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  1. ISET Training Agilent Restricted Page 1 August 17, 2012

  2. Agilent 1290 Infinity withIntelligent System Emulation Technology (ISET) ISET Training Agilent Restricted Page 2 August 17, 2012

  3. ISETIntroduction Intelligent System Emulation Technology (ISET) Trainings Material Course: AN-CE-LC-3-015-A (ISET) Course: AN-CE-LC-3-015-B (ISET 2) LC - Intelligent System Emulation Technology (ISET) (link to video about ISET features) ISET Training Agilent Restricted Page 3 August 17, 2012

  4. ISET Agenda ISET Training Agilent Restricted Page 4 August 17, 2012

  5. ISET Goal Running traditional HPLC methods using a 1290 Infinity LC with ISET system obtaining very similar results!  Method transfer Check specifications about ISET product  ISET Training Agilent Restricted Page 5 August 17, 2012

  6. ISET Needs Recently a number of UHPLC instruments have been introduced to the market. These systems are typically designed for the operation of small bore columns (2.1 mm - 3 mm internal diameter) packed with small particles to achieve high separation efficiencies. With regard to solvent delivery these systems usually differ significantly from standard HPLC systems, in particular the dwell volumes of such systems are minimized to allow fast gradient separations. When the attempt is made to transfer methods developed on standard HPLC systems to modern UHPLC systems, changes in retention times and often also in selectivity are observed. These changes are due to the differences in system (dwell) volumes, mixing characteristics and also properties of the pumping system (composition accuracy). ISET is to enable the transfer of methods from standard HPLC type instruments to UHPLC systems without changing the original method. ISET Training Agilent Restricted Page 6 August 17, 2012

  7. ISETImportant Parameters 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. Instrument to instrument method transfer is often problematic, especially in highly regulated industries, because any modifications of the instrument and original method should be avoided. Now, the 1290 Infinity LC with ISET extends capability by enabling method transfer between (U)HPLC systems with different instrument designs. Delay volumeGradient mixing behavior Pressure x flow rate Retention Time Resolution Delay volume Pump Sampler Column Thermostat Detector Delay volumeExtra column volumeInjection volume Retention Time Resolution Sensitivity Extra column volume Temperature profileExtra column volume Retention Time Resolution Data rateExtra column volumePath-length Resolution Retention Time Sensitivity ISET Training Agilent Restricted Page 7

  8. ISETActual situation Gradient Analyses with Different LC Systems- Impact of delay volume and mixing behavior 1200 Series LC Quaternary Pump 1290 Infinity LC • The result: • Difference in RT and Resolution • One peak is missing! ISET Training Agilent Restricted Page 8

  9. ISETEmulation! ISET Training Agilent Restricted Page 9 August 17, 2012

  10. ISET Delay volumes and Mixing What happens with a programmed gradient? response 90% 10% Programmedgradientstep 1200 Series Binary Pump SL 1290 Infinity Binary Pump Gradient slope/ Mixing behavior Delay volume time 1200 Series Total delay volume of the system(sum of capillaries, mixer, cells, valves..) 1290 Infinity ISET Training Agilent Restricted Page 10 August 17, 2012

  11. ISETHow it works • Simple and flexible (select the emulated LC system) • No determination of the dwell volume etc. required(perfectly covers pumps with damper and variable dwell volume) • Requires no modification of the methods (no conflicts in regulated environment) ISET Training Agilent Restricted Page 11 August 17, 2012

  12. ISETHow it works 1290 to 1200 mAU 400 350 Injection 300 Same gradient conditions by moving the gradient and by emulating the gradient mixing behavior. 250 200 150 Programmedgradient 1290 gradient 1200 gradient 100 50 0 3 min 0 0.5 1 1.5 2 2.5 3.5 ISET Training Agilent Restricted Page 12 August 17, 2012

  13. ISETWhy 1290? Two components Peak 3Overlay of 10 runsRSD = 0.005 % A. Best in class performance of the 1290 Infinity LCBroad power range, ultra-low delay volume, superior sensitivity, unmatched flow and composition accuracy B. The revolutionary ISET emulationalgorithm Full characterization of target LC systemsCreation of emulation function ISET Training Agilent Restricted Page 13 August 17, 2012

  14. ISETEmulation – What is? Method Transfer - Emulateother HPLC and instruments - Runexisting HPLC methods without modifying your method or system - Achievevery similarretention times and peak resolution for infinitely better method transfer   Check specifications about ISET product ISET Training Agilent Restricted Page 14 August 17, 2012

  15. ISETEmulation Models ISET 2 updates Generic Emulation WatersAlliance2690/95 WatersAlliance HT2790/95/96 1220 Infinity LC 1290 Infinity LC with ISET Added the Jet weaver V380 Characterization Pump Valve Cluster Support 1200 Series LC 1260 Infinity LC 1100 Series LC ISET Training Agilent Restricted Page 15 August 17, 2012

  16. ISETEmulation - Agilent Pumps G1311A 1100 Series Quaternary Pump G1311B 1200 Series Quaternary Pump 1260 Infinity Quaternary Pump G1311C 1260 Infinity Quaternary Pump VL G1312A 1100 Series Binary Pump G1312B 1200 Series Binary Pump SL 1260 Infinity Binary Pump G1312C 1260 Infinity Binary Pump VL G5611A 1260 Infinity Bio-inert Quaternary Pump Autosamplers G1313A 1100 Series Standard Autosampler G1329A 1100 Series Thermostatted Autosampler 1200 Series Standard Autosampler G1329B 1200 Series Standard Autosampler SL 1260 Infinity Standard Autosampler G1367A (100 μL syringe) 1100 Series Well Plate Sampler G1367B (100 μL syringe) 1100 Well Plate Sampler 1200 Series High Performance Autosampler G1367C (100 μL syringe) 1200 Series High Performance Autosampler SL G1367D (100 μL / 40 μL syringe) 1200 Series High Performance Autosampler SL+ G1367E (100 μL / 40 μL syringe) 1260 Infinity High Performance Autosampler G1377A 1100 Micro Well Plate Sampler 1200 Series Micro Well Plate Autosampler 1260 Infinity High Performance Micro Autosampler G5667A 1260 Infinity High Performance Bio-inert Autosampler LC Systems G4288A, G4289AA, G4290AA 1120 Compact LC G4288C, G4290C 1220 Infinity LC (VL) G4288B, G4290B, G4294B 1220 Infinity LC ISET Training Agilent Restricted Page 16 August 17, 2012

  17. ISETEmulation - Waters ISET Training Agilent Restricted Page 17 August 17, 2012

  18. ISETEmulation - Generic 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: The generic emulation using dwell volume does not compensate the following aspects: • Different mixing behaviors • Composition errors ISET Training Agilent Restricted Page 18 August 17, 2012

  19. ISETEmulation - Generic Dwell Volume Definition 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. ISET Training Agilent Restricted Page 19 August 17, 2012

  20. ISETEmulation - Generic 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 between the programmed and actual elution time of the gradient at 50 % of the composition. 4 - Measure tdwell (usually measured at 50 % of composition delta) Vdwell can be calculated from: Vdwell = tdwell × F ISET Training Agilent Restricted Page 20 August 17, 2012

  21. ISETCompatibility ISET will be included in a new RC.net driver for the 1290 Infinity pumps, thus any software supporting RC.net drivers can make use of this feature! Drivers Update only! ISET Training Agilent Restricted Page 21 August 17, 2012

  22. ISETProduct structure Delete option will be available to remove ISET ISET Training Agilent Restricted Page 22 August 17, 2012

  23. ISETActivation Firmware >= B.06.42 Activation or re-activation will be done plugging the USB stick intoPump ISET Training Agilent Restricted August 17, 2012

  24. ISETActivation Steps 1- Power off the Module 2- Plug the USB Dongle into the Infinity Pump USB Socket 3- Turn power on the G4220A/B 4- Once restarted you can choose to Remove the USB Dongle or not Store the USB Dongle in a safe place Must be used to re-activate ISET after a Mother Board replacement ISET Training Agilent Restricted Page 24 August 17, 2012

  25. ISETConfiguration ISET Training Agilent Restricted August 17, 2012

  26. ISETConfiguration ISET properly detected ISET Training Agilent Restricted August 17, 2012

  27. ISETConfiguration New Jet Weaver v380 Mixer support ISET Training Agilent Restricted August 17, 2012

  28. ISETConfiguration Importing future ISET Descriptions (emulated hplc's) ISET Training Agilent Restricted August 17, 2012

  29. ISETGraphical User Interface ISET Training Agilent Restricted Page 29 August 17, 2012

  30. ISETMethod Parameters 2 1 3 4 ISET Training Agilent Restricted August 17, 2012

  31. ISETSolvent Types 1 Do not deselect! Solvents selections-V.0x drop down menu) -Aqueous/Organic/Generic ISET Training Agilent Restricted August 17, 2012

  32. ISETEnabling ISET Enabling ISET to emulate target HPLC 2 ISET V1.0 still available To keep the compatibility ISET Training Agilent Restricted August 17, 2012

  33. ISETEnabling ISET Enabling ISET to emulate target HPLC 2 ISET 2 will ask for Manufacturer: -Agilent -Waters -Generic LC Instrument ISET Training Agilent Restricted August 17, 2012

  34. ISETSelect Target Pump Select the target HPLC pump - Agilent 3 ISET Training Agilent Restricted August 17, 2012

  35. ISETSelect Target Pump Select the target HPLC pump - Waters 3 ISET Training Agilent Restricted August 17, 2012

  36. ISETSelect Target Pump Select the target HPLC pump – Generic 3 ISET Training Agilent Restricted August 17, 2012

  37. ISETSelect Target Autosampler - Agilent 4 Select the target HPLC Sampler ISET Training Agilent Restricted August 17, 2012

  38. ISETSelect Target Autosampler - Waters 4 Select the target HPLC Sampler ISET Training Agilent Restricted August 17, 2012

  39. ISETAdvanced Parameters Fine tuning about compressibility Fine tuning to adjust chromatographic results ISET Training Agilent Restricted August 17, 2012

  40. ISETSetting the Time Table - Agilent Original Time Table ISET Training Agilent Restricted August 17, 2012

  41. ISETView “Emulated” Gradient - Agilent ISET Training Agilent Restricted August 17, 2012

  42. ISETISET Time Table - Agilent Initial conditions are updated Not possible to modify during RUN Note: the “Emulated” Timetable will NOT be printed ISET Training Agilent Restricted August 17, 2012

  43. ISETSetting the Time Table - Waters With Waters you HAVE to select first the “Manufacturer” ISET Training Agilent Restricted August 17, 2012

  44. ISETView “Emulated” Gradient - Waters Curve 6 means “Linear” Enter here the original Method parameters (Waters) …and populate here with the “Original” Waters Method ISET Training Agilent Restricted August 17, 2012

  45. ISETISET Time Table - Waters Initial conditions are updated Not possible to modify during RUN Note: the “Emulated” Timetable will NOT be printed ISET Training Agilent Restricted August 17, 2012

  46. ISETMethod printout – ISET Emulation Mode Enabled: Yes Model: ISET V1.0 ISET Parameter Delay Volume Offset: 0 µL Manually Select ISET Solvent Model: No Enable Tuning: No Manual Set Sampler Parameter: No Manual Set Pump Parameter: No Emulated Pump: G1311A V1.0 Solvent Model: Methanol (Channel A) - Water (Channel B) Emulated Sampler: G1367A - 100 µL Syringe V1.0 Pump Parameter Typical Operating Pressure: 200.00 bar Compressibility A: 100 10e-6/bar Sampler Parameter Sampler Seat Capillary: 2.30 µL Method printout (only the ISET part is reported here) ISET Training Agilent Restricted August 17, 2012

  47. ISETMethod printout – ISET 2 ISET Parameter Emulation Mode Emulation Enabled: Yes Emulation Model: ISET 2 V1.0 Emulation Manufacturer: Generic LC Instrument ISET Parameter Generic Emulation Dwell Volume: 350 µL Generic ISET Parameter Emulation Mode Emulation Enabled: Yes Emulation Model: ISET 2 V1.0 Emulation Manufacturer: Waters ISET Parameter Emulated Pump: Alliance 2690, 2695 V1.0 Solvent Model: Water (Channel A) - Acetonitrile (Channel B) Emulated Sampler: Alliance 2690, 2695 (100 µL Loop) V1.0 Manually Select ISET Solvent Model: No Dwell Volume Offset: 0 µL Enable Tuning: No Manual Set Sampler Parameter: No Manual Set Pump Parameter: No Sampler Parameter Pump Parameter Typical Operating Pressure: 200.00 bar ISET Parameter Emulated Pump: G1311A V1.0 Solvent Model: Water (Channel A) - Acetonitrile (Channel B) Emulated Sampler: G1367A - 100 µL Syringe V1.0 Manually Select ISET Solvent Model: No Dwell Volume Offset: 0 µL Enable Tuning: No Manual Set Sampler Parameter: No Manual Set Pump Parameter: No Sampler Parameter Sampler Seat Capillary: 2.3 µL Pump Parameter Typical Operating Pressure: 200.00 bar Compressibility A: 100 10e-6/bar Waters Agilent Method printout (only the ISET part is reported here) ISET Training Agilent Restricted August 17, 2012

  48. ISETMethod changed history The Method Resolution will appear because the ISET parameter was not present Method changed flag will be shown in the history ISET Training Agilent Restricted August 17, 2012

  49. ISETSolvents Best results with “Characterized Solvent” V.0x drop down menu Solvent lists are characterized for gradient compositions “Aqueous” “Organic” when buffers or solvent mixtures are used Aqueous (Channel A) – Organic (Channel B) or Organic (Channel A) – Aqueous (Channel B) for pure solvents as appropriate ISET Training Agilent Restricted August 17, 2012

  50. ISETDemo Function • Demo features will be available if a delete option is selected when ordering a New 1290 Pump ISET Training Agilent Restricted August 17, 2012