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Agilent Technologies 1260 Infinity Quick Start Manual

Purification solution
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  • Page 1 Agilent 1260 Infinity Purification Solution Method Developer's Quick Start Guide Agilent Technologies...
  • Page 2 Notices Warranty © Agilent Technologies, Inc. 2017 receive no greater than Restricted Rights as defined in FAR 52.227-19(c)(1-2) (June No part of this manual may be reproduced The material contained in this docu- 1987). U.S. Government users will receive in any form or by any means (including ment is provided “as is,”...
  • Page 3 In This Book In This Book This manual contains instructions for Method Developers on how to use the Automated Purification software. 1 The Role of the Method Developer This chapter describes the role of the user of the Automated Purification Software in Expert mode.

  • Page 4: Table Of Contents

    Contents Contents 1 The Role of the Method Developer 2 Preparing Default Purification Methods General Method Settings Analytical Method Settings Preparative Method Settings Agilent Active Splitter (G1968D/E/F) Critical Method Parameters 3 Setting Up an Analytical and Preparative System Setting up an analytical system Setting up a preparative system 4 Setting Up and Running a Purification Task Setting up a purification task...
  • Page 5 Contents UV/MS Workflow: Focused gradient in Purification software Purification Solution - Developer's Guide...
  • Page 6 Contents Purification Solution - Developer's Guide...

  • Page 7: The Role Of The Method Developer

    However, only method developers can accept a failed system suitability test to release a blocked instrument. • Instructing Operators on the work environment and how to set up and run purification tasks. Agilent Technologies...
  • Page 8 The Role of the Method Developer In This Book • Fixing problems (for example, adjusting parameters or target identification) of Operators' tasks, to which the Operators do not have access. Method Developers have full access to all features of the purification software and to the ChemStation while working within the main purification software user interface (the purification task screen).

  • Page 9: Preparing Default Purification Methods

    It is important for Purification software to mark the Save Method with Data check box in N O T E Method > Runtime Check List. Switch off all pumps while you are setting up the methods. N O T E Agilent Technologies...
  • Page 10 Preparing Default Purification Methods In This Book It is highly recommended that the pumps are not switched off in a pre- or post-run N O T E command of the method. Methods rely on the instrument configuration and driver revision. If you change the N O T E instrument configuration or upgrade the driver revision for which you originally set up your purification (base) method, you must at least save the method for the changed instrument...

  • Page 11: General Method Settings

    Preparing Default Purification Methods General Method Settings General Method Settings In all modules, set the Stoptime to As Injector/No Limit (infinite run time). The actual stoptime will be specified when the method is used. Isocratic Pump (G1310B) Make-up pump for splitter used with separated preparative MSD instrument. Stoptime Select As Injector/No limit Pressure Limits...
  • Page 12 Preparing Default Purification Methods General Method Settings Advanced Channel A Set Compressibility to 46 (Water) Advanced Channel B Set Compressibility to 115 (Acetonitrile) Prep Pump (G7161A) Solvents Select Aqueous and type the description text Water Mark the check box, enter , select Acetonitrile, and type description text Acetonitrile Pressure Limits...
  • Page 13 Preparing Default Purification Methods General Method Settings Dual Loop Autosampler (G2258A) Needle Wash Enable Needle Wash with Mode: Flush Port and Time15 s (to avoid contamination of injection seat, do not use less than 10 s). The wash procedure requires needle wash solution. Stoptime Select As Pump/No limit Advanced...
  • Page 14 Preparing Default Purification Methods General Method Settings UV (DAD, MWD,VWD) Wavelength DAD and MWD Set Signal AWavelength to 263 nm, Bandwidth: 4 nm, clear Reference wavelength. Set Signal to 263 nm. Peakwidth DAD and MWD 20 Hz (> 0.013 min) 40 Hz (>...
  • Page 15 Preparing Default Purification Methods General Method Settings Flow Modulator (G7170B) Splitting Operation Select Splitting disabled. Access by Method > Edit entire method or by right click on MSD icon Set Up MSD Signals General Peakwidth: 0.1 min (this defines the MSD data rate; make sure that it is sufficient).

  • Page 16: Analytical Method Settings

    Preparing Default Purification Methods Analytical Method Settings Analytical Method Settings Only for the combined analytical and preparative system. Load the general settings of the purification method: General_Purification(“General Method Settings” on page 11) Save the method as Analytical_Purification: • Method > Save Method As. Quaternary Pump (G1311B) Flow 1.0 mL/min...
  • Page 17 Preparing Default Purification Methods Analytical Method Settings Dual Loop Autosampler (G2258A) Injection Loop Lower Injection mode Depends on installed loop volume and injection volume. • Loop equal to injected volume: set Full loop with overfill factor 5, disable Plug Settings •...
  • Page 18 Preparing Default Purification Methods Analytical Method Settings Save the method changes: • Method > Save Method. Purification Solution - Developer's Guide...

  • Page 19: Preparative Method Settings

    Preparing Default Purification Methods Preparative Method Settings Preparative Method Settings Load the general settings of the purification method: General_Purification(“General Method Settings” on page 11) Save the method as Prep_Purification: • Method > Save Method As. Isocratic Pump (G1310B) Separated preparative MSD system — make-up pump Flow 1.5 mL/min Advanced...
  • Page 20 Preparing Default Purification Methods Preparative Method Settings Dual Loop Autosampler (G2258A) Injection Loop Upper Injection mode Partial Loop Filling Injector Cleaning Rinse volume: 1 (increase to 5 for injections below 200 µL) Advanced Draw speed and Eject speed: 8000.0 µL/min (decrease to 3000.0 for injections below 200 µL) Prep Sampler (G7157A) Advanced...
  • Page 21 Preparing Default Purification Methods Preparative Method Settings If the active splitter is activated by a contact other than contact A, use this contact N O T E instead. Fraction Collector (G1364B and its cluster unit) Fraction Trigger Mode Peak Based, max. peak duration: 1 min Peak Detector G1390B (UIB II) Mode: Off (if no auxiliary detector is connected to UIB II box)
  • Page 22 Preparing Default Purification Methods Preparative Method Settings Intended Pump Flow Set Main Flow and Makeup Flow. Split Mode Select the target split mode. Access by Method > Edit entire method or by right click on MSD icon Fraction Collection FC Mode Use sample target masses MS Signals Mark active signals (Signal 1, and Signal 2 if used)

  • Page 23: Agilent Active Splitter (G1968D/E/F)

    Preparing Default Purification Methods Agilent Active Splitter (G1968D/E/F) Agilent Active Splitter (G1968D/E/F) To start the Agilent active splitter automatically with the preparative method: • Set the external contacts and related timetable as in the UIB II method settings. • Set the Agilent active splitter to Local (communication with External Contacts).

  • Page 24: Critical Method Parameters

    Preparing Default Purification Methods Critical Method Parameters Critical Method Parameters In some situations, incorrect settings of the following parameters can cause incorrect functioning of the Purification software, incorrect fraction collection, or damage or contamination to some parts of the system. Therefore, if the purification system does not work correctly, ensure that the following parameters are set correctly: Configuration...
  • Page 25 Preparing Default Purification Methods Critical Method Parameters Preparative • Valve 2/10: Position > Use valve position > select correct preparative position default method • Iso. Pump (with splitter): Flow > target make-up flow must be set before use (Purification • UIB II (with MSD): Expected ERI Mode > MSD (for MS based fraction software base collection) method)
  • Page 26 Preparing Default Purification Methods Critical Method Parameters Purification Solution - Developer's Guide...

  • Page 27: Setting Up An Analytical And Preparative System

    Here, the meaning of a system is the relevant parameters in the purification workflow, which comprise both instrument configuration and operating parameters. These parameters are mandatory for the automatic purification run calculations. Agilent Technologies...

  • Page 28: Setting Up An Analytical System

    Setting Up an Analytical and Preparative System Setting up an analytical system Setting up an analytical system You can either create a new analytical system from scratch or, if you have already created a similar system, set up a clone of an existing system. 1 In the Chemstation Method and Run Control view, open the Purification menu and select Systems.
  • Page 29 • The tab is present only for flow-through samplers such as the G7157A 1260 Infinity II Prep Autosampler. There, the volumes of the sample loop and seat capillary contribute to the time that the sample needs to reach the column. If the analytical system is separate from the preparative system, such volumes are read from the method as specified in the Pumps &...

  • Page 30: Setting Up A Preparative System

    Setting Up an Analytical and Preparative System Setting up a preparative system Setting up a preparative system The process of setting up a preparative system is very similar to that for setting up the analytical system, but since the Automated Purification software is installed on the preparative system, it is able to determine the current instrument configuration and show a warning if the system does not match the instrument configuration.
  • Page 31 • The tab is present only for flow-through samplers such as the G7157A 1260 Infinity II Prep Autosampler. There, the volumes of the sample loop and seat capillary contribute to the time that the sample needs to reach the column. The volumes must be imported from the current preparative instrument configuration.
  • Page 32 Setting Up an Analytical and Preparative System Setting up a preparative system Purification Solution - Developer's Guide...

  • Page 33: Setting Up And Running A Purification Task

    Setting up a purification task Configuring a new task System Suitability Tests Running a task Processing partial data and cloning tasks Reviewing analytical results This chapter gives instructions for the most important steps for setting up and running a Purification Task. Agilent Technologies...

  • Page 34: Setting Up A Purification Task

    Setting Up and Running a Purification Task Setting up a purification task Setting up a purification task The purification task holds the complete data of the purification job, including the analytical and preparative systems, the analytical and preparative run conditions and parameters, and the analytical and preparative results once they are available.

  • Page 35: Configuring A New Task

    Setting Up and Running a Purification Task Configuring a new task Configuring a new task A new empty task can include information from an analytical run, which is either specified to run on a combined analytical and preparative instrument or which has been run on a separate analytical instrument. This is an analytical-to-preparative workflow task.
  • Page 36 Setting Up and Running a Purification Task Configuring a new task ChemStation sequence in the text input field or click Edit sequence to start editing a sequence from scratch or modifying a selected sequence. You can also import a sequence as a text (CSV or TXT) file. •...
  • Page 37 Setting Up and Running a Purification Task Configuring a new task Mark this check box, if: N O T E • you want to review the automatic target identification of individual samples and eventually correct it by manual target assignment or by changing the target identification parameters for all samples (in the Task Configuration tab) or for individual samples (in the Analytical Results tab) •...

  • Page 38: System Suitability Tests

    Setting Up and Running a Purification Task System Suitability Tests System Suitability Tests The system suitability tests are specific tasks of the preparative-only workflow. They are used to purge the autosampler, flush the instrument and submit a run of at least one known standard sample in order to verify that the compounds in the sample are still eluting within the expected retention time range and that their ion masses can still be detected.

  • Page 39: Running A Task

    Setting Up and Running a Purification Task Running a task Running a task 1 Click Run in the top toolbar of the Task dialog box to start the purification run. If the analytical sequence has not yet been run, it will be submitted to the ChemStation run queue.

  • Page 40: Processing Partial Data And Cloning Tasks

    Setting Up and Running a Purification Task Processing partial data and cloning tasks Processing partial data and cloning tasks You are dealing with partial data under the following conditions: • You stopped your analytical or preparative run, or your run was stopped due to an instrument error (such as solvent bottles running empty).
  • Page 41 Setting Up and Running a Purification Task Processing partial data and cloning tasks purification results and export fractions as described in “Reviewing Purification Results” on page 43. To process the missed samples of the purification run: 1 Create a clone of the partially processed task. 2 In the new cloned task, go to the Analytical Results tab and select the samples to be purified by marking the check boxes in the Purify? column.

  • Page 42: Reviewing Analytical Results

    Setting Up and Running a Purification Task Reviewing analytical results Reviewing analytical results 1 If you have selected to review the analytical results before starting the preparative run, the purification run stops after the evaluation of the analytical results. Select the Analytical Results tab to review the results. You can make any adjustments to the scale-up parameters and preparative run conditions before continuing with the purification step.

  • Page 43: Reviewing Purification Results

    The display of spectral data is an interactive process: clicking on a peak of the chromatogram displays the spectral data, or clicking on a collected fraction displays the spectral data and the chromatographic information. Select Agilent Technologies...
  • Page 44 Reviewing Purification Results Reviewing analytical results fractions to export by CTRL-clicking fractions in the graphics or the table. Selected fractions can be exported as a re-analysis sequence file or a liquid handler pooling file by clicking in the Purification Task toolbar. Purification Solution - Developer's Guide...

  • Page 45: Calibration Procedures

    (solvents, samples and others). The list is placed in the beginning of each document. The calibration procedures are a set of measurements and calculations that calibrate all required parameters for the Purification software. The work-flow is as follows: Agilent Technologies...
  • Page 46 Calibration Procedures Reviewing analytical results 1 Create default methods, see “Preparing Default Purification Methods” page 9 “Critical Method Parameters” on page 24 summarizes parameters of the purification LC system that are critical for correct function of the LC system and Purification software. Set up default methods for the purification instrument: •...
  • Page 47 Calibration Procedures Reviewing analytical results Do not forget to remove the column from the flow path. N O T E 3 Determine the column void volume, see “Characterizing the column volume” on page 63 Determination of the column Porosity parameter for Purification software. In the Purification software, the Porosity equals the column void volume divided by the column geometric volume.

  • Page 48: Characterizing The Delay Volumes: Mixing Point To Uv Detector And Column To Uv Detector

    Analytical Systems or Preparative Systems dialog box. Predefined systems are several default supported configurations defined in the M8368-90302 System User Guide for the 1260 Infinity Purification System, which have modules connected using the following capillary kits: • For the Combined UV/MSD and Preparative UV/MSD systems: 5067-6176 Capillary Kit for Mass-based Systems •...
  • Page 49 Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector For the combined preparative and analytical MSD default setup, the tubing volumes of the analytical flow path are dependent on pressure, solvent type and composition. This is due to the presence of the dampener in the quaternary pump G1311A/B/C used with this system.

  • Page 50: Calculation Of Delay Volume: Column To Uv Detector

    Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector “Calculation of delay volume: Mixing point to UV detector” on page 52. The capillaries are delivered in the respective capillary kit. If you have a custom tubing setup, use the tables as templates.
  • Page 51 Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector Table 5 UV/MSD-based combined analytical and preparative 1260 LC system Analytical Volume, mL Preparative with bypassed Volume, mL splitter Column to valve (0.17 × 400 0.009 Column to splitter (0.5 ×...

  • Page 52: Calculation Of Delay Volume: Mixing Point To Uv Detector

    Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector Calculation of delay volume: Mixing point to UV detector The tables provided in these sections describe the predefined capillaries, UV cells and sampler loop configurations, and can be used to calculate custom setups.
  • Page 53 Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector • If used, heat exchange capillary in the thermostat of the column compartment (TCC) • Column to detector • Inlet capillary of the UV detector cell •...
  • Page 54 Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector Autosampler Syringe: Volume, mL G7129A/B/C 40 µL: 0.04 + injected volume G7167A/B 100 µL: 0.062 + injected volume 900 µL: 0.177 + injected volume G4226A 20 µL: 0.08 + injected volume 40 µL: 0.118 + injected volume...
  • Page 55 Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector Part Description Volume, mL UV Cell Max-Light Cartridge, 10 mm cell 0.001 Total 0.258 + injected volume Preparative System The Mixing point to UV detector tubing volume of the preparative system or combined analytical and preparative 1260 system is calculated between the T-connection that connects the preparative pumps and the UV detector cell.
  • Page 56 Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector Table 9 Analytical flow path of combined instrument: Item Volume, mL Dual loop autosampler (G2258A), 50 µL loop 0.05 Autosampler to 2/10 valve (0.5 × 600 mm) 0.118 Valve to column (0.17 ×...

  • Page 57: Experimental Measurement Of Delay Volume: Mixing Point To Uv Detector

    Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector Because of piece-to-piece variations noted in preparative tubing, the calculated Mixing N O T E point to UV detector tubing volume can deviate from the actual value, typically by 5 – 10 % .
  • Page 58 Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector programmed profile of solvent B (is shown in red; the UV profile of the tracer is shown in blue. Instrument in analytical flow path of combined analytical and preparative system with 10 μL sample loop in flow path;...
  • Page 59 Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector • Save the Method as Prep_Mixing_Point_To_UV. • Set Stoptime to No Limit for all modules (infinite run time). • For the combined instrument, set the 2/10 port valve in the method to the preparative flow path.

  • Page 60: Determining The Elution Time Of Acetone

    Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector 9 Open the Sample Info dialog box, clear Vial/Location (blank run), enter a run name and click Run Method. 10 Stop the run 2 min after the acetone UV signal has reached maximum absorption.
  • Page 61 Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector • Zoom in to the signal after the elution of acetone and note the absorbance approximately 1 min after the signal rises as A The signal after the elution of acetone is typically not completely stable, so it is sufficient N O T E to have only an approximate reading.
  • Page 62 Calibration Procedures Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector Purification Solution - Developer's Guide...

  • Page 63: Characterizing The Column Volume

    Calibration Procedures Characterizing the column volume Characterizing the column volume Chemicals Both standard analytical system and preparative or combined system: • Solvent A: water (optionally with 0.1 % formic acid) • Solvent B: acetonitrile (optionally with 0.1 % formic acid) •...
  • Page 64 Calibration Procedures Characterizing the column volume • 3 mm flow cell: 0.5 mM thiourea or uracil in 75 % acetonitrile. • 10 mm flow cell: 0.3 mM thiourea or uracil in 75 % acetonitrile. • 60 mm flow cell: 0.04 mM thiourea or uracil in 75 % acetonitrile. 2 Recommended: Filter the sample before use with the regenerated cellulose syringe filter.
  • Page 65 Calibration Procedures Characterizing the column volume • Enter the Vial/Location of the sample vial • Enter a run name. • Run the method. 10 Stop the run after the delay marker peak has been detected. 11 Repeat twice. 12 Install the target column. 13 Equilibrate the column until the pressure and UV signal are stable.

  • Page 66: Combined Analytical And Preparative System

    Calibration Procedures Characterizing the column volume • Multiply it by the applied flow rate. • Subtract the elution volume without the column and one half of the injection volume. Combined Analytical and Preparative System Do not use the combined preparative system to determine the column volume of columns N O T E with ID smaller than 4.6 mm (column volume too low) and particle sizes below 3.5 µm (back pressure too high).
  • Page 67 Calibration Procedures Characterizing the column volume • Save the Method as Column_Volume. • Set the Stoptime to No Limit for all modules (infinite run time). • Pump: • Set Solvents: 75 % B. • Clear the TimeTable. • Injection volume 2 μL (important: increase the volume if the marker signal is too low).
  • Page 68 Calibration Procedures Characterizing the column volume • right-click on autosampler diagram • Start Purging 3 times 8 Set the flow to 1 mL/min. Use a restriction capillary of known volume to maintain the backpressure above 20 bar if N O T E necessary.
  • Page 69 Calibration Procedures Characterizing the column volume If the injection volume is close to or exceeds the volume of the sample loop, use Injection N O T E Mode Full loop with overfill factor and set the factor to 5. 16 Note the applied flow rate and injection volume. 17 Open the Sample Info window, update the run name and run the method.

  • Page 70: System Configuration And Delay Time Calibration

    Calibration Procedures System Configuration and Delay Time Calibration System Configuration and Delay Time Calibration Chemicals • Solvent A: water with 0.1 % formic acid • Solvent B: acetonitrile with 0.1 % formic acid • Make-up solvent: 80 % acetonitrile in water with 0.1 % formic acid •...

  • Page 71: Running Conditions For Calibration Procedures

    Calibration Procedures System Configuration and Delay Time Calibration If a flow splitter is used, then the delay calibration is valid only for the specific combination N O T E of preparative and make-up pump flows. If one of the flows is changed, or if tubing after the splitter is modified, then the delay calibration must be rerun.
  • Page 72 Calibration Procedures System Configuration and Delay Time Calibration The Purification software offers the function of a scale-up from the analytical to the preparative flow or back using parameters of analytical and preparative columns. Since the results of the FC delay calibration are required for the initial setup of the software, it is easier to do a manual flow scale-up using one of following equations: •...

  • Page 73: Configuring The Analytical And Preparative Systems

    Calibration Procedures System Configuration and Delay Time Calibration Example of F calculation for two columns of the same porosity: prep • Analytical column 4.6 x 50 mm (d = 4.6 mm) with d = 5 μm particle anal p,anal diameter •...

  • Page 74: Delay Time Determination For Analytical Systems (Msd And/Or Auxiliary Detectors)

    Calibration Procedures System Configuration and Delay Time Calibration • Initial Pressure: the 1260 quaternary analytical pump has a dwell volume dependent on the actual pressure. In order to minimize any possible discrepancy, enter the initial pressure on the pump at the gradient start conditions.

  • Page 75: Configuring The Preparative System Parameters

    Calibration Procedures System Configuration and Delay Time Calibration Configuring the Preparative System Parameters In the Purification menu of the Chemstation, select Systems to open the System Parameters dialog box. The preparative system parameters are on the right of the divided window. Click Add system to add a new system, and set the following: 1 List of systems: •...
  • Page 76 Calibration Procedures System Configuration and Delay Time Calibration • Select the instrument tubing setup from the drop-down list: • Selecting a predefined system fills tubing volumes and the position of the UV detector in relation to the make-up splitter if an MSD is used.

  • Page 77: Delay Time Calibration For Preparative Systems

    Calibration Procedures System Configuration and Delay Time Calibration Delay time calibration for Preparative Systems If a flow splitter is used, the calibrated values can be used only for runs with identical tubing and settings of preparative and make-up pump flow. In addition, a change of the data rate (that is, peakwidth for UV or cycle time for MSD), of the detector that is used for fraction collection, can cause previously measured delay times to be insufficient.
  • Page 78 Calibration Procedures System Configuration and Delay Time Calibration 5 For G1364B and G1364E: place an empty vial tray or well-plate tray in the fraction collector. FC Delay Calibration 1 From the Purification menu in the Chemstation, open the System Parameters dialog box.
  • Page 79 Calibration Procedures System Configuration and Delay Time Calibration • G7159B and G1364E with Agilent Delay and Checkout Calibrant (5190-8223): • Positive polarity: M+H (545.2), M+Na (567.2) and M+2Na (589.2). • Negative polarity: M-H (543.2). • If another ion mass is required for the evaluation under given conditions, select the relevant MSD signal in Selected signal and modify the Ionic mass value.

  • Page 80: Insufficient Uv-To-Fc Delay Time Or Msd-To-Fc Delay Time

    Calibration Procedures System Configuration and Delay Time Calibration after the preparative sequence when the delay value needs to be written to the collectors. Insufficient UV-to-FC delay time or MSD-to-FC delay time Insufficient UV-to-FC delay time The minimum required delay time for UV detector triggered collection is: 4/frequency + Response time.
  • Page 81 Calibration Procedures System Configuration and Delay Time Calibration • Decrease the volume of tubing between the splitter and the detector(s) in the make-up flow path. After one or more solutions have been applied, repeat the fraction collector delay calibration from the beginning. Insufficient MSD-to-FC delay time The minimum required delay time for MSD triggered collection either 5 sec or 6 ×...
  • Page 82 Calibration Procedures System Configuration and Delay Time Calibration also, a longer coil with the same inner diameter will increase back-pressure). • Decrease the preparative flow (influences the chromatographic resolution). • Increase the make-up flow (increases speed of the analytes when passing through detectors, which typically results in narrower and/or smaller peaks, so consider whether or not the detector data rate is sufficient for the signal response).

  • Page 83: Checkout Procedure

    3 mm UV cell (10 μL injection with dual-loop autosampler): Dilute 10× (1 mL = 100 μL sample + 900 μL solvent) • 10 mm UV cell (10 μL injection with dual-loop autosampler): Dilute 40× (1 mL = 25 μL sample + 975 μL solvent) Agilent Technologies...
  • Page 84 Checkout Procedure System Configuration and Delay Time Calibration • 10 mm UV cell (1 μL injection): Dilute 20× (1 mL = 50 μL sample + 950 μL solvent) • 60 mm UV cell (1 μL injection): Dilute 100× (1 mL = 10 μL sample + 990 μL solvent) •...

  • Page 85: Acquire An Analytical Run Using The Chemstation Sequence

    • Pump > Solvents > B: 2 % • Pump > Timetable: For an Agilent 1260 Infinity analytical run using a 4.6 × 50 mm, 5 μm column and a Flow of 1 mL/min: Time (min) A (%) B (%) 0.00...
  • Page 86 Checkout Procedure Acquire an analytical run using the Chemstation sequence Time (min) A (%) B (%) 0.00 0.17 1.83 2.20 2.30 Stoptime: 3.3 min Set the recommended Stoptime and Flow as given above. c Set the injection volume: • Stand-alone analytical system: 1 μL injection (increase if necessary). •...
  • Page 87 Checkout Procedure Acquire an analytical run using the Chemstation sequence • 60 mm UV cell: dilute 100× (1 mL = 10 μL sample + 990 μL solvent). 3 Place 1 mL filtered sample in the autosampler. 4 Create a new sequence: Sequence > New Sequence Template 5 Set Sequence >...
  • Page 88 Checkout Procedure Acquire an analytical run using the Chemstation sequence is too low (below 100 mAU) or too high (above 1500 mAU), adjust the injection volume or sample concentration and measure the sample again. 15 When the sequence is finished, turn off the pump(s). Purification Solution - Developer's Guide...

  • Page 89: Uv-Only Workflow: Focused Gradient In The Purification Software

    Checkout Procedure UV-only Workflow: Focused gradient in the Purification software UV-only Workflow: Focused gradient in the Purification software This procedure is the analytical-to-preparative workflow for the standalone UV-only preparative instrument. 1 Place at least 1.5 mL of filtered undiluted Prep LC Standards #1 sample in the autosampler.
  • Page 90 Checkout Procedure UV-only Workflow: Focused gradient in the Purification software • Review the analytical and preparative flows in the Pumps and Detectors tab if the target flows are set. • Analytical Run • Click Browse and select the acquired analytical sequence (Checkout_Sample) •...
  • Page 91 Checkout Procedure UV-only Workflow: Focused gradient in the Purification software 14 Click Run. 15 When the preparative task is completed, check in Preparative Results that the correct peak was focused and that it is among collected fractions. Purification Solution - Developer's Guide...
  • Page 92 Checkout Procedure UV/MS Workflow: Focused gradient in Purification software UV/MS Workflow: Focused gradient in Purification software This procedure is the analytical-to-preparative workflow for the combined analytical and preparative UV/MSD instrument and for standalone preparative UV/MSD instrument. 1 Place at least 1.5 mL of filtered undiluted Prep LC Standards #1 sample in the autosampler.
  • Page 93 Checkout Procedure UV/MS Workflow: Focused gradient in Purification software • Select the analytical and preparative systems from “Configuring the Analytical and Preparative Systems” on page 73. • Review the analytical and preparative flows in the Pumps and Detectors tab if the target flows are set. •...
  • Page 94 Checkout Procedure UV/MS Workflow: Focused gradient in Purification software • MS-based target identification: • Check in the chromatogram that the correct peak was selected: Ethyl 4-hydroxybenzoate (166.1) is the third major peak of the mixture to elute. • Also check that the ethyl 4-hydroxybenzoate peak was identified in both the UV detector and the MSD target ion species.
  • Page 95 Checkout Procedure UV/MS Workflow: Focused gradient in Purification software Minimal target candidate peak height in EIC default value was increased. Change the value if necessary. • Also check that the caffeine peak was identified in both the UV detector and the MSD target ion species. If the UV and MSD are not well aligned, then the problem can be in UV/MSD delay value or integration parameters.
  • Page 96 Glossary Glossary Administrator User who sets up the users of the purification software as Operators or Method Developers in the Agilent OpenLAB Control Panel. Analytical run The analytical (scouting) run scans your samples to find out if your expected target compound is present, and if it exists in a quantity sufficient to proceed with a purification run.
  • Page 97 Glossary Regular purification tasks can be submitted to run only if the last system suitability test was passed or accepted by a method developer. Task template A purification task template that is used to create other tasks for a certain combination of systems. Tasks templates are created in a special folder by the Method Developer.
  • Page 98 • how to set up the analytical and preparative systems • how to set up a Purification task and review the results • how to calibrate the system and run a checkout sample © Agilent Technologies 2017 Printed in Germany 11/2017 *M8368-90011* *M8368-90011* M8368-90011 Rev. D...

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