Technical Overview. Author. Abstract. A.G.Huesgen Agilent Technologies, Inc. Waldbronn, Germany

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Using the Agilent Instrument Control Framework to control the Agilent 1290 Infinity LC through Waters Empower software Instrument set up and performance Technical Overview Author A.G.Huesgen Agilent Technologies, Inc. Waldbronn, Germany Abstract The Agilent Instrument Control Framework (ICF) enables other providers of LC data acquisition and processing software to simplify the development of the control of Agilent LC instrumentation. In this Technical Overview we demonstrate how Agilent ICF facilitates enhanced control of the Agilent 1290 Infinity LC through Waters Empower chromatography data software versions 2 and 3. The combination of Agilent ICF and Waters Empower software provides easy access to advanced features of the 1290 Infinity LC such as priming and purging the pump, external needle wash, and acquisition of all eight MWD and DAD signals.

Introduction The Agilent Instrument Control Framework (ICF) is a software component that makes it easier and faster for software providers to implement control of Agilent LC equipment in their chromatographic data systems or workstations 1,2. Based on new standard instrument drivers from Agilent, ICF eliminates much of the delay and effort of using low-level instrument control codes and the need of software developers to write their own native drivers. In this Technical Overview we demonstrate: What prerequisites have to be fulfilled to ensure seamless interaction with Agilent 1290 Infinity LC systems, Waters Empower software and ICF Which modules and instrument features are supported How instruments are configured and methods are created for the Agilent 1290 Infinity LC system using Waters Empower software in combination with ICF That the performance of the Agilent 1290 Infinity LC system fulfills expectations using Waters Empower data acquisition and processing tools Experimental An Agilent 1290 Infinity Binary LC system with the following modules was used for the precision measurement: Agilent 1290 Infinity Binary Pump Agilent 1290 Infinity Thermosttated Column Compartment Agilent 1290 Infinity Diode Array Detector Agilent 1290 Infinity High Performance Autosampler Agilent ZORBAX RRHT Eclipse Plus C-18 column packed with 1.8-µm particles Chromatographic conditions for precision measurement Compounds: Uracil, phenol, methyl-, ethyl-, butyl-, and heptylparaben, toluene, and n,n-diethyl-mtoluamide Column: Agilent ZORBAX Eclipse C18 RRHT, 3 100 mm, 1.8 µm Mobile phases: Water/acetonitrile Gradient: 30% to 80% in 8 min, at 8.01 min 30% Flow rate: 0.8 ml/min Stop time: 11 min Column temperature: 30 C Injection volume: 3 µl DAD: 254/10 nm Ref. 360/100 nm, 20 Hz Software: Empower 2 Build 2154, Installed Service Packs: A-D, Installed Feature Releases: 1-4, with ICF version A.01.02 SP1 2 Prerequisites for the combination of Empower and ICF All Agilent LC modules must have firmware version A.06.32 or B.06.32 or B.06.41 or higher. All Agilent LC modules must have RC.Net drivers. Agilent Infinity LC ICS 1.0.0 Empower 2, feature release 3 or higher or Empower 3 software 3 Windows XP with service pack 3 or higher 3

Results and discussion Agilent ICF facilitates access to advanced features of Agilent LC instruments that were previously not supported by earlier revisions of Empower with drivers provided by Waters. Now, all features are supported and available through the new On Line screen, which has been added to the familiar Empower screen, see Figure 1. A rightclick on one of the module fields gives access to all control, method and other advanced features of the module. RFID tags can be seen if the cursor is moved over the label pictogram for the lamp and cell of the DAD module. For supported modules and functions, refer to the Appendix. Configuring the Agilent 1290 Infinity LC system 1. Set up the DHCP of the Empower node. 2. Set all DIP switches of the Agilent module to 0. This module is connected to the Empower node through LAN. 3. The LC receives an IP address from the DHCP server. 4. To connect the Agilent instrument, configure the DHCP server through Properties of the Empower Nodes. Note: For more details see Technical Overview in reference 4. 5. Use Edit to set the instrument type and a unique name. 6. Click File and New chromatographic system to make the new LC system accessible for data acquisition. After this last configuration step, the Agilent 1290 Infinity LC system is On Line and ready for use. These configuration steps have to be followed whenever a new module is added or removed. Previously, the old configuration had to be deleted from the DHCP server configuration. Then the Empower software had to be shut down and the LAN connection to the module had to be switched off and on again. When the LAN connection has been restored the new configuration procedure can be started. Figure 1 Agilent Instrument Status screen under Empower and ICF software. 3

Creating an instrument method and a method set Having configured the instrument, the instrument method and the method set can be created in the Empower Run Sample screen. The Instrument method is set up through Edit method. The pump screen for setting appropriate parameters is shown in Figure 2. All parameters that are available in the Agilent ChemStation are now accessible in Empower. To use Agilent well/vial plates, the dimensions of the plates have to be configured. The created instrument method is saved and used to set up a method set. The method set can then be used to create sequences. The Agilent Instrument Status screen implemented in the Run Sample screen of Empower is used to set up online methods, to switch the system on or off, to equilibrate columns, to view the status of single module, and to access special features. Control Tab functions such as purging and priming the pump and setting the minimum stroke are now available. See Figures 3 and 4. The method parameters filled in here are not saved as an instrument method. Only the method created in the Edit method screens is saved. Figure 2 Parameter selection screen for the binary pump of the 1290 Infinty LC. Figure 3 Control screen of Agilent 1290 Infinity Binary Pump. Figure 4 Method screen of the Agilent 1290 Infinity Pump selected from the new user interface. 4

Creating a sequence Create a sequence by filling the sample set table with the sample name, position of vials, number of injection per vial, method set to be used, and so forth. Performance of the Agilent LC systems using Waters Empower data processing tools To demonstrate that the Agilent 1290 Infinity Binary LC fulfills the expected performance, the following tests were done using a paraben sample: Precision of retention time Precision of areas Precision of retention time and areas The chromatogram of the paraben sample is shown in Figure 5. Precision of retention times and areas for a 3-µL injection are combined in Table 1. Data were evaluated using Empower Component summary report. The precision for the retention times for six consecutive runs is < 0.11% RSD, for the area the precision is < 0.30% RSD. Conclusion Empower software. The instrument was configured in Empower and data were acquired and processed. The combination of ICF and Empower software facilitates access to most available Agilent instrument features such as external needle wash, purging and priming of the pump, and acquisition of more than five signals. The Agilent Instrument Status screen is used to set up On Line methods, to switch the system on or off, to equilibrate columns, to view the status of single module, and to access special features using the Control function available for each Agilent LC module. As expected, the Agilent 1290 Infinity Binary LC system shows the same excellent performance for data acquired and processed using Empower and ICF. The Agilent Instrument Control Framework (ICF) is a software component that makes it easier and faster for software providers to implement control of Agilent liquid chromatography systems in their chromatographic data systems or workstations. In our application example, ICF was used to control the Agilent 1290 Infinity Binary LC system in combination with Waters Figure 5 Overlay of six chromatograms of paraben sample for evaluation of retention time and area precision. Peak name RSD RT (%) RSD Area (%) (3 µl injection volume) Phenol 0.108 0.125 Methyl- 0.108 0.227 Ethyl- 0.090 0.148 Toluamid 0.059 0.159 Butyl- 0.068 0.142 Toluene 0.060 0.278 Heptylparaben 0.036 0.197 Table 1 Precision of retention times and areas for six consecutive runs. 5

References 1. The Agilent Technologies Instrument Control Framework, Technical Overview, Publication number 5990-6504EN, November 2010. 2. The Agilent Technologies Instrument Control Framework Short overview, Publication number 5990-5756EN, June 2010. 3. Agilent Infinity LC Instrument Component software Version 1.0 for Empower software Waters Installation note, Publication number 716003453 Rev.A, August 2011. 4. Using the Agilent Instrument Control Framework to control the Agilent 1260 Infinity LC through Waters Empower software- Instrument set up and performance, Agilent Publication, Publication number 5990-9092EN, November 2011. Appendix See Table 2 for supported and tested Agilent modules. See Table 3 for supported and not yet supported functions and instrument features. Tested by Waters Tested by Agilent Agilent 1290 Pumps Binary pump G4220A OK OK Binary pump VL G4220B OK Sampler 1290 HiP G4226A OK OK TCC TCC G1316C OK OK 2-pos/6-port valve G4231A/B OK 6 column selection valve G4234A/B OK 8-pos/9-port valve G4230A/B OK 2-pos/10-port valve G4232A/B OK Detector DAD G4212A OK OK VWD G1314E OK Table 2 Supported and tested Agilent LC Modules. The following configurations and tasks are now accessible by Empower with ICF as tested in the application lab in Waldbronn, Germany. System with second pump, for instance for alternating column regeneration Dual detector application is possible to configure and run (this functionality is not officially supported by Waters) 2-pos/10-port, 2-pos/6-port, 8-pos/9-port, and 6 column selector valve built in column compartment, valve-heads Switching of these valve is possible External needle wash All eight signals for acquisition on a DAD or MWD are supported Early Maintenance Feddback (EMF) can be set up in the Agilent Lab Adviser. Warnings and limits are displayed in the Agilent System UI in Empower Overlapped injections RFID tags: Lamp and detector cell tags and column tags can be accessed in the Agilent Instrument Status screen by clicking on the label pictograms Features not yet supported in the current version used for this Technical Overview Configurations such as UIs needed for clustering of pumps and column compartments are not yet available Creation of additional compressibility curves by the user not yet available Manual injection as single sample run; i.e. Empower always requires a sequence when Agilent LC instruments are used Fraction collector Purge kit (G1373A) DAD data recovery card Clustered column compartments New external valve drives (G1170A) Clustered pump with Solvent Selection Valve Injector programming Post time available in the pump set up screen of Agilent pumps None or All spectra can be selected for DADs Module control functions Pump bottle filling Table 3 Supported and not yet supported instrument features. 6

Agilent 54 vial plate Tab: PLATE Tab: Rows and columns Tab: Referencing Configuring Agilent vial and well plates In Empower, go to Create New Plate Type and enter the dimensions in Table 4 for the Agilent vial/well plate types. 11.80 10.30 Agilent 96 well plate Tab: PLATE Tab: Rows and columns Tab: Referencing 14.30 11.30 Agilent 384 well plate Tab: PLATE Tab: Rows and columns Tab: Referencing 12.15 9.05 Table 4 Configuration of Agilent well/vial plate types in Empower. 7

www.agilent.com/chem/icf Agilent Technologies, Inc., 2012 Published in USA, January 1, 2012 Publication Number 5990-9093EN