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CEM Liberty Blue Manual

Automated microwave peptide synthesizer
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Liberty Blue
TM
Automated Microwave Peptide Synthesizer
Manual

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Summary of Contents for CEM Liberty Blue

  • Page 1 Liberty Blue Automated Microwave Peptide Synthesizer Manual...

  • Page 3: Table Of Contents

    Loading a Method Applications Support Standard Reagents and Concentrations Technical Support Recommended Reagents and Strategies for Microwave Requested Information SPPS on the Liberty Blue CEM Corporation Headquarters Preparing Reagents CEM International Subsidiaries Setting up the Liberty Blue Running the Liberty Blue...

  • Page 5: Introduction

    CEM’s website at http://cem.com/peptide- synthesis. A registered account is required for certain downloads. This manual refers to Liberty Blue Software version 2.2 for all software information, including screen shots and technical information.

  • Page 6: How To Obtain Support

    Firmware versions (both Liberty Blue and Discover) and Liberty Blue software version can be found within Liberty Blue Software from the About Liberty Blue menu (Help Tab). Serial numbers can be found on the silver tags on the rear of the Liberty Blue and on the side of the Discover.

  • Page 7: Cem International Subsidiaries

    Matthews, NC 28106-0200 Physical Address: 3100 Smith Farm Rd Matthews, NC 28104 Email (Applications Support): peptide.support@cem.com Email (Technical Support): service@cem.com CEM International Subsidiaries France: CEM mWaves S.A.S. Phone: (33-1) 69 35 57 80 Fax: (33-1) 60 19 64 91 Address: Immeuble Ariane Domaine Technologique de Saclay 4, rue René...
  • Page 8 CEM Distributors For a complete list of distributors of CEM products, including contact information, go to the CEM website (http:// www.cem.com), select Contact CEM, and then select your region to see a list of distributors by country. How To Obtain Support...

  • Page 9: Safety

    Personal Laboratory Safety Solvents and chemical reagents used on the Liberty Blue can be hazardous and should only be handled under a fume hood. Personal protective gear, including but not limited to safety glasses, chemical resistant gloves, and a laboratory coat are highly recommended when operating the Liberty Blue.

  • Page 10: Operating Precautions

    Operating Precautions The Liberty Blue must be grounded. In the event of an electrical short circuit, grounding reduces the risk of electric shock by providing an escape wire for electric current. This instrument is equipped with a cord having a grounding wire with a grounding plug.

  • Page 11: System Requirements

    Electrical Requirements The Liberty Blue requires electrical power of 120 VAC (60 Hz, 1.7 A) (or 240 VAC [50 Hz, 1.7 A] where applicable). Specific power requirements (120 VAC vs. 240 VAC) can be found on the nameplate affixed to the rear of the Liberty module and on the side of the Discover module.

  • Page 12: Introduction To The Liberty Blue

    Discover Module Components and Parts Liberty Blue Module: The Liberty Blue handles all of the fluid transfer into and out of the reaction vessel. In addition, the Liberty Blue manages communications with the computer controller and the Discover during peptide synthesis.
  • Page 13 Liberty Blue module. Optimal large capacity pressure rated bottles are available (PN 551240, 551245, 551250, 551255). Amino Acid Manifolds: There are 27 positions on the manifold for amino acids, 20 corresponding to a specific amino acid, and 7 extra positions.

  • Page 14: Quick Setup Guide

    Quick Setup Guide Create and Load the Liberty Method NOTE To load an existing method, skip to step 9. Create the Method 1. Open the appropriate folder or sub folder in the Methods box for the new method. 2. Click the New Method button.

  • Page 15: Prepare And Load Reagents

    8. When the method is complete, click Save to save the method, then close the Liberty Method Editor. Load the Method 1. From the Liberty Blue application software main screen, open the appropriate folder in the Methods tab. 2. Click on the desired method, and drag the method to the resin position.

  • Page 16: Run The Method

    2. Verify that the fiber optic probe is fully inserted into the thermowell. WARNING If the probe is not inserted all the way to the bottom of the vessel the Liberty Blue will not accurately measure the temperature, and significant overheating of the vessel will occur. This will result in poor synthesis quality and/or serious damage to the vessel.
  • Page 17 7.5. Connect the vessel body to the attenuator, then connect the drain line to the vessel body using the beige union connector. 7.6. Place the vessel back into the microwave cavity, securing the attenuator. 8. The peptide is now ready to be cleaved and analyzed, and the Liberty Blue is ready for its next run. Quick Setup Guide...

  • Page 18: Operation Of The Liberty Blue

    (individual coupling cycles for each residue, C-terminus type, final deprotection, etc.) are selected. Liberty Methods can be created or modified using the Liberty Method Editor. Run: A specific instance of a method being loaded and started in Liberty Blue Software. A Run History file is recorded for each run.
  • Page 19 Integrity command (which will verify there are no problems with the Liberty Blue Software database). Help: This menu contains the About Liberty Blue screen (which displays the software and firmware version numbers), and allows you to create a troubleshooting bundle.

  • Page 20: Microwave Editor

    Microwave Editor allows the user to select the microwave time, microwave power, maximum temperature, and sample agitation. To create a new Microwave Method: 1. Click on the filter buttons at the top of the Microwave Editor to open the filter list. Operation of the Liberty Blue...
  • Page 21 Duplicate button. 4. The new Microwave Method will appear named “Copy of (method name)”. Type a new name and press Enter. 5. Make any changes to the method, then click Save to save the modified method. Operation of the Liberty Blue...

  • Page 22: Cycle Editor

    Calibrating Rapid Liberty Blue Microwave Methods The rapid microwave methods used by the Liberty Blue employ rapid heating rates. For both the deprotection and coupling methods the set temperature of 90 °C should be reached within 20-30 seconds and maintained for at least 45 seconds and 100 seconds respectively.
  • Page 23 Creating a Cycle Cycles allow for control of each action taken in a given step of a run. The Liberty Blue application software comes with default cycles for each synthetic scale; however, cycles can be customized to accomplish a number of...
  • Page 24 5.1.1.2 To change the parameters, double-click on the parameter value in the Parameter Values table. A pop-up will appear to configure the parameter. Make any changes, then click OK to close the pop-up. Operation of the Liberty Blue...
  • Page 25 5.1.1.3 To add a pause after the operation is carried out, check the Pause box. NOTE The method will not resume until the user presses the Start button on the main screen of Liberty Blue Software. 5.2. To add a new step: 5.2.1 Click the...

  • Page 26: Liberty Method Editor

    Liberty Method Editor is used to program the specific conditions to be used for a given synthesis. Once the sequence is entered, several options for the run can be configured that will determine the specific steps to be used in the synthesis. The Liberty Method Editor can be accessed from the Edit menu. Operation of the Liberty Blue...
  • Page 27 5. The remaining options can now be selected. 5.1. C-Terminus: The C-terminus (acid or amide) is only used to calculate the molecular weight, and does Operation of the Liberty Blue...
  • Page 28 When multiple rows are marked (indicated by a blue highlight), any change made to one row will be made to all selected rows. When all changes are made, click the Clear button to deselect all rows. Operation of the Liberty Blue...
  • Page 29 5. If the sequence appears correct, click Import Sequence. The sequence will be loaded into the method. Importing a Batch of Sequences to the Method Editor 1. From the Liberty Blue Software, save the Batch Sequence Import Template (File -> Import -> Save Batch Sequence Import Template).
  • Page 30 To load the Liberty Methods into the HT Grid immediately upon import, input the desired HT position for each Liberty Method. 4. Import the Batch Sequence Import Template file into the Liberty Blue Software (File -> Import -> Sequences). The Liberty Method Editor window will open, revealing the recently imported sequences.
  • Page 31 Exporting Microwave Methods and Operations The Liberty Blue application software allows operations, microwave methods, and associated microwave cycles to be transferred from one Liberty Blue Software database to another. To export existing operations: 1.

  • Page 32: Calculators

    Importing Microwave Methods, Operations, and Settings The Liberty Blue application software allows settings, operations, Liberty methods, cycles, and associated microwave methods to be transferred from one Liberty Blue Software database to another. To import existing operations, settings, or Liberty methods: 1.
  • Page 33 Usage Calculator Usage Calculator can be used to determine the amount of each reagent needed for a given method. Multiple methods can be loaded into the calculator, so that sufficient reagents can be loaded onto the Liberty Blue for multiple peptides.

  • Page 34: Edit Users And User Group

    4. To create a new user, select the “+” icon. 5. Create user name, group type, and change password. 6. Follow the prompts. Password must be at least 8 character(s) and cannot contain any part of user name. 7. Select the green Save icon. Operation of the Liberty Blue...
  • Page 35 Import/Export methods Permitted Permitted Permitted Denied Results View results (all) Permitted Permitted Permitted Permitted View results (group) Permitted Permitted Permitted Permitted View results (lesser groups) Permitted Permitted Permitted Permitted Miscellaneous View camera Permitted Permitted Permitted Permitted Operation of the Liberty Blue...
  • Page 36 Data Integrity The Liberty Blue provides a software solution for automated microwave peptide synthesis users to aid in achieving compliance with the FDA’s guidance on electronic records and electronic signatures: 21 CFR Part 11. 1. Software allows viewing of records for validity. Records include a date of last modification and a revision number which is incremented upon modification.

  • Page 37: Setting Up A Synthesis

    3. The method name will appear in the Loaded Method indicator. The indicator will turn blue, meaning the method is loaded and ready to run. Standard Reagents and Concentrations The Liberty Blue uses stock solutions of all reagents. The table below details the concentrations used on the Liberty Blue and/or CEM’s preferred reagents. Reagents 0.005 mmol...
  • Page 38 Fmoc Rink Amide ProTide (LL) Mesh Size Only 100-200 mesh (or lower) resin (>75 micron) should be used with the Liberty Blue. The use of higher mesh resins will result in clogging of the reaction vessel frit and damage to the system.
  • Page 39 These conditions should be used for all onium activators (HBTU, 6-Cl-HBTU, HATU) and for the phosphonium activators (PyBOP, PyAOP). DIEA should be prepared in NMP. NOTE For assistance with cycle and microwave method programming, please contact CEM Applications Support at peptide.support@cem.com. Setting up a Synthesis...

  • Page 40: Preparing Reagents

    DIEA should be prepared in NMP. NOTE The rapid microwave methods used by the Liberty Blue employ rapid heating rates. For both the deprotection and coupling methods the set temperature of 90 °C should be reached within 20-30 seconds, and maintained for at least 45 seconds and 100 seconds respectively. The starting temperature for each reaction will be significantly warmer than room temperature as residual heat will remain from the previous reaction.

  • Page 41: Setting Up The Liberty Blue

    Piperazine may take up to an hour to go into solution. It is helpful to add a magnetic stir bar and stir the solution to speed dissolution. Setting up the Liberty Blue Loading Reagents onto the Liberty Blue 1. Connect amino acid centrifuge tubes to the appropriate positions on the system. Connect empty centrifuge tubes to all unused positions.
  • Page 42 3.4. Add the required volume of deprotection solution to the bottle. Ensure the reagent line has a main solvent filter in place (PN 551000), then connect the bottle to the cap. 3.5. Click OK to continue with the Change Bottle procedure. The Liberty Blue will automatically pressurize the bottle and click OK to prime the lines.

  • Page 43: Running The Liberty Blue

    3. Verify that the fiber optic probe is fully inserted into the thermowell. WARNING If the probe is not inserted all the way to the bottom of the vessel the Liberty Blue will not accurately measure the temperature, and overheating of the vessel can occur. This will result in poor synthesis quality and/or serious damage to the vessel.

  • Page 44: Generating A Run History Report

    2. The Loaded Method indicator will turn yellow to indicate the method is running. 3. The Liberty Blue will illuminate all required amino acids positions for the method. Ensure that all reagents are connected and have sufficient volume, empty centrifuge tubes are connected to any unused reagent positions, and that there is sufficient solvent and deprotection solution, then click OK to continue.

  • Page 45: Cleavage

    (aids in removal of Pbf protecting groups from Arg(Pbf), suppresses oxidation of Cys/ Met side chains). Cleavage can be performed heated, or at room temperature. The table below lists CEM’s recommended parameters for both heated and room temperature cleavages.

  • Page 46: Maintenance Of The Liberty Blue

    • Perform a timed addition calibration for all timed additions (see Timed Addition Calibration). • Verify the performance of the filtered drain (see Reaction Vessel Drain Test). • Review recent run history reports to ensure that the Liberty Blue is heating properly. Semiannual Maintenance •...

  • Page 47: Maintenance Procedures

    Click OK to continue. 7. A progress window will pop up showing the progress of the backflush procedure for all amino acids. Maintenance of the Liberty Blue...
  • Page 48 Timed Addition Calibration There are four timed additions that must be calibrated for optimal functionality of the Liberty Blue: Wash, Wash Thru Manifold, Deprotection, and Wash Thru RV Bottom. To calibrate a timed addition: 1. Place the reaction vessel into the volume calibration stand.
  • Page 49 Verification of Waste Sensor Operation The Waste Sensor is a float sensor located on the top of the waste bottle. It is connected to the Liberty Blue through a 9-pin connector cable plugged into the left side of the Liberty Blue module. Periodically, operation of the Waste Sensor should be verified.
  • Page 50 6. Release the float sensor, connect the cap to the waste container, then click OK to clear the Waste Full error. Standby Procedure If the Liberty Blue will be idle for a period of two weeks or more: 1. Remove all reagents (amino acids, activator, activator base) from the system.

  • Page 51: Maintenance Screen

    5. Click OK to continue. The Liberty Blue will pressurize the bottle and prompt to begin priming the lines. Click OK to begin the priming procedure, or click Cancel to skip priming the lines.

  • Page 52: Calibration Screen

    6. The Liberty Blue will prime the line to the reagent manifold (through valve MS1), purge the liquid to the reaction vessel, then drain the vessel. Next, the Liberty Blue will prime the direct wash line to the reaction vessel (through valve MS2), then drain the vessel.
  • Page 53 (Deprotection/Drain sensor), LS3 (Reaction Vessel Overflow sensor), LS4, and LS7 (Spill sensor). WARNING Liquid sensor calibration of LS3, LS4, and LS7 should only be done on installation of the Liberty Blue or as part of a Preventative Maintenance visit performed by a CEM-trained technician.

  • Page 54: Advanced Features

    Cycle Rules Default cycles for resin swelling, amino acids, and final deprotection are located in Cycle Rules. Please contact CEM Applications Support at peptide.support@cem.com for assistance assigning/changing default cycles. Reagent Library Reagent Library is a list of common reagents for peptide synthesis, including solvents, deprotection reagents, amino acids, activators, and activator bases.
  • Page 55 To create a new specific derivative: 1. Open the Amino Acid folder. 1.1. If the new derivative is a derivative of an existing amino acid, open the folder for the parent amino acid, then proceed to step 2. 1.2. If the new derivative is not a derivative of an existing amino acid, select one of the External (corresponding to external amino acid positions on the instrument) or Unusual - XXX folders in which to add your new derivative.

  • Page 56: Bottle Setup

    Bottle Setup Bottle Setup screen allows the user to assign any reagent to any position on the Liberty Blue from within the Liberty Blue software. Individual instrument setups can be saved to allow for quick and easy configuration of the instrument for a variety of syntheses. The Bottle Setup screen can be accessed from the Edit menu.

  • Page 57: Refill Reagents Button

    Diagnostics Screen The Diagnostics Screen allows a number of manual controls and operations that are not intended for normal use. The Diagnostics screen should only be accessed under the guidance of a CEM representative. Settings Screen The Settings Screen can be accessed from the Options menu. The Settings screen allows configuration of a number of important system parameters.

  • Page 58: Reaction Vessel Camera Option

    Camera Retrofit Kit (PN 730125). Camera Software The Liberty Blue with camera option includes a USB drive with the necessary software driver for operating the camera option. To download this driver, connect the USB drive to a computer and open the Dell Webcam folder.

  • Page 59: Warranty

    Limited Warranty Information What Is Covered: CEM Corporation warrants that the instrument will be free of any defect in parts or workmanship and will, at its option, replace or repair any defective part (excluding consumables) or instrument. For How Long: This warranty remains in effect for 365 days from date of delivery to the original purchaser.
  • Page 60 CEM Corporation hereby excludes and disclaims any warranty of merchantability or fitness for any particular purpose. No warranty, express or implied, extends beyond the face hereof. CEM Corporation shall not be liable for loss of use of instrument or other incidental or consequential costs, expenses or damages incurred by the purchaser or any other user.
  • Page 61 Warranty...
  • Page 63 Copyright 2018 by CEM Corporation CEM® is a registered trademark of CEM Part Number 600291 All Rights Reserved. This guide contains Corporation. Liberty Blue , CarboMAX November 12, 2018 11:53 AM proprietary information which shall not Razor™, and ProTide are a trademark of Rev.

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