No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or transla-tion into a foreign language) without prior agreement and written consent from Agi-lent Technologies, Inc. as governed by United States and international copyright laws. Manual Part Number G7120-90310 Edition 08/2017
User Manual Agilent InfinityLab LC with ISET Agilent Technologies Notices © Agilent Technologies, Inc. 2017 No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws. Manual Part Number G7120-90310 Edition 08/2017 Printed in Germany Agilent Technologies Hewlett-Packard-Strasse 8 76337 Waldbronn Warranty The material contained in this document is provided "as is," and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. 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It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met. WARNING A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met. ISET - User Manual In this book In this book The manual describes the Agilent InfinityLab LC Intelligent System Emulation Technology (ISET). It contains the following: 1 What is ISET? This chapter gives a definition and brief overview of ISET and its intended use. 2 Installing and Configuring ISET This chapter gives detailed step-by-step instructions for installing a new system and upgrading an existing system. 3 Setting Up ISET Parameters This chapter gives detailed step-by-step instructions for setting up the standard ISET parameters, and for setting up and using a verification method to confirm that ISET is functioning as expected. It also contains explanations of the advanced ISET parameters, with step-by-step instructions for setting them up. 4 Understanding ISET Functionality This chapter explains in detail how ISET works. 5 Application and Technical Notes This chapter gives an overview on additional literature. ISET - User Manual 3 Contents Contents 1 What is ISET? 5 What is Intelligent System Emulation Technology (ISET) 6 The solution for instrument to instrument method transfer: ISET 11 2 Installing and Configuring ISET 19 Preconditions for ISET 20 Installing and configuring ISET 25 ISET Removal and Reinstallation 27 3 Setting Up ISET Parameters 29 Preparing the system for emulation 30 Setting up the basic ISET parameters 31 Fine-tuning the emulation 38 Generic emulation using dwell volume 39 4 Understanding ISET Functionality 41 Understanding ISET functionality 42 Composition differences 43 ISET functionality 45 5 Application and Technical Notes 47 Application and technical notes 48 4 ISET - User Manual ISET - User Manual 1 What is ISET? What is Intelligent System Emulation Technology (ISET) 6 The solution for instrument to instrument method transfer: ISET 11 This chapter gives a definition and brief overview of ISET and its intended use. Agilent Technologies 5 1 What is ISET? What is Intelligent System Emulation Technology (ISET) What is Intelligent System Emulation Technology (ISET) Instrument-to-instrument method transferability is an important topic for all laboratories throughout all industries, where HPLC and UHPLC methods are transferred between different departments and locations with different LC instruments. In the pharmaceutical industry, the transfer of analytical methodology between R&D, contract research organizations and manufacturing is an essential part in the development of a new pharmaceutical product. Several hundred observations from the FDA and a proposal for a new chapter in USP 1224 Transfer of analytical procedures emphasize the actuality and importance of this topic. ISET use cases ISET offers the following options for instrument to instrument method transfer : · Perform legacy methods, see Figure 1 on page 7 · Method development, see Figure 2 on page 7 · Method robustness testing, see Figure 3 on page 7 6 ISET - User Manual What is ISET? 1 What is Intelligent System Emulation Technology (ISET) Perform legacy methods With ISET it is possible to perform legacy methods on modern (U)HPLC instruments without changes in retention time and resolution. /RDGRU SURJUDP OHJDF\ PHWKRG 6HOHFW LQVWUXPHQW WREH HPXODWHG ([HFXWH PHWKRGLQ HPXODWLRQ PRGH Figure 1 Backward compatibility Method development With ISET it is possible to develop methods for a variety of instruments on one high performance instruments while maintaining individual instrument characteristics. *HQHUDWH QHZ PHWKRG 6HOHFW LQVWUXPHQW WREH HPXODWHG ([HFXWH PHWKRGLQ HPXODWLRQ PRGH 0HWKRG RSWLPL]HG IRU HPXODWHG +3/& Figure 2 Method development Method robustness testing With ISET it is possible to test a method for robustness against variation in instrument characteristics using one single instrument. 6HOHFW PHWKRG 6HOHFW LQVWUXPHQW WREH HPXODWHG ([HFXWH PHWKRGLQ HPXODWLRQ PRGH 2EWDLQ LQIRUPDWLRQ RQPHWKRG UREXVWQHVV Figure 3 Method robustness testing ISET - User Manual 7 1 What is ISET? What is Intelligent System Emulation Technology (ISET) Which parameters affect method transfer? Design differences between LC instrumentation such as power range, delay volume, mixing behavior, temperature control, extra column volume and detector cell design all affect the ability to transfer a method from one system to another. Therefore identical LC methods used on different LC instrumentation could result in different retention time and chromatographic resolution. Figure 4 Parameters affecting method transfer A wide range of instrument parameters have a direct impact on the retention times, resolution and sensitivity of a separation. 8 ISET - User Manual What is ISET? 1 What is Intelligent System Emulation Technology (ISET) The impact of delay volume and gradient mixing The delay volume of an LC system determines how fast the gradient reaches the column. Further, the mixing behavior influences the gradient profile. Both these factors delay volume and mixing behavior are determined by the instrument design and the consequences for method transfer are differences in retention times and in resolution. *UDGLHQWVORSH0L[LQJEHKDYLRU 5HVSRQVH 'HOD\YROXPH 3URJUDPPHGJUDGLHQWVWHS 7\SLFDO8+3/&V\VWHP 6HULHV%LQDU\V\VWHP Figure 5 7LPH Comparison of delay volumes and gradient mixing behaviors between a typical UHPLC system and an 1100 Series Binary LC using a tracer experiment. On a typical UHPLC system the mixed solvents reach the column much earlier, and the set composition is also achieved earlier due to the steeper gradient slope. ISET - User Manual 9 1 What is ISET? What is Intelligent System Emulation Technology (ISET) Figure 6 Different solvent compositions at the column due to different delay volumes and gradient mixing behaviors result in different retention times and resolutions. Column: Mobile Phase: Gradient: Poroshell 120, 3.0 x 50 mm (2.7 µm). Flow rate: 0.85% mL/min Water, Acetonitrile 0 min (10% Acetonitrile), 3 min (90% Acetonitrile) 10 ISET - User Manual What is ISET? 1 The solution for instrument to instrument method transfer: ISET The solution for instrument to instrument method transfer: ISET Intelligent System Emulation Technology allows to run HPLC or UHPLC methods from other instruments and delivers almost the same chromatographic results without any changes in the instrument setup or the original method - all by a few mouse clicks. Performance Specification The InfinityLab LC Series delivers the key requirement for the implementation of ISET technology. ,QILQLW\,,/& ,QILQLW\,/& ,QILQLW\,,3ULPH/& DQG,QILQLW\,DQG,QILQLW\,,/& 6HULHV+3/& Figure 7 Agilent InfinityLab Series Power Range The wide power range of the InfinityLab LC Series ensures that both HPLC and UHPLC methods from narrow bore to standard bore columns can be executed. ISET - User Manual 11 1 What is ISET? The solution for instrument to instrument method transfer: ISET The ISET emulation algorithm With the detailed knowledge about the system behavior of the target LC instrument and the high accuracy of the InfinityLab LC Series instruments, ISET is able to create an emulation function, which delivers similar gradient conditions as the selected instrument. The results are similar retention times and similar chromatographic resolution. The InfinityLab LC Series instruments with ISET deliver a gradient very similar to the 1100 Series Binary LC emulating the delay volume and mixing behavior. $ % & Figure 8 Chromatograms of a gradient separation of a pesticide mixture run on (A) 1100 Series Quaternary Pump (G1311A) + Autosampler (G1367A), (B) 1290 Infinity II LC without emulation, (C) 1290 Infinity II LC in emulation mode 12 ISET - User Manual NOTE What is ISET? 1 The solution for instrument to instrument method transfer: ISET The result: similar retention times and similar resolution, without modifying the instrument or the original method. The sample and method conditions in the figure above are used to display a critical transfer situation. The method is not optimized, and not meant to be a good example for best resolution. For ISET it is key to always reproduce the original resolution. It delivers a good separation in C if the separation in A is good, and a compromised separation in C if the separation in A is compromised. ISET truly reproduces the original separation pattern. ISET - User Manual 13 1 What is ISET? The solution for instrument to instrument method transfer: ISET ISET support for emulation Table 1 List of modules, which can be emulated by ISET (Agilent) Pumps Autosamplers G1311A G1311B G1311C G1312A G1312B G1312C G4204A G7104A G7104C G4220A1 G4220B1 G7120A1 G5611A G1313A G1329A G1329B G1367A (100 µL syringe) G1367B (100 µL syringe) G1367C (100 µL syringe) G1367D (100 µL / 40 µL syringe) G1367E (100 µL / 40 µL syringe) G1377A G4226A (20, 40, 100 µL syringe) G7167A/B (40 µL / 100 µL / 900 µL syringe) 1100 Series Quaternary Pump 1200 Series Quaternary Pump 1260 Infinity Quaternary Pump 1260 Infinity Quaternary Pump VL 1100 Series Binary Pump 1200 Series Binary Pump SL 1260 Infinity Binary Pump 1260 Infinity Binary Pump VL 1290 Infinity Quaternary Pump 1290 Infinity II Flexible Pump 1260 Infinity II Flexible Pump 1290 Infinity Binary Pump 1290 Infinity Binary Pump VL 1290 Infinity II High Speed Pump 1260 Infinity Bio-inert Quaternary Pump 1100 Series Standard Autosampler 1100 Series Thermostatted Autosampler 1200 Series Standard Autosampler 1200 Series Standard Autosampler SL 1260 Infinity Standard Autosampler 1100 Series Well Plate Sampler 1100 Well Plate Sampler 1200 Series High Performance Autosampler 1200 Series High Performance Autosampler SL 1200 Series High Performance Autosampler SL+ 1260 Infinity High Performance Autosampler 1100 Micro Well Plate Sampler 1200 Series Micro Well Plate Autosampler 1260 Infinity High Performance Micro Autosampler 1290 Infinity High Performance Autosampler 1290 Infinity II Multisampler 14 ISET - User Manual What is ISET? 1 The solution for instrument to instrument method transfer: ISET Table 1 List of modules, which can be emulated by ISET (Agilent) LC Systems G7129A (40 µL / 100 µL / 900 µL syringe) G7129B (40 µL / 100 µL / 900 µL syringe) G7129C (40 µL / 100 µL / 900 µL syringe) G5667A G4288A, G4289AA, G4290AA G4288C, G4290C G4288B, G4290B, G4294B 1260 Infinity II Vialsampler 1290 Infinity II Vialsampler 1260 Infinity II Vialsampler 1260 Infinity High Performance Bio-inert Autosampler 1120 Compact LC 1220 Infinity II LC VL / 1220 Infinity LC VL 1220 Infinity II LC / 1220 Infinity LC 1 Emulation of this module is possible with G4220A/B or G7120A only. Table 2 List of instruments, which can be emulated by ISET (Waters) System Alliance 2690, 2695 Alliance 2790, 2795, 2796 Specifications Comment Total system delay volume: < 650 µL Injection range: 0.1 100 µL (standard), up to 2000 µL with optional sample loop standard configuration with 100 µL sample loop and 250 µL syringe. Sample loops available: 2000, 200, 193, 100 µL Syringes available: 25, 250, 2500 µL (syringe volume does not contribute to dwell volume). Total system delay volume: < 400 µL Injection range: 5 50 µL (standard), up to 1500 µL with optional sample loop standard configuration with 50 µL sample loop and 500 µL syringe. Sample loops available: 5, 20, 50, 100, 500, 2000 µL Syringes available: 100, 250, 500, 1000, 2500 µL (syringe volume does not contribute to dwell volume). ISET - User Manual 15 1 What is ISET? The solution for instrument to instrument method transfer: ISET Table 2 List of instruments, which can be emulated by ISET (Waters) System Acquity UPLC1 Acquity UPLC H-Class Specifications Total system delay volume: <120 µL 50 µL, 100 µL mixer Injection range: 0.1 5 µL, up to 50 µL with optional sample loop. Standard configuration with 5 µL sample loop and 100 µL syringe. Sample loops available: 1, 2, 5, 10, 20, 50 µL Total system delay volume: <400 µL 100 µL mixer Injection range: 0.1 10 µL, up to 1000 µL with optional sample loop. Standard configuration with 10 µL sample loop and 100 µL syringe. Sample loops available: 10, 50, 100, 250, 1000 µL 1 Emulation of this instrument is possible with G4220A/B or G7120A only. Comment Syringes available: 100, 250 µL (syringe volume does not contribute to dwell volume). Syringes available: 100, 250, 500 µL (syringe volume does not contribute to dwell volume). Table 3 List of instruments, which can be emulated by ISET (Shimadzu) Pumps Sampler LC-20 AB1 w/o mixer, 100 µL, 0.5 mL, 1.7 mL, 2.6 mL mixer LC-20 AT w/o mixer, 100 µL, 0.5 mL, 1.7 mL, 2.6 mL mixer LC-20 SIL A/AC Injection Range: 0.1 100 µL, up to 2000 µL with optional sample loop. Standard configuration with 100 µL sample loop. Sample loops available: 50, 100, 500, 2000 µL 1 Emulation of this instrument is possible with G4220A/B or G7120A only. Binary High Pressure Mixing Pump Quaternary Low Pressure Mixing Pump Sampler NOTE ISET supports the emulation of any LC instrument. For details see "Generic emulation using dwell volume" on page 39. 16 ISET - User Manual What is ISET? 1 The solution for instrument to instrument method transfer: ISET Specifications Table 4 Retention Time (RT) Deviation RT ±5% ±0.3 min RT ±5% ±0.1 min (for RT > 6 min) (for RT 6 min) (for RT > 2 min) (for RT 2 min) NOTE For other vendor systems these specifications are only valid for column operation at 40 °C. These specifications limits are not valid for the generic emulation and generic solvents. ISET - User Manual 17 1 What is ISET? The solution for instrument to instrument method transfer: ISET Demo/Trial A 700-hour demonstration/trial version of ISET is available with the following pumps: · Agilent 1290 Infinity II Flexible Pump (G7104A with firmware revision B.06.56 and later) · Agilent 1260 Infinity II Flexible Pump (G7104C with firmware revision B.07.20 and later) · Agilent 1290 Infinity Quaternary Pump (G4204A with firmware revision B.06.56 and later) · Agilent 1290 Infinity II High Speed Pump (G7120A) with firmware revision B.06.42 and later) · Agilent 1290 Infinity Binary Pump (G4220A with firmware revision B.06.42 and later) · Agilent 1290 Infinity Binary Pump VL (G4220B with firmware revision B.06.42 and later) A pictogram in the controller software indicates the use of the demonstration/trial version. Table 5 Active Pictograms of ISET versions Demonstration/trial Registered Inactive Unless the demonstration/trial version is upgraded to a registered version, the ISET function will be deactivated when the trial period has elapsed. 18 ISET - User Manual ISET - User Manual 2 Installing and Configuring ISET Preconditions for ISET 20 Installing and configuring ISET 25 ISET Removal and Reinstallation 27 This chapter gives detailed step-by-step instructions for installing a new system and upgrading an existing system. Agilent Technologies 19 2 Installing and Configuring ISET Preconditions for ISET Preconditions for ISET You need one of the Chromatographic Data Systems specified in Table 8 on page 23 (OpenLAB CDS, OpenLAB CDS ChemStation Edition, OpenLAB CDS EZChrom Edition, or Mass Hunter). Alternatively, you need one of those 3rd party CDS systems that support the Agilent Instrument Control Framework (ICF), plus you need this ICF installed in the specified revision. In addition to the CDS the installation of the Agilent LC and CE drivers ( A.02.04 see Table 8 on page 23) is required. 20 ISET - User Manual Installing and Configuring ISET 2 Preconditions for ISET Table 6 Modules needed for ISET Module P/N 1290 Infinity Binary Pump 1290 Infinity Binary Pump VL 1290 Infinity Quaternary Pump 1290 Infinity II Flexible Pump 1260 Infinity II Flexible Pump 1290 Infinity II High Speed Pump G4220A G4220B G4204A G7104A G7104C G7120A Firmware Comments Revision B.06.42 B.06.42 B.06.56 B.06.70 B.07.20 B.06.70 You need one of the pumps listed with either no mixer or one of the specified mixers (see Table 7 on page 22). NOTE ISET emulation is not possible when the dwell volume of the system to be emulated is smaller than that of the emulating system. In this case an error message Dwell volume of emulated system too small to be emulated with current configuration! is shown in the driver and the method cannot be downloaded to the instrument. NOTE In Method Scouting Wizard configuration either choose Generic as ISET solvent model which works always but leads to less accurate emulation results or make sure that the solvents combinations used in method scouting match with the solvent setting for the ISET solvent model. Currently ISET supports following resulting gradients: Water vs. Acetonitrile, Water vs. Methanol or Aqueous vs. Organic. 1290 Infinity Autosampler 1290 Infinity II Multisampler1 1260 Infinity II Multisampler1 1290 Infinity II Vialsampler 1260 Infinity II Vialsampler 1260 Infinity II Vialsampler G4226A G7167B G7167A G7129B G7129A G7129C B.06.42 D.06.70 D.06.75 D.06.75 D.06.75 D.07.20 ISET in Pump Valve Clusters is supported since ISET 2. With 20 µL, 40 µL, 100 µL loop 1 single needle is supported, dual needle is not supported ISET - User Manual 21 2 Installing and Configuring ISET Preconditions for ISET Table 7 ISET - supported mixers Module 1290 Infinity Binary Pump P/N G4220A 1290 Infinity Binary Pump VL G4220B 1290 Infinity II High Speed Pump G7120A 1290 Infinity Quaternary Pump G4204A 1290 Infinity II Flexible Pump G7104A 1260 Infinity II Flexible Pump G7104C Supported mixers · V35 JetWeaver · V100 JetWeaver · V380 JetWeaver · V35 JetWeaver · V100 JetWeaver · V380 JetWeaver · V35 JetWeaver · V100 JetWeaver · V380 JetWeaver · V35 JetWeaver · V100 JetWeaver · V380 JetWeaver · V35 JetWeaver · V100 JetWeaver · V380 JetWeaver · V35 JetWeaver · V100 JetWeaver · V380 JetWeaver 22 ISET - User Manual Installing and Configuring ISET 2 Preconditions for ISET Table 8 Software compatibility ISET ISET Revision ISET 1 ISET 2 ISET 3 ISET 4 Driver Revision A.02.04 A.02.06 A.02.09 A.02.11 Emulated LC Instruments OpenLAB CDS OpenLAB CDS ChemStation Edition OpenLAB CDS EZChrom Edition Mass Hunter ICF1 Agilent 1100 and 1200 Series, Agilent 1260 Infinity LC CS B.04.03 SP1 CS C.01.03 EE A.04.03 Q/TOF B.05.00 QQQ B.05.00 A.01.04 Scope of ISET 1 plus Waters Alliance, Agilent 1220 Infinity LC, Agilent 1120 Compact LC, Generic emulation Scope of ISET 2 plus Agilent 1290 Infinity Quaternary Pump Waters H-Class Waters Acquity Shimadzu LC 20 Scope of ISET 3 plus Agilent 1290 Infinity Binary Pump Agilent 1290 Infinity II Flexible Pump Agilent 1290 Infinity II High Speed Pump CS B04.03 SP2 CS C01.03 SP1 CS C.01.04 CS C.01.05 CS B04.03 SP2 EE A04.03 SP1 EE A.04.04 EE A.04.05 Q/TOF B.05.01 (September 2012) QQQ B.06.00 (October 2012) Q/TOF B.06.00 QQQ B.07.00 A.01.05 A.02.01 CS C.01.07 EE A.04.07 Q/TOF B.06.01 QQQ B.07.00 SP2 A.02.03 DU2 ISET - User Manual 23 2 Installing and Configuring ISET Preconditions for ISET Table 8 Software compatibility ISET ISET Driver Emulated LC Revision Revision Instruments ISET 4.1 A.02.12 Scope of ISET 4 plus Agilent 1260 Infinity Autosampler Agilent 1290 Infinity II Vialsampler ISET 4.2 A.02.18 Scope of ISET 4.1 plus 1260 Infinity II Flexible Pump 1260 Infinity II Vialsampler 1 Agilent Instrument Control Framework OpenLAB CDS OpenLAB CDS ChemStation Edition OpenLAB CDS EZChrom Edition CS C.01.07 EE A.04.07 Mass Hunter ICF1 Q/TOF B.07.01 QQQ B.08.00 A.02.03 DU2 2.2 C.01.07 A.04.08 QTOF A.02.05 SR4 SR1 B.09.00 QQQ B.09.00 Ultivo C.01.00 24 ISET - User Manual Installing and configuring ISET Installing and Configuring ISET 2 Installing and configuring ISET Hardware required Pump G4204A, G7104A/C, G4220A/B or G7120A The installation of ISET in the trial version is already complete if the prerequisits are met and the components have been properly installed. To upgrade to the registered version, you need to follow these steps: 1 Power up the pump. 2 Plug the ISET USB dongle into the USB port on the back of the pump. NOTE You must use the same ISET USB dongle for reinstallation as was originally used for installation. This, for example, is necessary after exchanging the mainboard of the pump. Thus it's advisable to keep the serial number information of the pump together with the ISET USB dongle. G4220A/B 86%3RUW G4204A 86%3RUW 6HULDOQXPEHULQIRUPDWLRQ ISET - User Manual 6HULDOQXPEHULQIRUPDWLRQ 25 2 Installing and Configuring ISET Installing and configuring ISET G7120A 86%3RUW G7104A/C 86%3RUW 6HULDOQXPEHULQIRUPDWLRQ 6HULDOQXPEHULQIRUPDWLRQ NOTE 3 Wait until blinking of USB dongle stops. 4 Switch off the pump. 5 Switch on the pump to finalize the activation procedure. Do not remove the ISET USB dongle at this stage. 6 Go to instrument configuration to display the pump configuration screen. The ISET installed check box in the Options section is marked to indicate that the installation is complete. 7 Remove the USB dongle. 26 ISET - User Manual ISET Removal and Reinstallation Installing and Configuring ISET 2 ISET Removal and Reinstallation NOTE ISET functionality will not be affected by a firmware update. If ISET was present before the update, it will remain active after the procedure. Replacement of the pump main board will completely remove the ISET functionality. Reinstalling ISET In the case of a replacement of the pump's main board you will need to reinstall ISET. To reinstall ISET, follow the installation procedure (see above). You must use the same ISET USB dongle for reinstallation as was originally used for installation. The ISET USB dongle has a counter that tracks the number of times it has been used. You can install and reactivate ISET a maximum of five times. ISET - User Manual 27 2 Installing and Configuring ISET ISET Removal and Reinstallation 28 ISET - User Manual ISET - User Manual 3 Setting Up ISET Parameters Preparing the system for emulation 30 Setting up the basic ISET parameters 31 Fine-tuning the emulation 38 Generic emulation using dwell volume 39 Definition of dwell volume 40 Determination of dwell volume 40 This chapter gives detailed step-by-step instructions for setting up the standard ISET parameters, and for setting up and using a verification method to confirm that ISET is functioning as expected. It also contains explanations of the advanced ISET parameters, with step-by-step instructions for setting them up. Agilent Technologies 29 3 Setting Up ISET Parameters Preparing the system for emulation Preparing the system for emulation NOTE NOTE 1 Set up the system with the correct column and mobile phases for the method to be transferred. If possible, transfer the column from the original system. The differences in selectivity between similar types of column, for example, C18 from different manufacturers, is likely to be greater than differences caused to the separation by parameters that ISET controls. 2 Download the method parameters for all modules from the original method. OR Enter the method parameters into the new method in the regular way. Ensure that all values are correctly transferred, and that appropriate values are provided for the data collection rate (detector PeakWidth setting). 3 Allow the system to equilibrate. 30 ISET - User Manual Setting Up ISET Parameters 3 Setting up the basic ISET parameters Setting up the basic ISET parameters Prerequisites NOTE In this section, you will transfer your original method to the system and activate ISET by selecting the original hardware configuration. All known differences in the behavior between the original LC instrument and the current target system will be addressed by ISET. · ISET is installed (see "Installing and Configuring ISET" on page 19). When ISET is installed but not enabled, the grey pictogram is displayed in the Pump Dashboard panel; the colored pictogram indicates that ISET is enabled. ,6(7SLFWRJUDP NOTE To create a new method, the latest ISET revision is always the best choice. For use of already established emulation methods, previous ISET versions are still available for backward compatibility. ISET - User Manual 31 3 Setting Up ISET Parameters Setting up the basic ISET parameters 1 Right-click in the Pump Dashboard panel and select Method from the context menu. The method setup dialog box is displayed. 2 Expand the Advanced section of the pump method parameters and ensure that the following check boxes are marked: 32 ISET - User Manual Table 9 Advanced pump method parameters 1290 Infinity II High Speed Pump (G7120A) 1290 Infinity Binary Pump (G4220A) 1290 Infinity Binary Pump VL (G4220B) NOTE ISET only works under the following preconditions: · Minimum Stroke: Radio button Automatic and check box Synchronized are selected. · Compressibility: Check box Use Solvent Types is selected. 1290 Infinity II Flexible Pump (G7104A) 1260 Infinity II Flexible Pump (G7104C) 1290 Infinity Quaternary Pump (G4204A) NOTE ISET only works under the following preconditions: · Minimum Stroke: Radio button Automatic is selected. · Compressibility: Check box Use Solvent Types is selected. · Primary Channel: Drop-down list Automatic is selected Setting Up ISET Parameters 3 Setting up the basic ISET parameters NOTE For ISET to work correctly, it is vital to ensure best accuracy of both flow and composition of the mobile phase. Critical performance factors are the stroke settings and the solvent correction. Only when the built-in solvent libraries are used is the operation accurate enough to expect correct results. ISET - User Manual 33 3 Setting Up ISET Parameters Setting up the basic ISET parameters NOTE 3 Verify that the following method parameters for the pump show the correct values as given in your original method: · solvent flow rate · G4220A/B, G7120A: Solvents A and B and solvent composition G4204A, G7104A/C: Solvents A, B, C, and D and solvent composition G4204A, 7104A/C: · Binary gradients with a constant addition of a additive in water are fully supported in ISET. · Quaternary and ternary gradients or the simulation of binary gradients, which are composed with more than one organic solvent, are only supported by the generic model. ISET will still compensate delay volume differences. NOTE · pressure limits · stop- and post-times · gradient timetable Dependent on selected manufacturer a gradient curve parameter can be defined in the time table. The default is a linear gradient. 4 Expand the ISET section of the method parameters. 34 ISET - User Manual NOTE Setting Up ISET Parameters 3 Setting up the basic ISET parameters 5 Mark the Enable ISET check box. Currently there are the following versions of ISET available: · ISET 4 Default - version of choice to establish a new method. ISET 4 offers downward compatibility - version of choice to use methods, already available and established with ISET 3. ISET 4 offers more flexibility (as more systems may be emulated) and bases on an optimized algorithm. · ISET 3 Offers downward compatibility - version of choice to use methods, already available and established with ISET 2. ISET 3 offers more flexibility (as more systems may be emulated) and bases on an optimized algorithm. · ISET 2 Offers downward compatibility - version of choice to use methods, already available and established with ISET 2. · ISET 1 Offers downward compatibility - version of choice to use methods, already available and established with ISET 1. The Model Parameter section of the method setup is displayed. ISET - User Manual 35 3 Setting Up ISET Parameters Setting up the basic ISET parameters NOTE NOTE Figure 9 Model Parameter section The Model Parameter section enables you to configure the ISET emulation by selecting the original hardware module that you used during the development of your original method. Currently, the ISET library holds parameters only for the pump and the sampler, since these are the major components that influence the mobile phase composition and its transient parameters as seen on the head of the column. With ISET 4, ISET 3, and ISET 2 under Model in the Emulation section there are different Manufacturer options. If using the Generic LC Instrument option, the parameter in the Dwell Volume must be provided by the user (see "Generic emulation using dwell volume" on page 39). 6 Click the Emulated Pump down arrow and select the pump used for the original method from the drop-down list, see Figure 9 on page 36. The drop-down list shows the module number of the pump and the revision of its characterization. To determine a pump's name by its specific module number, please refer to "ISET support for emulation" on page 14. 7 If the solvent system defined in the method includes only water, methanol or acetonitrile, no changes to the ISET solvent model are required. 36 ISET - User Manual NOTE Setting Up ISET Parameters 3 Setting up the basic ISET parameters In ISET, water, methanol and acetonitrile are fully characterized for gradient performance. 8 If solvents other than water, methanol or acetonitrile are defined in the method, mark the manually select ISET solvent model check box, click the down-arrow and select an appropriate solvent system from the drop-down list: · select Generic when buffers or solvent mixtures are used. · select Aqueous (Channel A) Organic (Channel B) or Organic (Channel A) Aqueous (Channel B) for pure solvents as appropriate. You can also select Acetonitrile/Water or Methanol/Water if the solvents used are similar to these. NOTE 9 Mark the manually set check box for compressibility and enter the compressibility settings that were used in the original method. This is especially important if the compressibility values in the original method are different from the default values. NOTE The parameters you set up to this step are those that ISET uses to correct for the behavior of the original pump module. 10 Click the Emulated Sampler down-arrow and select the sampler and configuration from the drop-down list. ISET calculates delay volumes using the factory-installed values of capillaries, syringes and needle seats. If other capillaries are installed, fine-tune the delay volume (see "Fine-tuning the emulation" on page 38). 11 If a needle seat for higher injection volumes is installed, enter the correct needle seat volume in the Seat field. 12 When all method settings are finished, click OK to close the method setup-screen. During the run of an ISET method it is not possible to change the method parameters. ISET - User Manual 37 3 Setting Up ISET Parameters Fine-tuning the emulation Fine-tuning the emulation NOTE When the original system and the ISET system both use standard configurations, no offset setting should be required. As a rule of thumb, only the excess dwell volume of the system with respect to the standard configuration contributes to the offset. The total dwell volume offset setting is then composed of the original system's contribution minus the ISET system's contribution. If the emulated method was already run under ISET, the results of those previous runs may then be used to accomplish a perfect match of the emulated method with respect to the original if there are still remaining retention time differences. In critical applications, where the gradient is too early or reaches the column head later than expected, the separation and selectivity are changed, and influence the resolution and retention times. For fine-tuning, the dwell volume may be adjusted to improve the emulation. 1 Mark the Enable manual fine tuning check box to display the fine-tuning parameters. 2 If necessary, enter a Typical Operating Pressure. During the formation of gradients using Water/Methanol or Water/Acetonitrile, the system pressure changes. This change in pressure affects the volume of the damper of the pump, which results in a change in volume. The Typical Operating Pressure can be used to compensate for this change in volume. 3 If necessary, enter a Dwell Volume Offset. The Dwell Volume Offset can be used to compensate for a difference in dwell volume between the original instrument and the ISET system caused, for example, by wider ID capillaries or an additional mixer (that is, non-standard system configurations). Positive Dwell Volume Offsets shift the gradient to a later position; negative Dwell Volume Offsets shift the gradient to an earlier position 4 When all method settings are finished, click OK to close the method setup-screen. During the run of an ISET method it is not possible to change the method parameters. 38 ISET - User Manual Setting Up ISET Parameters 3 Generic emulation using dwell volume Generic emulation using dwell volume The generic emulation using dwell volume can be used for LC instruments not listed as ISET supported modules/systems. In these cases the dwell volume has to be entered into the field Dwell Volume. NOTE Figure 10 Generic emulation using dwell volume Sometimes the dwell volume is specified by the vendor of the system to be emulated. If not, it can be determined (see "Determination of dwell volume" on page 40). The generic emulation using dwell volume does not compensate for following aspects: · Different mixing behaviors · Composition errors ISET - User Manual 39 3 Setting Up ISET Parameters Generic emulation using dwell volume Definition of dwell volume The system dwell volume includes the volume of the system flow path from the point of mixing of the mobile phase components to the head of the column. It has an important effect on the gradient that the pump delivers because every gradient has an initial isocratic segment as the mobile phase must travel through the dwell volume before any change made at the pump arrives at the head of the column. Any differences in dwell volume cause variations in retention times and often also in selectivity. Determination of dwell volume 1 Replace column by a zero dead volume connection or low volume capillary. 2 Run a linear gradient from 0 10 min from 5 95 % B at a flow rate (F) of 1 mL/min with water (channel A) and water with 0.2 % acetone (channel B) at 263 nm detection wavelength. 3 Determine the difference in time (tdwell) between the programmed and actual elution time of the gradient at 50 % of the composition. &RPSRVLWLRQ 7LPH 4 Vdwell can be calculated from: Vdwell = tdwell × F 40 ISET - User Manual ISET - User Manual 4 Understanding ISET Functionality Understanding ISET functionality 42 Composition differences 43 ISET functionality 45 This chapter explains in detail how ISET works. Agilent Technologies 41 4 Understanding ISET Functionality Understanding ISET functionality Understanding ISET functionality The transfer of a method from one liquid chromatography system to another usually involves a direct transfer of all instrument parameters, for example, flow rate, mobile phase composition and gradient timetable, injection volume, column temperature and UV detection wavelength. However, there can also be subtle differences such as system delay (or dwell) volume (the volume of the system flow path from the point of mixing of the mobile phase components to the top of the column), the design of the autosampler and column compartment flow paths and temperature, detector cell design, and the extra-column volume of the system, detector data collection rates and response times and the sensitivity of the detector. A closer investigation into the comparative performance of different designs of LC gradient pumps reveals that other hidden factors have an effect on how the pump delivers the gradient. Principally, this is characterized by the mixing behavior of the pump, which in turn is determined by the basic concept and design of the flow path, the volume and design of the mixer, the efficiency of the solvent mixing and the behaviour of the piston movement under different loads and compressibilities of solvents. This behaviour can be visualized by using tracer experiments (0.2 % acetone in channel B for example) to plot the profile of the pump as it delivers a stepwise gradient in the proportion of the solvents. In practice, there is always a delay, and the step gradient is smoothed and transformed into a curve that is characteristic of the pump. Every model of pump has its own characteristic profile, and this leads to slight differences in nominally similar gradients on different pumps. Often, these are sufficient to cause problems in the transfer of some methods, and the challenge is to measure the characteristics of different pumps and reproduce them on the Agilent 1290 Infinity LC system pump. This is the heart of the ISET system; however, the accurate performance of the 1290 Infinity is still needed to execute the settings. ISET reveals and translates those parameters to provide an accurate method transfer from older Agilent systems to the system that is used for ISET, and vice versa. 42 ISET - User Manual Composition differences Understanding ISET Functionality 4 Composition differences The primary and most visible aspect of compressibility settings is reduction of pressure ripple, but for predictable and accurate pumping, the flow accuracy is more critical. Additionally, with the high-pressure blending concept of binary pumps, this flow accuracy translates into compositional accuracy. The compressibility of the solvents in use affect retention-time stability and predictability, especially when the back-pressure in the system changes (for example, ageing of column). To minimize this effect, the pump provides a compressibility compensation feature that optimizes the stability of the flow accuracy according to the solvent type. The compressibility compensation is set to a default value and can be changed through the user interface. Without compressibility compensation, the following happens during a stroke of the first plunger: · The pressure in the plunger chamber increases, and the volume in the chamber is compressed depending on back-pressure and solvent type. · When dispensing a more compressible solvent against pressure, the displacement rate of the piston is reduced to compensate for the expansion of the solvent while it travels down the column. When a compressibility value is set, the processor calculates a compensation volume that is dependent on the back-pressure in the system and the selected compressibility. This compensation volume is added to the normal stroke volume and compensates for the loss of volume during the delivery stroke of the first plunger. Composition differences are generated by the HPLC by imperfect compensation of the solvent compressibility, for example, due to mismatch of the compressibility settings and the actual solvent compressibility. Accurate blending of mobile phase composition is vital for predictable retention. While historically the equipment was well-known for its reproducibility, in terms of accuracy of the mobile phase composition, offsets may have been allowed. However, in order to emulate the historic behavior, these systematic offsets, characteristic of the individual instrument classes, need to be taken into consideration. ISET - User Manual 43 4 Understanding ISET Functionality Composition differences As an example, consider volume contraction. When mixing water in an organic solvent, the basic pump concept, if it is low pressure proportioning or high pressure dispensing, may introduce a significant offset. This may even be different for various solvents, and may even change with runnning conditions such as %B or pressure and, of course, the compressibility settings that the original pump compensated for. The composition differences generated by a 1100 quaternary pump for different settings of solvent compressibility are shown in Figure 11 on page 44. Figure 11 Composition offset of an Agilent 1100 quaternary pump for acetonitrile-water at different compressibility settings. When a gradient timetable is executed on an (U)HPLC instrument, the shape of the resulting gradient formed at the top of the column depends mainly on · the system volume and geometry between the point of mixing (usually in the pump) and the column head. · the accuracy with which the programmed composition is delivered to the column. When an HPLC system is characterized with regard to gradient shape, it is important to separate the effects of the geometric volumes from those of static composition errors produced by the pump. Figure 11 on page 44 shows the composition offset generated by a 1100 quaternary pump for different settings of solvent compressibility. 44 ISET - User Manual ISET functionality Understanding ISET Functionality 4 ISET functionality ISET functionality The physical relationship between a programmed timetable and the system response can be described by a transfer function (Figure 12 on page 45). 7LPHWDEOH 6\VWHPUHVSRQV 7LPHWDEOH 6\VWHPUHVSRQV ,QSXW [ WUDQVIHUIXQFWLRQFRPSOH[ 2XWSXW Figure 12 Different gradient shapes resulting from the same time table for different instrument characteristics. For system B with a considerably lower delay volume than system A, it is possible to compute a (virtual) timetable from the transfer functions of the two instruments that corresponds to the programmed timetable of system A, and that generates a gradient response on system B that is equivalent to that of system A (Figure 13 on page 46). ISET - User Manual 45 4 Understanding ISET Functionality ISET functionality 0HWKRG 7LPHWDEOHD $ ,QSXW 7LPHWDEOHE % 2XWSXW Figure 13 Operation of the system in a mode that emulates the gradient response of a larger delay volume system. The transfer functions generated from thorough system characterizations are used by ISET to emulate another HPLC system. When the emulation mode is enabled, the system, emulating the original pump and auto-sampler can run the original gradient method and achieve a very similar separation to that produced on the emulated system (see Figure 8 on page 12). 46 ISET - User Manual ISET - User Manual 5 Application and Technical Notes Application and technical notes 48 This chapter gives an overview on additional literature. Agilent Technologies 47 5 Application and Technical Notes Application and technical notes Application and technical notes p/n 5991-7794EN 5991-6890EN 5991-6715EN 5991-6541EN 5991-6408EN 5991-6289EN 5991-5701EN 5991-5030EN 5991-4729EN 5991-2792EN 5991-2275EN 5991-2019EN Description Universal Analytical Method Development for Various HPLC Systems Using the Agilent 1290 Infinity II Method Development Solution - On-The-Fly Target System Emulation Using Intelligent System Emulation Technology ISET Agilent 1290 Infinity II LC with ISET - Method Development for Transfer to an Agilent 1260 Infinity LC with Autosampler and Integrated Column Compartment Agilent 1290 Infinity II LC with ISETEmulation of the Waters Acquity H-Class for Ternary Gradients Agilent 1290 Infinity II LC with ISET Emulation of the Agilent 1100 Series LC Through Waters Empower Software Agilent 1290 Infinity II LC with ISET Emulation of Waters Alliance 2695 LC Through Waters Empower Software Agilent 1290 Infinity II LC with ISET Emulation of the Agilent 1100 Series Binary LC for Analysis of Tricyclic Antidepressant Drugs QbD Based Method Development on an Agilent 1290 Infinity UHPLC System Combined with a Seamless Method Transfer to HPLC Using Intelligent System Emulation Technology Intelligent System Emulation Technology Transfers Methods from Binary to Quaternary LC Agilent 1290 Infinity Binary LC System with ISET Emulation of a Waters Alliance 2695 LC Applying Concave, Convex, and Linear Gradients Agilent 1290 Infinity Binary LC with ISET Emulation of the Waters Alliance 2695 LC System Analyzing Analgesics Agilent 1290 Infinity LC with ISET under Waters Empower control Emulation of Agilent 1100 Series LC Agilent 1290 Infinity Binary LC with ISET, emulation of the Waters Alliance 2695 LC system analyzing aromatic acids 48 ISET - User Manual Application and Technical Notes 5 Application and technical notes p/n 5991-1605EN 5991-1604EN 5991-1603EN 5991-1433EN 5991-1194EN 5990-9715EN 5990-9703EN 5990-9692EN 5990-9546EN 5990-9545EN 5990-9113EN Description Agilent 1290 Infinity Binary LC with ISET - Emulation of the Waters Alliance 2695 LC system analyzing endocrine disruptors Agilent 1290 Infinity Binary LC with ISET - Emulation of the Waters Alliance 2695 LC system analyzing antioxidants Agilent 1290 Infinity Binary LC System with ISET - Emulation of the Waters Alliance 2695 LC system analyzing -blockers Seamless instrument-to-instrument method transfer of an USP/EP method from an Agilent 1220 Infinity LC to an Agilent 1290 Infinity Binary LC using Intelligent System Emulation Technology (ISET) Seamless instrument-to-instrument method transfer of the EPA method 8330A/B for nitroaromatics from an Agilent 1200 Series LC to the Agilent 1290 Infinity Binary LC using ISET Method development on the Agilent 1290 Infinity LC using Intelligent System Emulation Technology (ISET) with subsequent transfer to an Agilent 1100 Series LC Analysis of an analgesic drug Transferring methods to the Agilent 1290 Infinity LC System using Intelligent System Emulation Technology (ISET) Analysis of paracetamol and its impurities Transferring methods to the Agilent 1290 Infinity LC using Intelligent System Emulation Technology (ISET) Analysis of metoclopramide hydrochloride and its impurities Comparing gradient transfer of isocratic hold and delay volume addition using the Agilent 1290 Infinity LC with ISET Seamless transfer of elution gradients from Agilent 1100/1200 Series LCs to an Agilent 1290 Infinity LC using ISET Seamless instrument-to-instrument method transfer from an Agilent 1100/1200 Series LC to an Agilent 1290 Infinity LC using Intelligent System Emulation Technology (ISET) Documents are available on the worldwide web site on the internet at http://www.agilent.com/en-us/promotions/applicationfinder?Focus=Intelligent%20System%20Emulation%20Technology ISET - User Manual 49 Index Index A automatic 33, 33, 33 autosamplers 14 C column temperature 42 column 30 compressibility 33, 33, 37, 43 D delay volume 42 demonstration version 18 dwell volume offset 38 dwell volume 40 linear gradient 40 E emulated pump 36 emulated sampler 37 enable ISET 35 extra-column volume 42 F firmware update 27 flow accuracy 43 flow path 42 flow rate 34, 42 G gradient performance 37 gradient timetable 34, 42 50 I injection volume 42 instrument parameters 42 ISET pictogram 31 M main board replacement 27 method parameters 30 method setup 32 minimum stroke 33, 33 mixing behavior 42 mobile phase composition 42 mobile phase 30 N needle seat 37 O offset 38 P peakwidth 30 posttime 34 pressure limits 34 pressure ripple 43 primary channel 33 pumps 14 R reinstallation 27 removal 27 ripple 43 S solvent composition 34 specifications 17 stoptime 34 synchronized 33 system delay volume 42 systems 15 T trial version 18 typical operating pressure 38 U use solvent types 33, 33 UV detection wavelength 42 ISET - User Manual www.agilent.com In This Book The manual describes the Agilent Intelligent System Emulation Technology (ISET). It contains the following: · Instructions for installing and configuring ISET, · Details on setting up the ISET parameters, · Detailed information about how ISET works. © Agilent Technologies 2017 Printed in Germany 08/2017 *G7120-90310* *G7120-90310* G7120-90310 Agilent TechnologiesAcrobat Distiller 11.0 (Windows)