com 1 2 017 min 0 054 AS External Area Lin Auto Auto 2 2 3 292 min 0 071 AS External Area Lin Auto Auto 3 4 400 min 0 079 AS External Area Lin Auto Auto 4 5992 min 0 104AS External Area Lin Auto Auto a5 6 767 min 0 079 AS External Area Lin Auto Auto 6 6 7 196 min 0 100 AS External Area Lin Auto Auto 4 Peak Table 4 Amount Table Calibration Peak Tracking spectra Library Screening FIGURE 3 13 The Peak Table tab from the method editor with Autogenerated Peak Table The Peak Table only lists properties for the active chromatogram The peak properties for other chromatograms e g retention time are determined automatically by Chromeleon and used in the report Operating Instructions 21 Automated Method Scouting 3 2 4 3 Defining Parameters to Find the Best Chromatographic Conditions Once all the peaks are labeled as described above the next step is to define the parameters of interest to find the optimized chromatographic conditions With Chromeleon there are three possibilities to find the results 1 With a method file made for Automated Method Scouting SCOUTING gqnt Section 3 2 4 3 1 This option is easy and quick to use but it is limited to a maximum of 25 peaks and can be used when the only chromatographic parameter of interest 1s the resolution of compo
29. Retention Flow min ml min O 000 2 000 2 000 4 000 2 000 4 000 9 000 27 000 3000 2 000 FIGURE 6 13 Program wizard Loading Pump options 8 The next step not shown allows to specify the mobile phase names pressure limits flow rate and maximum flow acceleration deceleration for the Analytical Pump The right pump if the system configuration is made as described in the Quick Installation Guide and set up according FIGURE 6 10 76 Operating Instructions Automated On Line SPE LC 9 After specifying the settings described above the gradient profile for the Analytical Pump is specified In this example FIGURE 6 14 the column switching times are marked as t V1 and t V2 The analytical column is equilibrated at 5 B before transferring the analytes from the SPE column to the analytical column t V1 During the transfer the gradient profile is already started These settings and mobile phases used here should be identical to the settings used with the experimental determination of the Matrix Depletion Time t M Analyte Break Through Time t A and Transfer Time t T Program Wizard Analytical Pump Options AC BB Flow ml min Retention Flow min ml min 0 00 1 200 4000 1 900 2 0900 71 500 9 000 1 500 9 000 1200 FIGURE 6 14 Program wizard Analytical Pump options 10 The last steps in the wizard not shown are used to specify the detector and additional acquisition channel setting
30. S 1 or UV VIS 2 FIGURE 3 20 SST Wizard New Single Test Peak amp Channel Condition Test Condition peak resolution ep next Aggregate lt none gt Peak Peak from Peak Table Amilonde l Detected Peak Number C Detected Peak with 3 Pesk Reta tt t lt i lt is sSY C All identified Peaks Channel Apply on Channel Prefered Channel lt Back Finish Cancel Help FIGURE 3 20 SST properties Peak amp Channel Condition where the compound and channel are specified 26 Operating Instructions Automated Method Scouting 5 In N A amp Fail Action parameters can be set to define an action if the SST fails FIGURE 3 21 The fail action is not applicable for Automated Method Scouting because the SST is generated after the batch has run In the case of peak resolution if the test cannot be evaluated it is recommended to set the N A Not Available value to Passed This is to avoid a failed SST in the case of the last or first compound whereby the resolution cannot be determined to the next or previous compound 5S1T Wizard New Single Test N A amp Fail Action NLA f Passed t Failed Fail Action What should the server do if the test fails f Nothing Abort Batch What should the test result be f the test can mot be evaluated FIGURE 3 21 SST properties N A amp Fail Action 6 Repeat the steps 1 to 5 to insert more tests in the SST for different co
31. SRD 3600 Solvent Rack and Degasser DGP 3600A Dual Gradient Analytical Pump or DGP 3600M Dual Gradient Micro Pump WPS 3000SL Analytical or Micro Split Loop Autosampler TCC 3100 1x2P 6P Thermostatted Column Compartment with 1x 2 Position 6 Port Switching Valve PDA 3000 Photodiode Array Detector or VWD 3x00 Variable Wavelength Detector 66 Operating Instructions Automated On Line SPE LC FIGURE 6 2 Stack of the On Line SPE LC system A complete description of the installation and configuration of the hardware is presented in the Quick Installation Guide The application kits P N 6037 0006 On Line Sample Preparation Capillary Kit Dual Ternary Analytical or P N 6037 0007 On Line Sample Preparation Capillary Kit Dual Ternary Micro contain all the tubing to make all the fluidic connections for a micro or analytical On Line SPE LC system TABLE 6 1 List of capillaries and accessories supplied with the Automated On Line SPE LC kit l Column Clips Kit Quick Installation Guide U3000 Application Kits i Operating Instructions 67 Automated On Line SPE LC 6 2 Configuring Software 6 2 1 Configuring Hardware in the Server Configuration The UltiMate 3000 Automated On Line SPE LC system is configured in the server configuration of Chromeleon Create a timebase in the server configuration and add the devices Details about adding a device to the timebase are presented in the manual of each device and in the C
32. adients One pump is used for the sample injection and SPE column the other pump is used for the analytical column e One TCC 3100 column compartment The 2 position 6 port valve allows easy switching between the SPE and analytical column Also a 2 position 10 port valve can be used in the configuration e One WPS 3000SL split loop autosampler e One PDA 3000 or one VWD 3x00 detector Automated On Line SPE LC experiment stages e Inject the sample matrix and analyte of interest e The analyte of interest is trapped on the SPE column the matrix is flushed to waste e After a valve switch the SPE column is back flushed and the analyte is transferred to the analytical column e After transfer from the SPE column to the analytical column the valves switches again and the separation of the analyte on the analytical column is done with isocratic or gradient elution e During the analysis on the analytical column the SPE column is reconditioned Operating Instructions 65 Automated On Line SPE LC 6 1 Preparation of the System The schematic of the HPLC set up for Automated On Line SPE LC is shown in FIGURE 6 1 FIGURE 6 1 Schematic representation for Automated On Line SPE LC All components except the columns that are required for a fully operable system are provided with the system modules or the Automated On Line SPE LC kit For a list of supplied capillaries and accessories in the Automated On Line SPE LC kit see TABLE 6 1
33. am file The program wizard has no option for Automated 2D LC A minimum of three programs is required for a 2D LC separation The example programs are based on the Technical Note 705 Automated Enrichment and Determination of Phosphopeptides Using Immobilized Metal Affinity and Reversed Phase Chromatography with Column Switching When programming the valve switching times the dead volume of the system is very important Because the sample is transferred from the first dimension to a loop the flow should be stopped in time to keep the fraction in the loop If the fraction is too big or the valve is switched too early too late the sample is partially lost Operating Instructions 81 Automated 2D LC 7 2 3 1 Sample injection program The first program loads the sample on the IMAC column with the valve in position 10 1 Details are provided in TABLE 7 2 TABLE 7 2 Sample injection program loading the IMAC column IMAC RP Time Events MA B Flow A B Flow 0 200 1 000 Valve position 10 1 Inject start acquisition 7 2 3 2 Second dimension analysis program The second program is the HPLC analysis of the breakthrough fraction with valve in position 1 2 In the middle of the program the flow on the IMAC column is resumed with the same solvent composition as before This is to make sure that all the trapped phosphopeptides remain on the IMAC column There is no solvent composition specified in the second program ther
34. ample A typical spectrum recorded by the PDA 3000 is shown in FIGURE 3 9 The option to copy the spectrum to the clipboard is highlighted From the clipboard the spectrum can be copied to the Spectra Library 2416 How to What s this 240 Full Size Autoscale Unzoom 4 260 Decoration Replot LWD PECL c Extract Chromatogram to file 280 Export Opt Int Path to file Library Search Opt Int Path PGM to clipboard Show Spectra 300 Show Chromatogram Show Report Load Report Definition Save Report Definition t 13 T 40 7 60 7 80 5 00 5 20 5 40 5 60 8 85 FIGURE 3 9 PDA Spectrum Recorded with the PDA 3000 The Spectra Library is a file used by Chromeleon to store the spectra of the known compounds The spectra recorded with the PDA 3000 are compared with a Spectra Library for peak identification 1 Create a new Spectra Library or use an existing one with the identified compounds of interest that will be used in the method file for automated peak tracking by clicking File New Spectra Library 2 After the Spectra Library is created it needs to be specified in the Method Editor where it will be used for Automated Peak Tracking FIGURE 3 12 The Automated Peak Tracking feature identifies the spectrum of a peak in a chromatogram by comparing it to the Spectra Library FIGURE 3 10 Operating Instructions 19 Automated Method Scouting Spectra Library to be searched ir ULT localKADO236_H Col
35. and 15 cm long columns stainless steel tubing 1s provided while 25 cm maximum length long columns are connected with PEEK tubing For other column lengths PEEK tubing or a combination of stainless steel tubing for the inlet and PEEK tubing for the outlet can be used If desired an eluent pre conditioner can be used in the column compartment If this option is selected two additional capillaries are supplied with the kit to support the installation of an eluent pre conditioner in the column compartment Operating Instructions Q Automated Method Scouting TABLE 3 1 List of capillaries supplied with the Automated Method Scouting Kit 4 WPS port 4 Pre heater in gt 4 TCC port 1r 5 cm Col 1 in gt 4 TCC port 1r 15 cm Col 1 in gt 4 TCC port 2r ValveRight Port 2 to Column 2 Inlet for 5 cm columns ants 1 5 cm Col 2 in gt 4 TCC port 2r ValveRight Port 2 to Column 2 Inlet for 15 cm columns Bn l l 15 cm Col 2 in gt 4 TCC port 3r 5 cm Col 3 in gt 4 TCC port 3r 15 cm Col 3 in gt ValveRight Port 1 to Column 1 Inlet for 5 cm columns 4 TCC port 4r 15 cm Col 4 in gt 4 TCC port 5r G ValveRight Port 5 to Column 5 Inlet for 5 cm columns ae t 1 5 cm Col 5 in gt 4 TCC Ea G Val aay l alveRight Port 5 to Column 5 Inlet for 15 cm columns 15 cm Col 5 in gt B tubing to replace the direct connection of WPS 3000 to the TCC in case an eluent preconditioner
36. anel tab for the Column Compartment has script buttons that can be changed by the user FIGURE 3 5 A script contains a list of commands to be executed per line The script buttons labeled Column 1 to Column 6 can be changed by right clicking on the button selecting the properties and selecting the Command tab The commands in the script in the example depicted in FIGURE 3 5 will equilibrate the 4 column with 100 mobile phase A for 500 seconds TABLE 3 2 lists a short explanation of the commands used in the example li Tip Changes in the script button properties will not be automatically saved in the Commands j Connect Disconnect More Options Service Qualification Wellness Relays Inputs Ret Time panel tabset Any changes will be lost as soon as the panel tabset 1s closed To permanently save changes locate the panel U tiMate_3000_TCC 3x00 MPV pan in directory Dionex Templates Panels Tabset_Panels UItiMate_3000 Open the panel and make the desired changes Before saving it may be necessary to unlock the Dionex Templates directory This is done by right clicking the directory and selecting Properties After you have made the changes save the file manually Temperature Control Columns Temperature Control fon 3 Column 1 Column 2 Column 3 Nominal Temperature Column 4 Column 5 Column 6 Temperature Ready Delta a 0 C as Actual Value Colum
37. arallel LC 3 The next step in the wizard is to set the temperature values for the column compartment Make sure that the same temperature settings are used for the column compartment when creating the program for Timebase_ 2 Program Wizard ColumnOven Options Temperature Control M Use Temperature Control Temperature 30 0 5 0 85 0 C Lower Limit 5 0 5 0 85 0 C Upper Limit 25 0 5 0 85 0 C Equilibration Time 2 0 None 30 0 min Ready Temp Delta li Q None 5 0 T Cancel l Help FIGURE 4 12 Program wizard column compartment temperature settings Operating Instructions 45 Parallel LC 4 The next step in the wizard is to set the pump flow rate flow acceleration minimum and maximum pressure and gradient type These options are only available for PumpLeft of the DGP 3600 because this is the pump connected to Timebase_1 Program Wizard PumpLeft Options Gradient Type Solvents Name m Pressure Limits r Column How Lower Limit 0 10 400 bar Stat 1 000 10 000 10 000 ml min Upper Limit 400 D 400 bar End 000010000 miimi Maximum How Acceleration Deceleration Up i 000 0 001 9999 999 ml min4 Down 1 000 linfinite 9999 999 ml min Next gt Cancel Help FIGURE 4 13 Program wizard pump settings The settings for PumpRight will be available when a program is made for Timebase_ 2 If t
38. art the wizard and select Timebase_1 to create a program that will use the hardware in Timebase_1 FIGURE 4 10 Similarly Timebase 2 should be selected to make a program for the hardware in Timebase_ 2 The manual will describe the program for Timebase_1 and indicate where changes apply when Timebase 2 is used Program Wizard Select Timebase Options The Program Wizard guides you through the creation of program files To start select the timebase where the program will run Timebase Timebase_1 3 some 5 Timebase_ 2 RD_MARCO ga Favorites Network Neighborhood Protocol Enter connection information manually or pick a timebase from the list at right FIGURE 4 10 Program wizard selection of the timebase A Note The wizard is not able to consider every possibility for Parallel LC Some manual changes may have to be applied at the end this fine tuning is explained in section 4 2 3 2 Operating Instructions 43 Parallel LC 2 Next specify the temperature settings for the autosampler Make sure that the same temperature settings are used when creating the program for Timebase_ 2 Program Wizard Sampler Options M Use Temperature Control Temperature 7 0 4 0 45 0 TC Max Deviation 5 0 None 10 0 TC m Satety Limits Lower Limit 4 0 4 0 45 0 TC Upper Limit 45 0 4 0 45 0 C FIGURE 4 11 Program wizard autosampler temperature settings 44 Operating Instructions P
39. at are commonly used in regular programs are not explained e g detector wavelength settings or injector settings 1 After selecting the timebase to use for the program the next step in the wizard FIGURE 6 7 allows selecting the type of program to create Select Program for On Line SPE LC Solid Phase Extraction Liquid Chromatography Program Wizard Wizard Options Which kind of Program do you want to create C Regular Program z Back Cancel Help FIGURE 6 7 Program wizard select the type of program to create Operating Instructions 71 Automated On Line SPE LC 2 After selecting the Program for On Line SPE LC Solid Phase Extraction Liquid Chromatography the wizard will display a schematic overview of the On Line SPE LC system and provide links to the Chromeleon online help to determine the Matrix Depletion Time t M Analyte Break Through Time t A and Transfer Time t T _ On Line SPE LC System Schematic Sample Transfer Separation Fractionation Separation 1 Dual Gradient Pump 4 Analytical Column 2 Autosampler 2 Detector 3 SPE Column 6 Six Port Valve FIGURE 6 8 Program wizard the schematic overview Program Wizard On Line SPE LC Options The On Line SPE LC wizard is used to simplify the creation of methods for matrix elimination and analyte ennichment During the wizard process you will be prompted for Matix Depletion Time t M Analyte Break Th
40. ate the column with the mobile phases of the second application Both applications should have a similar analysis time It is possible that the valve switching time and release exclusive access time need to be adjusted if the reproducibility between the two columns is not as good as expected Improve the reproducibility by increasing the isocratic time exclusive access time and valve switching time When using sticky samples it 1s also recommended to increase the isocratic time before the gradient start Exclusive Access Time and valve switching time Operating Instructions 51 Parallel LC 4 2 3 3 Sequences A new sequence for Timebase_1 is created by clicking File New Sequence using Wizard in Chromeleon To complete the creation of the sequence simply follow the Chromeleon wizard as with a normal sequence no other steps are required for Parallel LC Just make sure that the correct timebase is selected at the start of the wizard for a new sequence After creating the sequence for Timebase_1 create a sequence for Timebase_ 2 Batches are timebase specific therefore a separate batch is started for each timebase FIGURE 4 18 and FIGURE 4 19 First start the batch with the sequence for Timebase_ 1 After starting this batch it is recommended to start the batch for Timebase 2 with the sequence for Timebase 2 immediately The batch for Timebase 2 will be on hold as long as Timebase_1 has the exclusive access to the autosampler
41. ature Ready temp delta ColumnQwen Temperature Ready temp delta Valves Actre Columns Wavelength Noise amp drift limits Data settings U lamp Value s and or limit s F8 to edit 0 800 ml min Purge Via sampler SB 72 0 SC 0 0 So Duration 5 0 min 6 72 0 SC 0 0 Lower limit 0 bar upper limit 400 bar Ripple limit 3 0 1 000 mlmin Purge Via sampler w0 0 0 mC 0 0 Duration 5 0 min B 80 0 C 0 0 Lower limit 0 bar upper limit 400 bar Ripple limit 3 0 Prime syringe 1 Wash buffer loop 300 000 pl Nominal 10 0 C lower limit 4 0 C upper limit 45 0 C 10 0 C Nominal 35 0 C lower limit 20 0 C upper limit 45 0 C 1 0 C Left 61 Right 61 None 245 nm Note 0 10 mAU Drift 3 0 mAU hours Collection rate 5 0 Hz Time constant 1 00 s Minimum intensity Use performance limit 50 Maximum Equilibration Time 45 000 min Display Success Message Next gt FIGURE 8 6 SmartStartup wizard equilibration settings for the system devices 90 Operating Instructions Automated Application Switching 5 In FIGURE 8 7 the equilibration settings for the left pump are shown The lower and upper pressure limit ripple flow rate and solvent composition can be specified It is also possible to purge the pump and to program a flush with e g mobile phase B prior to equilibration PumpLeft Equilibration Conditions Flow 0 800 0 000 1 0 000 mlmin
42. base Timebase_1 Timebase 2 Select at least 1 and at most 2 timebase s to share this device Cancel Help FIGURE 4 6 Sharing of the WPS 3000SL autosampler between the timebases Segments Pump Link Well Plate Sampler Segment Types Red Green 40 Standard Vials Blue 40 Standard Vials Waming Ensure that these settings match the installed items Pump Link How through Sampler is delivered by pump 8 UMSPUMP_L_STRK UM3PUMP_R_STRK Note This is also the pump that the injection is synchronized l with if the pump is an LPG 2c00 or DGP 3600 lt Back Next gt Cancel Help FIGURE 4 7 Specifying both pumps to deliver flow to the WPS 3000SL autosampler Operating Instructions z Parallel LC 4 2 2 Direct Instrument Control from Chromeleon The panel tabset offers the interface to control the system It is automatically generated based on the hardware configuration and can be used by clicking the panel tabset button FIGURE 4 8 For each timebase a new tabset is created and within each tabset a panel tab for each device is added Each tab will allow direct control via the panel for that device The two panel tabsets will be displayed in a splitview FIGURE 4 9 For information about direct instrument control of each device refer to the user manual or the Chromeleon online help FIGURE 4 8 Panel Tabset Button Chromeleon Ultimate_3000 Jle Je g a x faz Fie Ed
43. cnscedusveateevestescevelastewnvcsssoteeweetotevescccuelevtewscssuboteeweeteteveses sade ues towsesssbotesees 4 Ded Fanden DO at E a 5 2 4 A tomated On Line SPE LC soscsceereccetccetsesestadcstcecistessucvecinsceut sevedtuscsadcieseddewvecinicetseas 5 Ze A tmated 2D LC cassis aena aea eaaa en oaa asa aE ETa 5 2 6 Automated Application Switching ecscocssssseccccccossseseccocoosssecccecoooosssscecesssssseseeeesso 6 3 Automated Method Scouting sxcsscccicccccscesectsevecsaceeceseacsacacnscceceseadtavessceeecbasccserdeniceeceeeecs 7 Sil Preparation ofthe SVStO Ml sisson A 8 3 1 1 Considerations for Sample Analysis c cc cccccssssecsesseeedesssesesssesesedosseosesssesesedesssesesssesesedesssesensseses 8 ge Eem ll Cy A fetter cat tases ee tes tac oe te tae ta casts eee ates hace Ae tae tae caste eee testa oe ate tae ta eats thas 8 32 COMMOUTIMNG Sotware seose a Eaa EEEa 12 3 2 1 Configuring Hardware in the Server Configuration ccccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 12 3 2 2 Direct Instrument Control fron Chromele Ont eressero tinamaan Meaeeatteterte 12 32o Proorams and Segue NCES aei a Sen ee Sn ee ee eee 14 3 2 4 Automated Data Analysis with Chromeleon cccccccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 18 4 Bear ACU LC rnas n a E shed a a E EEEa 35 Al Preparation Of the System sissien aas aa u aad aE 36 A2 COMMPUPING Sowar aseeni aeeai Na 38 4 2 1 Configuring
44. ct this box if you want to compute the test condition for more than the cument sample Use aggregation z Back Finish Cancel Help FIGURE 3 18 SST Properties Test Condition The Test Condition field is entered by clicking the button with dots FIGURE 3 19 depicts the available test conditions Edit Result Formula Categories Variables General Signal Value at Peak Retention Sequence P Signal Value at Peak End T Sample Baseline Value at Peak Start Audit Trail Baseline Yalue at Peak Retentic Preconditions Baseline Value at Peak End Chromatagram Detection Code at Peak Start Detection Parameters Detection Code at Peak End Peak Results Type Peak Calibration Modified Peak T able Manually Assigned Peak Purity and Identification Resolution Explain variable Mi sahin sb hank hii A ararnambr i hiia Formula peak resolution ep next Parameter FIGURE 3 19 Specification of the test condition for the SST Operating Instructions 25 Automated Method Scouting The Parameter button in FIGURE 3 19 gives the option to specify additional parameters such as resolution to next or previous peak when resolution is selected or the height at which the peak width has to be determined if peak width was selected as test condition 4 In Peak amp Channel Condition the compound can be selected to which the conditions should apply as defined in the previous steps including the channel like UV VI
45. d e The sample is injected on the second column e The above steps are repeated Operating Instructions 35 Parallel LC 4 1 Preparation of the System The schematic of the HPLC set up for Parallel LC is shown in FIGURE 4 1 Column 1 gt H FIGURE 4 1 Schematic representation for Parallel LC All components except the columns that are required for a fully operable system are provided with the system modules or the Parallel LC kit For a list of supplied capillaries and accessories in the Parallel LC Kit see TABLE 4 1 SRD 3600 Solvent PDA 3000 Photodiode Rack and Degasser Array Detector or VWD DGP 3600A M SL 3x00 Variable Wavelength Dual Gradient g Detector Analytical Micro TCC 3100 1x2P 6P Pump we thermostatted Column Compartment with 1x 2 PS L Hi sA es s gt Position 6 Port Switching Analytical or Micro Valve Split Loop Autosampler KS PDA 3000 Photodiode Array Detector or VWD 3x00 Variable Wavelength Detector FIGURE 4 2 Stack of the Parallel LC system A complete description of the installation and configuration of the hardware is presented in the Quick Installation Guide The application kits P N 6037 0004 Parallel Operation Kit Dual Ternary Analytical or P N 6037 0005 Parallel Operation Kit Dual Ternary Micro contain all the tubing to make all the fluidic connections for a micro or analytical Parallel LC system 36 Operating Instructions Parallel LC TABLE 4 1
46. d Method Scouting Automated Method Scouting is an LCi solution for investigating the effect of changing a chosen subset of the large number of parameters that are involved in HPLC method development It 1s designed to aid the analyst in the selection of the optimum stationary phase mobile phase and column temperature as well as other method parameters Automated Method Scouting will perform the experiments and present the optimum of the chosen set of conditions to the analyst The HPLC system that is used with the Automated Method Scouting program includes a pump with the ability to perform quaternary gradients a thermostatted column compartment with two 6 position 7 port valves to allow easy switching between columns a split loop well plate autosampler and a UV or a PDA detector that is fully controllable by Chromeleon Chromatography Management Software 2 2 Parallel LC Parallel LC is an LCi solution for doubling the throughput for both isocratic and gradient separations by efficient use of hardware The Parallel LC system uses two pumps and two detectors but only one autosampler and one column compartment This allows the user to operate one Parallel LC setup as two independent HPLC systems Parallel LC offers an increase in throughput without the need for new method development This eliminates the need to revalidate methods and revision of corresponding documentation when using other approaches to increase sample throughput The Parall
47. e The template contains UDCs To use the template in your own datasource it is necessary to import the UDCs To import the UDC in the target in your datasource 1 Right click the datasource green icon in the Chromeleon browser 2 Click properties and select the User Defined Columns tab FIGURE 3 6 3 Click the button Import Columns 4 Select the datasource from the LCi solutions CD 3 2 4 Automated Data Analysis with Chromeleon After all the data has been acquired it is possible to find the best separation conditions without manually going through all the generated data A PDA 3000 detector can be used with spectra libraries to include compound identification A VWD 3X00 can be used when only the resolution and number of peaks are of interest Use the method file SCOUTING gqnt and the Find Best Method rdf file supplied with the Chromeleon templates on the Intelligent LC Solutions Reference Library CD for automated data analysis 18 Operating Instructions Automated Method Scouting 3 2 4 1 Peak Tracking with Spectral Library Recorded with the PDA 3000 The PDA 3000 detector makes it easy to identify the various compounds in the sample The absorption spectra of the desired compounds should be sufficiently different and each compound should elute at a different retention time In addition it should be noted that compound identification is required if there 1s a need to have sufficient resolution for selected compound s in the s
48. e SQL SaL Result Restrictions Title guerram Datasource lt lt Selected Datasource gt gt General Into Created 9 11 2007 14 45 03 mkarsten Last Update 9 11 2007 14 45 03 mkarsten Prefered Report amp Channel Prefered RDF File NIL Ej Frefered Channel FIGURE 3 29 Last screen of the Query Wizard Operating Instructions 31 Automated Method Scouting The query will generate a virtual sequence in the directory where the search is performed SST Passed in FIGURE 3 30 In the virtual sequence only the samples that have passed the SST will be displayed Title AMSTERDAM S coutFinahNew Column _Scoutingsorted CO 5 all columns New _Column_Scouting a Smp sorted_COMBI_TEST_SCOUTING ey ff Diuretics pH3 ACN AMSTERDAM Scout Standard i ff Diuretics pH3 MeOH AMSTERDAM Scout Standard nal J i iE 5 Tm E EE ic era MEN fi Diuretics pH3 MeOH AMSTERDAM Scout Standard In assed 2 COLUMN SCOUTING DIURETICS 2mpv 4 fF Diuretics pHs ACN AMSTERDAM Scout Standard Find_Best_Hits 5 Diuretics pH3 ACN AMSTERDAM Scout Standard ral Al TOMATED MFTHON DEVE OPMENT imnv FIGURE 3 30 Results of the query showing the sample name sequence name and sample number 32 Operating Instructions Automated Method Scouting 3 2 4 5 The Report Graphically Depicting the Results The results can be graphically displayed by using the report Find_Best_Method rdf The method sample number is displayed o
49. e of the accompanying information may result in damage to the instrument li Tip Indicates general information intended to optimize the performance of the instrument Safety Precautions Warning The following precautions should be followed to minimize the possibility of personal injury and or damage to property i Tip Make sure that you are familiar with the contents of this manual and the operating instructions before working on the system The operator should follow all safety precautions warnings etc provided with the instruments in addition please note the items presented below All components of the system should be plugged into a common power line that is directly connected to a true ground Repair or replace faulty power cords and all communication cables If a leak occurs turn off power to the instrument and remedy the situation immediately If the mobile phase includes volatile or flammable solvents avoid open flames and sparks Many organic solvents and buffers are toxic Make sure that you know the toxicological properties of all mobile phases that you are using The toxicological properties of many samples may not be well known If you have any doubt about a sample treat it as if 1t contained a potentially harmful substance Wear protective eye goggles when handling mobile phases or operating the instrument An eye wash facility and a sink should be close to the unit If any mobile phase splashes on t
50. e switch between the applications is performed by an extra program To combine both applications in one system startup programs and switching programs between the applications have to be made as described below If the system is installed according the Quick Installation Guide the pump and valve settings are listed in TABLE 8 2 TABLE 8 2 Device settings for the applications and wash step ae Application 1 Wash Step Application 2 PumpRight PumpLeft PumpLeft If a 10 port valve is installed in the column compartment replace the valve position 6 1 with 10 1 The recommended method of operating would be to use a separate sequence for each application including standards samples etc Between those two sequences a sequence is programmed to flush the autosampler fluidics and to switch to the next application The three sequences are combined in a Batch by Chromeleon to be run unattended one after the other Application 1 2 Programs FIGURE 8 4 Schematic overview of sequences for Automated Application Switching Create sequences and program files for both applications In the program files for the application set the valves to lt do not switch gt in the ColumnOven Options step of the wizard The valves are not used during the run of one application and there are no settings required for the inactive pump Save the programs and sequences for later use Each application sequence will use a startup program to prepare the system for
51. eara a E 78 Tek Preparation ofthe SysteM cecornisiesnnsen ea EE aaa 79 Ta COMMBUPING Sotware aicese EE EEES 81 7 2 1 Configuring Hardware in the Server Configuration cccccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 81 7 2 2 Direct Instrument Control from Chromeleon cccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 81 U2 Programs ANG CeO Ne NC CS eeni Facets neta ha tage te N ees at asa ae tae hake ee vata aca a et tas has dee 81 8 Automated Application Switching ccccccsssssssssscccccssssssssssssccccsssssssssssscccssssseeees 84 SL Preparation ofthe Systemics iiel 85 2 Conhcurino SOWAT assire a e R EEEa 87 8 2 1 Configuring Hardware in the Server Configuration cccccccceeeseeeeeeeeeeeeeeeeeeeeseeeeeeeeeeeeeeeeeeeeees 87 8 2 2 Direct Instrument Control irom C hromeleon cernere E A E E A A 87 52 9 Programs ANG SSCUCIICES aissa a a a e Siesta 88 2 Operating Instructions LCi Solutions 1 How to use this Manual The material included in this manual is provided as an introduction to Dionex LCi solutions It is assumed that the individual using this manual has sufficient training in the use of analytical instrumentation has a basic knowledge of Chromeleon Chromatography Management Software and is aware of the potential hazards including but not limited to electrical hazards chemical solvent hazards exposure to UV radiation and the exposure to pressu
52. efore the solvent composition for the IMAC column will be left unchanged from the previous program Details are provided in TABLE 7 3 TABLE 7 3 Second dimension program HPLC analysis of first fraction Time Events 1 000 Valve position 1 2 ON 0 2 31 0 0 1 000 Autozero detector and start 82 Operating Instructions acquisition of data Automated 2D LC 7 2 3 3 First dimension elution program The third program elutes the phosphopeptides from the IMAC column by increasing the B The valve is again in position 10 1 to capture the eluting phosphopeptides Details are provided in TABLE 7 4 TABLE 7 4 First dimension elution program elute from the IMAC IMAC Ro Time Events Valve position 10 1 Inject start acquisition 7 2 3 4 Sequence The second dimension program is also used to analyze the phosphopeptides on the RP column and has to be run after the third program The example sequence is shown in FIGURE 7 4 No Name Type Pos Inj Wol Program 1 A Sample Name Start of injection Unknown RES 20 0 Sample injection 2 B RF analysis of breakthrough Blank GA 20 0 Second dimension analysis 3 a Elution of phosphopeptides Blank GA 20 0 First Dimension Elution 4 a RP analysis of phosphopeptides Blank GA 20 0 Second dimension analysis FIGURE 7 4 Example sequence for the analysis of phosphopeptides It is possible to use more steps to elute the sample from the first dimension when usi
53. el LC solution is designed to allow maximum sample throughput while saving resources and reducing investment Chromeleon Chromatography Management Software treats the configuration as two complete independent systems and manages seamless autosampler and column compartment sharing The HPLC system that 1s used with the Parallel LC program includes a dual pump with the ability to perform two ternary gradients a thermostatted column compartment with one 2 position 6 port valve to allow easy switching a split loop well plate autosampler and two UV or two PDA detectors that is fully controllable by Chromeleon Chromatography Management Software 4 Operating Instructions LCi Solutions 2 3 Tandem LC Tandem LC is an LCi solution for increasing the throughput with 50 up to 100 for gradient separations With Tandem LC two different flow paths are used allowing off line equilibration of one column while another column is used for the analysis Tandem LC offers an increase in throughput without the need for new method development This eliminates the need to revalidate methods and revision of corresponding documentation when using other approaches to increase sample throughput The HPLC system that is used with the Tandem LC program includes a pump with the ability to perform two ternary gradients a thermostatted column compartment with one 2 position 10 port valve to allow easy switching a split loop well plate autosampler and a UV or
54. eration Deceleration Lower Limit 0 0 400 bar Up 1000 0 001 999 999 ml min Upper Limit 400 0 400 bar Down 1 000 0 000 9999 9399 ml min FIGURE 5 7 Program wizard Tandem pump options Li Tip It is recommended to set the maximum flow acceleration deceleration to a value between 1 3 or 3 times the flow rate This avoids a very fast increase or decrease in flow rate and pressure A too sudden increase or decrease in pressure could disturb the column bed 60 Operating Instructions Tandem LC 5 In this step of the wizard the Tandem Gradient Options the gradient profile and Off Line Reconditioning Start Time are specified The gradient profile 1s made by specifying the time percentage of mobile phase and flow rate in this step of the wizard The gradient profile created here should be made as for a single column The off line reconditioning start time is the time after which the acquisition will end in the program Set it to a time after which no more components will elute from the column In the example depicted in FIGURE 5 8 the gradient runs for 5 minutes followed by a 2 minutes wash step and a 5 minutes equilibration step In the wash step and equilibration no components of interest elute of the column therefore the off line reconditioning start time is set to 5 minutes Program Wizard Tandem Gradient Options Specify the complete single column gradient profile and an off4ine reconditioni
55. f your UitiMate parallel system Used Switching Valve Valve Right Valve Position 12 Capillary Void Volume 30 0 0 1000 yi lt Back Cancel Help FIGURE 4 14 Program wizard Parallel Operation Options li Tip For the program file for Timebase 2 the same valve is used ValveRight but the valve position should be set to 6 1 Typically the Capillary Void Volume is the same in both program files 6 The next step in the wizard Sampler Options allows to specify the injection parameters No special considerations are necessary for a Parallel LC system in this step of the wizard Operating Instructions 47 Parallel LC 0 010 83 333 p s Draw Delay 000 0 300000 ms Dispense Speed 2 000 0 010 83 333 pls Dispense Delay 2000 D300000m Dispense to Waste Speed 4 000 0 010 83 333 pis Sample Height 2 000 0 000 30 000 mm Inject Wash 7 Wash Volume 0 000 5000 000 yi Wash Speed m 0 010 83 333 pis coed e FIGURE 4 15 Program wizard autosampler options injection parameters 48 Operating Instructions Parallel LC 7 The next step in the wizard continues with the Sampler Options Program Wizard Sampler Options Inject Mode Noma it Connected Pump Device Pump Lett i Synchronize injection with pump Bypass Flush Out Factor i 0 1 0 10 0 Maximum Inject Volume 100 000 0 007 250 000 ui Flow ml min Bypass Time min
56. gram file and will use the value entered in the corresponding UDC ColumnOven Temperature Nominal 50 0 C change it to ColumnOven Temperature Nominal Sample ColumnOvenTemp 16 Operating Instructions Automated Method Scouting Sampler Temperature UpperLimit 45 0 c Sampler ReadyTempDelta 10 0 ColumnOven TempCtrl ColumnOven Temperature Nominal ColumnOven Temperature LowerLimit ColumnOven Temperature UpperLimit EquilibrationTime 0 5 min ColumnOven ReadyTempDelta ValveLeft ValveRight Sample ColumnSelector Sample ColumnSelecto No Type ColumnSelector column_name Pos Inj Wol Dil Facto STD Select Active Column example Celeius 1 Standard 684cclaim C18 RC1 30 0 1 0000 1 0 2 Standard Acclaim C18 RC1 30 0 1 0000 1 0 3 Blank Acclaim C18 RC1 30 0 1 0000 1 0 4 Blank Acclaim C18 RC1 30 0 1 0000 1 0 5 Standard 684cclaim C18 RC1 30 0 1 0000 1 0 6 Standard 684cclaim C18 RC1 30 0 1 0000 1 0 T Blank 1 4cclaim Polar4dyvantage Il RC 30 0 1 0000 1 0 6 Standard cclaim PolarAdyvantage Il RC 30 0 1 0000 1 0 9 Standard cclaim Polar4dvantage Il RCI 30 0 1 0000 1 0 FIGURE 3 8 UDC example in a program file and a sequence A UDC 1s recognizable in the sequence by the asterisk in the column header In FIGURE 3 8 a comment is used to identify the column for a valve position Chromeleon will not accept string values when integers for the UDC are expected in the progra
57. he applications on both systems are not identical make sure the mobile phases are fully miscible This is to prevent precipitation of buffers in the shared fluidics of the system li Tip It is recommended to set the maximum flow acceleration deceleration to a value between 1 3 or 3 times the flow rate This avoids a very fast increase or decrease in flow rate and pressure A too sudden increase or decrease in pressure could disturb the column bed 46 Operating Instructions Parallel LC 5 The next step in the wizard provides the Parallel Operation Options These are used to select the fluidic pathway of the autosampler When the hardware is configured according to the Quick Installation Guide valve right is used to switch between the two systems Position 1 2 includes the autosampler in Timebase_1 and position 6 1 includes the autosampler in Timebase_ 2 The Capillary Void Volume is the volume of the tubing From the valve to the autosampler From the autosampler back to the valve The volume from the valve to the column The volumes of the grooves from the injection valve and the valve in the column compartment also need to be considered by the user The value entered here will be used to calculate the valve switching time in the program The volume of the tubing is determined by the user the volume depends on the length and ID of the tubing Program Wizard Parallel Operation Options Please specify the configuration o
58. he eyes or skin wash the affected area and seek medical attention Dispose of all waste mobile phase in an environmentally safe manner that is consistent with all local regulations Do not allow flammable and or toxic solvents to accumulate Follow a regulated approved waste disposal program Never dispose flammable and or toxic solvents through the municipal sewage system Operating Instructions Page III LCi Operating InstructionsLCi Solutions e Wear protective eye goggles when handling fused silica tubing i e installation cutting etc e Ifa buffer is used as a part of the mobile phase flush the system with several volumes of a methanol water 50 50 solution before it is shut down This will prevent salt buildup inside the unit e Do not use the instrument in ways other than those indicated in the instructions given in the documentation provided with the instrumentation Page IV Operating Instructions LCi Solutions Table of Contents W APM GS acdc adwaven tuadesonnt EEE E EE Il Safety Precautions 45045 tnscachawdsdoriaenanuduuinadasenenesaana aa Il 1 How to use Unis Wi aniw alos sccscccitcaseousceccceseeuaseasacccccibeuaseeasccecacet ueuautaddiacceissuadeisedinnceet bees 3 2 Introduction to LCi Solutions sissacessscccccseaceevienchescosescestecssecccassadesstencesecesucessteesiesceeceses 4 2 1 Automated Method Scouting ccsssssscccccccssssscccccocssssescecosoosssesccccoscsssseseseosossseseecesso 4 22 MAG AMC UNG we aele
59. hromeleon online help Use the default device names in the server configuration Enable column A and column B in the TCC 3x00 configuration allowing easy selection between the two columns It is recommended to use column identification chips P N 6710 1505 set of 5 The column identification chip allows storing column specific data More information regarding the column identification chips can be found in the Chromeleon online help or the manual of the TCC 3100 6 2 2 Direct Instrument Control from Chromeleon The panel tabset offers the interface to control the system It is automatically generated based on the hardware configuration and can be used by clicking the panel tabset button FIGURE 6 3 A panel tab for each device is automatically added to the tabset each tab will allow direct control via the default panel for that device For information about direct instrument control of each device refer to the user manual or the Chromeleon online help for that device FIGURE 6 3 Panel Tabset Button 68 Operating Instructions Automated On Line SPE LC 6 2 3 Programs and Sequences 6 2 3 1 Preliminary measurements In preparation of the Automated On Line SPE application three constants have to be determined experimentally The values will be used by the program wizard to calculate the valve switching times To perform these experiments the same mobile phase tubing and flow rate as for the sample analysis should be used Howeve
60. it View Workspace Qualification Control Batch Window Help E D cm tel EB odb 2 e nT Qe Timebase_1 7 gt l Timebase_2 7 gt l Home Sequence Control Sampler Cal Comp Pump wD Home Sequence Control Sampler Col Comp YWD Commands Start Up Tray Control Commands Signals P coma Prime Syringe gt Eee BES vine constant Raa Disconnect Cycles ho 40_Vials Disconnect 190 nm ii E Start Tray To Front P _Acq On Vials To Front _Aca off JP a Wash Buffer Loop _Autozero Aaaa 300 pl Ready a a On line Plot 1 000 yl perme i e ee Mins amn FIGURE 4 9 Splitview of the panel tabset The splitview allows easy switching between direct control for the instruments in the timebases The active tabset 1s outlined in red by Chromeleon F A a This icon allows a fullscreen view of one panel tabset ole This icon will set the panel tabsets in splitview mode 42 Operating Instructions Parallel LC 4 2 3 Programs and Sequences 4 2 3 1 Programs A new Program File can be created by clicking File New Program File in Chromeleon A wizard will appear allowing the user to set the various program settings such as column compartment temperature flow rate etc The steps of the wizard are described below Program settings that are unique for Parallel LC are explained in detail Regular program settings are not shown e g detector wavelength settings 1 St
61. itions Temperature nominal 20 5 0 85 0 C Lower Limit 50 5 0 85 0 C Upper Limit 850 0 5 0 85 0 C Ready Temp Delta Wo Mone 5 0 C Valve Left 61 ha E_1 1_2 Valve Right Active Columns Column 6 Cancel Help FIGURE 8 9 SmartStartup wizard column oven equilibration conditions 92 Operating Instructions Automated Application Switching 8 The UV equilibration conditions are shown in FIGURE 8 10 and allow to specify the wavelength data collection rate time constant minimum lamp intensity and noise and drift limits UV Equilibration Conditions Equilibration Channel Channel Uy VIS wavelength 245 090 500 rm Data Collection Rate 5 0 0 2 100 0 Hz Time Constant 1 00 000 455 3 Moise Limit O10 003 050 mA rift Lirnit 30 0 8 20 0 mA amp U hours Lamps e Minimum Intensity Use Performance Limit 50 Custom IL io G Cancel Help FIGURE 8 10 SmartStartup wizard UV equilibration conditions 9 If all the equilibration settings for the devices are finalized click Next in the equilibration settings for the system devices FIGURE 8 6 Make sure that all devices are switched on before the equilibration is started FIGURE 8 11 Operating Instructions 93 Automated Application Switching SmartStartup Wizard Start Equilibration of Timebase dgp3600a Start Equilibration Assure all devices are switched
62. izard FIGURE 5 10 Click the Open TCC 3x00 x2 Valve Settings Panel button to open a small dialog window with the option to set the valve position and columns for Tandem LC FIGURE 5 11 The same settings as used in the panel FIGURE 5 4 can be applied here The active column is column A with the valve position 10 1 The analytical and reconditioning pumps are already set with the wizard in the Tandem Pump Options step of the wizard FIGURE 5 7 Program Wizard Valve Settings Options The Ultimate Tandem System requires to specify which columns to use and which valve port is connected to which column Please press the button below to open the TCC 3100x2 Valve Settings Panel where you can adjust those settings Open TCL 3c00 x2 Valve Settings Panel lt Back aa Dee FIGURE 5 10 Program wizard Valve Settings Options Operating Instructions 63 Tandem LC Fal TCC 3x00 x2 Valve Settings mmx Left Valve Right Valve Column A Column B Column C Column D Columns to Use AB Active Column A Next Column FIGURE 5 11 Program wizard panel for the valve settings After making the changes for the valve settings the TCC 3x00 x2 Valve Settings panel can be closed The wizard can be continued by clicking next then the options to save and or review the program file is offered indicating that the program has been completed It is recommended to verify that the analytical pump is on line with the active column bef
63. l LC Parallel LC is an LCi solution for doubling the throughput for both isocratic and gradient separations by efficient use of hardware The Parallel LC system uses two pumps and two detectors but only one autosampler and one column compartment This allows the user to operate one Parallel LC setup as two independent HPLC systems Parallel LC offers an increase in throughput without the need for new methods The system includes e One DGP 3600 pump that can make two ternary gradients independently and simultaneously The left pump of the DGP 3600 is used in one timebase while the right pump is used in the other e One TCC 3100 column compartment The 2 position 6 port valve allows easy switching between columns on which the sample will be injected Both columns are installed in the column compartment e One WPS 3000SL split loop autosampler When the injection is done in the first timebase the autosampler is idle for the remainder of analysis time and can be used to inject in the other timebase The sharing of the autosampler between the timebases 1s performed by providing one timebase with exclusive access to the sampler e Two PDA 3000 or two VWD 3x00 detectors Each timebase has its own detector to allow simultaneous detection on the two timebases Parallel LC experiment stages e Injection of the sample on the first column e Analysis start on the first column e While the analysis is running on the first column the valve is switche
64. l the peak table with the peak properties of the active chromatogram e g retention time for a compound The peak properties for other 20 Operating Instructions Automated Method Scouting chromatograms e g identification retention time are determined automatically by Chromeleon and used in the report No Peak Name Ret Time Window Reference Spectrum Match Crit afsmioride O 2 042 min 0 042 AS LAA _ Diu retics pH3 AC Least Square ie Chiortalidone 4 458 min 0 075 AS h Diu retics pH3 AC Least Square E Furosemide 6 142 min 0 129 AS fla Diu retics pH3 AC Least Square 4 Ethacrynic acid 7 925 min 0 079 AS Diuretics pos AG Least Square 5 Probenecid 6 192 min 0 054 AS Diuretics pH3 AC Least Square 6 Triamterene 15 925 min 0 142 AS Diuretics pH3 AC Least Sgu Peak Table 4 Amount Table A Peak Tracking A Calibration A Spectra Library Screenine FIGURE 3 12 The Peak Tracking tab from the Method Editor with Autogenerated Peak Table from the Spectra Library 3 2 4 2 Peak Tracking with the VWD 3x00 If a UV detector such as the VWD 3x00 is used it is not possible to identify the peaks automatically If an overall average resolution for the compounds 1s the criterion for the method validation there is no need to identify the compounds in the sample It is possible to use Autogenerate Peak Table in the Peak Table of the method editor without any Spectra Library Select Enumerate peaks of current chromatogram F
65. laies Malar A dsenntoane Il a me otis od Cada FIGURE 3 24 The SST Result column in a part of the sequence li Tip It is recommended to put the sequence s in a separate directory before performing the query This has the advantage that you limit the query to the sequence s of interest The query results will also be available in the directory making it easy to retrieve the same data again After the creation of the report column for the SST perform a query by right clicking the sequence and selecting Query The Query Wizard will ask several questions to define the criteria for the query The various steps from the Query Wizard are explained below Press Next to continue to the next step 1 Select the appropriate datasource and the field types The next steps depend on the selection of the field types Select all field types in FIGURE 3 25 Query Wizard Query Wizard r Datasource Selected datasource curently ULTZ local 0 Fed datasource Field types IW Sequences f you click the Next button the Query Wizard will ask you Tor more information on Sequences Samples and W Samples Results W Results Cancel Help FIGURE 3 25 Datasource and Field type selection in the Query Wizard Operating Instructions 29 Automated Method Scouting 2 The search results can be limited to a directory containing the sequence Select the directory containing the sequence s used for Automated Method Scouting in FIGURE
66. m file For information an additional UDC named column_name is created to link column information to a valve position The column_name UDC will not be used by the program but will help to relate a valve position in the sequence to a chosen column 3 2 3 3 Sequence A new sequence is created by clicking File New Sequence using Wizard in Chromeleon To complete the creation of the sequence simply follow the Chromeleon wizard After creation of the sequence enter desired values in the User Defined Columns e g for the position of the multi position valve or the column oven temperature Li Tip It is recommended to set up the sequence to run a set of various conditions e g different pH or organic modifier on each analytical column on a sequential basis before changing the column oven temperature The sequence is now ready to start Open the batch and add the sequence s to the batch It is recommended that you use SmartStartup a Chromeleon feature to equilibrate the system before running the sequence The SmartStartup is started from the Batch menu in Chromeleon The batch can be started now Operating Instructions 17 Automated Method Scouting 3 2 3 4 Importing Example Data The Intelligent LC Solutions Reference Library CD contains templates for the Automated Method Scouting application This datasource can be mounted by clicking File Mount Datasource and selecting the correct drive letter for the CD or select brows
67. mated 2D LC kit see TABLE 7 1 SRD 3600 Solvent Rack and Degasser DGP 3600A Dual Gradient Analytical Pump or DGP 3600M Dual Gradient Micro Pump WPS 3000SL Analytical or Micro Split Loop Autosampler TCC 3100 1x2P 6P Thermostatted Column Compartment with 1x 2 Position 10 Port Switching Valve PDA 3000 Photodiode Array Detector or VWD 3x00 Variable Wavelength Detector FIGURE 7 2 Stack of the Automated 2D LC system Operating Instructions 79 Automated 2D LC A complete description of the installation and configuration of the hardware is presented in the Quick Installation Guide The application kit P N 6037 0008 2D LC Kit Dual Ternary Analytical contains all the tubing to make all the fluidic connections for an analytical 2D LC system TABLE 7 1 List of capillaries and accessories supplied with the Automated 2D LC kit Description Capillary Left Pump TCC port 8 ca Capillary WPS Port 4 Col 1 RO oC Pa capitan c26 OOOO o Pa ei cot sem Tope H Capillary Col 2 25 cm TCC port 9 e cat 106 pon a ooo a F Capillary TCC port 1 VWD Outlet 1 casino TOC 7 106 oe PEEK capillary for VWD waste connection 1 Capillary TCC PDA analytical M Finger tight 33 mm fitting set 5 pcs E Quick Installation Guide U3000 Application Kits ra 80 Operating Instructions Automated 2D LC 7 2 Configuring Software 7 2 1 Configuring Hardware in the Server Configuration The UltiMate 3000 Aut
68. meters are set on the SST tab in the method editor FIGURE 3 15 1 Right click on a line select Lines and then select Insert Line or Append Line to start with a new condition for the SST properties A wizard will assist to create a new SST parameter If SST properties are edited a screen will appear with tabs for the settings instead of the wizard Each next step for the SST is a step in the wizard Click Next in the wizard to advance one step Predefined tests for the SST are presented in FIGURE 3 16 Ho Name Sample Condition Test Condition Peak Operator Value Result a Number of Peaks sample Type Standard chm nPeaks i Passed _2 PRoextProbenecid Sample Type Standard peak resolution ep next 1 5 n a gt Failed Ea PR previous Probenecid Sample Type Standard peak rezolution ep previous 1 5 n a gt Failed 4 PR next Chlortalidone Sample Type Standard peak resolution ep next 1 5 n a gt Failed 5 PR previous Chlortalidone Sample Type Standard peak resolution ep previous 1 5 n a gt Failed 6 PR next Furosemide sample Type Standard peak resolution ep next gt 1 5 n a gt Failed By PR previous Furosemide Sample Type Standard peak rezolution ep previous 1 5 n a gt Failed FIGURE 3 15 The SST in the Method Editor SST Wizard New Single Test SST Wizard This Wizard guides you through the creation of a Sy
69. mns are already defined ColumnOvenTemp and ColumnSelector To define a new UDC in the datasource properties 1 Select lt New user defined column gt and specify the Properties e Name is the name used for the UDC in the sequence and in the program It may only contain letters numbers or an underscore e The Value type depends on the type of command to be used e g integer for integer values like a valve position or column compartment temperature or string for command parameters If an integer is selected define the minimum maximum and default values e g 5 C 70 C default 25 C for the column oven temperature In the case of a string for Value type anything can be used in the UDC it will be enclosed between quotation marks automatically 2 Press Append Column to add the new UDC to the existing columns in the sequence 3 Stop and start the Chromeleon server to use the UDC in the programs Operating Instructions 15 Automated Method Scouting 3 2 3 2 Programs A new Program File can be created by clicking File New Program File in Chromeleon and then following the Chromeleon wizard to complete the creation of the program file The program wizard has no option for Automated Method Scouting therefore the program has to be modified manually in a second step The new program file or alternatively an existing program file will be used as a template which is modified for the use of the UDC Sampler TempCtrl on
70. mpounds or for different test parameters The lines in the SST tab of the method editor are depicted in FIGURE 3 22 No Name Sample Condition a Number of Peaks Sample Type Standard 2l PR next Probenecid Sample Type Standard 3 PR previous Probenecid sample Type Standard 4 PR next Chlortalidone Sample Type Standard 5 PR previous Chiortalidone Sample Type Standard 6 PR next Furosemide sample Type Standard Ea PR previous Furosemide sample Type Standard FIGURE 3 22 List of different parameters for the SST In the example in FIGURE 3 22 more than eight compounds should be found in the sample Test Condition chm nPeaks peak resolution ep peak resolution ep peak resolution ep peak resolution ep peak resolution ep peak resolution ep lt Back HEr Cancel Help next previous next previous next previous Peak Probenecid Probenecid Chiortalidone Chiortalidone Furosemide Furosemide Operator Value a 15 15 1 5 1 5 15 1 5 UV _VIS_1 UY VIS 1 UV VIS 1 UV VIS 1 UV VIS 1 UV VIS_1 UV VIS 1 Failed Fassed Fassed Passed Passed Passed Passed and the compounds probenecid chlortalidone and furosemide should be baseline separated 7 Perform a query as described in Section 3 2 4 4 to find the results of interest Operating Instructions 27 Channel WLA Result Passed Passed Passed Passed Passed Passed Passed
71. n Details Leak Sensor Settings Left Valve Right Valve Gas Leak Sensor standard 4 izi Leak Alarm Off 3 3 Humidity Leak Sensor Low Audit Trail 4915 19 57 Chromeleon server stopped for f 30 0 TCC 3200 11 _U2000M_RO RD_MARCO ColumnOven_Temp configuration 15 19 2284 Script Button Properties 15 19 tempd Gener itosize Button Command SEQ General Autosize Button 15 20 config message the pump flow rate will be set to 0 ml min bef Check 15 20 pump flow 0 00 2284 delay 60 TTE 15 20 ColumnOven ValveRight 4 _Tryt Now tempi ColumnOven ValveLeft 4 SEQ pump flaw 2 00 08 14 pump B 0 acqui pump C 0 06 17 pump D 0 Config delay 500 0 24 message column 4 is equilibrated FIGURE 3 5 Panel for the TCC 3200 with Multi Position Valves with the Script Button Properties The available commands used in a script are the same commands as used in Chromeleon program files The syntax of the typed command can be checked by clicking the Check button The Try it Now button will execute the commands in the script right away Warning When creating or modifying a script make sure that the flow rate is set to 0 00 ml min and there is enough time for the column pressure to be released before switching a valve This will prevent possible damage to the column bed Operating Instructions 13 Automated Method Scouting TABLE 3 2 List of Commands U
72. n the X axis and the elution time of the last compound is displayed on the Y axis The minimum resolution for the previous or next compound is displayed in the graph but only for the compounds of interest as specified in the SST or SCOUTING gqnt method file other resolutions may be larger but are not displayed in the graph FIGURE 3 31 The size of the circle is an indication of the resolution A bigger circle indicates a better minimum resolution The position of the circle on the Y axis corresponds with the speed of the method A lower circle indicates a faster method The report Find Best _Method rdf also lists the results in text below the graph listing the method number sample name elution time of last compound and minimum resolution Depending on the preference for a fast method or a method that provides a better resolution the user can select the best method s from the report Find Best Separation r a ta 4 B i7 3 D Method Development Sample Number FIGURE 3 31 Graph from the Find_Best_Method rdf report showing 12 results Operating Instructions 33 Automated Method Scouting TABLE 3 3 Part of the results table from the Find_Best_Method rdf report Method Development Sample Elution Time Global Minimum Sample Number Name of Last Peak Resolution BLANK ad n a 10 n 15 L1 1 19 1 11 MS_Cl n a MS Cl n a BLANK n a 34 Operating Instructions Parallel LC 4 Paralle
73. n the server configuration and add the devices Details about adding a device to the timebase are presented in the manual of each device and in the Chromeleon online help Use the default device names in the server configuration Enable column A and column B in the TCC 3x00 configuration allowing easy selection between the two columns It is recommended to use column identification chips P N 6710 1505 set of 5 The column identification chip allows storing column specific data More information regarding the column identification chips can be found in the Chromeleon online help or the manual of the TCC 3100 5 2 2 Direct Instrument Control from Chromeleon The panel tabset offers the interface to control the system It is automatically generated based on the hardware configuration and can be used by clicking the panel tabset button FIGURE 5 3 A panel tab for each device is automatically added to the tabset each tab will allow direct control via the default panel for that device For information about direct instrument control of each device refer to the user manual or the Chromeleon online help for that device In the tab for the column compartment the valve position active column and analytical recondition pump can be specified FIGURE 5 4 These specifications can also be done when using the program wizard FIGURE 5 3 Panel Tabset Button Columns Left Valve Right Valve ColumnA f hoi Column B shez lt Columnc fi
74. nature of the sample and a general understanding of the various stationary phases are used to select the column to be used to separate the sample Once the appropriate column is selected a number of variables must be studied to optimize the resolution and selectivity of the stationary phase to be sufficient for the compound s of interest Typical parameters that should be considered when using a reversed phase column include the selection and amount of the organic modifier the pH the ionic strength the temperature and the nature of the gradient A very large number of test separations could be required to obtain the desired separation The combination of the UltiMate 3000 and Chromeleon Chromatography Management Software makes the task of HPLC method development easier by automation The quaternary gradient pump provides up to four different user selected mobile phases In addition the use of two 6 position 7 port valves allows the operator to select which of the columns in the system should be tested with chosen mobile phases Chromeleon Chromatography Management software automatically presents the method s that provides the best resolution between a chosen critical peak pair or the best average resolution The system includes e One LPG 3400 pump that can make quaternary gradients e One TCC 3200 column compartment with two 6 position 7 port valves to allow easy switching between up to six different columns e One WPS 3000SL split
75. ng e g an IEX column Several first dimension elution programs 7 2 3 3 are required each with a slight increase in the eluting solvent This can be achieved by making several distinct programs or use a UDC to specify the percentage of eluting solvent similarly to what is described in section 3 2 3 1 Because the solvent composition for the first dimension column is not specified in the second dimension analysis program this program can be used for all second dimension analysis Operating Instructions 83 Automated Application Switching 8 Automated Application Switching Automated Application Switching is an LCi solution for increased efficiency by using two applications on one UltiMate 3000 Automated Application Switching eliminates the manual equilibration that has to precede any application change The system will equilibrate and perform a set of runs with one method After this first application the system will wash and prepare for the second application The second application will be started automatically when the equilibration 1s complete Columns solvents and samples can be completely different for the two applications The system includes e One DGP 3600 pump that can generate two ternary gradients independently and simultaneously One pump is used for the first application while the other pump 1s used for the second application e One TCC 3100 column compartment The two 2 position 6 port valves allow easy switching bet
76. ng start time Flow ml min Off Line 25 Reconditioning Start Time ni Loo 5 000 min Rete nti F Ta F 7 Insert Line min ml min i E ni 0 000 1 000 5 f Delete Line 5 000 1 000 60 0 C J 5100 1 000 Clean Up 000 1 000 1 interpolate FIGURE 5 8 Program wizard Tandem Gradient Options Operating Instructions 61 Tandem LC 6 Next the Void Volume Purge is specified and optionally the Reconditioning Flow can be set as depicted in FIGURE 5 9 Determine the void volume between the pump and switching valve Purge the related solvent lines 2 to 3 times to make sure that the solvent composition corresponds to the starting composition After off line reconditioning has been started the analytical pump flushes the solvent lines between the pump and the switching valve The solvent line has to be purged before the switching valve can be switched The Flow is the flow rate used for the gradient Increasing the flow rate will shorten the purge cycle The Volume is the volume that needs to be purged it depends on the type of system and pump used The Time is calculated automatically depending on the flow rate and volume specified Chromeleon provides default volumes for the following systems These volumes are three times the delay volume UltiMate 3000 analytical LPG 3400A and WPS 3000SL analytical autosampler 2 5 mL delay volume approx 850 uL up to the valve UltiMate 3000 analytical HPG 3x00A
77. ni f Column O o SSF hoi lt Details Active Column A Analytical Pump PumpLeft Columns to Use JAB 4 Recondition Pump PumpRight FIGURE 5 4 Panel configuration for tandem of pumps column and valve position 56 Operating Instructions Tandem LC In FIGURE 5 4 the settings apply to an installation as described for Tandem LC in the Quick Installation Guide With the valve in position 10 1 the active column is column A the PumpLeft is specified as analytical pump The Next Column button is used to switch between columns the valve switches in the program files this command is executed by writing NextColumn Operating Instructions 57 Tandem LC 5 2 3 Programs and Sequences A new Program File can be created by clicking File New Program File in Chromeleon A wizard will appear allowing the user to set the various program settings such as column compartment temperature flow rate etc The steps of the wizard are described below Program settings that are specific for Tandem LC are explained in detail Program settings that are more commonly used are not explained and can be found in the online help of Chromeleon 1 First select the timebase to use for the program 2 Next the wizard will ask to select the type of program to create Select Program for x2 Tandem Operation to get a wizard that will offer additional options especially for Tandem LC FIGURE 5 5 Program Wizard Wizard Options x
78. olumn Switching Times Sample Transfer Fractionation separation Separation bu ee oro pump _Srecmment uo Analytical Analytical column Pung equilibration AL rs Lil tute oe AMAlyiCH FIGURE 6 12 Program wizard On Line SPE LC Column Switching Times 6 The next step in the wizard not shown has the option to specify the mobile phase names pressure limits flow rate and maximum flow acceleration deceleration for the Loading Pump The left pump if the system configuration is made as described in the Quick Installation Guide and set up according FIGURE 6 10 Operating Instructions 75 Automated On Line SPE LC 7 After specifying the settings described above the gradient profile for the Loading Pump is specified In this example FIGURE 6 13 the column switching times are marked as t V1 and t V2 The loading of the sample is done with 100 mobile phase A after t V1 the sample is transferred from the SPE column to the analytical column with the Analytical Pump After t V2 the SPE column is washed with 100 mobile phase B for 2 5 minute and equilibrated with 100 mobile phase A to prepare for the next sample injection These settings and mobile phases used here should be identical to the settings used with the experimental determination of the Matrix Depletion Time t M Analyte Break Through Time t A and Transfer Time t T Program Wizard Loading Pump Options iE Hc B Fow ml min
79. omated 2D LC system is configured in the server configuration of Chromeleon Create a timebase in the server configuration and add the devices Details about adding a device to the timebase are presented in the manual of each device and in the Chromeleon online help Use the default device names in the server configuration Enable column A and column B in the TCC 3x00 configuration allowing easy selection between the two columns It is recommended to use column identification chips P N 6710 1505 set of 5 The column identification chip allows storing column specific data More information regarding the column identification chips can be found in the Chromeleon online help or the manual of the TCC 3100 7 2 2 Direct Instrument Control from Chromeleon The panel tabset offers the interface to control the system It is automatically generated based on the hardware configuration and can be used by clicking the panel tabset button FIGURE 7 3 A panel tab for each device is automatically added to the tabset each tab will allow direct control via the default panel for that device For information about direct instrument control of each device refer to the user manual or the Chromeleon online help for that device FIGURE 7 3 Panel Tabset Button 7 2 3 Programs and Sequences A new Program File can be created by clicking File New Program File in Chromeleon and then following the Chromeleon wizard to complete the creation of the progr
80. or Automated Method Scouting is shown in FIGURE 3 1 The heart of the system is the Dionex TCC 3200 Column Compartment with two 6 position 7 port valves Column 2 ro l J 6 i Column3 gt 2 x eo __ Column 4 ee T i Column5 M G Column6 N H FIGURE 3 1 Schematic representation for Automated Method Scouting All components except the columns that are required for a fully operable system are provided with the system modules or the Automated Method Scouting Kit For a list of supplied capillaries and accessories in the Automated Method Scouting Kit see TABLE 3 1 8 Operating Instructions Automated Method Scouting SRD 3600 Solvent Rack and Degasser LPG 3400A Quaternary Analytical Pump or LPG 3400M Quaternary Micro Pump WPS 3000SL Analytical or Micro Split Loop Autosampler TCC 3200 2x6P 7P Thermostatted Column Compartment with two 6 Position 7 Port Switching Valves PDA 3000 Photodiode A Detect f otodiode Array Detector or VWD 3x00 Variable Wavelength Detector FIGURE 3 2 Stack of the Automated Method Scouting system A complete description of the installation and configuration of the hardware is presented in the Quick Installation Guide The application kits P N 6722 0100 Automated Method Scouting Kit Quaternary Micro or P N 6722 0101 Automated Method Scouting Kit Quaternary Analytical contain tubing to connect columns from 5 up to 25 cm to the switching valves For 5
81. ore running the sequence To calculate the throughput increase the number of runs in one hour for single gradient Operation is compared to the number of runs in tandem operation The normal run time is 12 min including wash and equilibration steps allowing 5 runs in one hour In tandem mode the run time is 7 5 min allowing 8 runs in one hour The throughput increase is 3 runs in one hour which equals a 60 higher throughput 64 Operating Instructions Automated On Line SPE LC 6 Automated On Line SPE LC Automated On Line Solid Phase Extraction LC is an LCi solution allowing easy and automated isolation of analytes of interest from a complex matrix The Automated On Line SPE LC reduces time labor and cost thus increases productivity After injection of an untreated sample the On Line SPE LC allows automated sample cleanup and or analyte enrichment Samples can run unattended increasing the workload per system The setup of an Automated On Line SPE LC system includes a dedicated SPE column and an analytical column The target analytes are selectively extracted from the matrix integrated sample cleanup and pre concentrated on the SPE column The trapped analytes are transferred in backflush mode by switching the 2 position 6 port valve to the conventional analytical column for analyte separation and detection The system includes e One DGP 3600 pump that allows the independent and simultaneous generation of two ternary gr
82. p is generating the gradient profile on line for the separation e One TCC 3100 column compartment The 2 position 10 port valve allows easy switching between the columns e One WPS 3000SL split loop autosampler e One PDA 3000 or VWD 3x00 detector Tandem LC experiment stages e Injection is done on the first column e After the gradient the valve is switched e The first column is equilibrated off line e The sample is injected on the second column while the first column is equilibrated e After the gradient the valve is switched e The second column is equilibrated off line Operating Instructions 53 Tandem LC 5 1 Preparation of the System The schematic of the HPLC set up for Tandem LC is shown in FIGURE 5 1 F A l E Column 1 p O g ee Waste 3 D A OS 7 l C H FIGURE 5 1 Schematic representation for Tandem LC All components except the columns that are required for a fully operable system are provided with the system modules or the Tandem LC kit For a list of supplied capillaries and accessories in the Tandem LC Kit see TABLE 5 1 a ei x Ma x f ioa T URNE 3000 SRD 3600 Solvent Rack and Degasser DGP 3600A Dual Gradient Analytical Pump or DGP 3600M_ Dual Gradient Micro Pump WPS 3000SL Analytical or Micro Split Loop Autosampler TCC 3100 1x2P 10P Thermostatted Column Compartment with 1x 2 Position 10 Port Switching Valve PDA 3000 Photodiode Array Detector or VWD 3x00 Variable Wa
83. r the analytical column is not required The system setup used for the experiments is similar to the setup as depicted in FIGURE 6 1 The valve is set to position _2 before starting with the experiments and both pumps are purged The three constants are e The Matrix Depletion Time t M is the time it takes at a given flow rate to completely elute the sample matrix from the SPE column The waste line in port 4 on the 2 position 6 port valve FIGURE 6 1 should be replaced with a connection to the detector to be able to monitor the elution profile of the sample matrix The Matrix Depletion Time is the time after the elution profile when the signal of the detector has returned to the baseline WL 260 nm FIGURE 6 4 Matrix Depletion Time t M is the time after the elution profile when the signal of the detector has returned to the baseline Flow 2 0m Limir hii nutes Operating Instructions 69 Automated On Line SPE LC 70 The Analyte Break Through Time t A is the time it takes at a given flow rate to start eluting the analyte from the SPE column The waste line in port 4 on the 2 position 6 port valve FIGURE 6 1 should be replaced with a connection to the detector to be able to monitor the elution profile of the analyte The analyte should have a concentration similar to the expected analyte concentration in the sample and t A should be larger than t M The Analyte Break Through Time is the time just before the
84. rized solvents The layout of this manual is designed to provide quick reference to the sections of interest to the user However we recommend that you review the manual thoroughly before starting to operate the instrument in order to obtain full understanding of the LCi solutions This manual is provided as 1s Every effort has been made to supply complete and accurate information and all technical specifications and programs have been developed with the utmost care However Dionex assumes no responsibility and cannot be held liable for any errors omissions damage or loss that might result from any use of this manual or the information contained therein We appreciate your help in eliminating any errors that may appear in this document The Intelligent LC Solutions Reference Library CD contains a digital version of this LCi Solutions manual the Quick Installation Guide posters for applications templates for applications in the form of Chromeleon back up files and a Chromeleon datasource Operating Instructions 3 LCi Solutions 2 Introduction to LCi Solutions Dionex has developed a number of Intelligent LC LCi solutions to allow the analyst to optimize the performance reliability and ease of operation of the HPLC system These solutions combine UltiMate 3000 hardware Chromeleon software and Dionex column chemistries to solve typical analytical challenges such as method development or increasing throughput 2 1 Automate
85. rogram file Manual Input gt Select lt Manual Input gt to use the cument instrument settings as startup conditions Or select any saved startup conditions Press Next gt to review the choosen startup conditions and modify them as needed lt Back l A wf Cancel Help FIGURE 8 5 SmartStartup wizard specify a program or manual input for equilibration 3 Select lt Choose Program gt and select the previously created program file for the application The wizard will use the settings from the program file for initial equilibration settings such as solvent composition for the pump and wavelength for the detector 4 In the next step the wizard provides the option to fine tune the equilibration FIGURE 8 6 The Display Success Message option has to be deselected to prevent a pause of Operating Instructions 89 Automated Application Switching the sequence until the success message dialog is confirmed by the user The Maximum Equilibration Time should be set to allow sufficient time for the system to equilibrate Make sure to deselect the pump that is not used for the application equilibration Select a device and click Edit to modify the settings for the device SmartStartup Wizard Equilibration Conditions for Timebase DGP3600A Device Equilibrate Properties PumpLett m Flow wB C Flush system Pressure limits FumpRight Flow wB C Flush system Pressure limits Sampler Purge settings Temper
86. rough Time t 4 Transfer Time t T which have to be evaluated before creating an On Line SPE LC method Take etra caution when specifying these values as mistakes may lead to damage of the analytical column Please read the Chramoleon Help for more detailed information FIGURE 6 9 Program wizard links to Chromeleon Online Help about the On Line SPE LC Options 72 Operating Instructions Automated On Line SPE LC 3 The next step in the wizard has the option to specify the configuration of the On Line SPE LC system The settings for pumps and valve in FIGURE 6 10 are applicable if the instrument is setup as described in the Quick Installation Guide The SPE Column and Analytical Column are specified as Column A or Column B depending on how the column identification chips are inserted in the TCC 3100 Program Wizard System Configuration Options Please specify the configuration of your On Line SPE LC system Columns SPE Column Analytical Column Column_6 Pumps Loading Pump Fumplet gt Analytical Pump FumpRight Switching Valve Use Two Position Switching Valve Valve Right bi Valve Position for Loading Step 1 lt Back Cancel Help FIGURE 6 10 Program wizard On Line SPE LC system configuration 4 The next steps in the wizard not shown specify the temperature settings for the column compartment and the autosampler Operating Instructions 73 Automated On Line SPE LC
87. rovides the option to turn off the UV lamp The batch can be started and the system will run fully automated 96 Operating Instructions
88. s It is possible to start with the data acquisition after t V1 At the end the option to save and or review the program file is offered After saving the program file it 1s ready to be used in a sequence Use the wizard to create a sequence and select the just created program file Operating Instructions 77 Automated 2D LC 7 Automated 2D LC 2D LC is an LCi solution for separating complex samples It enables the combination of two orthogonal chromatographic techniques for example IEX and RP The sample is injected on the first dimension column Fractions of sample eluting from the first dimension are transferred to the second dimension for further analysis In the presented example for Automated 2D LC system an Immobilized Metal Affinity Chromatography IMAC column for the first dimension and a reversed phase RP column in the second dimension are shown The IMAC column is used for isolating and enriching phosphopeptides from a peptide digest and the RP column is used to separate the peptide fractions A loop is used to capture a fraction of the sample eluting from the first dimension and to introduce it in the second dimension The system includes e One DGP 3600 pump that can generate two ternary gradients independently and simultaneously One pump is used for the injection of the sample and the first dimension of the separation the other pump is used for the second column e One TCC 3100 column compartment The 2 position 10
89. sed in the Script Shown in FIGURE 3 5 Command in the box Explanation The message command will create a pop up with the text between quotes and an OK button After reading press OK to continue message the pump flow rate will be set to 0 ml min before switching the valves Set the pump flow rate to 0 00 ml min 3 delay 60 Wait 60 seconds 4 before continuing to the next command 4 ColumnOven ValveRight 4 Switch the valve right in the specified position pump flow 0 00 5 ColumnOven ValveLeft 4 es me ae the specified position Set the pump flow rate to x Set the percentage of mobile 0 pump AB lt Y phase B to 0 x Set the percentage of mobile 0 PUUD OEN phase C to 0 x Set the percentage of mobile 0 pune phase D to 0 Wait 500 seconds before continuing to the next command 10 delay 500 The message command will create a pop up with message column 4 is equilibrated the text between quotes and an OK button After reading press OK to continue Percentage A for mobile phase is not specified It is the remainder of 100 B C D 3 2 3 Programs and Sequences 3 2 3 1 User Defined Columns For Automated Method Scouting a different program is used for each different condition e g different valve positions or temperature To minimize the number of programs that need to be created a variable in the program file is defined by the User Defined Column UDC For example if a separation has to be performed
90. stem Suitability Test First you can choose from a list of predetined tests Predefined Tests Copy Previous Test gt Copy Previous Test gt A Maximum Peak Amount Minimum Peak Amount Peak Asymmetry Peak Height Peak Width 5 Peak Width 10 Peak Width 50 Peak Width baseline Resolution EP Resolution USP Retention Time RSO of Peak Areas RSD of Peak Heights RSD of Peak Retention Time Signal Noise Signalto Noise Ratio Theoretical Plates EP Theoretical Plates JP Ta ee el ee l S FIGURE 3 16 Start of the wizard for the SST parameters where a predefined test can be selected from the drop down menu Operating Instructions 23 Automated Method Scouting 2 The conditions for the sample can be set e g sample type sample name or sample position Sample Type C Sample Number s C Sample Position s Sample Property User defined Condition lt Back Finish Cancel Hep FIGURE 3 17 SST properties Sample Condition 24 Operating Instructions Automated Method Scouting 3 The test parameters can be set in Test Condition FIGURE 3 18 In this example the resolution to the next peak is specified SST Wizard New Single Test Eg Test Condition Test Name 7 Resolution EP Test Conaition peak resolution ep next Operator Value Round Test Condition and Compare Value to decimals before comparing them kia lI Sele
91. tion sequence in the batch should run This sequence will turn on the devices of the system and the UV and or VIS lamp 94 Operating Instructions Automated Application Switching 8 2 3 1 Creating the switch sequence A flush wash sequence is created by using two programs 1 The first program in the sequence is used to e g flush the system with mobile phase C in case buffers are used and to stop the left pump in a controlled manner for the first application It is also possible to use a program file that reduces the flow rate for a continuous flow on the column for the first application For example e Switch to mobile phase C with the same flow rate as used in the program for the left pump The right pump is not used yet e Continue pumping with the left pump for at least 15 min with mobile phase C to flush out any buffers e Reduce the flow rate of the left pump to zero 2 The second program in the flush wash sequence is used to set the valve in the correct position and to purge the lines of the fluidics from and to the autosampler with the right pump to prepare for the second application This purging of the fluidic autosampler lines should be done with a mobile phase that is miscible with the mobile phase used for the first application For example e Valve Left is switched from position 6 1 to position 1 2 e Right pump flow rate is started The mobile phase from the right pump must be fully miscible with the mobile phase of
92. ts after the Formula input box and select Quantification Method in the Categories section 3 Select SST result in the Variables section FIGURE 3 23 Formula gnt sst_result Alt Identifier 55TResult unique name e g Weed for column layo Header SST Result visible headline Dimension ee ea Chromatogram Matis Blank Subtraction Detection Parameters Number of Detection Parameter ty Statist Peak Results Number of Peaks In the Peak T Peak Calibration Select Peak in the Peak Table Sy Peak T able Number of Amount Columna l Peak Purity and Identification Amount Column Hame 28 Categories Yarables Delay time value ra Delay time detector t Trai Blank Run Subtraction m Preconditions Blank Run Sample Record ANGE Quantification Method Mel SST Result m Explain variable Dankan Ciak bilihs Tank COT Dann Forrnula ignt sst_result Operating Instructions Automated Method Scouting FIGURE 3 23 Report column for the sequence The result of the Quantification Method SST Result will be displayed in the SST Result column FIGURE 3 24 ColumnSetector column_name ColumnQventemp Method ool Result Select Active Column example Celsius 6 Acclaim C18 SCOUTING Passed 6 Acclaim C18 SCOUTING Failed 6 Acclaim C18 SCOUTING Failed 1 Acclaim Polar Advantage Il SCOUTING Failed 1 Acclaim Polar Advantage Il SCOUTING Passed 4 amp uae
93. ual Left TCC 3x00 Column Compartment Virtual Oven Column E fq WPS 3000 Autosampler Virtual fq VW0 3x00 Detector 2 For Helo press Fi FIGURE 4 3 Server configuration Parallel LC with two timebases To each timebase one detector is added The detector is not shared between timebases and each detector can only be controlled from the timebase were the detector is installed 38 Operating Instructions Parallel LC The DGP 3600 is shared between the two timebases by specifying which pump is used for a timebase The right pump is used in Timebase_1 the left pump is used in Timebase 2 The pumps can be purged via the autosampler which can be specified when following the wizard of the DPG 3600 configuration FIGURE 4 4 depicts the configuration for the DGP 3600 with the default device names The DGP 3600 will appear in both timebases after configuration DGP 3600 Pump Let Solvents Relays Inputs Eror Levels Heneral Devices Right Pump Right Solvents Left Pump Device Name Pump Left Pump PumpLet on Timebase 7 purges via WP S 30005L UM3WPS_PURGEO Right Pump Pump Right on Timebase 2 purges via WFP S5 30005L JUMSWPS_PURGEO IY Share eluent bottles M Share waste bottle M Pressure Signalis FIGURE 4 4 Sharing of the DGP 3600 pump between the timebases Operating Instructions 39 Parallel LC The TCC 3100 column compartment is shared between the t
94. umn ScoutingsDiuretics Browse Apply Compare Conditions Restrict Library Spectra Match Criterion Least Square Hit Threshold 800 O 1 000 Use Spectrum Derivative P Restrict Wavelength Range fram 210 0 450 0 Check Greatest Rel Mas Allowed Deviation ma Masimum Retention Time Deviation oo Check Number of Relative Extrema Masimum Retention Index De viatior oo Masimum Kovats Index Deviation o 0 Amount Table A Peak Tracking A Calibration A Spectra Library Screening ASST F E FIGURE 3 10 The Spectra Library Screening tab from the Method Editor where the Library for Automated Peak Tracking is selected 3 Right click on a row in the Peak Tracking tab of the Method Editor and selecting the Autogenerate Peak Table option The window in FIGURE 3 11will be shown Autogenerate Peak Table Type C Enumerate peaks of curent chromatogram Use Spectra Library Screening results m Options W Apply only to peaks with Height l greater thar 5 000 Signal Apply only to peaks in current time interval Copy reference spectrum from Multiple hits C Allow multiple best hits f Unique identification Check the best hits only Cancel Help FIGURE 3 11 Autogenerate Peak Table options 4 Select Use Spectra Library Screening results to use the Spectra Library Specify the other options as desired for optimal results Confirm the settings by clicking OK 5 Chromeleon will fil
95. unds 2 With a System Suitability Test SST in combination with a query Section 3 2 4 3 2 This option offers more flexibility but 1t requires more parameters to be specified 3 With a combination of the method file and the SST combined with a query Section 3 2 4 3 3 When this option is selected the SST and query limit the number of results at first then the method file is used to find the compounds of interest with baseline separation This is the preferred option Each option will present the results graphically in the Find_Best_Method rdf report The SCOUTING gnt method file and Find Best_Method rdf report file are both available in the template sequence found on the Intelligent LC Solutions Reference Library CD 3 2 4 3 1 Finding the Best Chromatographic Conditions with the Method File for Automated Method Scouting If the only interest is the resolution of all compounds the easiest way to find the best chromatographic conditions is using the SCOUTING gqnt method file in combination with the Find_Best_Method rdf report Peaks are listed in the method file in the automatically generated peak table The method file has the option to select for which peaks the resolution needs to be checked The Find _Best_Method rdf report shows the results in a graphical tones al SCOUTING 1 YG 1 317 min in 5 000 AG Least Square 4 950 OF OF Autogenersted No SCOUTING 2 3625 min min 5 000 AG i Least Square 0 950 OTT
96. velength Detector FIGURE 5 2 Stack of the Tandem LC system 54 Operating Instructions Tandem LC A complete description of the installation and configuration of the hardware is presented in the Quick Installation Guide The application kits P N 6037 0001 Tandem Operation Capillary Kit Dual Ternary Analytical or P N 6037 0002 Tandem Operation Capillary Kit Dual Ternary Micro contain all the tubing to make all the fluidic connections for a micro or analytical Tandem LC system TABLE 5 1 List of capillaries and accessories supplied with the Tandem LC Kit e A Capillary Left Pump TCC port 8 C D Capillary TCC 6p port 2 1 or 10p port 7 Col 2 Col 1 5 cm ao Capillary TCC 6p port 4 r or 10p port 5 Col 2 5 cm D Capillary TCC 6p port 2 1 or 10p port 7 Col 2 Col 1 15 cm Capillary TCC 6p port 4 r or 10p port 5 Col 2 15 cm Capillary TCC 6p port 2 1 or 10p port 7 Col 2 Col 1 25 cm l D E ca ena TCC 6p port 4 r or 10p port 5 Col 2 25 cm Go o fe forcon OOO a fowr SSS e feme o Capillary TCC 6p port 2 r or 10p port 1 Det PEK epi fr WWD waste comei ooo E SC d e T oss mstar Guid W000 sopies i Operating Instructions 55 Tandem LC 5 2 Configuring Software 5 2 1 Configuring Hardware in the Server Configuration The UltiMate 3000 Tandem LC system is configured in the server configuration of Chromeleon Create a timebase i
97. ween the first application autosampler flush and second application e One WPS 3000SL split loop autosampler e One PDA 3000 or one VWD 3x00 detector Automated Application Switching experiment stages Sample and mobile phase preparation for the first and second application Startup the instrument Run the samples from the first application Stop the first application flush the autosampler path Prepare for second application Run the samples from the second application Standby or shutdown of the instruments 84 Operating Instructions 8 1 Preparation of the System Automated Application Switching The schematic of the HPLC set up for Automated Application Switching is shown in FIGURE 8 1 FIGURE 8 1 Schematic representation for Automated Application Switching All components except the columns that are required for a fully operable system are provided with the system modules or the Automated Application Switching kit For a list of supplied capillaries and accessories in the Automated Application Switching kit see TABLE 8 1 Operating Instructions SRD 3600 Solvent Rack and Degasser DGP 3600A Dual Gradient Analytical Pump or DGP 3600M Dual Gradient Micro Pump WPS 3000SL Analytical or Micro Split Loop Autosampler TCC 3200 2x2P 6P Thermostatted Column Compartment with 2x 2 Position 6 Port Switching Valves PDA 3000 Photodiode Array Detector or VWD 3x00 Variable Wavelength Detector 85
98. wo timebases by specifying which column is used for a timebase Column_A is used in Timebase_1 Column_B is used in Timebase_2 The option can be specified when following the wizard FIGURE 4 5 depicts the configuration for the TCC 3100 The TCC 3100 will appear in both timebases after configuration Components m Installed components Right Valve 6 ports positions V Column A Column A on Timebase1 ts F Column B Column B on Timebass 2 Column E Column e on Timebase 2 II Column D Comm D on Timebase 2 tis lt Beck Coa He FIGURE 4 5 Sharing of the TCC 3100 column compartment between the timebases It is recommended to use column identification chips P N 6710 1505 set of 5 The column identification chip allows storing column specific data More information regarding the column identification chips can be found in the Chromeleon online help or the manual of the TCC 3100 40 Operating Instructions Parallel LC The WPS 3000SL autosampler is shared between the two timebases by specifying both timebases in the configuration The option can be specified when following the wizard also specify that both pumps left and right deliver flow to the WPS 3000SL FIGURE 4 6 depicts the configuration for the WPS 3000SL which allows sharing between timebases FIGURE 4 7 depicts the pump selection that delivers flow to the autosampler The WPS 3000 will appear in both timebases after configuration Time

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