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1. WINDOW TITLE BAR 4 WINDOWS CONTROLS 4 DROP DOWN 4 BioLogic Duo Flow user name project name method name run name MENUS File Edit View Utilities Options Window Help gt MAIN New Edt New w RA KA io lethod Method Run Browser Report 2 Setup 55 Run Notes PostRun Eeg Settings ey Web MENUS TOOLBAR Maximizer Gradient Pump F 10 Fraction Collector BioFrac QuadTec Detector Econo Gradient Pump 1 D Mode System Local Mode System Local Mode System Local System Local Flowrate 1 00 HH Rack F1 12 13 mm tubes v xk Zero Baseline Flowrate 1 00 ml min Tris 20 mM Rack Tube Grid ON OFF n A 1 2 Deuterium EGP 0 CONTROL PH 205 m MINI oo InetB o25 HH End B Range 190 370 Split PANELS FOR Wavelength Selection raction size 1 INSTRUMENTS High limit 1000 psi ub X 280 nm Fw OG OO CONNECTED TO
2. Ay BIOLOGIC DUOFLow CHROMATOGRAPHY SYSTEM INSTRUCTION MANUAL BioLogic DuoFlow Software Version 5 0 Copyright 2003 Bio Rad Laboratories Inc All rights reserved TABLE OF CONTENTS TABLE OF CONTENTS SAFETY SECTION 1 Chapter 1 0 1 1 1 2 1 3 1 4 1 5 Chapter 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 SYSTEM OVERVIEW Introduction einn ee eee trece e ooo E e de Ee e Ed ee dac rae 1 1 i i rec LAE 1 1 E 1 2 Unpack ma MEET 1 3 System Configurations aron iiini ernir a dian ANEN EAN AREE EAE NA 1 4 Quick Start Proced rTe srini aaa a ae ea aa aaa eiiie aan aada 1 5 Description of System Components eee 2 1 Controller and USB Bitbus Communicator 2 2 24 1 y Controller neo on e bodie ton eios 2 2 2 1 2 USB Bitbus Communicator sssssssssssssssessseeeneeem 2 5 2 6 BioLogic Maximizer Valve System 2 11 bim PEE MP 2 18 PA SEM PX C eR eee ei eet ence
3. Event mark UV Detector ayn Zero Baseline 0 00 i 0 00 y AU Milliliters QuadTec indios m m Zero Baseline Scout Method 7 Scout Run S Scout Buffer A A1 1 of 1 S Scout Buffer A A1 Protocol gt 1 0 00 0 Collection Fractions of size 2 00 ml during entire run Run Time Run Volume Step Time Left Fraction Vol Left WL1 280nm 0 00 0 0 0 ml 0 00 0 0 0m Port 2 User Valve Position 1 WL1 280nm WL2 260nm WL3 214nm WL4 405nm Econo Gradient Flow Rate EGP B Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 B 0 Maximizer Gradient Pump F10 UV Conductivity SIM1 SIG _SIM1 pH 1 00ml min 0 B2 438 psi 1 003 AU 1 23 mS cm 0 548 Volt 7 00 pH QuadTec Figure 7 5a Run Screen showing a Run in Progress with a Volume based Chromatogram and Rack and Tube fraction numbering 7 30 SYSTEM OPERATION MODES OF OPERATION ACTIVE PULL DOWN MENU ACTIVE TO SELECT LEFT TRACE PULL DOWN MENU TO SELECT RIGHT TRACE
4. MODEL 2110 FRACTION COLLECTOR Lm mm Figure 2 20 Model 2110 Fraction Collector with Optional Dust Cover The Model 2110 is connected to the Maximizer or Workstation with system cable 5 bare wires to DB 9 via the AUX connector black wire to pin 5 and white wire to pin 9 To prevent shorting cut or tape wires not in use If a Maximizer is being used the Model 2110 should be connected to it rather than to the Workstation The Model 2110 is plumbed to the DuoFlow system using 1 16 1 6 mm OD inlet tubing connecting directly to the fraction collector s drop former without the need for additional fittings The SVT3 2 diverter valve is required by the Model 2110 for collection schemes other than Collect All When this valve is configured as the fraction collector diverter valve in the Setup screen additional collection parameters Collect All Threshold Collection Windows and Threshold Collection Windows become available in the Protocol screen When controlled by the DuoFlow system fraction collection is specified by volume The fraction collection scheme is programmed in the Protocol screen of the software where a Delay Volume feature is standard see Chapter 7 for discussion of Delay Volume Note Collection by drop count is not available with the DuoFlow system This instrument is described in detail in its separate documentation 2 37 DESCRI
5. m SORT r SORT CHRONOLOGICALLY ALPHABETICALLY EARLIEST TO LATEST A Z Z A LATEST TO EARLIEST USER ICON BioLogic Duo Flow user rjame project name method name run name gt BROWSER ORE iew Utilities Options Window Help New Ed New Ea LE TOOLBAR Method Method Run Browser Report Setup Run nore 2 Segings E Name Date e New m n 11 55 42AM 1 SB Open 11 55 42AM 8 SCOUT METHOD 2 12 2002 10 04 05AM 1 ICON Scout Run 1 12 12 2002 10 04 09AM 1 e Edit i i WISI Scout Buffer A Buffer A1 Running gt 12 12 2002 10 04 23AM METHOD Method 6 08 04 2001 12 17 42AM 0 ICON T Delete 08 04 2001 12 29 14 0 RUN BROWSER SUMMARY ICON Pint T 08 04 2001 11 55 42AM 1 DATABASE Demo Chromatography 08 12 2002 01 04 22PM 1 TREE PROJECT ICON Project for Demo Chromatography 07 03 2002 03 32 09 1 WINDOW WE Copyou i Compare2 10 12 09AM 0 itandard UV detector 09 01 16AM 2 COMPARE ICON zn e Wy Run 1 Not Run 11 17 10 1 if Comparet 12 23 21 4 QUEUE ICON Eel Demonstration Queue 08 22 2002 11 20 31AM 1 1 00 Demonstration Queue 1 08 22 2002 01 52 22PM 4 Cu e ESC H B RUN ICON Pun Q Queue Method 4 08 22 2002 01 55 12PM Queue l
6. Volume ml Pass 1 0 Sample 1 Sample 2 Sample 3 Fow rli Sample 4 Y This operation will occur BEFORE injecting sample on column Step 4 Volume 1 00 ml OK Cancel The Fill Before Inject and Rinse After Inject tabs are used to automatically fill a sample loop before an injection step or to rinse the loop after an injection has occurred The Fill Before Inject tab is active for both static loop and dynamic loop injections if an auxiliary pump is defined in the device setup The Rinse After Inject tab is active only for static loop injections if an auxiliary pump and auxiliary pump inlet valve are defined in the device setup e Enable Fill Before Activates the Fill Before Inject controls e Enable Rinse After Activates the Rinse After Inject controls e Flow Direction Sets the flow direction for an Econo Gradient Pump For an EP 1 pump or non Bio Rad pump the flow direction is set at the pump e Sample Used to select a sample or wash solution for use in the Load Inject step Active only if an Aux Pump Inlet valve has been defined for the method e Flow Used to enter the auxiliary pump flow rate Note that for an EP 1 pump or non Bio Rad pump the flow rate must be set at the pump itself Volume Used to set the amount of solution loaded into the loop from the auxiliary pump 7 14 SYSTEM OPERATION MODES OF OPERATION
7. 2 56 IN 1 INDEX Controller cable connections 3 2 CD ROM 2 3 description ao im Ter 2 2 floppy disk drive 2 2 function KEYS 2 3 keyboard c an dari 2 3 keyboard 2 4 MOUSE ei 2 3 mouse 2 4 parallel 2 4 power 2 4 power switch eessssssseses 2 2 USB 2 3 USB 2 4 video 2 4 D Detectors See UV detector and QuadTec UV Vis detector Genel C E E 2 25 Drop down menus s e 5 5 Edit menu Post Run Screen Copy zoom chromatogram to clipboard 5 8 Delete all tags 5 8 Delete selected tag 5 8 Edit Activity Trace 5 8 Full View in zoom chromatogram 5 8 Print zoom chromatogram 5 8 Tag and Trace Options 5 8 Trace
8. FR PR 5wm AE m C o T E us m DELL CONTROLLER MAXIMIZER BIOFRAC KEYBOARD Figure 1 1 BioLogic DuoFlow System 1 1 INTRODUCTION SYSTEM OVERVIEW 1 2 FEATURES BioLogic DuoFlow systems provide the following features 1 2 Setup Flexibility A space saving modular design that is stackable and easily configured to meet your exact needs and fit into your desired bench space Trays and vertical bars are moveable and removable Horizontal bars can be placed in the optimal position for valves columns detectors etc The system fits easily into a cold box Modular components provide an easy upgrade path to fit all applications and financial requirements For example the BioLogic DuoFlow Basic system may be purchased and upgraded to a BioLogic DuoFlow QuadTec BioLogic DuoFlow Maximizer or BioLogic DuoFlow Pathfinder System as needed See Section 1 4 for a description of the DuoFlow systems and upgrades Intuitive user friendly software programming Users become an expert in only a few runs e Four easy steps to set up new devices instruments create a new method and start to run samples Step 1 Browser to enter new user name project name and method name Step 2 Setup to specify devices required for the method Step 3 Protocol to enter seq
9. valve connections B Backpressure BioFrac fraction collector connecting to system collection description racks available software BioLogic configuration utility software BioLogic DuoFlow system See DuoFlow system Browser Name and Date bars Browser screen bolded text nes creating and running a queue creating and viewing a compare icon colors ir d doen OVerVIeW DEED procedure for creating run tree hierarchy buttons Browser Tabs and Browser Tab window Compare tab COPYIN List Error List tab Information tab MOVE List Queue tab Browser toolbar Buffer Blender See Maximizer INDEX Buffer blending sianie eee 10 1 B ffer editor iie eese o ete tenus 7 9 10 2 C Chart recorder See Model 1327 chart recorder Chromatogram exporting data
10. L SYSTEM CABLE 4 OS nun WORKSTATION SYSTEM CABLE 17 18 19 21 OR 30 Lr BIOFRAC UV DETECTOR Figure 3 5 System Cable Connections with Maximizer SYSTEM SETUP SYSTEM INSTALLATION AND SETUP 3 4 SYSTEM RACK SETUP Assemble the system rack before placing it on the Workstation Detailed discussion of system rack assembly is provided in Section 2 9 1 Keep in mind that the illustration is only an example Alternative rack arrangements include setting up the rack to use only one or two trays To mount the system rack on the Workstation remove the four green caps covering the holes at the four corners on top of the Workstation Place the rack into the four corner holes COLUMN CLAMPING ARRANGEMENT 0 1 TRAY TAPERED COLLAR Figure 3 6 Rack Assembly 3 6 SYSTEM INSTALLATION AND SETUP SYSTEM SETUP 3 5 MIXERS Two mixers have been designed for use with the DuoFlow system the MX 1 mixer and the higher capacity Maximizer mixer The mixers are shipped pre assembled with their mixer barrel The mixer barrel may be removed or replaced increasing or decreasing the internal volume to provide the mixer capacity appropriate to the flow rate Refer to table
11. 5 3 buffer 7 5 Setup manual screen 5 4 conductivity monitor 7 5 Troubleshooting QE p Buffer Blending s 12 11 fraction 7 5 Conductivity flow cell and trace 12 9 gradient pump eee 7 6 Controller and software 12 1 7 5 UV detector and UV 12 7 f ngtioning And Fins 7 6 Workstation pump 12 3 setup information 7 7 U Signal Import Module SIM Uninterruptible Power Supply UPS 2 53 cable connections 2 50 3 description 2 50 USB Bitbus Communicator software setup 7 5 bus power LED sese 2 5 cable connections 3 3 Special function keys description 2 5 Abs oie teet eter 5 2 instrument bus connector 2 5 ESCh en REINES 5 2 ONILEDdS eS nemen heres mir ee 2 5 Help EE E 5 2 power 2 5 Hold until 5 2 power select switch 2 5 Starter Kit tdeo 2 47 setup with SIM seeen 2 5 Status coe att so tes 5 2 setup without SI
12. 2 50 2 30 Cable Connections to the Signal Import Module seee 2 50 2 31 Model 1327 Chart Recorder eene nennen nnne ener nen 2 52 3 1 Example of a DuoFlow Pathfinder System 3 1 3 2 DuoFlow Pathfinder Setup in Non Condensing 3 2 3 3 USB Bitbus Communicator Cabling seriene Raa A E A nnn 3 3 3 4 System Cable Connections without Maximizer sseeen nn 3 4 3 5 System Cable Connections with Maximizer 3 5 3 6 Rack Assembly eterni ree eee etu eve dune Pa a a uec Pe e Re ecd 3 6 3 7 3 8 UV Detector Conductivity Monitor esessssseeeeneenen mene 3 9 3 9 QuadTec Detector oh i eme io EO diate atin nice Ede EO 3 10 3 10 Instrument Control Module ICM ssssseeeeeeeennnm nennen nennen 3 10 1 ERUNT 3 12 3 12 51 2 2 ere oer ee du oa unn DV ego n mde e edet 3 12 3 13 DUOFlOW rn t eet N ete a UN etes 3 14 3 14 Connecting an EP 1 Econo Pump to the BioLogic DuoFlow Workstation 3 16 3 15 Example of Direct Inject Sample Loading using
13. AVR7 3 SAMPLE QUADTEC DETECTOR INJECT VALVE MAXIMIZER MIXER H PEFBAOIIIORS USB BITBUS COMMUNICATOR CONDUCTIVITY MONITOR E WORKSTATION a 0 i ooo 0 e B e o o an po RN 00 A FE GS 2 m WD Ww s DELL CONTROLLER MAXIMIZER BIOFRAC MOUSE KEYBOARD Figure 2 1 BioLogic DuoFlow Pathfinder System Components DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW 2 14 CONTROLLER AND USB BITBUS COMMUNICATOR The DuoFlow system is controlled by a PC computer referred to throughout this document as the Controller The Dell Controller available from Bio Rad includes a color display monitor a keyboard a mouse device a CD ROM drive and a floppy disk drive The Controller communicates with the workstation and other external devices through its USB connector The USB Bitbus Communicator serves as the link between the Controller s USB port and the DuoFlow s instrument bus This link allows the Controller to control the Maximizer and Workstation as well as any devices connected to the Maximizer or Workstation such as automatic valves UV detector and conductivity monitor and peripheral instruments suc
14. Integ L AAN Settings Set p Opens the Protocol screen for creating and editing a method Opens the Run screen from which you can start a run of the currently open method You will be prompted for a new run name prior to launching the run Opens the Notes screen that allows you to enter additional information about your method and or run Notes are always editable even after a run is executed Opens the PostRun screen that allows you to view and customize your chromatogram prior to printing Opens the run log Read Only for the current run and displays the events that occurred for the run The button for the EZLogic integration program replaces this button when it is installed EZLogic Integration button appears when the EZLogic integration software is installed this button replaces the Log button Enables the selection of up to 8 instrument traces to display on the screen and on the printed report The trace options are UV detector Conductivity monitor pH system backpressure theoretical B concentration four QuadTec wavelengths and detector traces acquired via the System Interface Module SIM Available only in the Manual screen Displays a screen that enables the following inputs Econo Gradient Pump EGP split time period when the EGP is used QuadTec UV VIS detector time constant when the QuadTec detector is used Pump purge rate
15. 11 7 TS DCUM 11 8 11 6 Valve IE 11 10 11 6 1 SVT3 2 Diverter Valve k Aetnae eap anae e aaaea e aeaa ha an na iais 11 10 11 6 2 AVR7 3 and AVR9 8 11 11 11 7 Maximizer Valves etr t Mr ene e rec Ret a xe eaten eher end 11 13 Chapter 12 0 Troubleshooting DuoFlow 12 1 12 1 Troubleshooting the DuoFlow Controller and Software 12 1 12 2 Troubleshooting the DuoFlow Workstation 12 3 12 3 Troubleshooting the UV Detector and UV Trace 12 7 12 4 Troubleshooting the Conductivity Flow Cell and 12 9 12 5 Troubleshooting the Maximizer Buffer Blending 12 10 12 6 Troubleshooting Other Bio Rad Instruments and 12 11 SECTION 6 APPENDICES Appendix A A 1 Appendix B Pressure Conversion B 1 Appendix C Warranty Statement 00 0 ccc ce cece cece cece eet eee cece aeeeeeeeeeeeeeeeeeeccencaeeeeeeeeeeeeeeteeeees C 1 Appendix D
16. 2 54 2 10 3 Anion Exchange DEAE Weak Anion 2 55 2 10 4 Cation Exchange Carboxy Methyl CM Weak Cation Exchange 2 55 2 10 5 Ceramic Hydroxyapatite 2 55 2 10 6 Size Exclusion Chromatography 2 56 2 10 7 High Pressure Reversed Phase Columns 2 56 2 10 8 Hydrophobic Interaction Chromatography 2 56 2 10 9 Affinity Chromatography ssseessseeenenenm mene 2 57 2 10 10 Empty Golumns rte Gaetan 2 57 2 10 11 Column Fittings cecinere 2 58 SECTION 2 SYSTEM INSTALLATION AND SETUP Chapter 3 0 System Setup te icc eine gene defe desea Hehe ese dura en death 3 1 3 1 Controller Cable Connections sseseseeennneeeenn eene 3 2 3 2 USB Bitbus Communicator Cable Connections seen 3 3 3 3 Workstation Cable Connections sssssseeeeeeneeeneeenmeeeennn nennen 3 4 3 3 1 Systems without a MAXIMIZED sese 3 4 3 3 2 Systems with a 3 5 3 4 System Rack Setups e
17. d eR ONES 3 19 3 11 3 BioLogic Configuration Utility 3 20 Chapter 4 0 System eene nennen nnne nnns 4 1 4 1 General Guidelines for Creating Your Own Tubing Connections 4 2 4 2 Plumbing a DuoFlow System eee eene enne 4 3 4 3 Priming the System sineret dece a ete n 4 8 iv TABLE OF CONTENTS SECTION 3 SYSTEM OPERATION Chapter 5 0 Introduction to the System Software 5 1 5 1 System Interface enics e dede Lo dee Pe eel ve Rave d eve gue 5 1 5 2 Standard Mouse and Keyboard Functions 5 2 5 3 System Men s eer igo aded e apta eee ee de ee aue 5 3 5 941 Toolbar Buttons tenerte ode Basted acaba aia tatoo ees 5 3 5 3 2 Drop down nicae enhn nnn deiae kaeni 5 5 Chapter 6 0 Introduction to the Browser 6 1 6 1 OVERVIGW tele EE 6 1 6 2 Method Templates initiieren 6 7 6 3 Creating and Running a Queue ssssssssssseeeeeeeeenn m nennen 6 9 6 4 Creating and Viewing a Compare sssssssssssseeeeneeeeen nm ennemis 6 11 6 5 T
18. exporting in Manual in Post RUN settings in Run screen Cleaning instrument surfaces Column formats Affinity Chromatography Anion Exchange DEAE Weak Anion Exchange Q Strong Anion Exchange Cation Exchange Carboxy Methyl CM Weak Cation Exchange nee een S Strong Cation Exchange Ceramic Hydroxyapatite CHT Empty Column seii High Pressure Reversed Phase Columns eere es Hydrophobic Interaction Chromatography HIC Size Exclusion Chromatography SEC Columns and column fittings ADOUE eee ect Econo Column eene Econo Pac rente Examples FPLC HPLC Column eese Low pressure columns and flow adapters i rere est RESOURCE Column UNO Column to FPLC system UNO Column to HPLC system Compare creating and viewing in Browser SCION Lie ROO Conductivity monitor attaching to cleaning the flow cell description iet nd
19. Values at Cursor Run Time 00 01 56 6 Gradient 2196 Buffer B QuadTec 1 280 nm 0 0345 AU QuadTec 2 260 nm 0 0228 AU QuadTec 3 214 nm 0 4762 AU QuadTec 4 405 nm 0 0021 AU Conductivity 8 47 mS cm GP pressure 375 53 psi No Trace Current Tagging Trace QuadTec 3 214 nm QuadTec 3 214 nm Y GP pressure A BL i Rack Pos A Fractions Tube 1 2 18 14 0 100 f 100 o Conalb L Conalbumin B Min Tenth QuadTec WL1 280nm WL2 260nm WL3 214nm WL4 405nm Econo Gradient Flow Rate EGP B Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 B 0 Maximizer Gradient Pump F10 UV Conductivity SIM1 SIG v SIM1 pH Y 1 00ml min 0 B2 438 psi 0 03775 AU 359 mS cm 0 953 Volt 0 00 pH ooo o EEE IIIT Figure 7 14 Activity Trace 7 42 SYSTEM OPERATION MODES OF OPERATION 7 5 5 Exporting Chromatogram Data to other Software Applications To export data go to the Post Run screen of the desired run Select Export Data from the File drop down menu See Figure 7 15 Export Data Setup screen From this screen choose the run time and the data trace s that will be exported Alth
20. 5 11 Protocol Editor Mode 5 11 Run 5 11 Status Lines 5 11 Use Time Use Volume 5 11 Utilities menu Calibrate Gradient Pump 5 10 Check Hardware connections 5 10 Conductivity Flow Cell Constant 5 10 Diagnostics Information 5 10 EGP Calibration 5 10 pH Probe Calibration 5 10 System Information 5 10 Validate Method 5 10 View menu Bio Rad webpage 5 9 BioFrac tube format 5 9 Manial 1 cedente den 5 9 Post Run eee 5 9 giis ET 5 9 RUN e ideer a dis 5 9 E ete 5 9 Run 5 9 EE 5 9 TraceCompare 5 9 Volume Time based chromatogram 5 9 Window menu BioLogic 5 12 BioLogic Online 5 12 DuoFlow software description 5 1 Manual screen See Manual screen Post Run screen See Post Run screen Protocol screen See Protocol screen Run screen See Run screen Setup screen See Setup screen Troubleshooting
21. description rete petente Divert Valve Fraction Collector Advance button Gradient Pump button Hold Log button epe New Method Notes Pause button Settings button UV drop down Zero Baseline button Runs editing a method during post run See Post Run screen working offline during S Sample loading options Sample Loading Automatic loop fill and rinse AUX pump direct Gradient pump direct injection Dynamic loop injection Set Browser Options window Enable Methods Enable Project Runs Enable INDEX IN 5 INDEX Enable Runs eed 6 6 Protocol deeds 5 4 Enable User Methods 6 6 UD iei 5 4 Setup screen Settings nec deci 5 4 auxiliary pump eem 7 5 Setup devices
22. 4 2 plumbing from the mixer to the AVR7 3 VAIVG 4 5 plumbing from the transducer to the IiXef 3 at et 4 5 plumbing from the Workstation pumpheads to the transducer 4 5 plumbing the AVR7 3 valve 4 6 plumbing the Maximizer 4 4 plumbing the UV detector and conductivity monitor 4 6 plumbing the Workstation pump inlets 4 5 priming the 4 8 DynaLoop desCcriptior c ra rette Rie 2 40 example 8 6 plumbing for use with inject valve 2 41 E EGP Econo Gradient Pump connecting to Workstation 3 17 description iicet 2 43 examples of sample loading using 3 17 8 2 EP 1 Econo pump See Model EP 1 Econo pump F F10 Pump 2 51 F40 Pump 2 51 Ferrule installation 2 48 Fittings Kit etie Rena 2 47 Fittings tightener ssssssessesssss 2 48 Fraction collectors See BioFrac fraction collector Model 2110 fraction collector and Model 2128 fraction collector eot E ER Eod 2 39 INDEX Fraction collection schemes 7 20 G Gradient pumps See Workstation pumps TOT
23. tute ete eid eade rid Pete eodd 7 35 Protocol Screen during a nnne nn nnne nennen 7 36 Run Notebook Screen tes irit de tee Ee odore teehee 7 37 Log Screen iei coe Ue aao E de ED ede edu 7 37 POStRUM SChOGM pe 7 38 Post Run Tags for UV Detector mene nnne nennen nene 7 40 Activity Trace Editor iioii e ESSI GR EAAS AA 7 41 Activity Trace 7 42 Export Data Setup Screen ii Ce ee etae 7 43 Exporting a Chromatographic Image 7 44 Multiple Sample Loading with an Auxiliary Load Pump and an AVR9 8 Valve 8 2 Plumbing an AVR7 3 Inject Valve with an Auxiliary Load 8 3 AVR7 3 Valve Positions During a Run with Direct Sample Loading and Injection 8 4 Sample Loading through the Workstation Pump sessen 8 5 Plumbing the DynaLoop for use with an Inject Valve 8 6 Valve Positions During a Run using the 8 8 Column Switching using Two AVR7 3 Valves sssssssssseeeeeeeene mener 9 1 Column Switching using two AVR9 8 Valves ssssssssseeeneeeene nennen nere 9 3
24. 7 18 Step Time or Volume 7 18 Threshold sssssssss 7 18 Threshold Detector 7 18 Time Out MIN eects 7 18 Time Out 7 18 Isocratic Flow button pcr 7 11 B lffers 7 11 Canc6l x iate 7 11 Composition 7 11 Flow rate ml min 7 11 OK C 7 11 pH aetate ed 7 11 Step Time or Volume 7 11 Volume Time 7 11 Linear Gradient button 7 15 gt sterne pen 7 15 eL ns 7 15 oe e Cere 7 15 Initial and Final 96B 7 15 7 15 7 15 Step Time or Volume 7 15 Volume nre tete ene 7 15 Load Inject Sample button eB eoim eid 7 12 Cancel be ette 7 13 Flow Rate enmt 7 13 Injection Buffers and 96 Composition 7 12 Load Inject Sample 7 13 OK c E 7 13 nid cite RES 7 12 Step Time or Volume 7 13 Type Direct Inject svarnir 7 13 Dynamic Loop 7 13 Static 7 13 Volume veera 7 13 Pause
25. 5 8 Protocol Screen COPY is eerte doe eite tp 5 7 Qul iei la ee te 5 7 Delete enn ee 5 7 anon 5 7 lac 5 7 Select All 2 2 1 5 7 Run Screen Full view in Run Chromatogram 5 7 Hold gradient 5 8 Multiple runs 5 7 Pause buffer pump 5 8 Start te 5 8 Setup Screen Delete oen 5 7 oou 5 7 Select All eet 5 7 File menu 1 through 4 5 6 Buffer 5 6 Change Name Author 5 5 ClOSG eie 5 5 Copy and Edit Method 5 5 Data Management 5 6 reete ite Ue 5 6 Export Chromatogram Image 5 6 Export 5 6 IN 2 Integration 5 6 5 5 New 5 5 NeW 5 5 ODOREM 5 5 Print 5 6 Save setup 5 5 Select User and Project 5 5 Options menu Browser settings 5 11 Buffer Blender Setup 5 11 Chromatogram settings 5 11 Edit User Preferences 5 11 Manual
26. 7 19 Repeat Steps button 7 19 Scouting Creating a scout experiment 7 28 Selecting method steps to scout 7 26 Setting scouting parameters 7 27 Types of scout experiments 7 25 UV Lamp button ossee 7 19 Zero Baseline button 7 19 Pumps See Model EP 1 Econo pump EGP Econo Gradient Pump and Workstation pumps Q QuadTec UV Vis detector connecting to Workstation with Maximizer ienen ieta connection to Workstation without MaXiM Z r isi i i description installing the PEEK flow cell software Queue icons Delete Edit Method Edit Queue Open Method Run Q eue ricki rers aere reden dt Verify Queue ene R Reverse flow chromatography Run Log screen eie get unies Run Notebook screen Run procedure overview Run screen Abort ce tree e Browser button Chart Recorder button Cond Range eee Event UV Range ien Conductivity drop down menu
27. The BioLogic DuoFlow software allows you to continue working while a run is in progress Offline A complete discussion of this function is provided in section 7 4 2 Working Offline e BioLogic Online Allows you to observe and control the run in progress e BioLogic Offline Allows you to write a protocol analyze data and export data while a separate run is in progress 5 12 SYSTEM OPERATION INTRODUCTION TO THE BROWSER SCREEN 6 0 INTRODUCTION TO THE BROWSER SCREEN The Browser is the organizational tool for Users Projects and chromatography data It is a database that is displayed as a tree hierarchy which can be sorted by Users Projects Methods and Runs Figure 6 1 shows the layout of the Browser screen A User must be selected or a new user added to the list and then selected in order to gain access to any program function except Manual All menus and toolbar functions will be grayed out and inacessible until you select or enter a User name in the Browser
28. esses 12 1 DuoFlow system cleaning outer surface of instruments 11 1 connecting system to network 3 19 features ooi eie e s 1 2 ISO 9001 certificate E 2 ordering information D 1 OVOIVIOW REPE 1 1 pressure limit settings 7 2 T 3 safety and compliance i storage of system long term 11 1 storage of system overnight 11 1 system configurations 1 4 System power 3 19 System setup 3 1 system specifications A 1 npacking nee m 1 3 warranty statement C 1 DuoFlow system configurations DuoFlow 1 4 DuoFlow Maximizer 20 1 4 DuoFlow Maximizer 80 1 4 DuoFlow Pathfinder 20 1 4 DuoFlow Pathfinder 80 1 4 DuoFlow Standard 1 4 DuoFlow QuadTec Basic 1 4 DuoFlow QuadTec Standard 1 4 DuoFlow system plumbing connecting to a fraction collector 4 7 ferrule installation 4 2 guidelines for creating tubing
29. BioLogic Duo Flow user name project name method name run name File Edit View Utilities Options Window Help 4 Z 38 AA EA X Method Method Run Browser Report Settings Delete Available Devices Devices in setup UV Detector Aux Load Fraction Pump Collector Conductivity Monitor AVR7 3 Valve Sample Inject Buffer Blender xg Detectors SVT3 2 85 4 Valve St Valve AVR7 3 AVR9 8 Valve Ba Valve Maximizer Gradient Pump F10 Inlet A1 Buffer A1 Is assigned to buffer blender Inlet A2 Buffer A2 Is assigned to buffer blender Inlet B1 Buffer B1 Is assigned to buffer blender Inlet B2 Buffer B2 Is assigned to buffer blender Buffer Editor QuadTec WL1 280nm WL2 260nm 214nm WL4 405nm Econo Gradient Flow Rate EGP Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 B 0 Maximizer Gradient Pump F10 UV Conductivity SIM1 SIG Y SIM1 pH 1 00ml min 0 B2 438 psi 1 003 AU 1 23 mS cm 0 548 Volt 7 00 pH Figure 7 3 Setup Screen 7 4 SYSTEM OPERATION MODES OF OPERATION
30. E AURE UV DETECTOR AND FRACTION COLLECTOR REVERSE FLOW SAMPLE LOOP SAMPLE INJECT 3 X UV DETECTOR AND FRACTION COLLECTOR Figure 9 3 Reverse Flow Affinity Chromatography with an AVR7 3 Valve System Setup 1 Plumb an AVR7 3 reverse flow valve as shown in Figure 9 3 The column should be connected between port 4 column inlet and port 6 column outlet Plumb a short piece of tubing between ports 1 and 2 of the reverse flow valve Plumb port 5 of the reverse flow valve to port 4 of the upstream sample inject valve Plumb port 3 of the reverse flow valve to your detector In the device setup configure the AVR7 3 valve as a user define valve and name the valve Change Flow Valve Name valve positions 1 2 and 3 as Forward Flow Reverse Flow and Purge respectively Writing a Flow Switching Protocol ode XO NS The following three steps should be included as part of a reverse flow experiment 1 Add a Change Valve step at the beginning of the protocol and place the Reverse Flow valve in the Forward Flow position 2 Just before the elution step add a Change Valve step to your protocol and place the Reverse Flow valve in the Reverse Flow position ADVANCED SYSTEM APPLICATIONS MULTIPLE COLUMNS 9 3 MULTI DIMENSIONAL CHROMATOGRAPHY Multi dimensional chromatography is used to perform multi step chromatographic purifications in a single automated run This technique is particularly useful for purification
31. UP WITH PORTA SCREWS 2 RACK CLAMP B amp SCREWS 2 Figure 11 7 SVT3 2 Valve Assembly 11 10 MAINTENANCE AND TROUBLESHOOTING MAINTENANCE Use the 0 Phillips cross head screwdriver to remove the two screws and washers that secure valve bodies A and B to the actuator Set the actuator aside taking care not to lose the spring Use your fingers to remove the plunger diaphragm assembly from the valve body Remove the label A new label is provided in the rebuild kit Note that ports A and B are on one side the pump common port is on the other side of valve body A When reassembled the new label will not fit properly if ports A amp B are not adjacent to each other Carefully separate the two valve bodies Note the location of the O ring Inspect the disassembled valve Make sure there are no scratches or foreign material on the sealing surfaces of the two valve bodies Clean the valve bodies if necessary by soaking or sonicating in a bath containing a mild detergent Replace all required parts if necessary To reassemble the valve 1 Insert the plunger diaphragm assembly into valve body A Invert it and install the sealing disk onto the plunger tip Make sure the disk inserts into the plunger tip Install the O ring on valve body B Replacing the O ring is easily done with your finger Put the two valve bodies together so that the two ports are next to each other The two valve bodies must
32. B New Manual Method Manual screen is for individual Popup window indicates user amp control of instruments and devices project name The user then in the system enters the method name des cription and author A method is defined by its name setup and protocol A method may contain any number of runs Setup Protocol Run Run screen lets you run the steps in the protocol Press Start 5 to begin the run Figure 7 1 Relationships between Modes of Operation MODES OF OPERATION SYSTEM OPERATION 7 1 MANUAL SCREEN The Manual screen is used to control and monitor the operation of the DuoFlow Workstation and each of its peripheral devices This interface is divided into three basic groups of dialogs and includes control panels a real time chromatogram display and a status bar BioLogic Duo Flow user name lt name method name run name File View Utilities Options Window Hel New Method Edit Method New Z is Run Report TI was Toad Mangal E Setup Protocol Run BA tes PostRun Eag Settings Setup Bio Rad W
33. Direction Low limit 0 psi 260 nm INSTRUMENT Tube zu Em BUS Sat Advance Vol left Set 405 START STOP f START Set f START STOP Workstation Valves Y SVT3 2 at port 1 SV5 4 at port 2 AVR7 3 at port 4 AVR9 8 at port 5 WORKSTATION CONTROL 8 2 gt VALVE CONTROL O 19 OPFl7O Os 4Q A B Conductivity Rack Pos Tube Fractions 2 00 11009 Buffer B CHROMATOGRAM 1 50 4 DISPLAY CONTROL 3 i E Resize i T T TT 71 TX T T IY I TT 7 7 Clear 2 4 6 8 Traces Min Tenth ai WL1 280nm WL2 260nm WL3 214nm WL4 405nm Econo Gradient Flow Rate EGP Split QuadTec B 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 0 STATUS Maximizer Gradient Pump F10 UV Conductivity SIM1 SIG Y SIM1 pH f BAR HINT 1 00ml min 0 2 438 psi 1 003 AU 1 28 mS cm 0 548 Volt 7 00 pH BOX STATUS BOX Figure 5 1 Layout of the Screen Display showing the Manual Screen INTRODUCTION TO THE SYSTEM SOFTWARE SYSTEM OPERATION The DuoFlow system is controlled and monitored using the following e Toolbar The toolbar is the primary navigation tool for the system software e Drop down menus The drop down menus provide access to advanced functions Some functions found on the toolbar are duplicated in the drop down menus e Status bar The status bar provides realtime information about active instruments and devices connected to the system for example system
34. Macro Prep DEAE Support This is a weak anion exchanger containing diethylaminoethyl functional groups with a 50 um particle size It is ideal for purification of acidic and neutral proteins and peptides Refer to bulletin 1840 A 400 2 10 4 Cation Exchange Carboxy Methyl CM Weak Cation Exchange The CM weak cation exchanger chemistry is available in the following formats e Econo Pac CM Low Pressure Chromatography Cartridges These cartridges are available in 1 ml and 5 ml formats to accommodate most sample loads They are recommended for method scouting and for first step purification of crude samples They are based on 50 um Macro Prep9 supports 2 10 5 Ceramic Hydroxyapatite CHT The CHT chemistry is available in the following formats Bio Scale CHT Type I Prepacked Medium Pressure Columns These columns are designed for high resolution separations of proteins peptides and polynucleotides in analytical to semipreparative medium pressure applications They are available in four column sizes Methods developed on the Bio Scale columns can be transferred to production scale using the Macro Prep 10 um supports Refer to bulletins 1929 1946 2079 and 2156 Econo Pac CHT II Low Pressure Chromatography Cartridges These cartridges are available in 1 ml and 5 ml formats to accommodate most sample loads They are recommended for method scouting and for first step purification of crude samples They are based on 20 um Macro Prep9
35. 2 10 pumphead flow rates 2 6 purge A amp B 2 7 software 7 5 solenoid valves 2 9 test 2 10 UV chart 2 10 UV lamp connector 2 9 UV optics 2 9 INDEX IN 7 Life Science Group Bio Rad Laboratories Inc Web site www bio rad com Bio Rad Laboratories Main Office 2000 Alfred Nobel Drive Hercules CA 94547 Ph 510 741 1000 Fx 510 741 5800 Also in Australia Ph 02 9914 2800 Fx 02 9914 2889 Austria Ph 01 877 89 01 Fx 01 876 56 29 Belgium Ph 09 385 55 11 Fx 09 385 65 54 Brazil Ph 55 21 507 6191 Canada Ph 905 712 2771 Fx 905 712 2990 China Ph 86 21 63052255 Fx 86 21 53964775 Czech Republic Ph 420 2 4141 0532 Fx 420 2 4143 1642 Denmark Ph 45 44 52 1000 Fx 45 4452 1001 Finland Ph 358 0 9 804 2200 Fx 358 0 9 804 1100 France Ph 01 47 95 69 65 Fx 01 47 41 9133 Germany Ph 089 318 84 177 Fx 089 318 84 123 Hong Kong Ph 852 2789 3300 Fx 852 2789 1257 India Ph 91 124 6398112 113 114 6450092 93 Fx 91 124 6398115 6450095 Israel Ph 03 951 4127 Fx 03 951 4129 Italy Ph 39 02 216091 Fx 39 02 21609399 Japan Ph 03 5811 6270 Fx 03 58
36. 3 10 MODEL 1327 CHART RECORDER CONNECTIONS The Model 1327 chart recorder is optional It may be positioned on the rack shelf or on the bench 1 Connect System Cable 2 between the Workstation and the recorder as follows a The mini DIN connector is connected to the connector marked UV Chart on the rear of the Workstation Refer to Figures 3 4 and 3 5 b The DIN connector is connected to the single DIN connector on the side of the chart recorder This cable provides system control of pen up down event marks and paper advance but chart speed MUST be set on the recorder faceplate itself 2 Conductivity signals are recorded on channel 2 of the recorder using System Cable 4 as follows a The mini DIN connector is connected to the connector marked Cond Chart on the rear of the Workstation Refer to Figures 3 4 and 3 5 b The banana plugs are connected to the connectors marked CH 2 on the side of the recorder red wire to black wire to ground 3 Set both channel inputs on the recorder to 1V Set all other switches to their position marked in green Connect the power adapter to the chart recorder 3 11 COMPLETING SYSTEM SETUP Once you have completed the system setup turn the units around Connect the USB cable to the USB Bitbus communicator and connect the instrument bus cables Plug in the power cords and turn on the system In certain laboratory environments an uninterruptable power supply UPS may be required Bio
37. 7 2 1 Device Selection The devices that can be defined in the device setup are described below The actual devices available on your DuoFlow system may vary All DuoFlow systems are delivered with a detector conductivity monitor and an AVR7 3 valve All other devices are optional See Appendix D for information about ordering additional devices Auxiliary Pump Used to define the type of auxiliary pump connected to the DuoFlow system Econo Gradient Pump EGP Model EP 1 Econo or generic non Bio Rad pump These pumps may be defined as a sample load pump or as a user defined pump EGP only See Chapters 2 and 8 for ways to use an auxiliary load pump Generic pumps should be defined as a Model EP 1 pump Fraction Collector Used to define the type of fraction collector connected to your system BioFrac Model 2128 Model 2110 or generic non Bio Rad The fraction collection options available depend on the type of fraction collector connected to the system All of the fraction collectors can be synchronized with a detector signal by checking Synchronize Fraction Collection with the Detector and entering a delay volume The delay volume is defined as the system fluid volume including tubing and inline devices between the significant detector and the fraction collector drop head See the BioLogic software online Help for help determining the delay volume The BioFrac and Model 2128 options are used to control these fraction collectors over th
38. BioLogic Duo Flow user name lt project name method name run name GE e File Edit View Utilities Options Window Help New Edit New Z a Pe gres ANA Method Method Run Browser Report Manual Setup Protocol Run Notes PostRun Eeg Frac Collector UV M Bio Rad AAN M 5 WA Settings Abort Pause Hia Full View Web Conductivity Y Fractions 1 7 Advance RackPos A Divert Valve E Grid i1F 2F 4F 5F 6F 7F 8F 10F 11 12F 1 14 I 1 Collect 0 100 0 B Waste Grad Pump High psi 1000 4 0 075 Low psi 0 p44 4 T 4 Set Chart Recorder UV Range 0 01 y Cond Range 0 025 0 050 4 Lp Event mark 0 000 a o o 50 0 40 0 30 0 UV Detector M EIC Zero T Baseline 0 00 2 00 4 00 T AU Min Tenth QuadTec Protocol gt 1 0 00 0 Collection Fractions of size 2 00 ml during entire run M Zero Baseline Scout Method 7 Scout Run S Scout Buffer A A1 1 of 1 S Scout Buffer A A1 Run Time Run Volume Step Time Left Fraction Vol
39. 2 00 1 50 i 1 00 i 0 50 4 0 00 4 A BL Fractions Conductivity 1 10094 Buffer 50 4 6 Min Tenth Resize E Traces QuadTec WL1 280nm WL2 260nm WL3 214nm W L4 405nm Econo Gradient Flow Rate EGP Split 0 0041 AU 0 00001 AU 0 00021 AU 0 00004 AU Pump 0 00 ml min 0 0 10 UV Conductivity SIM1 SIG SIM1 pH 1 00ml min 0 2 438 psi 1 003 AU 1 23 mS cm 0 548 Volt 7 00 pH L CONTENTS MAY VARY DEPENDING ON CONFIGURATION Figure 7 2 Manual Screen for the BioLogic DuoFlow system connected to a Maximizer BioFrac Fraction collector QuadTec Detector Econo Gradient Pump and Four Valves From the Manual screen you can control the following Gradient Pump This panel is used to control buffer composition flow rate and pressure limits see Table 2 3 as well as to start and stop the pumps The Start Stop buttons are used to start and stop the pumps The Set button is used to make changes to the composition flow rate or pressure limits while the pumps are running SYSTEM OPERATION MODES OF OPERATION Maximizer Gradient Pump This panel is displayed in place of the
40. 3 2 mm OD 0 062 1 6 mm ID PTFE tubing with flat bottom fittings which are supplied in the Fittings kit Pumphead Outlet and Pressure Transducer Inlet and Outlet ports All plumbing following the pumphead outlet ports uses standard 1 4 28 flat bottom fittings and the following tubing Orange PEEK tubing 1 16 1 6 mm OD 0 020 0 51 mm ID Used with pressures up to 5000 psi Green PEEK tubing 1 16 1 6 mm OD 0 030 0 76 mm ID Used with pressures up to 3000 psi usually for flow rates greater than 20 ml min Pumphead Priming ports This port on each pumphead is used to prime the pump The port accepts any size syringe with a luer fitting a syringe is included in the Fittings kit This draws buffer through the inlet line and to the pumphead Twist the port counter clockwise one turn to open it Pumphead Washout Inlet ports The port on top of each pumphead is used to rinse the piston to remove crystallized salts It accepts any syringe with a luer fitting A syringe for this purpose is included in the Fittings kit The rinse output is the open trough between the pumpheads The pumpheads should br rinsed daily SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM Table 2 6 Workstation Rear Panel Connectors n uv CHART COND CHART Zl e e e SOLENOID VALVES MIXER INSTR BUS INJECT uv s OPTICS AUTOMATED VALVES FC ADV
41. 7 17 MODES OF OPERATION SYSTEM OPERATION Table 7 8 Hold Edit Hold Until Event Hold Until Event r Hold Until Threshold Detector Key Pressed Time Out Reqd UV Detector v Start of Inject Time Out min Threshold End of Inject 1000 AU Q Above Threshold The method will automatically continue after the selected Below Threshold time out duration Sound Alarm Step 1 Volume 0 00 ml OK Cancel Inserts a Hold step into the method The Hold step stops the progression of the method the pumps continue pumping at the Hold step B composition The method time or volume advances When the run resumes the current fraction collection condition is maintained The hold condition is maintined until a specified activity occurs e g Hold until Start of Inject or Hold until Keypress For example if the hold starts at time 2 minutes then the method resumes at time 2 no matter how long the Hold was in effect Hold Until and Threshold Detector Choose one of five events to discontinue the hold Key Pressed Press the F2 key on the keyboard to end the programmed hold Start of Inject The run will continue when the manually controlled device which must be connected to the Workstation AUX connector at pin 1 Inject is moved to its desired position End of
42. A UPS may be required in laboratory environments that experience power outages or where the quality of power varies Bio Rad can supply a UPS in both 110 V and 220 V configurations For questions about this UPS consult your local Bio Rad representative 2 9 11 Printers Any Windows 2000 compatible printer may be used with the DuoFlow systems The printer must include a connection cable and a printer driver To install a printer driver exit the DuoFlow software If the printer driver disk is available follow the instructions that are included with that disk If the printer driver disk is not available click on the Windows Start button and from Settings select Control Panel In the Control Panel double click on the Printers icon to open the Printers window Then double click on the Add Printer icon and follow the on line instructions If your printer does not appear in the list of printers available contact the printer manufacturer for a recommendation on which driver to use 2 53 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW 2 10 COLUMNS AND COLUMN FITTINGS Bio Rad offers a variety of column chemistries and formats The following pages provide a summary of the different column types and column fittings for use with BioLogic DuoFlow systems Bio Rad columns for the DuoFlow use the following matrices e UNO columns use the new Continuous Bed matrix which contains an advanced polymer matrix that is completely homogeneous
43. Econo Pac Protein A Low Pressure Chromatography Cartridges These cartridges are available in 1 ml and 5 ml formats to accommodate most sample loads They are recommended for monoclonal antibody purification Refer to bulletin 1946 Econo Pac Blue and DEAE Blue Low Pressure Chromatography Cartridges These chemistries are available in a 5 ml format and are recommended for albumin and protease removal Refer to bulletin 1946 2 10 10 Empty Columns The following columns are available e Bio Scale MT empty columns These columns allow for easy packing of a chromatographic media bed height adjustment sample application and equilibration The availability of four column sizes 2 5 10 and 20 ml allows easy scale up of separation and purification protocols Request bulletin 1970 Glass Econo Column columns Econo Column chromatography columns are the standard for high quality affordable low pressure chromatography columns They accept both Econo Column funnels and flow adapters 2 57 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW 2 10 11 Column Fittings Table 2 15 Columns and Column Fittings Econo Pac The Econo Pac cartridge has one male and one Catalog 732 0113 Cartridge female luer lock fitting To connect it to the Econo Pac Cartridge to BioLogic system use 1 4 28 to male and 1 4 28 to Fittings Kit female luer adapters provided in the kit shown to the right One set is included in the Fittings kit Econo Co
44. Note the piston seal in each piston well Always replace both seals at the same time Using the piston seal removal tool pry the seal from the piston well To do this you insert the tip of the seal removal tool into the seal and gently pry the seal away Once a piston seal is removed it cannot be reused 11 3 MAINTENANCE MAINTENANCE AND TROUBLESHOOTING 11 4 BACK OF PUMP HEAD ASSEMBLY PISTON SEAL O RING A PISTON GUIDE cS SPRING GUIDE VERTICAL POSITION SPRING PISTON Figure 11 2 Piston Assembly and Access to the Piston Seal view showing the back of the Pumphead Module Using your finger press each new piston seal into its recess Use the piston guide to press the seal completely into its recess Wipe the pistons with a dampened cloth before returning them to the piston wells This ensures that no crystallized salt residues are present which could damage the new seals When returning each piston assembly to its piston well make sure that the seals in the piston wells on the pumphead assembly are securely mounted in place The piston assemblies should be oriented so that the flat faces on the piston guides are in the vertical position Using the four long mounting screws removed earlier remount the pumphead module to the Workstation so that it is secure Insert and tighten the mounting screws Do not over tighten Condition the seal by pumping 100 ml of 100 methanol through each pumphead with
45. OD x 0 03 ID rated to 3000 psi Installation instructions For a complete discussion of the Pump kits refer to the separate documentation supplied with the kits 2 51 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW 2 9 8 Model 1327 Chart Recorder The Model 1327 chart recorder is a dual pen chart recorder that is compatible with many detection devices The chart recorder includes the following features e Two channel dual pen capability e Compact in size 31 x 23 x 7 6 cm Voltage ranges from 1 mV to 20V Twelve chart speeds for recording methods Figure 2 31 Model 1327 Chart Recorder The DuoFlow Controller outputs the UV analog data signal pen up down and Stop Start commands to the Model 1327 chart recorder via the Workstation s UV Chart connector cabled to the chart recorder using System Cable 2 mini DIN to standard DIN The Conductivity analog data signal is sent from the Workstation s Conductivity Chart Cond Chart connector to channel 2 of the chart recorder using System Cable 4 mini DIN to banana plugs The chart recorder should be set to all green settings 1V The chart recorder is described in detail in its separate documentation Note The chart speed is set at the recorder The BioLogic DuoFlow does not control this function Event marks are recorded on the chart recorder for the following Fraction Collector Advance When the fraction collector advances to the next tu
46. e e UVLAMP 10 25V 0 3A MAX e BLUE TO PIN 9 SYSTEM CABLE 7 Figure 3 14 Connecting an EP 1 Econo Pump to the BioLogic DuoFlow Workstation 1 On System Cable 7 locate the red and blue wires at the wire end of the cable and connect them to the DuoFlow Workstation Cut short all other wires and insulate with tape a Connect the red wire to pin 6 of the Aux connector b Connectthe blue wire to pin 9 of the Aux connector 2 Plug the 8 pin mini DIN connector into the Aux connector on the rear panel of the Econo pump PharMed tubing is recommended for use in the pumphead of the EP 1 Econo pump For additional information on this pump consult its user manual The illustration below shows the Aux pump connected to port 3 of the Inject Valve 3 16 SYSTEM INSTALLATION AND SETUP SYSTEM SETUP WASTE WORKSTATION PUMP WASTE LOW PRESSURE COLUMN _ EP 1 PUMP EP 1 PURGE ECONO PUMP Figure 3 15 Example of Direct Inject Sample Loading using an Econo Pump The EP 1 Econo pump can be used to load up to 7 samples sequentially when used with an AVR9 8 at the pump s inlet valve as shown below WORKSTATION PUMP WASTE AVR7 3 1 LOW PRESSURE LOOP OVERFILL COLUMN TO WASTE EP 1 ECONO PUMP 8 SAMPLE 2 SAMPLE 3 Figure 3 16 Example of Multiple Sample Loading using an Econo Pump 3 9 2 Econo Gradient Pump EGP Refer to section 2 8 3 for more information The EGP is co
47. i Project for Mary Jones 03 13 2001 07 35 01AM f Joe Smith 03 27 2001 05 05 48PM Poh Project for Joe Smith 03 28 2001 02 49 39PM i E Default 03 27 2001 05 05 48PM 4 Edit A Delete E Del 03 28 2001 02 49 39PM Demo Chromatagraphy 02 13 2001 07 34 43AM 7 Project for Demo Chromatagraphy 03 13 2001 07 35 01AM Demonstration Queue 03 27 2001 05 05 48PM POE iji Demonstration Queue 1 03 27 2001 05 05 48PM CAU Run Q Queue Method 3 03 28 2001 02 49 39PM Run Q Queue Method 2 03 28 2001 02 42 54PM Copyln i 0 Run Q Queue Method 1 03 28 2001 02 36 06PM standard UV detector 03 27 2001 05 05 48PM i 03 28 2001 02 49 39PM Baw amp CopyOut Demo Queue with QuadTec 1 QUEUE QuadTec Starter 2 03 27 2001 05 05 59PM lt Open Method NAME T 03 27 2001 05 05 59PM Eu Edit Method Compare 2 Verify Queue Run Queue 4 Edit Queue Queue Reset Information MOVE List QUEUES COMPARE COPYIN List Error List WL1 280nm WL2 260nm 214nm WL4 405nm Econo Gradient Flow Rate EGP Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 B 096 M
48. vial of Anion Exchange Protein Standards catalog number 125 0561 1 ml disposable sample injection syringe One 50 ul sample loop 50 ml of Maximizer solution A1 10x concentrate 50 ml of Maximizer solution A2 10x concentrate 50 ml of Maximizer solution B2 2 5x concentrate The UNO Q1 Column catalog number 720 0001 is not included with the Starter kit but is included with each DuoFlow system For a complete discussion of the Starter kit refer to its separate documentation 2 9 3 Fittings kit including Tubing kit The DuoFlow Fittings kit is included with all DuoFlow system It contains the following components PEEK and PTFE Tubing Tefzel Plugs Adaptors Unions and Caps e Tubing Cutter ml Syringe and 10 ml Luer Slip Syringe Molded Bottle Caps BioLogic Fittings Tool Screwdriver PEEK F to M and F to F Luers e Delrin Nuts Super Flangeless Ferrules F10 Tubing Kit Optional tubing kits include the following F10 Tubing Kit The F10 Tubing Kit includes precut 1 4 28 fitted PTFE Tefzel and 0 02 ID PEEK tubing for easy installation of a DuoFlow basic systems An installation chart indicates suggested placement of each piece of labeled tubing see Section 4 0 The kit includes a length of PTFE tubing 1 8 OD for connecting solution bottles to the Workstation pumps A and B a length of Tefzel tubing 1 16 OD for connecting AVR7 3 waste lines and 8 additional pieces of P
49. where two solutions of fixed composition i e Buffer A 25 mM Tris pH 8 1 and Buffer B 25 mM Tris pH 8 1 1 M NaCl are mixed to produce a solution with a specific salt concentration Buffer Blending In Buffer Blending proportioning valve A is used to control the amount of acid and base added and valve B is used to control the amount of water and salt added The effluent from pump A and B are then combined in the mixer In Buffer Blending the effluent from pumps A and B dilute each other and So each solution must be prepared at a 2X concentration Buffer Blending allows buffers of any pH within the buffer pH range to be produced Buffer Blending is discussed further in Section 10 2 Non Blending In Non Blending both proportioning valves have the same function and are each used to control the percent Buffer A and Buffer B in the pump effluent In this case the effluent from pump A and pump B are identical so no dilution occurs when they are mixed Therefore the buffers can be prepared at their 1X concentration The Buffer Blender setup dialog includes a High Flow Non Blending 1x buffer system for use in situations where a high flow rate is required but where Buffer Blending is not needed When using this method inlet tubes A1 and B1 are placed in Buffer A and inlet tubes A2 and B2 are placed in Buffer B A second high flow non blending buffers system titled High Flow Non Blending has also been included in Buffer Blender buf
50. 0 090 4 0 080 4 2 A 2F 00 00 30 00 01 00 500000 00 00 01 00 00 01 30 0 00 0 0704 0 060 4 0 050 0 040 4 0 0354 A 4F 00 01 30 00 02 00 700000 00 OK o 5F 00 02 00 00 02 30 0 00 Y Cancel Show activity trace Reset Activity Values 0 020 0 010 10 0 0 000 1 700 0 010 10 0 00 00 00 00 02 00 00 04 00 00 06 00 Hr Min Sec WL1 280nm WL2 260nm WL3 214nm WL4 405nm Econo Gradient Flow Rate EGP Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 B 0 Gradient Pump F40 UV Conductivity SIM1 SIG Y SIM1 pH Y 1 00ml min 0 B 438 psi 0 03775 AU 359 mS cm 0 953 Volt 0 00 pH QuadTec Figure 7 13 Activity Trace Editor 7 41 MODES OF OPERATION SYSTEM OPERATION BioLogic Duo Flow user name project name method name run name File Edit View Utilities Options Window New dt Ne c a 0 FAI 209e mW Ex Method Method Run Browser Report Manual Setup Protocol Run N al Tags FullView Print Del Activity p
51. 50 mM 7 00 1 0 Dynamic Loop Solution A2 Tris 50 mM Load Inject Sample Screen Solution B1 Degassed Water B Flow ml min with a Maximizer in Use Solution B2 Sodium Chloride 2 00M 0 1 00 O Direct Inject Molarity at 100 B 1 0 M Y Auto Inject Valve will move to INJECT at start of step and return to LOAD at end of step Step 2 Volume 1 00 ml OK Cancel Sample E The Edit Load Inject Sample dialog is used to program sample injection The appearance of this dialog and its associated tabs depend on the injection type selected and the devices configured in the device setup e Injection Buffers Displays all the available buffers The names displayed for Buffer A and Buffer B are the names assigned to the pump inlets or inlet valves in the device setup see Chapter 7 2 2 e Composition Allows you to set the buffer composition used to push the sample onto the column e g 7596 A and 25 B When Buffer Blending is defined in the device setup the Buffer System pH and B fields are displayed e pH Allows you to enter the pH for the current step Displayed when Buffer Blending is included in the device setup e B Allows you to set the buffer composition for the step as a percentage of the 1x salt concentration Displayed when Buffer Blending is included in the device setup 7 12 SYSTEM OPERATION MODES OF OPERATION Table 7 4 continued Load Inject Sample The following selections are available
52. ID tubing rated to 3000 psi for higher flow rates 2 9 4 Fittings Tightener The fittings tightener is designed to apply appropriate tightness to the nut stainless steel lock ring and the ferrule when installing 1 4 28 fittings on the end of tubing The flattened end of the lock ring should face towards the nut with the tapered end facing the tapered end of the brown ferrule Place the tubing and fittings into the green fittings tightener Do not allow the tubing to slip out of the ferrule Tighten the fitting to seat the ferrule onto the tubing but do not over tighten Once the fitting is made it can be inserted into the port Refer to instructions on next page 2 48 FITTINGS TIGHTENER PEEK FERRULE STAINLESS STEEL 7 COMPRESSION RING CUT THE TUBING Figure 2 27 Making 1 4 28 Flat Bottom Fittings SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS The following procedure describes ferrule installation 1 Slide the nut stainless steel compression ring and the ferrule in that order onto the tubing as shown at left The flattened end of the ring should face the nut The tapered end of the ferrule should face the ring 2 Allow tubing to extend slightly beyond the end of the ferrule Place the fitting into the green fittings tightener Do not allow the tubing to slip out of the ferrule Insert the tubing and fitting into the fittings tool to tighten to seat the ferrule onto the tubing do not overtighte
53. Maximizer Mixer Barrel Extender 12ml Detectors QuadTec DuoFlow Detector Kit 100 110 200 240 V Includes QuadTec Detector 3mm flow cell System Cable 25 QuadTec RS232 cable 4x10 32 Fingertight fittings Instrument Control Module ICM System Cable 26 ICM power cable System Cable 17 instrument bus cable QuadTec Instruction Manual US power cord QuadTec Deuterium Lamp QuadTec Halogen Lamp and Holder The holder is required for first time use QuadTec Halogen Lamp QuadTec Flow Cell PEEK 3mm path length QuadTec 4x10 32 Long fittings for 3mm flow cell QuadTec Flow Cell PEEK High speed for 80 ml min flow rates QuadTec 4x10 32 Long fittings for Hi speed flow cell QuadTec Instruction Manual UV Detector Kit Includes UV Detector with 254 280nm filter 5mm flow cell conductivity flowcell and backpressure regulator 214 nm Conversion Kit Zinc lamp housing 214 nm filter for Model OM II UV Detector optics modules beginning with serial number 362 BR 214 nm Conversion Kit Zinc lamp housing 214 nm filter for Model OM IO UV Detector optics modules beginning with serial number 345 BR and prior to 362 BR UV Detector Flow Cell 2 mm path length UV Detector Flow Cell 5 mm path length Mercury Lamp replacement Zinc Lamp 214 nm replacement Filter 280 and 254 nm Filter 214 nm ORDERING INFORMATION APPENDIX D 750 0223 Filter 365 nm 750 0224 Filter 405 nm 750 0225 Filter 436 nm 750 0226 Filter
54. Remote Setup Manual Program Arrow Keys Figure 2 25 EGP Econo Gradient Pump Consult the Econo gradient pump user manual for additional information 2 43 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW 2 8 4 Other Gradient Pumps The BioLogic DuoFlow system sends a TTL signal to control an external pump This signal is normally TTL high 5 volts The DuoFlow system commands the external pump to run by holding Pin 4 low Any pump accepting this signal may be used with the DuoFlow system as a sample loading pump Refer to your pump s separate documentation when connecting the pump to the DuoFlow system 2 44 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS 2 9 SYSTEM PERIPHERALS A number of system peripherals are available for use with DuoFlow systems The following system peripherals are discussed in this section e System rack e Starter kit Fittings kit Fittings tightener Backpressure regulator e SIM F10 and F40 Pump kits e Chart recorders including the Model 1327 chart recorder e Uninterruptable power supply UPS Printers 2 9 1 System Rack The BioLogic rack is an adaptable and durable racking system made of solvent resistant polypropylene stainless steel and glass filled nylon The rack can stand alone or be placed directly on the Workstation and supports various columns and cartridges valves detection modules buffer bottles and peripheral equipme
55. Signal Import Module SIM signal parameters Available only when the Maximizer is used with the system This allows you to switch between buffer blending and non buffer blending modes In buffer blending mode a window listing buffer systems available for use is displayed If your controller has internet access selecting this button takes you to the Bio Rad web page SYSTEM OPERATION INTRODUCTION TO THE SYSTEM SOFTWARE 5 3 2 Drop down Menus The drop down menus are discussed in the following tables Many menus and tool bar functions will be grayed out and inacessible until you select or enter a User name in the Browser refer to chapter 6 Table 5 3 File Drop down Menu BioLogic Duo Flow user name project name 4 Options View Tools Window Hel New Method Copy and Edit Method New Run Open Close Select User and Project Change Name Author Save setup Load setup Print Report Data management Export Data Export Chromatogram Image Integration Buffer Editor Exit 1 Method 1 2 Method 2 3 Method 3 4 Method 4 The File menu consists of the following New Method Opens the New Method dialog used to create new methods or load method templates The new method is saved into the selected User and Project e Copy and Edit Method Allows you to use an existing method as a template for a new method A copy of the method is made and you will b
56. These columns are superior to beaded supports in resolution binding capacity speed and value Bio Scale chemistries are based on 10 um Macro Prep supports They are offered in various sizes and allow for scale up separation and purification These medium pressure columns are ideally suited for use on the DuoFlow system The Econo Pac cartridge chemistries are based on 50 um Macro Prep supports and are ideal for first step purification 2 10 1 Anion Exchange Q Strong Anion Exchange The Q strong anion exchanger chemistry is available in the following formats UNO Q Biochromatography Columns These columns are designed to handle separations at high flow rates with low back pressure Instead of a traditional bed of packed beads or particles each column contains an advanced polymer matrix called the Continuous Bed matrix which is nonporous and homogeneous The matrix is designed to maximize resolution binding capacity and speed Refer to bulletins 2116 and 1946 Bio Scale Q Prepacked Medium Pressure Columns These columns are designed for high resolution separations of proteins peptides and polynucleotides in analytical to semipreparative medium pressure applications They are available in four column sizes Methods developed on the Bio Scale columns can be transferred to production scale using the Macro Prep supports Refer to bulletins 1880 1946 and 2079 Econo Pac High Q Low Pressure Chromatography Cartridges These cartridg
57. This button starts the Buffer System Wizard and is used to edit view or copy existing buffer systems Buffers and salts that are in the current buffer and salts lists can be used in a buffer system e Edit Buffer List This button starts a dialog used to create buffer salts that are used in buffer systems e Edit Salt List This button starts a dialog used create salts that are used in buffer systems e Buffer Editor Report This button allows you to print out buffer system information e Export Buffer System This button allows you to export buffer system information to a text file e Close Exits the Buffer Editor e Help Opens the Buffer Editor online help e Delete Buffer System Deletes the currently selected buffer system Bio Rad buffer systems cannot be deleted e View Table Used to display the pH as a function of Inlet A2 and B Used to determine the pH range for a buffer The buffer pH range depends on the salt concentration and should be limited to the table pH valves shown between 10 and 9096 A2 7 9 MODES OF OPERATION SYSTEM OPERATION 7 8 PROTOCOL SCREEN The protocol screen is used to create a new method or edit an existing method Isocratic and linear gradient step duration may be programmed in units of volume or time The default duration units time or volume can be set using the drop down menu item Options Edit User Preferences The menu items Use Time min and Use Volume ml may be used to toggle bet
58. Use the Windows drop down menu b Use the Windows 2000 taskbar c Simultaneously hold down the Alt Tab keys With the cursor over the DuoFlow offline button at the bottom of the screen click on the right mouse button and from the menu that is displayed select Restore Problems using Browser functions If you are using the right mouse button rather than the toolbar buttons make sure you have first used the left mouse button to highlight the user project method or run name Using the left mouse button click on a user project method or run name Using the right mouse button select a function from the drop down menu that is displayed 12 1 TROUBLESHOOTING Problem Instrument face plates eg pump UV fraction collec tor drop off the screen when in Manual mode Possible Cause communication problem has occurred MAINTENANCE AND TROUBLESHOOTING Solution Exit the DuoFlow application and turn off the Controller Turn off the Workstation Make sure all Instrument Bus phone jack style connections are secure in all the instruments Turn the Controller and Workstation back on If the problem persists contact Bio Rad Technical Service Controller monitor displays unusual colors or characters The monitor or the Controller video card may be defective and need replacement Exit the DuoFlow application and restart the Controller If the problem persists contact
59. WL2 260nm WL3 214nm WL4 405nm Econo Gradient Flow Rate EGP Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 B 096 Maximizer Gradient Pump F10 UV Conductivity SIM1 SIG SIM1 pH 1 00ml min 0 B2 438 psi 1 003 AU 1 23 mS cm 0 548 Vol 7 00 pH QuadTec Figure 6 2 The Copyln Window Copyln Allows you to copy in Methods and Runs from a backed up or archived ZIB file Refer to CopyOut page 6 3 To copy in a Press the button and select the ZIB file you wish to copy in Figure 6 2 above b From the COPYIN List in the Browser Tab window select the desired Methods and Runs All Methods and Runs in the file are displayed in the COPYIN List Figure 6 3 c From the database tree window click the left mouse button to select the Project to which you want the Methods and Runs to be copied d Again click on the Copyln button or click on the right mouse button and select Copy to project name Specify where to copy the file to Note that clicking the right mouse button displays all the available Browser toolbar options for the highlighted selection e Once you finish the copy in procedure use the Clear List button on the Browser tab window to delete the methods and runs in the COPYIN List 6 4 SYSTEM OPERATION INTRODUCTION TO THE BROWSER SCHEEN BioLogic Duo Flow
60. first unplug the instrument Use a damp cloth to wipe down the outer case Avoid wetting the power switch located below the front panel and the connectors on the rear of the unit The system rack tray should be mopped up and any residues rinsed away with water via the drain 11 2 STORAGE OF THE DUOFLOW SYSTEM For overnight storage please note the following 1 The system tolerates low salt buffer overnight but sample and high salt buffers need to be washed out of the tubing 2 Use deionizied DI water and the 10mL syringe provided in the Fittings kit to wash behind each pumphead with at least 10 ml of water This applies to both the F10 and 40 pumps 3 Ifthe system is in a cold environment keep it powered on to prevent condensation If it is at room temperature it may be left off or on When it is at room temperature the DuoFlow Controller computer and monitor can be turned off to conserve energy and prolong monitor life 4 Thoroughly flush all valves with water especially the Maximizer valves and the SVT3 2 and SV5 4 solenoid valves For long term storage please observe the following precautions 1 Follow the column manufacturer s instructions to clean and store the column Remove the column from inline 2 Run deionizied DI water through the entire system Be sure to wash all wetted parts of both the Maximizer valves and the solenoid and automated valves to remove any contaminants Note It is particularly im
61. need calibration Use the Conductivity Flow Cell Constant Calibration function from the Utilities drop down menu Either input the constant value which is shown onthe tag attached to the flow cell or calibrate the flow cell using 0 5M sodium chloride NaCl Note that the relationship between the conductivity reading and salt concentration is not linear The curve will flatten out at higher salt concentrations If a Maximizer is connected make sure the Conductivity monitor is plugged into it 12 9 TROUBLESHOOTING Problem Conductivity trace does not follow exactly the theoretical gradient trace B Possible Cause MAINTENANCE AND TROUBLESHOOTING Solution The Conductivity flow cell is typically placed after the UV flow cell The Conductivity trace will be offset from the theoretical gradient based upon the column and system volume Distorted conductivity trace Mixer capacity is incorrect Refer to Section 2 4 Mixers for discussion of mixer capacities 12 5 TROUBLESHOOTING THE MAXIMIZER BUFFER BLENDING Problem No values for pH are displayed on the Controller status bar Possible Cause There may be a loose or incorrect cable connection Solution Make sure the cable from the pH monitor is connected to a SIM or Maximizer and that the SIM is connected to the system bus Noisy or unstable readings Air bubble on pH probe membrane Remove air bubble Dr
62. nnns 9 4 9 3 Multi dimensional Chromatography eene 9 5 TABLE OF CONTENTS Chapter 10 0 Buffer Blending nennen nennen nennen nns 10 1 10 1 Doubled Flow Rate Capacity Using a Maximizer 10 1 10 2 Buffer Blending with the 2 10 2 10 3 pH Measurement and Corrections sssssssssssssseseseeeeeeeeme nn 10 4 SECTION 5 MAINTENANCE AND TROUBLESHOOTING Ghapter 11 0 Maintenance eie ode tnde ek ace cepe ea FEE REESE REESE 11 1 11 1 Care of the Outer Surfaces of the Instruments 11 1 11 2 Storage of the DuoFlow 11 1 11 3 Care and Maintenance of the Workstation Pumps 11 2 11 3 1 Priming the Workstation Pumps and Removing Trapped Air Bubbles 11 2 11 3 2 Daily Maintenant Oss ieira iniii N E eene nnne 11 2 11 3 3 Routine Maintenance of the Workstation 11 3 11 4 Maintenance of the UV Detector and the Conductivity Flow Cell 11 6 11 4 1 Cleaning the UV Detector and the Conductivity Flow Cell 11 6 11 4 2 Replacing the Lamp in the UV
63. oi ERR qiie 2 11 enter and arrow 2 13 firmware version screen 2 17 inlet selection screens 2 16 instrument bus connector 2 15 LCD 2 12 mixer 2 15 pH calibration screens 2 16 pH monitor 2 14 power cord connector 2 15 power on off orsin 2 12 SIM calibration screens 2 16 SIM device connector 2 14 SOfIKeys Lecce ca ee aii den 2 13 software 7 4 solenoid valves connector 2 14 valve control screen 2 16 valve inlet LEDS 2 12 valve inlet select buttons 2 12 valves A amp 2 12 IN 3 INDEX Methods pausing and stopping one in ee tL teet 7 33 Method Templates 6 7 Mixers See MX 1 mixer and Maximizer mixer Model 1327 chart recorder connecting to system 3 19 description 2 52 Model 2110 fraction collector c
64. when using more fragile low pressure columns or flow sensitive detectors e g refractive index A valve rebuild kit is available BIO RAD Auto Selection Figure 2 13 AVR9 8 Stream Select Valve The valve s use dictates its plumbing For example e When two AVR9 8 valves are used as a column switching valve up to eight columns may be run sequentially An AVR9 8 expands the number of available buffers or solvents when used as an Inlet Valve When used in conjunction with an auxiliary peristaltic pump such as the Bio Rad Model EP 1 Econo pump or EGP Econo Gradient Pump for loop filling and an AVR7 3 inject valve the AVR9 8 valve may be used to select up to eight samples for consecutive chromatography runs 2 29 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW Position 1 on the AVR9 8 is the default position when the system is powered up or at the end of a method run unless configured differently from the Edit User Preferences window available from the Options menu The valve uses 1 4 28 fittings and 1 16 1 6 mm OD PEEK tubing supplied with the fitting kit when plumbing these valves Connect the AVR9 8 valve signal cable to any of the available automated valve connectors on the back of the Workstation ports 4 5 or 6 If a Maximizer is in use connect to ports 7 8 or 9 before those on the Workstation If more than three valves are desired you may connect additional valves to ports 4 5 and 6 on
65. 11 6 3 Wipe the inside of the mixer body with a paper towel To clean the mixer barrel or mixer barrel extension the O rings and the magnetic stir bar soak or sonicate in a bath containing a mild detergent Replace damaged O rings Note The stir bar is magnetized To remove it turn the mixer base upside down and tap it on your hand 4 Reassemble the mixer To re insert the magnetic stir bar place it on a tabletop and carefully place the mixer base over it 5 Insert a 1 4 28 plug into one inlet and a 10 ml syringe into the other inlet port and flush the system with DI water or mild detergent solution Reverse the location of the plug and syringe and repeat MAINTENANCE AND TROUBLESHOOTING MAINTENANCE Refer to Figure 11 6 for re assembling the mixer Make sure that the magnetic stir bar lies flat and that the O ring groove in either the mixer barrel or the mixer barrel extender faces up Place an O ring in each O ring groove If you are using only the mixer body then only one O ring is required If either the mixer barrel or the mixer barrel extender is used then two O rings are required 9 oH SCREWS lt SCREWS OUTLET PORT OUTLET PORT 12 ml MIXER O RING GROOVE O RING BARREL 5 O RING GROOVE 750 pl MIXER 5 MIXER N 4 BARREL EXTENDER BARREL EXTENDER O RING GROOVE O RING GROOVE MAGNETIC MAGNETIC STIR BAR STIR BAR INLET PORT MIXER 4 NEET PO
66. 2110 fraction collector 2 Connect the bare wires to the AUX connector on the Workstation as follows Black wire to pin 5 and white wire to pin 9 3 8 3 Model 2128 Fraction Collector The Model 2128 fraction collector is controlled by the DuoFlow Controller via the Instrument Bus The Model 2128 fraction collector is connected to the system as discussed below Connect the USB Bitbus to the Controller as discussed in section 3 2 2 Use the instrument bus connector on the rear of the fraction collector to connect the Model 2128 fraction collector to the instrument bus Since the Model 2128 has only one instrument bus connector it should be the last device daisy chained to the instrument bus 3 Select Model 2128 in the BioLogic Configuration Utility 3 15 SYSTEM SETUP SYSTEM INSTALLATION AND SETUP 3 9 PUMP CONNECTIONS This section discusses the connections for the following instruments and devices Model EP 1 Econo pump Econo Gradient Pump EGP 3 9 1 Model EP 1 Econo Pump The EP 1 Econo pump is connected to the system as discussed below To connect the EP 1 Econo pump with the DuoFlow system make the following cable connections ECONO PUMP REAR PANEL WORKSTATION REAR PANEL Uv CHART COND CHART MIXER INSTR BUS w OPTICS AUTOMATED VALVES ED EDN SEBS RED TO PIN 6 COND FLOWCELL e
67. 3 Connect the mixer signal cable mini DIN connector to the connector marked Mixer on the rear of the Maximizer if available Otherwise connect to the Workstation Refer to Figures 3 4 and 3 5 4 Plug the unused inlet port using the plug provided 3 8 SYSTEM INSTALLATION AND SETUP SYSTEM SETUP 3 6 DETECTION SYSTEM CONNECTIONS This section discusses how to connect the UV detector the Conductivity monitor the QuadTec detector and non Bio Rad UV detectors 3 6 1 UV Detector and Conductivity Monitor Two flow cells are available for use with the UV detector TO CONDUCTIVITY Analytical 5 mm flow cell for high resolution protein chromatography applications and low flow rates It has a path length of 5 mm for maximum sensitivity and a volume of only 16 ul It can be used with flow rates from 0 1 to 10 ml min Preparative 2 mm flow cell for work not requiring high sensitivity or when working with high protein concentrations and for flow rates greater than 10 ml min It has a path length of 2 mm and a volume of 30 ul UV FLOW CELL FILTER TRAY WITH 254 nm AND 280 nm UV FILTER MERCURY LAMP CONDUCTIVITY MONITOR FLOW CELL SIGNAL CABLE FLOW CELL CONNECTOR SIGNAL CABLE ON REAR OF TO REAR OF UV LAMP WORKSTATION WORKSTATION POWER CABLE Figure 3 8 UV Detector and Conductivity Monitor To attach the UV Detector and Conductivity Monitor to the rack 1 Using the information provided above conf
68. 3 1 when selecting a mixer volume SCREWS 4 SCREWS lt OUTLET PORT OUTLET PORT CO on ne 12 ml MIXER O RING GROOVE Qo orine BARREL 5 O RING GROOVE 750 ul MIXER 5 ml MIXER Nc BARREL EXTENDER BARREL EXTENDER O RING O RING GROOVE O RING GROOVE MAGNETIC MAGNETIC STIR BAR MIXER STRBAR MIXER INLET PORT BODY INLET PORT BODY ASSEMBLY OF THE MAXIMIZER MIXER ASSEMBLY OF THE MX 1 MIXER Figure 3 7 Mixers Table 3 1 Mixer Flow Rates Flow Rate Barrel Extension Capacity Assembly Screws MX 1 Mixer for use without Maximizer less than 1ml min none 10 32 x 5 8 1 6 cm 1 to 10 ml min 750 yl mixer barrel extender 10 32 x 7 8 2 2 cm 10 to 40 ml min 2 ml mixer barrel extender 10 32 x 1 1 2 3 8 cm Maximizer Mixer for use with Maximizer 0 5 to 10 ml min none 1 4 20 x 1 2 1 3 cm 10 to 40 ml min 5 ml mixer barrel extender 1 4 20 x 1 1 2 3 8 cm 40 to 80 ml min 12 ml mixer barrel extender 1 4 20 x 3 1 4 8 2 cm SYSTEM SETUP SYSTEM INSTALLATION AND SETUP To attach the Mixer to the rack 1 Confirm that the selected mixer and its capacity volume is appropriate for the flow rate If it is not refer to the previous chapter for the procedure for changing the mixer capacity 2 Attach the mixer to a vertical bar using its rod clamp The mixer should be positioned between the pump and the AVRT 3 inject valve Attach the mixer to the rack so that the mixer outlet port faces upward
69. 7 6 To select which traces to display in the chromatograms Refer to Table 5 7 SYSTEM OPERATION MODES OF OPERATION 7 4 4 Pausing Stopping a Method in Progress The Run screen provides three options for pausing or stopping a method in progress as discussed in the figure below E Abort Stops the current run and saves the data A confirmation message is presented to prevent accidents The run cannot be continued Pause Stops progression of method method time and pumps Valves remain in paused position From Pause you can Continue Abort or Edit Protocol Hold Stops progression of method and method time Pumps continue pumping at Hold concentration and valves remain in Hold position From Hold you can Continue Abort or Pause You cannot edit Setup Setup screen lets you view the list of devices available to the method From Setup you can select Protocol or Run From Run select Abort or Continue Method Name Method Description Method Author If there s a run already associated with the method the method is copied and you must rename it 1 wash 21 load Protocol Protocol screen View only lets you view the steps in the run Select Run or Edit Protocol From Run select Abort or Continue Edit Protocol involves the buttons shown below was loa Edit Protocol Pre
70. Annotating tagging the Chromatogram Tags can be placed on any trace To assign tags to a trace and to view all assigned tags and their data use the Post Run Tags window shown below To display the Post Run Tags window click once on the Tags button in the toolbar or from the Edit drop down menu select Tag and Trace Options In the Post Run Tags window select a trace by clicking on an Active Trace radio button Trace visibility and tag visibility can also be selected from this box Tag styles are specified in the upper right box Peaks can be annotated with a sequential tag number 1 2 a user defined name user tag name with the trace value or the run time Post Run Tags Trace Trace Visibility Tag Visibility Active Trace Tag Style for active trace 96 Buffer B x UV AU Conductivity mS cm GP pressure psi no trace no trace no trace no trace Sequential Tag Number User Tag Name Q Trace Value Y axis value Run Time X axis value SYSTEM OPERATION Les Cancel Tags for UV trace Tag Tagname Retention time h m s Volume Buffer B ml B Uv AU Cond mS cm GP pres notrace notrace no trace psi notrace Tube Myoglobin 00 00 32 2 2 0 07992 1 496 N A Conalbum
71. Barrels and Mixer Capacity for the Maximizer Mixer Flow Rate Barrel Extension Capacity Assembly Screws 0 5 to 10 ml min 750 14 20 x 1 2 1 3 cm 10 to 40 ml min 5 ml Barrel Extender 14 20 x 1 1 2 3 8 cm 40 to 80 ml min 12 ml Barrel Extender 14 20 x 14 8 2 cm The procedure for changing the mixer capacity is provided in the following section OUTLET PORT MIXER TOP o 5 MIXER BARREL EXTENDER MIXER INLET PORT BODY 12 ml MIXER BARREL EXTENDER MAXIMIZER MIXER Figure 2 3 Maximizer Mixer and Mixer Barrel Extender 2 19 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW 2 4 3 Changing Mixer Capacity Be sure to follow these directions carefully The mixer may leak if it is assembled incorrectly or if an O ring is not correctly placed in the O ring groove Note Flush any hazardous material from the system Drain fluid from the mixer and disconnect the mixer plumbing and cables 1 If you will be changing the mixer capacity use the information in Table 2 10 and Table 2 11 to select the appropriate screws and barrel extension Use the hex key provided to remove the four screws from the top of the mixer see Figure 2 4 Remove the mixer top and turn it upside down to remove the O ring If the O ring does not easily dislodge use your fingers to remove it 4 Referto Figure 2 4 for re assembling the mixer Make sure that the magnetic stir bar lies flat If the standard mix
72. Bio Rad Technical Service No sound from monitor speakers Cables not connected properly Mute switch is activated Consult the monitor instruction manual to ensure that the cables are correctly connected Press the mute button which is located on the front of the monitor to turn on the speakers Controller displays General Protection Fault message or locks up during normal operation A communication problem has occured Exit the DuoFlow application and restart the Controller If the problem persists contact Bio Rad Technical Service BioFrac Fraction collector will not go into system mode The BioFrac is not in Local mode and in the main screen Placed the BioFrac in Local mode so that it displays the main screen start up screen Press the System option in the BioFrac panel of the BioLogic software s manual screen A method or run is colored red in the browser and cannot open it 12 2 The file is open in either the BioLogic Online or Offline window The BioLogic Hardware server is hung Go to the BioLogic Online or Offline window where it is open and from the File menu select File Close Exit the BioLogic software press Alt Ctrl Del and then select the Task List button Select the Processes tab and kill any of the following processes that may be running BL HWSRV exe biologic exe biolog offline Restart the BioLogic software MAINTE
73. Cables System Cable 17 System Cable 18 instrument bus communication 4 feet 1 2 m instrument bus communication 12 feet 3 7 m System Cable 19 instrument bus communication 30 feet 9 2 m System Cable 21 instrument bus communication 100 feet 30 m System Cable 24 QuadTec analog to bare wire includes two cables to connect to two SIMs System Cable 25 QuadTec RS232 connects QuadTec to ICM System Cable 26 ICM power cord connects to DuoFlow Workstation System Cable 30 bus communication 1 foot 30 5 cm System Cable 31 USB Bitbus Communicator to controller System Cable 2 mini DIN to Model 1327 DIN System Cable 4 mini DIN to rec banana System Cable 5 Workstation AUX bare wires to DB 9 System Cable 7 mini DIN to bare wires System Cable 20 mini DIN to DIN Miscellaneous Starter Kit UNO Q1 Column BioLogic DuoFlow Instruction Manual INDEX A AVRT 3 sample inject valve description i dorsesi ieii eante aa cleaning the valve Plumbing examples software valve valve AVR9 8 stream select valve description oerte cleaning the valve examples using software
74. Editor Create New Edit Current Edit Buffer Buffer System Export Buffer Buffer System Buffer System List Edit Salt List Report System Close Help Buffer System V Read Only Author Acetate 20 mM Y Delete Buffer Bio Rad System Buffer System Recipe Text Acetate view Tabe acetate acia 40 mm Prepare by dissovling pH Range Reference Temperature 80 ml of 1M Acetic Acid in water and diluting to 2 0 L 3 60 to 5 60 at 250 O Sodium Acetate Trihydrate 40 mM Buffer s Prepare by dissovling 10 9 g of Sodium Acetate Trihydrate MW 136 1 g mol in water and diluting to 2 01 Name 2X Concentration Acetate 0 04 M Degassed Water M Degass water under vacuum for at least 15 minutes while stirring M Sodium Chloride 2 0 M Prepare by dissovling 233 6 g of Sodium Chloride MW 58 4 g mol in water and diluting to 2 0 L Salt Name 2 x Concentration Sodium Chloride 2 00 M The Buffer Editor main screen is used to view information about currently defined buffer system and to create and edit buffer systems e Create New Buffer System This button starts the Buffer System Wizard that is used to create new buffer systems Buffers and salts that are defined in the current buffer and salt lists can be used in a buffer system e Edit View Buffer System
75. Inject The run will continue when the manually controlled device which must be connected to the Workstation AUX connector at pin 1 Inject is returned to its original position Above Threshold The run will continue when the selected detector signal exceeds the specified threshold Threshold Detector Select the desired threshold detector absorbance conductivity RI etc Threshold Specify a threshold value Below Threshold The run will continue when the selected detector signal falls below the specified threshold Threshold Detector Select the desired threshold detector absorbance conductivity RI etc Threshold Specify a threshold value Time Out Reqd and Time Out min To hold for a specified length of time Sound Alarm When this box is checked an alarm will sound at the beginning of the step to remind you that the system requires an action on your part to allow the method to advance OK Adds the step to the protocol This is the same as pressing the Enter key on the keyboard Cancel Does not add the step to the protocol This is the same as pressing the Esc key on the keyboard Step Time or Volume Identifies current step number and calculates the elapsed time or volume from all previous steps This is not user editable 7 18 SYSTEM OPERATION MODES OF OPERATION Button Pause Repeat Steps V Alarm 3 Zero Baseline Lamp Q EGP Chat 27 Recorder Add Step V Tab
76. LAMP CONDUCTIVITY MONITOR FLOW CELL SIGNAL CABLE x2 TO CONDUCTIVITY FLOW CELL CONNECTOR ON REAR OF SIGNAL CABLE UV LAMP WORKSTATION TO UV OPTICS POWER CABLE CONNECTOR ON REAR OF WORKSTATION Figure 2 5 UV Detector with Mercury Lamp 254 amp 280 nm Filters and Conductivity Flow Cell The UV detector consists of the optics bench a filter tray a flow cell and a lamp It is designed to hold the Conductivity monitor flow cell The following configurations are available Mercury Lamp and Filters The mercury lamp comes installed in the optics module along with 280 nm and 254 nm filters The filters are both held by a single tray To switch filters rotate the filter holder Filters for different wavelengths are also available from Bio Rad These include 365 nm 405 nm 436 nm and 546 nm filters Zinc Lamp and 214 nm Filter The zinc lamp and 214 nm filter are optional The zinc lamp attaches directly to the optics module in place of the mercury lamp Refer to Figure 2 6 2 21 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW Two flow cells are available with the detector Both use 1 4 28 flat bottom fittings Analytical 5 mm flow cell This flow cell is recommended for high resolution detection This flow cell has a 5 mm path length a volume of 16 ul and is rated to 750 psi at flow rates between 0 1 and 10 ml min To reduce the risk of an entrapped air bubble causing an unstable baseline the 5 mm flow cell sho
77. Module SIM is connected to DuoFlow instrument bus The universal AC DC inline adapter catalog number 760 2034 must be used to supply the power Use of other power adapters may damage the USB Bitbus Communicator otherwise it is not required PWR SELCT Used to select whether power for the USB Bitbus Communicator is drawn from the Controller INT or the AC DC inline adapter EXT An external power source must be used if a Signal Import Module SIM is connected to the instrument bus BUS PWR Indicates that the instrument bus is receiving power from an external power source LED The light is off when the PWR SELCT switch is set to INT The light will turn on when the USB Bitbus Communicator is switched to EXT and power is being received INSTR BUS Used to connect the USB BitBus Communicator to the DuoFlow instrument bus DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW 2 2 WORKSTATION The Workstation contains the following Dual pumpheads each consists of two biocompatible dual piston pumpheads A built in pressure transducer is located on the workstation at the pump outlet The pressure transducer measures system pressure which is displayed on the lower status bar of the software Manual and Run screens Purge and Pause buttons Purge A Purge B and Pause are present on the front of the Workstation There are two types of pumpheads the F10 and the F40 All DuoFlow systems have F10 pumpheads except DuoFlow Maximizer 80
78. OPERATION Table 7 10 continued Fraction Collection MODES OF OPERATION Description Threshold and Collection Windows Fraction Collection Scheme BioFrac Rack Type F1 12 13 mm tubes Threshold Dectector Q Below Threshold O Collect Al UV Detector Above Threshold Threshold Tiresto Rack Row Col Collection Windows Strt A v 1 v 1F End B v 90 1A Close Fraction Collector Scheme CollectionWindows Start End Frac Size Thresh Non Peak Frac Size Close ml ml ml AU ml ADD MODE 8 00 100 100 0200 a 001 E p mes Save Window 1 4 00 5 00 0 60 0 100 Waste 2 7 00 8 00 1 00 0 200 Waste ndon Finished Adding Fraction Collector Scheme CollectionWindows Start End Frac Size Thresh Non Peak Frac Size Close ml ml ml AU ml 1 4 00 5 00 0 60 1 00 Waste SELECT MODE Add Window 2 7 00 8 00 1 00 0 000 Waste Fraction Collector Scheme CollectionWindows Start End Fra
79. Pore reversed phase columns These columns are commonly used for the purification and analysis of small proteins lt 50 kd peptides oligonucleotides and amino acids Refer to bulletin 1946 2 10 8 Hydrophobic Interaction Chromatography HIC The HIC chemistry is available in the following formats 2 56 Econo Pac t Butyl HIC Low Pressure Chromatography Cartridges These cartridges are available in a 5 ml format to accommodate most sample loads They are recommended for method scouting and for first step purification of crude samples They are based on 50 um Macro Prep supports Refer to bulletins 1946 and 2079 Econo Pac HIC Low Pressure Chromatography Cartridges HIC is offered in both methyl and t butyl chemistries These cartridges are available in 1 ml and 5 ml formats to accommodate most sample loads They are recommended for method scouting and for first step purification of crude samples They are based on 50 um Macro Prep supports Refer to bulletin 1946 Macro Prep HIC Support HIC is offered in both methyl and t butyl chemistries The Macro Prep methyl HIC support is ideal for purification of proteins with strongly hydrophobic regions The Macro Prep t butyl HIC support is ideal for purification of proteins with few or weakly hydrophobic regions Refer to bulletin 1841 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS 2 10 9 Affinity Chromatography Affinity chromatography is available in the following formats e
80. Queue These discrepancies must be corrected to run the queue SYSTEM OPERATION INTRODUCTION TO THE BROWSER SCREEN Browser toolbar buttons New Open Edit Delete Print CopyOut To create a new User Project Method Run Queue or Compare depending on which icon within the Browser is highlighted Allows you to open a selected Method or Run from the database tree Opening a Run allows you to view analyze and print the run data When a User or Project is selected Edit allows you to change that User name or Project name and or description When a Method or Run is selected Edit copies the currently open method renames it and places it in the Protocol Editor for editing The new method is assigned a default name lt method name gt lt version number gt that can be changed by the user Summary items are not editable Eliminates Users Projects Methods and Runs from the Browser To delete a Method you must first delete all of the Runs associated with the method You cannot delete methods or runs from method and run summaries To generate and print the report for a run Allows you to copy Methods and Runs to floppy disk or to another location on your computer s hard drive The CopyOut function is used for backup and archiving purposes and to transfer data between DuoFlow systems Copied out files are stored in a file with the default name biologic zib which is a compressed file format to conserve dis
81. Queue Method 2 WU Run Q Queue Method 1 03 02 2001 10 04 56AM 01 07 2001 07 08 34PM 01 13 2001 07 34 43AM 03 13 2001 07 35 01AM 03 27 2001 05 05 48PM 03 28 2001 02 49 39PM 03 27 2001 05 05 48PM 03 28 2001 02 49 39PM 02 13 2001 07 34 43AM 03 13 2001 07 35 01AM 03 27 2001 05 05 48PM 03 27 2001 05 05 48PM 03 28 2001 02 49 39PM 03 28 2001 02 42 54PM 03 28 2001 02 36 06PM 03 27 2001 05 05 48PM 03 28 2001 02 49 39PM standard UV detector 0 0 Sample Run A COED COMPARE NAME Queue 03 27 2001 05 05 59PM 3 27 2001 05 05 59PM 3 27 2001 05 05 59PM Open Run Remove Run Compare 232 Compare Traces Reset jormation f MOVE List QUEUES COMPARE List Error List X Edit Compare WL2 260nm 0 40 0 15 Maximizer Gradient Pump F10 1 00ml min 0 2 438 psi WL3 214nm 2 00 UV 1 003 AU WL4 405nm 0 00 EGP B 0 B SIM1 SIG SIMt pH Y 0 548 Volt 7 00 pH Flow Rate 0 00 ml min Split 0 Econo Gradient Pump QuadTec Conductivity 1 28 mS cm METHODS IN COMPARE Figure 6 7 Compare Displayed in the Browser Window To create a compare
82. SAMPLE LOADING OPTIONS The BioLogic DuoFlow system supports several methods for sample loading Typically a sample is loaded through a static fixed volume loop on the AVR7 3 inject valve When large volume sample injections are required the following options are available Sample loading through the Workstation pumps The SV5 4 SV3 2 or AVR9 8 valve may be used to load large sample volumes through the Workstation pumps Refer to Sections 2 62 through 2 64 for the discussion of these valves e Sample loading through a DynaLoop sliding piston loop When used with an AVR7 3 inject valve the DynaLoop may be used for large volume sample loading or repetitive injections of smaller volumes of sample The DynaLoop comes in two sizes 25 and 90 ml volume Sample loading through an auxiliary pump such as the EP 1 Econo pump EP 1 or the Econo Gradient Pump EGP When used with an AVRT 3 inject valve an EP 1 or EGP pump may be used for large volume sample injections Placing an SV5 4 or AVR9 8 valve before the pump inlet allows loading of multiple samples 2 8 1 DynaLoop Large Volume Sample Injection Loop The DynaLoop available in 25 ml and 90 ml sizes is a sliding piston dynamic loop for injection of a large sample volume or repetitive injections of smaller samples The DynaLoop has a sliding piston that functions like a syringe The loop is installed on the AVRT 3 in place of the static loop see Figure 2 23 Sample is loaded into the Dyn
83. Tris 50 mM 5 5 to 7 5 5 Citrate 20 mM 2 6 to 6 0 6 Citrate 50 mM 2 4 to 6 0 7 Formate 20 mM 3 0 to 4 6 8 Formate 50 mM 2 6 to 4 6 9 HEPES 20 mM 6 4 to 8 4 10 HEPES 50 mM 6 4 to 8 4 11 MES 20 mM 5 1 to 7 1 12 MES 50 mM 5 1 to 7 1 13 N Methylpiperazine pH 4 2 to 5 5 4 2 to 5 5 14 Phosphate 20 mM 5 8 to 7 5 15 Phosphate 50 mM 5 6 to 7 5 16 Phosphate 100 mM 5 5 to 7 5 17 Phosphate with Ammonium Sulphate 5 6 to 7 2 18 TAPS 20 mM 7 4 to 9 4 19 Triethanolamine 20 mM 6 8 to 8 8 20 Triethanolamine 50 mM 6 8 to 8 8 21 Tris 25 mM 7 1 to 9 1 22 Tris 50 mM 7 1 to 9 1 Broad Range Buffers 23 Bis Tris Tris 4 6 2 to 9 4 4 C 24 Bis Tris Tris 25 5 8 to 8 9 25 N methylpiperazine Bis Tris Tris 25 4 7 to 9 4 26 Formate Acetate Phosphate 25 3 1 to 6 8 pH range is at 25 C unless otherwise noted 10 2 ADVANCED SYSTEM APPLICATIONS MULTIPLE COLUMNS The Buffer Editor is used to create new buffer systems or to edit existing buffer systems Note that Read Only buffer systems cannot be modified To modify a Read Only buffer system uncheck the Read Only box or Edit a Copy of the buffer system The following procedure should be used to create or edit a buffer system 1 Define a buffer salt a b From the Buffer Editor main screen press Edit Buffer List Use the Buffer Salt Name list to select and view the desired buffer If the buffer is in the list and itis made from reagents avai
84. Valve Outlets to the Workstation Pump Inlets c Connect the two preformed fittings provided between the Maximizer valve ports and the Workstation pump inlet ports Connect to the Workstation first d Because the tubing is rigid you will need to lower the Maximizer valves in order to connect the tubing This requires loosening the two screws at the base of each valve so that you can tilt the valve downward Refer to the illustration below e Insert the tubing fitting into the Maximizer outlet port and screw in the fitting Figure 4 4 Maximizer Plumbing SYSTEM INSTALLATION AND SETUP SYSTEM PLUMBING 2 Plumbing the Workstation pump Inlets a PUMP A OUTLET PRIMING PORT A Workstation pump inlets attach two fittings to 1 8 3 2 mm OD PTFE tubing as described earlier See Section 4 1 General Guidelines for Creating Your Own Tubing To connect the SV5 4 buffer select valve or the SVT3 2 valve before the pump inlet 1 8 3 2 mm OD PTFE tubing is used Screw the tubing into the inlet connectors on the bottom of the Workstation pump housing Ensure a firm connection but do not over tighten Place opposite end of tubing in solution bottles A and B Refer to figure 4 1 If an SV5 4 valve is in use attach the pump inlet tube to its common port Refer to section 2 6 3 TO MIXER INLET PORT PUMPHEAD WASHOUT PORT INLET PUMP B OUTLET PRESSURE TRANSDUCER HOUSING PRIMING PORT B PUM
85. WASTE LL SYRINGE OR AUXILIARY LOAD PUMP Figure 8 5 Plumbing the DynaLoop for use with an Inject Valve ADVANCED SYSTEM APPLICATIONS SAMPLE LOADING Writing the Protocol Before running a protocol which involves the use of a DynaLoop be sure to purge air from the lines as discussed in the DynaLoop Instruction Manual Chapter 2 Installation Then follow the guidelines which apply to your application The following steps should be programmed within the method protocol to manually load a sample 1 2 5 Ensure that the column is properly equilibrated by using an Isocratic Flow step From the Manual screen set the AVR7 3 sample inject valve to its Inject I position and allow the sliding seal to contact the sample end fitting on the DynaLoop using the purge procedure discussed in the Dynaloop Instruction Manual Then set the valve to the Load L position Load sample into the DynaLoop by using a syringe As sample is loaded into the loop it displaces the buffer that was used to purge the unit This design means the DynaLoop cannot be overfilled When sufficient volume of sample for a run or series of runs is loaded into the DynaLoop stop the filling process For a series of partial volume injections after one loading sequence fill the DynaLoop with about 2 percent extra sample Leave the syringe in the Inject port to minimize the introduction of air into the DynaLoop The sample is now ready for inject
86. and that the correct wavelength filter is chosen before replacing the lamp Refer to the previous section Note As indicated during the following procedure make sure the DuoFlow Workstation is turned off This will eliminate the risk of electric shock thermal burns or UV light exposure to you or damage to the detector during cable connection MERCURY LAMP HOUSING Figure 11 4 Replacing a Mercury Lamp a Model OM II UV Detector ZINC LAMP HOUSING ZINC LAMP FIXTURE Figure 11 5 Replacing a Zinc Lamp in a Model OM II UV Detector FLOW CELL FILTER 11 7 MAINTENANCE MAINTENANCE AND TROUBLESHOOTING 1 At the DuoFlow Controller go to the Manual screen and turn off the lamp Turn off power to the Workstation and unplug the UV Lamp cable which is plugged into the UV Lamp connector on the rear of the Workstation 2 Without disconnecting the lamp housing from the UV bench undo the two small Allen screws securing the lamp fixture and gently pull the lamp out of the housing 3 Remove the lamp unit from its packing material WARNING Do not touch the glass part of the lamp Oils from you fingers will degrade the lamp over a period of time 4 Carefully insert the replacement lamp fixture The replacement lamp can be installed only in one orientation due to a small guide which fits into the housing Secure it using the Allen screws for that purpose Note that extra screws are provided with the kit Note Ch
87. appearance of the Buffer Blending setup dialog depends on whether a single component see Figure 10 1 or multiple component buffer is selected see Figure 10 2 Place a check in the pH correction box and select the Two Point Correction option Enter the desired pH in the Desired pH box Enter the low salt and high salt pH s that were measured in Step 6 in the Observed pH box and then enter the B that was used to collect the high salt pH For multi component buffers repeat this for each buffer in the buffer system The software will calculate the correction Optional Check the pH Correction 12 13 14 10 6 From the manual screen press the Buffer Blender Setup button and select the desired Buffer System from the recipe list Turn pH correction on and enter the Desired and Observed pH from step 10 above Set the pH Salt composition and flow rate Press run After the system has equilibrated collect some effluent and measure the pH using a high quality pH probe MAINTENANCE AND TROUBLESHOOTING MAINTENANCE 11 0 MAINTENANCE The BioLogic DuoFlow system requires minimal maintenance to assure reliable operation Note Maintenance of the QuadTec UV Vis detector and the Econo Gradient Pump EGP is discussed in their separate documentation 11 4 CARE OF THE OUTER SURFACES OF THE INSTRUMENTS During normal operation spills and splashes may cause residues to form on the component cases To clean the case of an instrument
88. backpressure and detector AU readings Items shown in gray are not currently active 5 2 STANDARD MOUSE AND KEYBOARD FUNCTIONS The DuoFlow system is supplied with a Dell PC computer The left mouse button is used with system software except as noted Table 5 1 Special Function Keys Special Function Description Keys Hold until Keypress Used during run to continue a method i e satisfy the Hold when the method includes a Hold until Keypress step Help Displays the Help menu for the currently displayed screen Esc Functions as an alternative to the Cancel button in a dialog box Alt Some system commands can be executed either by selecting them from a drop down menu or by holding down the Alt key and then pressing the appropriate character key 5 2 SYSTEM OPERATION INTRODUCTION TO THE SYSTEM SOFTWARE 5 3 SYSTEM MENUS The system menus consist of both drop down menus and the toolbar In some cases identical functions are found in both areas Advanced features are located only in the system drop down menu 5 3 1 Toolbar Buttons The function of each toolbar button defined in Table 5 2 is duplicated in the File and View drop down menus Table 5 2 Toolbar Buttons ee New Opens the New Method dialog used to create new methods or load method Method templates The new method is saved into the selected User and Project eat Copies the currently open method renames it and places i
89. be used with or without a mixer barrel the mixer body mixer top and mixer barrel are provided with the system an optional mixer barrel extender is available The mixer barrels fit between the mixer body and the mixer top and are used for higher flow rates Refer to the following table to determine the appropriate mixer volume Table 2 10 Mixer Barrels and Mixer Capacity for the MX 1 Mixer Flow Rate Barrel Extension Capacity Assembly Screws less than 1ml min 263 yl 10 32 x 5 8 1 6 cm 1 to 10 ml min 750 ul Barrel Extender 750 ul 10 32 x 7 8 2 2 cm 10 to 40 ml min 2 0 ml Barrel Extender 10 32 x 1 1 2 3 8 cm The procedure for changing the mixer capacity is provided at the end of this section MIXER TOP OUTLET PORT O 750 ul MIXER CS BARREL EXTENDER INLET PORT 2 0 MIXER MIXER BODY BARREL EXTENDER MX 1 MIXER Figure 2 2 MX 1 Mixer and Mixer Barrel Extender 2 18 SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM 2 4 2 Maximizer Mixer The Maximizer mixer provides the larger capacity required when the Maximizer is used The Maximizer mixer may be used with or without a mixer barrel the mixer body mixer top and mixer barrel are provided with the system an optional mixer barrel extender is available The mixer barrels fit between the mixer body and the mixer top and are used for higher flow rates Refer to the following table to determine the appropriate mixer volume Table 2 11 Mixer
90. eee tt 2 18 2 4 2 Maximizer MIXGF eme ead eae tied en enu du 2 19 2 4 3 Changing Mixer Capacity essen enm 2 20 D etectiori Systems utendo DG RECO eens 2 21 2 5 1 UN DetectoE 3 ree ev evoca ee deed 2 21 2 5 2 Conductivity Montor nennen 2 23 2 5 3 BioLogic QuadTec UV Vis 2 23 2 544 PH MORItOE dea ot teet oth oe Lo Ee cutee 2 25 2 5 5 Other Detectors neret tree einai vere ote daga nes denne Lag ev 2 25 2 26 2 6 1 AVRT 3 Sample Inject Valve sssssssseeee emen 2 26 2 6 2 AVR9 8 Stream Select Valve eee 2 29 2 6 8 SV5 4 Buffer Select and Automated Sample Loading Valve 2 31 2 6 4 SVT3 2 Diverter Valve idia ada mennad eaa 2 33 Fraction 87e Toyo s 2 35 2 7 1 BioFrac Fraction Collector sssssssses ee 2 35 2 7 2 Model 2110 Fraction Collector sssssssseemRe 2 37 2 7 3 Model 2128 Fraction Collector 2 38 2 7 4 Generic Fraction Collectors ssssssssssesne 2 39 Sample Loading 2 40 2 8 1 DynaLoop Large Vol
91. for the experiment Enter the collection technique e g collect all threshold etc fraction size and any required threshold or collection windows parameters Note that the dialog displays the number of tubes required for the currently defined protocol Press the New Run button to create a new run enter a run name and open the Run screen Set the appropriate pressure limits for the column being used and then press Start on the system tool bar Refer to Chapter 7 4 for more information about the Run screen 1 5 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS 2 0 DESCRIPTION OF SYSTEM COMPONENTS The DuoFlow s modular design supports many types of system components and allows for a wide variety of system configurations This section discusses in detail the function of each component and its connection to the system Dell PC Computer Controller and USB Bitbus Communicator Section 2 1 Workstation Section 2 2 Maximizer Section 2 3 Mixers MX 1 and Maximizer mixers Section 2 4 Detection Systems UV detector Conductivity monitor pH monitor and QuadTec detector Section 2 5 Valves AVR7 3 AVR9 8 SV5 4 and SVT3 2 valves Section 2 6 Fraction Collectors BioFrac Model 2128 and Model 2110 Section 2 7 Sample Loading Options DynaLoop EP 1 Econo pump and EGP Econo Gradient Pump Section 2 8 System peripherals Section 2 9 Columns and Column Fittings Section 2 10 UNO Q1 COLUMN
92. from the Buffer System list Make sure the pH correction box is not checked Press OK 2 Setthe desired pH salt composition and flow rate and then press Start After the system has equilibrated collect some effluent Measure the pH using a high quality pH probe connected to a desk top pH meter that has been calibrated at the run temperature 5 Load your experimental method from the Browser screen and press Setup Double click on the Buffer Blending icon and select the desired buffer The appearance of the Buffer Blending setup dialog depends on whether a single component see Figure 10 1 or multiple component buffer is selected see Figure 10 2 7 Place a check in the pH correction box and make sure the two point correction option is not selected 8 Enter the pH that was measured in Step 4 in the Observed pH box and the desired pH in the Desired pH box and press OK For multiple component buffers enter the measured pH for each buffer component in both the 0 B and user set B pH correction boxes The software will calculate the correction 10 4 ADVANCED SYSTEM APPLICATIONS REVERSE FLOW CHROMATOGRAPHY Optional Check the pH Correction 9 From the manual screen press the Buffer Blending Setup button and select the desired buffer system from the Buffer System list Turn pH correction on and enter the Desired and Observed pH from step 8 above 10 Set the pH Salt composition and flow rate Press run 11 After the system
93. has equilibrated collect some effluent and measure the pH Edit Buffer Blender Buffer Blender r Buffer Blender pH Correction Values Buffer System Sort By Name CO pH Range Tris 25 mM pH 7 1 to 9 1 Use pH Correction pH Range at 25 Celcius 7 10 to 9 10 Desired Molarity at 100 B 1 0 Observed at 0 Temperature Compensation Coefficient Use two point correction r Pump Head Selection F10 Flow Rate Range 0 20 20 0 ml min Max Pressure i Observed at 100 0 B 4 48 Recipe Solution A1 Acetic Acid 40 mM Prepare by dissolving 80 ml of 1M Acetic Acid in water and diluting to 2 0 L Solution A2 Sodium Acetate Teihydrate 40 mM Prepare by dissolving 10 9 g of Sodium Acetate Trihydrate MW 136 1 g mol in water and diluting to 2 0 L Solution A3 Sodium Chloride 2 0 mM Prepare by dissolving 233 6 g of Sodium Chloride MW 58 4 g mol in water and diluting to 2 0 L Y Cancel Figure 10 1 Buffer Blending Setup Dialog for a Single Component Buffer r Buffer Blender pH Correction Values Buffer System By Name CO pH Range Z Use pH Correction Formate Acetate Phosphate pH 3 10 to 6 80 Y Buffer System For
94. in both dialogs Load Inject Sample Tab Selects injection type The AVR7 3 sample inject valve will move to the Inject position at the start of the step and return to the Load position at the end of the step Static Loop Standard fixed volume loop for sample loading Used with the AVR7 3 valve sample can be loaded with a syringe through port 2 and the valve can rinse the sample An auxiliary AUX pump and valve such as the SVT3 2 SV5 4 or AVR9 8 may be used to fill the loop on the AVR7 3 prior to injection onto the column Refer to Chapter 8 for discussion of how to select volume fill and rinse Dynamic Loop Uses Bio Rad s DynaLoop or other sliding piston sample loop Both partial loop and full loop injections can be programmed The dynamic loop can be filled manually through Inject valve port 2 An auxiliary AUX pump and valve such as the SVT3 2 SV5 4 or AVR9 8 may be used to fill the loop on the AVR7 3 prior to injection onto the column The Rinse function is not available See Chapter 8 for DynaLoop loading instructions Direct Inject Allows direct injection of sample through either the Workstation pump not available when Buffer Blending is turned on or auxiliary load pump Pre pump valves such as the SVT3 2 SV5 4 or AVR9 8 automate direct injection Use of an auxiliary pump such as the Bio Rad Model EP 1 Econo pump or Econo Gradient Pump allows direct injection of sample onto a low pressure column Use of an auxiliary A
95. name When selected this feature automatically generates run names based on the current Run 1 run name Refresh updates the run names e Auto increment Automatically generates scout run parameters by incrementing the Run 1 scout parameter by the Increment Value The scout Increment Value can be positive or negative Note you should create your protocol with the appropriate starting value before defining the scout e Finish Saves the scout setup Note the protocol cannot be edited once a scout has been defined unless the scout step is deleted 7 27 MODES OF OPERATION SYSTEM OPERATION Table 7 11 continued Scouting Creating a Scout Experiment Setting up a Scout Method 1 gt oO Connect and plumb all the required hardware components to the DuoFlow system Define the required devices in the device setup Prepare sufficient buffers for the number of runs to be performed Prime the system and equilibrate the column s that will be used Adding a Scout Step 1 7 28 Write a new method copy an existing method or use a Bio Rad method template This method will be used for the first scout run Make sure all parameters are set correctly and the necessary devices have been defined in the device setup Once a Scout step has been added to a protocol it cannot be modified unless the scout step is deleted Press the Scout button on the lower left corner of the protocol screen to add a scout step to the protocol using the
96. nm steps When using the deuterium lamp for the monitoring of wavelengths gt 380 nm an automatic cutoff filter is activated Because very little light of wavelengths greater than 400 nm is emitted from a deuterium lamp the halogen lamp is required for routine detection above 380 nm The QuadTec includes the following 3mm Peek Flow Cell 1 ul flow cell volume 4 ul including flow cell inlet and outlet tubes 10 32 Fingertight fittings quantity 4 2 23 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW e System Cable 25 RS232 To connect to the Instrument Control Module ICM If a Maximizer is in use connect to its COM1 port instead of the ICM Power cord An optional 2 mm PEEK flow cell 2 2 ul flow cell volume 18 pl including flow cell inlet and outlet tubes is available for flow rates up to 80 ml min including 10 32 long Fingertight fittings quantity 4 AUTO ZERO KEY ARROW KEYS SCAN KEY NUMERIC KEYS FLOW PATH PORT FLOW PATH PORT FLOW CELL Pos Figure 2 8 QuadTec UV Vis Detector For a complete discussion of the QuadTec detector refer to its separate documentation 2 24 SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM 2 5 4 pH Monitor The BioLogic DuoFlow pH monitor enables direct monitoring of pH conditions during a run The pH monitor is optional for all DuoFlow systems The pH monitor consists of the following Flow cell The PEEK flow ce
97. nnne nnnerr nn en nnne 2 29 2 14 Two Examples Using AVR9 8 Valves ssssssseseneeenm eene nnne nnne 2 30 2 15 SV5 4 Buffer Select Valve eee dei edet ena dne a eee 2 31 2 16 Two Examples Using SV5 4 Valves sssssssssssseeeeeeneeneeen nennen nemen nnne 2 32 2 17 SVT3 2 Diverter Valvatne nennen nnne EENE nnt 2 33 2 18 Two Configurations of the SVT3 2 Valve ssssssessssseseeeeenen eene nemen 2 34 2 19 BioFrac Fraction Collector sorarsan 2 35 2 20 Model 2110 Fraction Collector with Optional Dust 2 37 2 21 Model 2128 Fraction Collector nennen nemen nnne nen 2 38 2 22 DytlaboOp ec er e tdeo ke NR e Ede RR 2 40 2 23 Plumbing the DynaLoop for use with an Inject Valve 2 41 2 24 Model 1 PUMP 2 42 2 25 EGP Econo Gradient 2 45 2 26 Rack Assembly 5 ee ica eite Rc ne RR 2 46 2 27 Making 1 4 28 Flat Bottom enne 2 48 2 28 Backpressure Regulator sssssssssssssssesseeneeeeneneeen nnne enhn ennt nnne nennen 2 49 2 29 Signal Import Module front and rear
98. place the backpressure regulator between the pump outlet and mixer Equilibrate the column for at least 30 minutes to allow all bubbles to clear Always degass buffers before use Make sure all fittings are tight to prevent leaks If liquid leaks down between the flow cell and the optics module case the cell should be removed and dried with a stream of pure nitrogen never use compressed air The case should be dried using a warm air blower or left for several hours to completely dry Air bubbles trapped in the Workstation pump give rise to erratic flow rates that are sometimes interpreted as problems with the UV detector Refer to the previous section If cleaning of the UV detector and the Conductivity flow cell is desired note the following Filters The standard filter tray contains both 280 nm and a 254 nm filter To clean the filters loosen the filter s thumbscrew and lift out the filter holder The filters should be cleaned only when necessary using a dry lens tissue To remove greasy fingerprints use dilute ethanol or isopropanol solutions UV Flow Cell To remove the flow cell undo the flat bottom fittings at the top and bottom of the flow cell Then loosen the thumbscrews and push out the flow cell from the bottom The flow cell is keyed to mount in one way only so that it cannot be incorrectly mounted Avoid touching the quartz windows with fingers If the flow cell is visually dirty it should be cleaned with one or mor
99. plugged filters There may be air bubbles trapped in the pumpheads causing erratic liquid delivery Note Always degas buffers before use Re prime the pumps and purge the lines a Re prime the pumps and ensure that the priming port is shut tight b Make sure the Inject Valve is set to Purge position and purge all lines Refer to the Maintenance section on how to prime the pump and remove trapped air bubbles To minimize the problem of air bubbles degas buffers by stirring vigorously under vacuum for approximately 20 minutes Use a heavy wall side arm Erlenmeyer flask as standard flasks may implode under vacuum 12 3 TROUBLESHOOTING MAINTENANCE AND TROUBLESHOOTING Problem Possible Cause Solution Pump is not Check valves are fouled 5 Clean or replace the check valves see delivering the should be replaced Section 11 3 3 Routine Maintenance of the correct flow rate Workstation Pumps continued The check valves should be a Cleaning Sonicate check valves in a warm replaced on a regular basis detergent solution rinse with DI water and depending on the usage and re install type of solution or sample Replacing Replace badly clogged or pumped through the damaged check valves pumphead Refer to the b Recalibrate the pumps after Maintenance section for more cleaning replacing check valves Select information on how to replace Gradient Pump Calibration from the a check valve Utili
100. plugs Caps 5 pack 1 4 28 to M6 FPLC column adapter 2 1 4 28 to 1 4 28 union 5 1 4 28 plug 5 HPLC Column to DuoFlow System Fittings Kit 1 set Econo Column to DuoFlow System Fittings Kit 1 set Econo Pac Cartridge to DuoFlow System Fittings Kit 1 set Econo Column to DuoFlow System Fittings Kit 1 set Tubing PTFE tubing 1 8 3 2 mm OD x 0 062 1 6 mm ID x 15 feet 4 6 m for pre pump connections PEEK tubing 1 16 1 6 mm OD x 0 020 0 51 mm ID x 30 feet 9 2 m for flow rates less than or equal to 10ml min PEEK tubing 1 16 1 6 mm OD x 0 030 0 76 mm ID x 30 feet 9 2 m for flow rates greater than 10ml min DuoFlow F10 Tubing Kit Includes premade tubing for basic installation of DuoFlow with F10 Workstation system DuoFlow F40 Tubing Kit Includes premade tubing for basic installation of DuoFlow with F40 Workstation system Maximizer Tubing Kit Includes premade tubing for buffer connection DuoFlow pH Monitor Tubing Kit Fraction Collectors BioFrac Kit 110 120V Includes the base unit 100V power cord Fittings kit Diverter valve Instruction manual and Rack set F1 2 x flatpack 12 13 mm BioFrac Kit 220 240V Includes the base unit Fittings kit Diverter valve Instruction manual and Rack set F1 2 x flatpack 12 13 mm BioFrac Diverter valve BioFrac Rack set F1 2 x flatpack 10 13mm BioFrac Rack set F2 2 x flatpack 15 16mm BioFrac Rack set F3 2 x flatpa
101. sleeves to the middle of all four long vertical bars Remember that the sleeves should be oriented so that the wide part of the taper is nearest the rod s bottom end COLUMN CLAMPING ARRANGEMENT TRAY TAPERED COLLAR Figure 2 26 Rack Assembly For a 2 or 3 tray rack place the second tray on top of the four sleeves and press firmly to seat the tray Repeat these instructions to add the upper tray Mount the 2 piece column clamping arrangement across the two long rods using the attached thumbscrews Two horizontal bars with rod clamps and an Allen wrench are provided with the system These are used to hold devices such as valves Position them between any of the upright bars Remove the four green caps covering the holes at the four corners on top of the Workstation Place the rack into the four corner holes SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS 2 9 2 Starter Kit The Starter kit is included with each DuoFlow system The kit includes step by step instructions for programming and running a separation of a premixed anion exchange standard containing equine myoglobin conalbumin chicken ovalbumin and soybean trypsin inhibitor using a 1 3 ml UNO Q1 Column The Starter kit includes the following items for running a separation 50 ml of Buffer A 250 mM Tris buffer pH 8 1 10 X concentrate 50 ml of Buffer B 250 mM Tris buffer pH 8 1 plus 5 0 M NaCl 10 X concentrate
102. supports Refer to bulletin 1946 2 55 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW Macro Prep CHT Support Macro Prep ceramic hydroxyapatite CHT overcomes the physical and chemical instability of crystalline hydroxyapatite and is available in two types Type has a high protein binding capacity and better binding capacity for acidic proteins Type 11 is better suited for proteins that elute early and for nucleic acids Refer to bulletins 1842 C 100 1927 1971 and 2156 2 10 6 Size Exclusion Chromatography SEC The SEC supports are available in the following formats e HPLC SEC Columns Bio Sil and Bio Select 5 um silica based columns separate compounds by the mechanism of size exclusion The technique is based on diffusion in and around highly porous spherical silica beads The columns are recommended for the separation of peptides proteins and nucleic acids for desalting or buffer exchange and for molecular weight or molecular constant determination Columns are available in both stainless steel Bio Sil columns and biocompatible PEEK plastic hardware Bio Select columns Each column is shipped with a free vial of Bio Rad s protein standards Refer to bulletins 1737 and 1946 Econo Pac P6 Low Pressure Chromatography Cartridges This 5 ml cartridge is used for desalting and buffer exchange Refer to bulletin 1946 2 10 7 High Pressure Reversed Phase Columns The following type of column is available e Hi
103. taken to prevent cross contamination Purge steps should be included before placing a column inline to ensure that the tubing is clean and filled with the appropriate buffer The following three steps should be included as part of a column switching step 1 Add a Column Switching step to your protocol and select the Column Bypass position 2 Add an isocratic flow step to wash the tubing and mixer with 3 5 volumes of buffer 3 Add a Column Switching step to your protocol and select the desired column Column switching steps are usually placed at the beginning of a protocol or at the start of each dimension in a multi dimensional chromatography experiment SAMPLE LOADING ADVANCED SYSTEM APPLICATIONS 9 2 REVERSE FLOW CHROMATOGRAPHY Reverse flow chromatography is generally used in affinity chromatography experiments to prevent peak broadening In affinity chromatography most protein sticks to the top of the column and is eluted off through the column This results in band broadened due to diffusion In reverse flow chromatography molecules are eluted directly off the top of the column and do not pass through the column This results in sharper bands For this application an AVR7 3 reverse flow valve in addition to the AVR7 3 sample inject valve is required AVR7 3 VALVE AVR7 3 VALVE FOR SAMPLE APPLICATION AS CHANGE FLOW VALVE WORKSTATION PUMP FORWARD FLOW COLUMN 2 SAMPLE LOOP 1 SAMPLE E INJECT
104. that any hazardous material has been flushed from the system the pumps are not running and any residual pressure has been bled from the system Disconnect the Workstation power cord Wear eye protection gloves and other protection as appropriate 1 Replacing a piston seal Refer to Figures 11 1 and 11 2 Each pump consists of PEEK pump blocks and screws piston seals O rings and check valves This procedure will require new piston seals See Appendix D Ordering Information a Soakthe piston seals in 5096 methanol to thoroughly wet them sonicate in 5096 methanol if possible b Disconnect all tubing from the pumphead c Using the pumphead disassembly Allen wrench or any standard 9 64 Allen wrench remove the four long screws on the face of the pumphead center block see Figure 11 1 Pull the pumphead away from the Workstation d Note that there are two piston assemblies per pumphead see Figure 11 2 Each piston assembly consists of a piston piston guide and spring Use a lab tissue to avoid getting fingerprints or oils onto the piston assembly Carefully remove each piston assembly from its piston well in the pumphead assembly and set it aside PUMP HEAD ASSEMBLY OUTLET BLOCK CENTER BLOCK LONG BA C SCREWS DUM 8 32 X 1 5 NOTE FOR BOTH SHORT AND LONG SCREWS USE PUMPHEAD DISASSEMBLY ALLEN WRENCH OR STANDARD 9 64 ALLEN WRENCH INLET BLOCK Figure 11 1 Workstation Pump Mechanism Parts
105. the 40 psi backpressure regulator in place Flush the system with water and then fill and flush with the buffers to be used for the separation Cleaning and replacing a check valve a Soak the new check valves in 50 methanol to wet them Sonicate in 50 methanol if possible Remove the two short screws at the top of the pumphead assembly and the two long screws at the bottom of the pumphead assembly Each pump separates into three blocks revealing four check valves and two O rings Replace the check valves It is not necessary to replace the O rings as they are not subjected to high pressure Reassemble the pumphead Refer to Figure 11 3 Check valves fit into the pumphead module in one direction only Wipe the pistons with lab tissue before returning them to the piston wells and rinse the piston guides in running water The piston assemblies should be oriented so that the flat faces on the piston guides are in the vertical position MAINTENANCE AND TROUBLESHOOTING MAINTENANCE f Using the four long mounting screws removed earlier remount the pumphead assembly to the Workstation so that it is secure Insert and alternately tighten the mounting screws Do not over tighten g Using the priming port on the front of the pumphead purge the pump with 5096 methanol and then flush with water 3 Care of the Pistons To maintain pump performance the pumphead should be rinsed on a nightly basis The pump may be rinsed a
106. the Windows clipboard and pasted into another Windows program Choose Copy zoom Chromatogram to clipboard from the Edit drop down menu Export Chromatogram Image File Save in amp Export v File name Save Save as type Export Image wmf Y C ancel Figure 7 16 Exporting a Chromatographic Image 7 44 ADVANCED SYSTEM APPLICATIONS SAMPLE LOADING 8 0 SAMPLE LOADING The BioLogic DuoFlow system is capable of running both simple and complex experiments types This Section describes how the optional hardware components can be used to incorporate a variety of advanced features into a method The BioLogic DuoFlow system supports several methods for sample loading Typically a sample is loaded through a static fixed volume loop as described in Chapter 4 However when automation or large volume sample injection is required the following options are available e Automatic loop fill and rinse using an auxiliary pump inlet valve and an auxiliary pump Direct sample loading onto low pressure columns using an auxiliary pump such as the Econo Gradient Pump EGP or EP 1 Econo pump Direct sample loading through the Workstation pump Sample loading using the DynaLoop sliding piston loop 8 1 AUTOMATIC LOOP FILL AND RINSE With the addition of an Econo Gradient Pump EP 1 Econo pump or other compatible pump as an auxiliary load pump along with an auxiliary pump inlet v
107. ul injection loop Nominal pressure limit of 4000 psi 750 0493 250 ul injection loop Nominal pressure limit of 4000 psi 750 0494 500 ul injection loop Nominal pressure limit of 4000 psi 750 0495 1 ml injection loop Nominal pressure limit of 3000 psi 750 0496 2mlinjection loop Nominal pressure limit of 3000 psi 750 0497 5 ml injection loop Nominal pressure limit of 3000 psi 750 0451 DynaLoop 25 for 25ml samples 750 0452 DynaLoop 90 for 90ml samples D 2 APPENDIX D ORDERING INFORMATION 760 0550 750 0560 750 0553 750 0554 750 0570 750 0571 750 0569 750 0556 750 0703 750 0704 750 0566 750 0559 750 0561 750 0562 750 0563 750 0564 750 0565 732 0113 750 0471 750 0603 760 0604 760 0605 760 0650 760 0652 760 2002 760 2046 741 0002 741 0003 741 0008 741 0010 741 0011 741 0012 741 0013 741 0014 741 0015 741 0016 741 0017 741 0018 Fittings DuoFlow Fittings Kit DuoFlow Fittings Tool 1 8 3 2 mm Pre pump Fittings includes nut ferrule and lock ring 10 1 16 1 6 mm Post pump Fittings includes nut ferrule and lock ring 10 1 8 3 2 mm Nut 5 1 8 3 2 mm ferrule and lock ring 5 1 16 1 6 mm Nut 10 1 16 1 6 mm ferrule and lock ring 10 Inline Filter Kit Includes one inline filter two replacement Ultra High Molecular Weight PolyEthylene UHMWPE filter frits Filter frits replacement 5 Bottle Cap Kit includes two bottle caps two cap
108. user name project name method name run name Edit File View Utilities Options Window Help a Me New E mae De Settings 6 All Users 1 user Open ppm 5 20 2001 Copyin Selected Items to Project f Default 5 20 2001 1 project Edit Delete Print CopyOut Copyln Move B af Select All Queue Compare N Clear List O Q PO 0 ear LIS Reset Information f MOVE List QUEUES COMPARE COPYIN List Error List QuadTec WL1 280nm WL2 260nm J WL3 214nm WL4 405nm Econo Gradient Flow Rate EGP Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 0 Maximizer Gradient Pump F10 UV Conductivity SIM1 SIG SIMt pH Y 1 00ml min 0 B2 438 psi 1 003 AU 1 23 mS cm 0 548 Volt 7 00 pH Move Queue Figure 6 3 Copyln with Information in Browser Tab Window Allows you to transfer a selected Project or Method from one location to another For example a Project can be moved from one User to anot
109. which method protocol a particular run is tied to For example assume you make three runs under a method named lon exchange UNO Q1 During the fourth run you may decide to pause the run and enter the Protocol screen to increase the length of the salt gradient In this case you will first be prompted to rename the method e g lon exchange UNO Q1 Rev 1 before proceeding with the edit Upon completion of the run the database of methods and runs will show three runs associated with the method lon exchange UNO Q1 and one run associated with the method lon exchange UNO Q1 Rev 1 e A fraction collection scheme is editable but it cannot be added to a protocol once run has been started In such cases you should abort the run and select Edit the Method e currently programmed fraction collection scheme may be edited during a run but note that changing the fraction size of a Collect scheme will first require selection of Collection Windows This is because the editing process effectively turns the initial Collect All scheme into the first collection window 7 35 MODES OF OPERATION SYSTEM OPERATION BioLogic Duo Flow offline File Edit View Utilities Options Window g BmsD pm u MW Bio Rad eq Settings Cut Copy Paste Delete Web Description Parameters Coll
110. zoom Chromatogram to clipboard Delete selected tag Delete all tags Note The Edit menu is not available in the Manual or Browser screens The contents of the Edit menu depends upon the displayed screen as indicated above In most instances the item in the drop down menu also appears in the system toolbar exceptions are noted below Setup Screen Edit Allows you to edit the selected device in Setup Delete Deletes the currently highlighted device in Setup To delete all devices first select Select All as described below Select All Highlights all devices in Setup Not available from the toolbar Protocol Screen Edit Displays the Edit window for the step selected in the protocol Cut Cuts deletes the currently highlighted step from the protocol A cut step may be pasted elsewhere Copy Copies the currently highlighted step so that it can be pasted elsewhere in the protocol Paste Pastes the cut or copied step into the protocol Delete Deletes the currently highlighted step from the protocol Select All Highlights all protocol steps Cut Copy and Delete then act on all steps To remove the highlighting from individual steps hold down the Ctrl key and click the mouse over the desired step Not available from the toolbar Run Screen Full view in Run Chromatogram Zooms out to show the full view for the run Multiple runs Specifies the number of times the method is to run INTRODUCTI
111. 0100 0 2962 14 5038 0 9869 1 0197 751 88 1 29 5300 Inches Hg 0 4912 0 0334 0 0345 25 400 0 03376 Example 1 Convert 521 psi to Kg cm 521 x 0 0703 or 521 psi 36 63 Kg cm Example 2 Convert 60 Bar to psi 60 x 14 5038 or 60 Bar 870 psi Example 3 Convert 42 70 Bar to Kg cm 42 70 x 1 0197 42 70 Bar 43 54 Kg cm 1 PRESSURE CONVERSION TABLE APPENDIX B B 2 APPENDIX C WARRANTY APPENDIX C WARRANTY STATEMENT Bio Rad Laboratories warrants that every instrument it sells will be free from defects in materials and workmanship when it leaves the factory and that if such defects appear within the first year following purchase the defective part s will be replaced or the entire unit will be replaced at Bio Rad s option free of any charges to the buyer other than expenses incurred in returning the unit to the factory Bio Rad s obligation under this warranty is specifically limited to the aforementioned replacement or repairs However the following defects are specifically excluded Defects caused by improper operation Repair or modification done by anyone other than Bio Rad Laboratories or their authorized agent Use with fittings or other spare parts not specified by Bio Rad Laboratories Damage caused by deliberate or accidental misuse Damage caused by disaster Damage due to use of improper solvent or sample Tubing fittings lamps pistons check valves piston seals and other consu
112. 1 In the Browser screen select the user name and open the method that contains the runs you wish to compare Select the NEW icon from the left screen sidebar and from the displayed menu select New Compare To place runs into the compare dialog highlight the runs and click the Compare icon on the sidebar Repeat until all desired runs are in the lower screen Project Compare window This places each run into your Project Compare and the Compare Tab window at the bottom of the screen e When you highlight the Compare icon under the Project Compare the list of runs will appear in the Tab window e Multiple methods may be selected in the Browser by either shift click or control click 6 11 INTRODUCTION TO THE BROWSER SCHEEN SYSTEM OPERATION Description of the Compare Icons Open Run Opens highlighted run in the PostRun screen ey Open Run Remove Run Deletes a highlighted method from the compare Remove Run Compare Traces Opens the Trace Compare window Edit Compare Permits editing of the compare name description and 27 Edit Compare author 6 5 TRACE COMPARE Trace Compare is a tool used to simultaneously visualize and compare chromatography data In Trace Compare run data can be compared side by side in Tiled mode or overlaid on top of each other in Overlay mode The Trace Compare screen consists of five functional regions as described in the following Sections s
113. 11 6272 Korea Ph 82 2 3473 4460 Fx 82 2 3472 7003 Latin America Ph 305 894 5950 Fx 305 894 5960 Mexico Ph 52 5 534 2552 to 54 Fx 52 5 524 5971 The Netherlands Ph 0318 540666 Fx 0318 542216 New Zealand Ph 64 9 4152280 Fx 64 9 443 3097 Norway Ph 47 23 38 41 30 Fx 47 23 38 41 39 Poland Ph 48 22 8126 672 Fx 48 22 8126 682 Portugal Ph 351 21 472 7700 Fx 351 21 472 7777 Russia Ph 7 095 721 1404 Fx 7 095 721 1412 Singapore Ph 65 2729877 Fx 65 2734835 South Africa 00 27 11 4428508 Fx 00 27 11 4428525 Spain Ph 34 91 590 5200 Fx 34 91 590 5211 Sweden Ph 46 0 8 55 51 27 00 Fx 46 0 8 55 51 27 80 Switzerland Ph 061 717 9555 Fx 061 717 9550 United Kingdom Ph 0800 181134 Fx 01442 259118 Sig 0402 4006229 Rev B
114. 15 750 0210 750 0212 750 0216 750 0217 750 0220 750 0221 F40 Pump Kit Includes fully assembled F40 pumpheads with check valves and seals installed four F40 piston assemblies one mixer barrel extender two mixer O rings a 2mm UV flow cell PEEK tubing 0 030 0 76 mm ID and necessary tools F10 Pump Kit Includes fully assembled F10 pumpheads with check valves and seals installed and four F10 piston assemblies F10 Pump Maintenance Kit services one F10 pumphead Kit includes two piston seals four check valves seal removal tool and two O rings F10 Piston Seals 2 and Seal Tool Two piston seals required to service one F10 pump F10 Piston Assembly 2 Two piston assemblies required to service one F10 pump F40 Pump Maintenance Kit services one 40ml pump Kit includes two piston seals two check valves and two pistons F40 Piston Seals 2 and Seal Tool Two piston seals required to service one F40 pump Check Valve 1 Four check valves required to service one pump for F10 and 40 pumps EZ Logic Integration Software Package USB Bitbus Communicator Mixers MX 1 Mixer Mixer includes mixer body 263yl and expansion barrel 750ul MX 1 Mixer Barrel Extender 2ml MX 1 Mixer Service Kit MX 1 Mixer Barrel Extender 1 750yl Maximizer Mixer Includes mixer body 750ul 5ml and 12ml expansion barrels five O rings stir bar and installation screws for all barrel sizes Maximizer Mixer Barrel Extender 5ml
115. 16 0 5 Tools Drop down Meri reete rer prem tee aee ka ee aun eda eH Ree Ru raea 6 16 6 6 Window Drop down rns nsns nsn nnne ness nnns 6 16 7 1 Valve Setup Information nennen E a aaea a onani 7 7 7 2 i cimo 7 9 7 3 Socratic somete etie oet e ae in t es uat do icu Feed Re taa 7 11 7 4 Load Inject Sample aerei 2 eee ide ceu rea ad n eee 7 12 25 Linear Gradient cito t cto b d et itte 7 15 7 6 Change Valve A E 7 16 ethene 7 17 7 0 Hold iacente 7 18 7 9 7 19 7 10 Fraction Collection BUON 7 20 FeV SCOUT ec 7 25 7 12 Protocol Screen s Editing Toolbar 7 29 7 13 Run Screen s Control ButtOns ccccccccceceeeeeeeeececenneeeeeeeeeeeeceeeeeeccaaaeeeeeeeeeeeeeeeeeeeecnneeeeeeeeess 7 32 9 1 Common Multidimensional Chromatography 9 5 10 1 Buffer Blending Buf
116. 2 8 3 and flow direction as well as start and stop the pump For more detail see the EGP instruction manual Workstation Valves Maximizer Valves These two panels are used to control any of the solenoid and automated valves connected to the workstation and Maximizer The down arrow in the upper right corner of the panel is used to toggle between the workstation and Maximizer valves Chromatogram This panel is used to view up to 8 instrument traces during a manual run Traces may include UV detector conductivity pH pressure theoretical B four QuadTec wavelengths and signals from two external detectors see Chapter 2 9 6 and Table 2 7 for more information about the SIM HR and the Maximizer SIM respectively The chromatogram display is controlled through a combination of the Chromatogram Settings dialog Settings button or Options pull down menu the Resize and Clear Traces buttons and the scroll bars Each scroll bar is assigned to an individual trace by the drop down menu above it Status Bar This panel is used to display numerical data from the BioLogic DuoFlow workstation and its attached devices Parameters include gradient pump flow rate B and pressure UV detector absorbance QuadTec UV Vis wavelength and absorbance conductivity SIM data from a pH probe or an external detector as well as the Econo Gradient Pump flow rate B and Split 7 3 MODES OF OPERATION SYSTEM OPERATION 72 SETUP SCREEN The Setup screen see Fi
117. 25mM Tris 55 100 Volume 2 0 min S ERNS 7 250 Linear Gradient 25mM Tris Salt 45 0 Flow 2 0 ml min 280 Isocratic Flow 25mM Tris 100 Volume 3 0 min Hold 25mM Tris Salt 0 Flow 2 0 ml min End of Protocol Pause Zero Baseline Lamp Q EGP L Repeat Steps Alarm Recorders Y Fraction Collection 22 Scouting WL1 280nm WL2 260nm WL3 214nm WL4 405nm Econo Gradient QuadTec Flow Rate EGP B Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 B 0 Maximizer Gradient Pump F10 Uv Conductivity SIM1 SIG SIM1 pH 1 00ml min 0 B2 438 psi 1 003 AU 1 23 mS cm 0 548 Volt 7 00 pH Figure 7 4 Protocol Screen j EDIT BUTTONS SYSTEM OPERATION MODES OF OPERATION Table 7 3 Isocratic Flow Edit Isocratic Flow Edit Isocratic Flow pH View Isocratic Flow socratic Flow pH View Buffer System Tris 25 mM Buffers 96 competion V
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119. 546 nm 750 0240 Conductivity Flow Cell replacement 750 0230 Backpressure regulator 750 0502 Signal Import Module SIM Valves 760 2008 Maximizer Proportioning Valve 760 0406 AVR7 3 Sample Injection Valve 760 0401 AVR7 3 Valve Rebuild Kit 750 0471 AVR7 3 Sample Injection Port 760 0408 AVR9 8 Stream Select Valve 760 0403 AVR9 8 Valve Rebuild Kit 760 0410 SVT3 2 Diverter Valve 760 0411 SVT3 2 Valve Rebuild Kit 750 0415 SV5 4 Select Valve 125 0224 Inject Needle 22 gauge blunt fits sample injection port 750 0251 Rack 750 0268 Rack expansion kit two trays two vertical bars sixteen sleeves 750 0260 Column clamp set 750 0261 Rack tray includes eight sleeves one drain plug 750 0262 Vertical bar long 2 750 0263 Vertical bar short 2 750 0264 Horizontal bar kit two tie bars four bar clamps 750 0265 Bar clamps 5 750 0266 Cable Manager clips 4 750 0269 System wrench set pH Monitor 760 2040 DuoFlow pH Monitor 760 2042 pH Electrode replacement 760 2044 Flow Cell replacement 750 0502 Signal Import Module SIM 910 1828 O ring pH Flow Cell size 2 009 910 3014 O ring pH Flow Cell size 2 018 760 2046 BioLogic pH Monitor Tubing Kit Sample Injection Loops 750 0490 Small Volume Sample Loop Kit 100 250 and 500yl loops Nominal pressure limit of 4000 psi 750 0491 Large Volume Sample Loop Kit 1 2 and 5ml loops Nominal pressure limit of 3000 psi 750 0492 100
120. 6 ethanol isopropanol and acetonitrile 0 1 trifluoroacetic acid 1 detergents including sodium dodecyl sulfate SDS Triton X 100 Sound level Workstation 70 dBa e Dimensions Workstation approx 35 x 41 x 18 cm W x D x height with 3 tray rack 76 cm Dimensions Maximizer approx 36 x 47 x 11 cm W x Dx H Weight Controller Workstation monitor rack inject valve and detection modules 34 5 kg 76 Ibs approximate Weight Maximizer 4 8 kg Operating temperature excluding Controller and USB Bitbus Communicator 4 40 C Detection Fixed wavelength mercury lamp with 280 254 nm filter auto zero 0 0001 2 0 AUFS detection range Analytical 5 mm pathlength 16 ul total volume flow cell e Preparative 2 mm pathlength 30 ul volume flow cell Optional 214 nm detection kit includes zinc lamp housing and 214 nm filter e Optional wavelength filters include 214 313 365 405 436 and 546 nm e Conductivity detection 500 mS cm 6 ul flow cell accuracy 2 full scale e Optional flow through pH monitor includes flow cell pH electrode and SIM Cold room compatible Sample Loading Fixed sample loops 100 ul to 5 0 ml automated injection valve included Large volume loading through the Gradient Pump Optional inlet select valves SV5 4 AVR9 8 for sanitation and multiple sample use e Variable volume sample loading through the DynaLoop sliding piston sample loop Soft
121. ANNEL 1 pH NON BIO RAD MONITOR UV DETECTOR Figure 3 12 SIM Connections SYSTEM INSTALLATION AND SETUP SYSTEM SETUP 3 6 4 Non Bio Rad Detectors The Signal Import Module SIM available from Bio Rad allows you to connect a variety of additional devices such as UV fluorescence and RI detectors Before connecting the detector to the SIM consult the documentation provided with the detector to determine the cable requirements for connecting to the 3 pin connector Gnd on the SIM The SIM connects to the DuoFlow Workstation and Controller through the use of the Instrument Bus cables System Cables 17 18 19 21 or 30 as shown above Channel 1 signals are sent to the chart recorder by connecting the UV detector to the chart recorder using bare wires to banana plugs System Cable 20 transmits start stop pen up down and event mark signals 3 13 SYSTEM SETUP SYSTEM INSTALLATION AND SETUP 3 7 VALVE CONNECTIONS The DuoFlow system is shipped with an AVR7 3 inject valve The connection of all valves is similar as discussed below To connect either the AVR7 3 inject valve or an AVR9 8 stream select valve mount the valve to a vertical bar on the system rack and connect its cable to any of the connectors labeled Automated Valves 10 11 or 12 on the rear of the Maximizer if available Otherwise connect it to connectors 4 5 or 6 on the Workstation connect an SVT3 2 diverter valve or the SV5 4 buffer select val
122. AUX PUMP e e m e OVTAMP 10 25 03A MAX Solenoid Valves To connect DuoFlow low pressure solenoid valves SV5 4 and SVT3 2 to the system If a Maximizer is in use connect to its solenoid valve connectors before those on the Workstation Automated Valves To connect DuoFlow high pressure automated valves AVR7 3 Inject and AVR9 8 Stream Select to the system If a Maximizer is in use connect to its automated valve connectors before those on the Workstation Cond Flowcell To connect the Conductivity monitor flow cell to the system If the Maximizer is being used you must use its Cond Flowcell connector rather than the connector on the Workstation COND FLOWCELL O BEANO BEES 9 2 e UV Lamp This specialized 6 pin square connector provides electrical power to the mercury or zinc lamp in the UV detector lamp housing This connector is not available on the Maximizer Mixer To connect the mixer to the system If the Maximizer is in use connect to its mixer connector before those on the Workstation 10 25V 0 3A Max Provides electrical power to the Model 1327 chart recorder or Instrument Control Module ICM UV Optics To connect the UV detector to the system Cond Chart For conductivity signal output to a single or dual pen chart recorder An 8 pin mini DIN to banana plug cable System Cable 4 for connection to the Model 1327 chart
123. C iei DETECTOR ICM MODULE BIOFRAC Figure 3 4 System Cable Connections without Maximizer SYSTEM INSTALLATION AND SETUP 3 3 2 Systems with a Maximizer SYSTEM SETUP If the Maximizer is to be part of the system follow the procedure below to connect the Maximizer to the Workstation and the USB Bitbus Communicator 1 2 Place the Workstation on top of the Maximizer Note the two connectors marked Instr Bus These connectors are identical either may be used when connecting instrument bus cables With the back side of the Workstation and Maximizer facing you connect System Cable 30 between the Workstation and the Maximizer Select a System Cable of sufficient length to reach the USB Bitbus Communicator System Cables 17 18 19 21 and 30 are different only in their lengths Connect the power cable to the Maximizer Do not turn on this device yet SYSTEM CABLE 30 DELL CONTROLLER S AVR7 3 INJECT USB BITBUS VALVE COMMUNICATOR MAXIMIZER e 5 U FF MAXIMIZER MIXER SYSTEM CABLE 25 CONDUCTIVITY MONITOR pH MONITOR QUADTEC DETECTOR SYSTEM CABLE 17 18 19 OR 21 MODEL 1327 CHART RECORDER SYSTEM CABLE 2
124. DESCRIPTION OF SYSTEM COMPONENTS 2 7 FRACTION COLLECTORS The DuoFlow features several options for fraction collection BioFrac fraction collector Model 2110 fraction collector e Model 2128 fraction collector e Non Bio rad fraction collectors The BioFrac fraction collector the Model 2110 fraction collector and the Model 2128 fraction collector are programmable and can be run via BioLogic software version 4 0 or higher 2 7 1 BioFrac Fraction Collector The BioFrac functions with the BioLogic DuoFlow system and as a stand alone fraction collector with the BioLogic HR and non Bio Rad chromatography systems It is suited for both analytical and preparative applications Figure 2 19 BioFrac Fraction Collector Features of the BioFrac include It be used with any chromatography system at flow rates up to 100 ml min It can collect in time drop or volume mode time and drop mode are only available in stand alone mode Adiverter valve mounted on either side of the vertical bars diverts unwanted eluant to waste e Can be fitted with an optional drop former 25 yl drops designed for collecting small sample sizes 2 35 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW BioFrac collects fractions in a serpentine pattern for all racks but may be changed to a row or column pattern for microplates and microtiter tubes Fourteen collection choices are possible with a total of nine racks The BioFr
125. EEK orange 1 16 OD x 0 02 ID tubing to connect the pump mixer column detector and other components 2 47 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW F40 Tubing kit The F40 Tubing kit is identical to the F10 kit described previously however all orange PEEK tubing is replaced with PEEK green 1 16 OD x 0 03 ID tubing The large bore tubing is designed for use with the higher flow rates when using the Maximizer or FA0 Workstation pumps Maximizer Tubing kit The tubing kit that accompanies the Maximizer for connecting the reagent bottles to the valves is composed of FEP PTFE 1 8 OD x 0 062 ID Each length of tubing is color coded to identify its connection site and solution Inlet A1 uses red tubing and is used for acid solutions Inlet A2 uses blue tubing and is used for base solutions Inlet B1 uses yellow tubing and is used for water solutions Inlet B2 uses green tubing and is used for salt solutions The kit includes an installation chart Maximizer Interconnect Tubing kit This kit contains cut and fitted tubing to connect the Workstation pumps A and B inlets to the Maximizer valves A and B outlets There are two preformed PEEK 1 8 OD sets of tubing in this kit pH Montior Tubing kit The pH monitor includes cut and fitted tubing to connect the pH flow cell to the Conductivity flow cell There is a length of PEEK orange 1 16 OD x 0 02 ID tubing rated to 5000 psi and a length of PEEK green 1 16 OD x 0 03
126. EM OVERVIEW LARGE VOLUME SAMPLE LOADING WORKSTATION WASTE PUMP 6 LOW PRESSURE COLUMN SAMPLE LOADING AND AFFINITY CHROMATOGRAPHY AVR7 3 VALVE FOR SAMPLE LOADING WORKSTATION PUMP 6 oe AVR7 3 VALVE AS CHANGE FLOW VALVE JER 2 3 SAMPLE LOOP 1 SAMPLE INJECT EZ WORKSTATION PUMP UV MONITOR amp FRACTION COLLECTOR COLUMN SAMPLE LOOP SAMPLE INJECT H UV MONITOR amp FRACTION COLLECTOR AVR7 3 AS COLUMN SWITCHING VALVE SAMPLE LOADING COLUMN 2 UV CONDUCTIVITY FLOW CELLS Figure 2 12 Examples of AVR7 3 Valve Tubing 2 28 SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM 2 6 2 AVR9 8 Stream Select Valve The AVR9 8 stream select valve is an 9 port 8 position valve This valve is optional for all DuoFlow systems The valve is rated at 3500 psi 233 bar and is designed with non metallic wetted parts for bio compatibility and minimal internal dead volumes e The AVR9 8 is ideal for stream selection column switching and large volume fraction collection The AVR9 8 stream select valve uses a patented make before break design MBB that prevents pressure spikes when the valve rotates from one port to another eliminating baseline interferences and prevents pump shutdowns due to transient over pressure situations This is especially beneficial
127. F Loosen the two knurled screws on the front of the flow cell and gently pull them out This allows the flow cell housing to slide out Remove the dummy cell by gently pulling it upward Save the dummy cell in a secure place It is required for measuring signal and reference output values for the lamps See Section 5 1 Checking the Status of D2 Lamp in the QuadTec instruction manual for instruction on recording signal and reference values Insert the new flow cell and make sure that the engraved specifications point towards the user and that the fixing hole on the back side of the cell meets the corresponding metal pin of the detector s housing Slide the complete system towards the detector insert the two knurled screws and tighten them by hand Attach the 10 32 Fingertight fittings and tubing to the inlet and outlet ports and use a syringe to rinse the flow cell with 10 20 ml of 10096 analytical grade methanol followed by 5 ml distilled deionizied DDI water Connect the QuadTec to the DuoFlow system If the Maximizer is to be used the QuadTec should be connected to the Maximizer rear panel Com 1 connector using System Cable 25 If the Maximizer is not in use connect the QuadTec via the Instrument Control Module ICM as discussed on page 3 11 FLOW PATH PORT FLOW PATH PORT L FLOW CELL Ee om Figure 3 9 QuadTec Detector FLOW CELL HOUSING SYSTEM INSTALLATION AND SETUP S
128. Gradient Pump panel when a Maximizer is connected to the system and is used to control buffer composition flow rate and pressure limits see Table 2 3 as well as to start and stop the pumps When Buffer Blending is turned on composition is determined by pH and Inlet B When Buffer Blending is turned off composition is determined by percent Inlet A1 or A2 and percent Inlet B1 or B2 The Start Stop buttons are used to start and stop the pumps The Set button is used change the composition flow rate and pressure limits while the pumps are running The System and Local option buttons are used to toggle the Maximizer between System and Local mode Fraction Collector This panel is used to control a fraction collector Its appearance depends on the type of fraction collector connected to the system BioFrac or Model 2128 From this panel the Start Tube End Tube Fraction size and rack type are set and the fraction collector can be started or stopped Also displayed on this panel are the current Tube number and the tube Volume left to fill The System Local option buttons are used to toggle a BioFrac or Model 2128 fraction collector between System and Local mode Only fraction collection by volume is supported in manual mode When a BioFrac is connected to the system the rack grid numbers for the start and end tubes are also displayed UV Detector Chart Recorder Signal Import Module This panel is used to control the standard UV monitor and char
129. Indications of valve damage include valve leaks and valve switching problems To replace a valve you ll need a standard 9 64 hex key one has been provided for this purpose Safety Note Before removing a valve be sure to flush any hazardous material from the valve Wear protective clothing as appropriate To remove the valve follow the procedure below and refer to Figure 11 9 1 Use the 9 64 hex key to remove the two screws that attach the valve to the Maximizer Set the screws aside 2 Lower the front of the valve to disengage the outlet tubing from the top of the valve Slowly pull away the valve to expose the wiring behind the valve Using your fingers press the connector latch to separate the valve wiring from the Maximizer wiring REMOVE MAXIMIZER VALVE DISCONNECT MAXIMIZER VALVE Figure 11 9 Replacing a Maximizer Valve To attach the new valve follow the procedure below 1 Connect the valve wiring to the Maximizer wiring 2 Place the valve against the Maximizer but do not reattach the screws at this time Rotate the valve slightly downward until you can engage the valve with the output tubing to the Workstation Reattach the output tubing using its fittings 3 Reattach the valve mounting screws 11 13 MAINTENANCE MAINTENANCE AND TROUBLESHOOTING 11 14 MAINTENANCE AND TROUBLESHOOTING TROUBLESHOOTING 12 0 TROUBLESHOOTING DUOFLOW SYSTEMS The following section lists potential problems and some s
130. Inject Protocol The protocol Load Inject step should be programmed as follows 1 In the protocol Load Inject step select Direct Inject as the injection type select the Pump A or Pump B option select a sample and set the sample volume and flow rate 2 Add an isocratic step after the load step to ensure that the entire sample is loaded onto the column The inlet tubing and mixer should be rinsed with at least 5 volumes of running buffer to ensure that the entire sample is loaded onto the column 3 Add protocol cleaning steps to ensure that all the residual protein has been removed from the tubing and mixer Add a Change Valve step to move the AVR7 3 sample inject valve to the Purge position a b Add an isocratic flow step and run 3 5 volumes of Buffer B through pump B 9 Add an isocratic flow step and run 3 5 volumes of Buffer A through pump A d Adda change Valve step to move the AVR7 3 sample inject valve back to the Load position System Sanitization For sanitization 1 0 M sodium hydroxide NaOH may be used Be sure to wash with water to remove the base Refer to Chapter 11 Maintenance for information on how to store the DuoFlow system SAMPLE LOADING ADVANCED SYSTEM APPLICATIONS 8 4 DYNALOOP SAMPLE INJECTION The DynaLoop which is available in 25 ml and 90 ml sizes is used to load large volumes of sample directly onto chromatography columns These loops have a sliding piston and function very much like a syringe
131. It is recommended that valves peripheral devices and flow cells be connected to the Maximizer rather than the Workstation unless more valving capability is needed 2 11 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW Table 2 7 Maximizer Front Panel Controls LCD DISPLAY VALVESA amp B VALVE INLET SELECT BUTTONS ON OFF VALVE INLET SOFT KEYS STATUS LEDs Power On Off Controls power to the unit Valves amp B Proportioning valves used for Buffer Blending and inlet selection Each valve has two inlet ports and one outlet port For Buffer Blending acid is placed at inlet A1 base at inlet A2 water at inlet B1 and salt and inlet B2 The valve outlet ports are connected directly to their respective Workstation pump inlet ports Color coded tubing is supplied with the Maximizer for plumbing the inlet ports A1 red A2 blue B1 yellow and B2 green Valve Inlet Used when the Maximizer is in Local mode to change the position of valves A and B Select Buttons Valve Inlet Status LEDs LCD Display Displays the Maximizer status When the Maximizer is operated from its front panel controls the LCD displays status and control information including valve positions pH and conductivity calibration information and current temperature Indicates which valve port is open 2 12 SYS
132. LE AVR9 8 AS COLUMN LOOP SWITCHING VALVE SAMPLE TO RECEIVE SAMPLE INJECT UP TO 8 DIFFERENT COLUMNS COLUMN OUTLET SECOND AVR9 8 AS COLUMN SWITCHING VALVE TO DIRECT ELUENT TO DETECTORS AND FRACTION COLLECTOR UV DETECTOR AND FRACTION COLLECTOR Figure 9 2 Column Switching using Two AVR9 8 Valves For this application two AVR9 8 valves are required 9 2 ADVANCED SYSTEM APPLICATIONS SAMPLE LOADING System Setup 1 Plumb two AVR9 8 valves as shown in Figure 9 2 Each column inlet should be connected to the column inlet valve and each column outlet to the column outlet valve Unless eight columns will be connected a length of tubing should be plumbed between the two AVR9 8 valves at position 1 to be used as a column bypass This facilitates washing of the system between column switching events Column bypass tubing or 74 28 plugs should be placed at each unused valve port 2 Plumb the common port of the column switching inlet valve to port 4 of the sample inject valve 3 Plumb the common port of the column switching outlet valve to your detector In the device setup define an AVR9 8 valve as a Column Switching valve In the AVR9 8 valve dialog identify the ports where the two AVR9 8 valves are connected to the Workstation or Maximizer and name each valve positions e g Column Bypass Column 1 Column 2 etc Writing the Column Switching Protocol When using multiple columns care should be
133. Left WL1 280nm 0 00 0 0 0 ml 0 00 0 0 0 ml Port 2 User Valve Position 1 WL1 280nm WL2 260nm 214nm WL4 405nm Econo Gradient QuadTec Flow Rate EGP Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 0 Maximizer Gradient Pump F10 UV Conductivity SIM1 SIG Y SIM1 pH 1 00ml min 0 B2 438 psi 1 003 AU 1 23 mS cm 0 548 Volt 7 00 pH Figure 7 5b Run Screen showing a Run in Progress with a Time based Chromatogram and Rack and Grid fraction numbering 7 31 MODES OF OPERATION SYSTEM OPERATION Frac Collector Vr Advance Divert Valve Collect Waste Grad Pump High psi 400 Low psi 20 Set Chart Recorder UV Range 0 2 Y Cond Range 500 Y Event mark a E Zero Baseline UV Conductivity New z Method Browser Nare Pause Hold EAN ettings 7 32 Table 7 13 Run Screen s Control Description Advances the fraction collector to the next tube Pressing this button does not modify the method The event is recorded in the Run Log Immediately changes the position of the diverter valve Pressing this button does n
134. M 2 5 Storing the system 11 1 i UV detector SV5 4 buffer select and automated sample loading valve attaching to 3 9 description 2 31 changing the flow cell or filter 2 22 examples using een 2 32 8 5 cleaning 11 6 software setup E DH UHR 7 5 7 6 cleaning UV flow 11 6 valve 3 14 description 2 21 SVT3 2 diverter valve replacing the 11 7 rhet 2 33 software 7 5 cleaning the valve 11 10 example configurations 2 34 V software 7 5 7 6 Valves valve connections MEE 3 14 See AVR7 3 sample inject valve AVR9 8 stream with Mogel 2110 and generic select valve SV5 4 buffer select and automated fraction collectors 7 20 sample loading valve SVT3 2 diverter valve System rack and Maximizer valves assembly ee 2 46 dsseniblyz uer ciem peg 3 6 description 2 45 Workstation pumps T care of 11 5 cleaning and replacing a check
135. Maintenance The pumpheads should be washed daily with water when high salt buffers are used This can be automated through the use of an SVT3 2 valve connected between the high salt buffer buffer B and pumphead B Use one of the valve inlet ports to run the buffer and use the other inlet port to run water through the pumphead If the system is configured with a Maximizer non blending mode use the B1 and B2 inlet valves to run water B1 or buffer B2 through the pumphead Washing behind the piston seal extends seal life Insert a 10 ml syringe filled with deionized water in the hole at the top of the pumphead A 10 ml syringe is provided in the Fittings kit Each pumphead should get a 10 ml washout at the end of the day s operation Run off water exits through the washout drain between the two pumpheads It can be collected in a small beaker 11 2 MAINTENANCE AND TROUBLESHOOTING MAINTENANCE 11 3 3 Routine Maintenance of the Workstation Pumps The Workstation pumps require minimal maintenance to stay in good working condition The DuoFlow F10 Pump and F40 Pump Maintenance kits include the piston seals check valves and O rings for routine maintenance of the pumps Indications that maintenance is required include flow rates that are below the specified rate an erratic delivery of liquid liquid running out of the washout drain during normal operation often seen as salt crystal accumulation and poor gradient performance Safety Make sure
136. NANCE AND TROUBLESHOOTING TROUBLESHOOTING 12 2 TROUBLESHOOTING THE DUOFLOW WORKSTATION PUMP Pump is not delivering the correct flow rate Possible Cause The pumps may not be calibrated The incorrect pumpheads may be mounted or the BioLogic Configuration software utility may not have been run after the pumpheads were changed Solution Recalibrate the pumps Select Gradient Pump Calibration from the Utilities dropdown menu Make sure the correct pumpheads labeled F10 or F40 on the lower right corner of the center block are mounted Then run the BioLogic Configuration software utility and select the correct pumphead There may be problems with the fittings or tubing sizes It is important to use the wide bore 1 8 OD tubing for the pump inlets and from all pre pump buffer selection valves to the buffer containers Check pumps A amp B and pre pump valves a Make sure the inlet lines into pumps A and B are tight b If pre pump valves are used e g for buffer selection make sure all tubing connections are secure and any unused ports are plugged The pump may not be receiving buffer Check that there is buffer flowing to the pump a Ensure that the pump inlet lines are immersed in buffer b Ensure that the buffer bottles are positioned at or above the level of the Workstation Ensure all inlet fittings are secure Ensure that any inlet filters are clean Remove temporarily to test for
137. NS 1 Confirm the voltage setting for the Workstation power supply A red switch on the back of the Workstation allows you to switch between 110 VAC and 240 VAC 2 Connect the power cable to the Workstation Do not turn on this device yet 3 3 1 Systems without a Maximizer If the Maximizer is NOT to be part of the system follow the procedure below to connect the Workstation to the USB Bitbus Communicator 1 Note the two connectors marked Instr Bus These connectors are identical either may be used when connecting bus communication cables 2 Select a System Cable of sufficient length to reach the USB Bitbus Communicator System Cables 17 18 19 21 and 30 differ only in their lengths SYSTEM SYSTEM CABLE CABLE 31 17 18 19 21 OR 30 USB BITBUS DELL CONTROLLER COMMUNICATOR MODEL 1327 CHART RECORDER UUJ AVR7 3 00 d INJECT VALVE SYSTEM SYSTEM CABLE 2 CABLE 4 WORKSTATION ammi nuin q USB CABLE MX 1 MIXER SYSTEM CABLE 26 CONDUCTIVITY d SYSTEM CABLE 17 18 19 21 OR 30 SYSTEM EU i CABLE 25 SYSTEM CABLE 17 18 19 21 OR 30 JL QUADTE
138. OAD PUMP Figure 8 6 Valve Positions During a Run using the DynaLoop ADVANCED SYSTEM APPLICATIONS SAMPLE LOADING 9 0 COLUMN AND BUFFER FLOW SWITCHING APPLICATIONS 9 1 COLUMN SWITCHING A Column Switching valve is used to connect multiple columns to the BioLogic DuoFlow system Inclusion of a column switching valve in the setup can be an advantage if you routinely use a variety of columns on your system One application of this feature includes the running of multiple samples see Section 8 1 and 8 2 where each sample requires its own column Another application is the running of two and three dimensional chromatography experiments where the sample eluted from one column is subsequently injected onto a second or third column There are two types of column switching valves that can be used with the DuoFlow system AVR7 3 two column switching valve and an AVR9 8 eight column switching valve requires 2 AVR9 8 valves 9 1 1 AVR7 3 Two Column Switching AVR7 3 AS INJECT VALVE SAMPLE LOOP AV AS COLUMN SWITCHING VALVE COLUMN SAMPLE INJECT UV DETECTOR AND WORKSTATION FRACTION COLLECTOR Figure 9 1 Column Switching Using Two AVR7 3 Valves For this application an AVR7 3 column switching valve is required in addition to the AVR7 3 sample inject valve System Setup 1 Plumb an AVR7 3 column switching valve as shown in Figure 9 1 Column 1 should have its inlet connected
139. ON CONNECTING TWO SIMs INSTRUMENT BUS DEVICE POWER NUMBER STATUS INSTRUMENT BUS DEVICE POWER NUMBER STATUS TO USB BITBUS COMMUNICATOR TO WORKSTATION Figure 2 30 Cable Connections to the Signal Import Module 2 50 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS 2 9 7 Pump Kits The Workstation pumps are easily converted to either a F10 or 40 pumphead to expand functional pump flow rates as indicated in the table below Table 2 14 Workstation Pump Configuaration Flow Rates Workstation Pump Flow Rate Flow Rate with Maximizer F10 0 01 10 ml min 0 5 20 ml min F40 0 5 40 ml min 1 0 80 ml min There are two kits the F10 Pump kit and the 40 Pump kit Each kit contains fully assembled pumpheads with seals and check valves installed The F10 Pump kit contains the following items Two F10 pumpheads assembled with check valves seals and O rings e Four F10 piston assemblies e Installation instructions PEEK tubing orange 1 6 OD x 0 02 ID is supplied in the Fittings kit of the DuoFlow system The F40 Pump kit contains the following items Two F40 pumpheads assembled with check valves seals and O rings e Four F40 piston assemblies One 2 ml mixer barrel extender e Two mixer O rings One 2mm UV flow cell PEEK tubing green 1 16
140. ON TO THE SYSTEM SOFTWARE SYSTEM OPERATION Table 5 4 Edit Drop down Menu continued Run Screen continued Start run Starts the run Pause buffer pump Pauses progression of method s protocol Stops gradient pumps and the method time volume does not advance From Pause you can Abort Continue or Edit During Run Hold gradient Holds the current B gradient pump conditions and halts the advance of the method s protocol including fraction collection The method time volume does not advance From Hold you can Abort Pause or Continue Post Run Screen 5 8 Edit Activity Trace Allows you to input post run sample activity data obtained off line refer to Section 7 5 4 Tag and Trace Options Displays the Post Run Tags window that allows you to specify which traces are to be displayed and name peaks Also available from the Tag button in the Toolbar refer to Section 7 5 3 Full View in zoom chromatogram Allows you to view the complete chromatogram Also available from the Full View button in the system toolbar Print zoom chromatogram Allows you to print the complete chromatogram Also available from the Print button in the toolbar Copy zoom chromatogram to clipboard Copies to clipboard the complete chromatogram From the clipboard it can then be copied into other applications Not available from the toolbar Delete selected tag To delete a tag highlight the selected tag before using this function Also ava
141. ONS APPENDIX A SPECIFICATIONS System Power Requirements BioLogic DuoFlow Controller Workstation and Maximizer e 100V 53A e 230V 3 5A e 50 60Hz DuoFlow Dell Controller e Pentium IV9 Processor 20 GB hard drive 256 MB RAM SoundNIC and video 4MB graphics performance accelerator Dell M781 multimedia 16 monitor 3 5 floppy drive CD RW CD ROM drive e Windows 2000 operating system e Microsoft PS 2 IntelliMouse DuoFlow software version 5 0 or higher Networkable for printer and file access Maximizer Size 18 5 x 14 25 x 4 33 47 x 36 x 11 cm Lx W x H e Weight 11 165 4 8 kg Environmental Cold room compatible e Solvent compatibility 1M sodium hydroxide NaOH hydrochloric acid and organic acids 0 1M sulfuric acid 7M urea and guanide hydrochloric acid 10096 methanol MeOH isopropanol IPA and acetonitrile 0 1 triflouracetic acid TFA 1 detergents including sodium dodecyl sulfate SDS and TritonX 100 Workstation pump Dual piston positive displacement solvent pumps with interchangeable solvent delivery pumpheads F10 and F40 made of bio compatible PEEK e F10 pumpheads Without Maximizer 3 500 psi 233 bar 23 MPa from 0 01 to 10 0 ml min in 0 01 ml min increments With Maximizer 3 500 psi 233 bar 23 MPa from 0 5 to 20 0 ml min in 0 02 ml min increments Flow accuracy 0 01 to 10 0 ml min 2 Flow reproducibility 0 01 rsd F40 pumpheads Without Maximiz
142. ORKSTATION PUMP WASTE LOW PRESSURE 2 LOOP OVERFILL COLUMN TO WASTE AUXILIARY Figure 8 1 Multiple Sample Loading with an Auxiliary Load Pump and an AVR9 8 Valve Writing an Automatic Loop Fill and Rinse Protocol The protocol may be written as a single protocol containing back to back experiments or may be written as a series of queued runs The latter option generally produces a simpler protocol that may be copied into a queue once for each experiment to be run The Load Inject step can then be edited for each method in the queue so that the appropriate sample is loaded Several methods that include the Automatic Loop Fill and Rinse feature have been included in the BioLogic DuoFlow Method Templates see Section 6 2 and the online help for more information This feature may be added to any protocol using a static loop 1 Inthe protocol load inject step select Static Loop as the injection type 2 Inthe Fill Before Inject tab check the Enable Fill Before box select the sample and set the sample volume and flow rate Note that if an EP 1 or non Bio Rad pump is used the flow rate must be set both in this dialog and at the pump Note that the fill time must be less than the duration of the preceeding step 3 If the sample loop and tubing are to be rinsed between sample injections select the Rinse After Inject tab check the Enable Rinse After box select the Rinse buffer and set the rinse volume and flow ra
143. Ordering Information D 1 ING OX cis MEE ERN EIE IN 1 vi TABLE OF CONTENTS LIST OF FIGURES 1 1 BioLogic DuoFlow Systemi a nnne 1 1 2 1 BioLogic DuoFlow Pathfinder System Components 2 1 2 2 MX 1 Mixer and Mixer Barrel Extender sess enne nemen 2 18 2 3 Maximizer Mixer and Mixer Barrel eem 2 19 2 4 Assembly of MIX6rs edet a E eat athe e ete teet a 2 20 2 5 UV Detector with Mercury Lamp 254 amp 280 nm Filters and Conductivity Flow Cell 2 21 2 6 UV Detector with Zinc Lamp 214 nm Filter and Conductivity Flow Cell 2 22 2 f Conductivity MOnltor ciiin tede ie egeat e en d dae see deae e da adu 2 23 2 0 QuadTec UV Vis Detector mete reete bee reet exea rebns d EORR xar Ee RR 2 24 2 9 edere Le HE aie on da an esae aea ee 2 25 2 10 AVR7 3 Sample Inject ValVe ecd ei eames tua ee Fete ci ena 2 26 2 11 Sample Load Positioris tei ide dede nid detur dede deat Ve Reo ea esses Reus 2 27 2 12 Examples of AVR7 3 Valve Tubing sssssseeeneeeneeen nennen nmn nnne 2 28 2 13 AVR9 8 Stream Select Valve sssssssssssssssesseseseee nennen
144. P OUTLET PORT WASHOUT PORT OUTLET TUBING FROM BUFFERS OR MAXIMIZER VALVE OUTLETS Figure 4 5 Plumbing Connections to the Workstation Pump 3 Plumbing from the Workstation pumpheads to the Transducer Figure 4 3 a b Use the two pieces of tubing labeled PUMP from the tubing kit or make two fittings using 1 16 1 6 mm OD orange PEEK tubing and 1 4 28 fittings as described in section 4 1 Connect the tubing to each pumphead outlet port and to each transducer inlet port Ensure a firm connection but do not over tighten 4 Plumbing from the Transducer to the Mixer Figures 4 2 and 4 3 a Use the tubing labeled 1 from the tubing kit or make a fitting using 1 16 1 6 mm OD orange PEEK tubing and 1 4 28 fittings Connect the tubing to the transducer outlet port and to either of the inlet ports on the mixer Plug the unused mixer inlet port using the plug provided for this purpose Ensure a firm connection but do not over tighten 5 Plumbing from the Mixer to the AVR7 3 Inject Valve a Use the tubing labeled 2 from the tubing kit or cut a suitable length of 1 16 1 6 mm OD orange PEEK tubing that will reach from the mixer outlet port to port 5 of the inject valve Attach 1 4 28 fittings to each end Connect the tubing to the mixer outlet port and to port 5 on the inject valve Ensure a firm connection but do not over tighten SYSTEM PLUMBING SYSTEM INSTALLATION AND SETUP Plumbing th
145. PTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW 2 7 3 Model 2128 Fraction Collector The Model 2128 provides X Y motion drop dispensing across 5 available racks which accommodate a wide range of tube diameters and lengths microtiter plates microtubes and bottle size fractions It is suited for both analytical and preparative applications Figure 2 21 Model 2128 Fraction Collector The following racks are available Table 2 13 Model 2128 Racks Available Rack Name Tube Description Capacity Software ID Number Rack 1 1 12 and 13 mm diameter 128 16 X 8 tubes 180 mm height max Rack 2 2 16 to 18 mm diameter 78 13 X 6 tubes 180 mm height max Micro Adapter 3 Microplate 3 plates 288 wells 96 wells in 8 X 12 format Micro Adapter 4 Microtubes 128 microtubes capless tubes required Prep Adapter 5 Bottles of any size 10 bottles 2 38 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS The Model 2128 is controlled by the DuoFlow Controller via the Instrument Bus See Chapter 3 8 3 for connection instructions The Model 2128 is plumbed to the DuoFlow system using 1 16 1 6 mm OD inlet tubing and either of the following fittings To connect directly to the dispenser head use 1 4 28 flat bottom fittings To connect to the optional on arm diverter valve use the 10 32 nut and ferrule fittings supplied with the valve When the Model 2128 is configured in the Setup scr
146. RE MIXER BODY BODY ASSEMBLY OF THE MAXIMIZER MIXER ASSEMBLY OF THE MX 1 MIXER Figure 11 6 Mixer Assembly MAINTENANCE MAINTENANCE AND TROUBLESHOOTING 11 6 VALVES The following sections discuss the procedures for cleaning and repair of the SVT3 2 AVR7 3 and AVR9 8 valves 11 6 1 SVT3 2 Diverter Valve The SVT3 2 can be disassembled for cleaning or to replace parts which may become damaged over time Indications of damage include valve leaks and valve switching problems Items labeled in bold in Figure 11 7 are included in the SVT3 2 Valve Rebuild kit catalog 42760 0411 You will need a 1 Phillips cross head screwdriver and a 0 Phillips cross head screwdriver Note These parts are rather small Be sure you are working on a clean tabletop with room to lay out these parts and that your hands are clean Safety Note Before disassembling the valve be sure to flush any hazardous material from the valve and to disconnect it from the Workstation Wear protective clothing as appropriate To disassemble the valve follow the procedure below 1 Remove the large screw that attaches the clamp to the rack 2 Use the 1 Phillips cross head screwdriver to remove the two screws that attach the clamp to the valve Set the clamp and screws aside B SPRING PLUNGER AND DIAPHRAGM ACTUATOR MUST EINE VALVE BODY A UP WITH PORT B O DISK CO o rine EDS PORT B VALVE BODY B MUST LINE
147. ROREM 2 44 l Instrument Control Module ICM 3 11 M Manual screen chart 7 3 CeSCcripllON ae 5 1 description 7 2 Econo Gradient 7 3 fraction 7 3 gradient pump 7 2 QuadTec 7 3 Signal Import Module SIM 7 3 UV detector 7 3 ValVesxi 7 3 Maximizer mixer attaching to 3 8 changing mixer capacity 2 20 CICANING 4 nsec been 11 8 description 2 19 mixer barrels and mixer capacity 2 19 Maximizer valves tubing to Workstation pumps 4 4 replacing valves 11 13 Maximizer connector 2 14 automated valves connector 2 14 aux connector ssseeeeeee 2 15 Beeper screens senes 2 17 buffer blending troubleshooting 12 10 Com 1 2 15 Com 2 2 15 cond flowcell connector 2 14 conductivity calibration screens 2 17 description
148. Rad offers both 110 V and 220 V UPS configurations consult your local Bio Rad representative 3 11 1 DuoFlow System Network Connections The DuoFlow system can be connected to an Ethernet network allowing you to print reports and export files over the network Refer to the documentation provided with your PC computer for details on cable connection and software setup Contact your site administrator to ensure proper communication and access with the existing network 3 11 2 System Power Up Power up the system The following is intended as a checklist of instruments and devices that may be connected to the system The sequence is not important although it is best to power on the Controller last 1 Turn on power to the DuoFlow Workstation 2 Turn on power to the Maximizer if in use 3 Turn on power to the following devices and instruments if they are available a Power up the QuadTec using the power switch on the rear of the detector The QuadTec detector goes through a startup routine self test and lamp calibration routine b Power up the auxiliary pump Auxiliary pumps include the EP 1 Econo pump and the Econo Gradient Pump EGP c Power up the fraction collector Fraction collectors include the BioFrac the Model 2110 and the Model 2128 4 Power up the Controller At this point the DuoFlow software application may be launched When the DuoFlow Controller establishes communication with the system the faceplate in the Ma
149. Reverse Flow Affinity Chromatography with an AVR7 3 9 4 Buffer Blending Setup Dialog for a Single Component 10 5 Buffer Blending Setup Dialog for a Multi Component 10 5 Workstation Pump Mechanism Parts sssssssssssssssssssseeeeneeee meer 11 3 Piston Assembly and Access to the Piston Seal 11 4 Pumphead 2 eee Lace eame e ede Pe Dee eei ote ba ee dine van 11 5 Replacing a Mercury Lamp in a Model OM II UV 11 7 Replacing a Zinc Lamp in a Model OM II UV 11 7 Mixer Assembly rte ae Sal 11 9 SVT3 2 Valve Assembly titer ne 11 10 AVR7 3 and AVR9 8 Valve Assembly ssssssssssseseeeeeeeneeeeeeen nemen nnne nnne 11 12 Replacing a Maximizer 11 13 TABLE OF CONTENTS LIST OF TABLES 1 1 DuoFlow System nnns 1 4 2 1 Front View of the Dell PC Computer as the DuoFlow 2 2 2 2 Rear Vie
150. STEM COMPONENTS SYSTEM OVERVIEW 2 8 2 Model EP 1 Econo Pump The Model EP 1 Econo pump is a two channel bi directional variable speed peristaltic pump for low pressure chromatography The EP 1 offers a full range of features to facilitate ease of use as a stand alone pump or as an accessory to the DuoFlow System to load large volumes onto low pressure columns This instrument is described in detail in its separate documentation Features of the EP 1 include EP 1 has preset calibration for common tubing sizes and manual calibration for nonstandard size tubing e Jt controls various fraction collectors including the BioFrac Model 2110 and Model 2128 fraction collectors t can control the run length with a programmable total run volume e Itis coldroom compatible BIO RAD ECONO PUMP n me min ml min 0 O iS oO e 6 0 e e32 Svo 8 Figure 2 24 Model EP 1 Econo Pump The EP 1 Econo pump is ideal for loading samples onto a low pressure column When using a low pressure column such as an Econo Pac cartridge remove the 40 psi backpressure regulator from the post column position Place it at the outlet of the Workstation pump This will allow the check valves to properly seat and ensure pump flow performance The EP 1 Econo pump must contain firmware version 2 12 or higher to function with the BioLogic DuoFlow system To confirm the firmware version simultaneously press and hold down the Direc
151. SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS Econo Pac High S Low Pressure Chromatography Cartridges These cartridges are available in 1 ml and 5 ml formats to accommodate most sample loads They are recommended for method scouting and for first step purification of crude samples They are based on 50 um Macro Prep supports Refer to bulletin 1985 Macro Prep High S Support This is a strong cation exchanger containing sulfonic acid functional groups with a 50 uim particle size It is ideal for purification of basic and neutral proteins and peptides Refer to bulletins 1840 A 200 1917 and 2079 2 10 3 Anion Exchange DEAE Weak Anion Exchange The DEAE weak anion exchanger chemistry is available in the following formats Bio Scale DEAE Prepacked Medium Pressure Columns These columns are designed for high resolution separations of proteins peptides and polynucleotides in analytical to semipreparative medium pressure applications They are available in four column sizes Methods developed on the Bio Scale columns can be transferred to production scale using the Macro Prep 10 um supports Refer to bulletins 1930 1946 and 2079 Econo Pac DEAE Blue Low Pressure Chromatography Cartridges These cartridges are available in 1 ml and 5 ml formats to accommodate most sample loads They are recommended for method scouting and for first step purification of crude samples They are based on 50 um Macro Prep supports Refer to bulletin 1946
152. Scout Wizard In the Scout Wizard Step 1 dialog select the parameter type to be scouted As a general rule those parameters that are expected to have the greatest impact on the chromatography results should be scouted first In the Scout Wizard Step 2 dialog select the protocol step or steps to be scouted Steps that cannot be scouted are grayed out and steps selected for scouting are highlighted In the Scout Wizard Step 3 dialog enter the number of runs name the runs and for each run enter a value for the scouted parameter The run name can be auto generated or entered manually Automatically generated run names use the Run 1 name as the base name The value of the scout variable can be auto generated or entered manually Auto generated values use the Run 1 value as the start value and increment the value by the Increment Value amount The Increment Value can be a positive or negative number and is always defined as 1 when scouting valve positions e g Buffer A Buffer B Sample Name and Column SYSTEM OPERATION MODES OF OPERATION The following Toolbar menu options are available only when creating and editing a protocol Table 7 12 Protocol Screen s Editing Toolbar cee p Edit buttons These buttons are available from the System menu Edit To edit the currently highlighted step s in the protocol i To cut the currently highlighted step s from the protocol It places the step in the Cut clipboard which m
153. TEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM Table 2 7 continued Maximizer Front Panel Controls BECHER Softkeys For limited local operation of the Maximizer This is discussed further in Table 2 9 Enter and Arrow To operate the Maximizer in conjunction with the softkeys as discussed above Keys 2 13 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW Table 2 8 Maximizer Rear Panel Connectors POWER ENTRY MIXER COM1 SVPORT7 SVPORT8 SVPORT9 AVPORT10 AVPORT11 AV PORT 12 AUX PORT ww vw 100 240V INSTR BUS 2 14 MAXIMIZER REAR Solenoid Valves To connect DuoFlow low pressure solenoid valves SV5 4 and SVT3 2 to the system If a Maximizer is in use connect to its solenoid valve connectors before those on the Workstation Automated Valves To connect DuoFlow high pressure automated valves AVR7 3 Inject and AVR9 8 Stream Select to the system If a Maximizer is in use connect to its automated valve connectors before those on the Workstation ATC Reserved for future use pH monitor To connect the DuoFlow pH electrode The pH monitor is described in greater detail in Section 2 5 4 SIM device The Signal Import Module SIM enables connection of a detector or device that outputs an analog signal between 2 5 Volts to 2 5 Volts Instruments that may be connected in this way could include a variable wavelength UV det
154. Table 7 5 Linear Gradient Edit Linear Gradient Linear Gradient Buffers Composition Initial Final Volume ml Buffer A1 100 10 Linear Gradient Screen Flow ml min A Buffer B1 Y B 0 0 1 00 Step 8 Volume 2 00 ml OK Cance Edit Linear Gradient pH View Linear Gradient pH View Buffer System Tris 25 mM pH Range 7 20 to 9 20 3 pH Volume ml Linear Gradient Screen Solution A1 Tris HCI 50 mM 7 00 1 00 with a Maximizer in Solution A2 Tris 50 mM Buffer Blending Mode Solution B1 Degassed Water Initial Final B Flow ml min 9 Solution B2 Sodium Chloride 2 00M 0 100 1 00 Molarity at 100 B 1 0 M vi Step 2 Volume 1 00 OK Cancel T Gradient Use the Edit Linear Gradient screen to deliver buffer gradients The Linear Gradient screen sets the initial and final composition of the buffers and the period over which the change in composition is to occur There is no limit to the number of gradient steps in a protocol The following selections are available e Buffers Allows you to choose two buffers from which to make a binary gradient Drop down menus display all buffers that are are assigned to the pump inlets
155. The sample is inserted into the DynaLoop s sample end connector The loading of the sample pushes the DynaLoop s sliding seal assembly towards the buffer end connector Note While other dynamic loops may be used the application discussed in this section applies specifically to the DynaLoop System Setup When plumbed directly to the AVR7 3 inject valve the DynaLoop functions just like a static sample loop The sample end of the DynaLoop should be plumbed to port 3 and the buffer end to port 6 of the Inject valve The valve s operation and sample loading are controlled automatically simplifying the sample injection process and insuring precise sample loading and gradient formation The DynaLoop may be filled either manually with a syringe or automatically using an auxiliary pump such as an Econo Gradient Pump EGP or EP 1 Econo pump that is controlled by the DuoFlow system When using a syringe to fill a DynaLoop the syringe should be connected to port 2 of the AVR7 3 valve However using a 1 4x28 to female Luer adapter between the syringe and the valve makes filling the loop much easier When using the auxiliary pump to fill the loop the pump s outlet plumbing should be connected directly to port 2 of the inject valve Consult the DynaLoop Instruction Manual or DuoFlow online help for additional information The figure below shows how to set up a DynaLoop WORKSTATION PUMP WASTE DYNALOOP LOOP OVERFILL TO
156. UM RUNNING RUNNING HYDROXIDE BUFFER BUFFER SV5 4 SV5 4 SAMPLE LOADING BUFFER SELECT VALVE amp BUFFER SELECT VALVE SV5 4 AS FRACTION COLLECTOR UV MONITOR CONDUCTIVITY FLOW CELL FRACTION FRACTION COLLECTION FH COLLECTION FRACTION WASTE COLLECTION FRACTION COLLECTION Figure 2 16 Two Examples using SV5 4 Valves 2 32 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS 2 6 4 SVT3 2 Diverter Valve The SVT3 2 diverter valve is a low pressure 3 port 2 position valve This valve may be used in a number of ways To divert the eluant stream from the detectors to a fraction collector or a waste container As a sample select valve when placed before Workstation pump The Maximizer may not be used in this application As a water rinse valve when placed before Workstation pump As a user define valve when included as part of the plumbing setup Figure 2 17 SVT3 2 Diverter Valve Connect the SVT3 2 valve s signal cable to any of the available solenoid valve connectors on the back of the Workstation ports 1 2 or 3 Plumbing connections depend on the valve s use Plumbing as a diverter valve Use 1 16 1 6 mm OD PEEK tubing and 1 4 28 fittings Use orange PEEK 0 020 0 51 mm ID tubing for the F10 pumps and green PEEK 0 030 0 76 mm ID tubing for the FA0 pumps e Plumbing before a pump Use 1 8 3 2 mm OD tubin
157. UP arrow is pressed the display responds with RESET to PREV show that the calibration was reset pH Calibration Displays the current pH and temperature CAL pH ENTER Y PREV NEXT ENTER causes the Maximizer to enter pH calibration mode 2 16 SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM Table 2 9 continued Maximizer Screens Sereen Function and Description 1ST pH PT _4 00 CURSOR CANCEL 1ST 35 22 mV CANCEL SET 2ND pH PT _7 00 CURSOR CANCEL 2ND 35 22 mV CANCEL SET SET CELL C ENT Y PREV NEXT NEW CELL C 43 00 CURSOR CANCEL BEEPER ENTER Y PREV NEXT VOLUME UP DOWN ENTER TO FINISH MAXIMIZER pH Calibration continued CURSOR moves the cursor to the next digit of the pH set point UP DOWN arrows adjust the pH set point CANCEL aborts the calibration ENTER accepts the pH value of the first calibration buffer and moves to the next screen Display of the current pH voltage for monitoring probe equilibration SET or ENTER sets the first calibration point CANCEL aborts the calibration CURSOR moves the cursor to the next digit of the pH set point UP DOWN arrows adjust the pH set point CANCEL aborts the calibration ENTER accepts the pH value of the second calibration buffer and moves to the next screen Displays the current pH voltage for monitoring probe equilibration SET or ENTER sets the second calibration point and ex
158. UX pump and valve allows multiple direct injections onto a low pressure column Refer to Chapter 8 for discussion of a direct inject applications Volume Allows you to specify the volume of the sample to be injected The smallest volume selectable is 0 1 ml the largest volume selectable is 9999 ml for larger volumes use another Load Inject Sample step Actual load volume is defined by the sample loop size Flow Rate Allows you to specify the flow rate for the sample injection step OK Adds the step to the protocol This is the same as pressing the Enter key on the keyboard Cancel Does not add the step to the protocol This is the same as pressing the Esc key on the keyboard Step Time or Volume Identifies current step number and calculates the elapsed time or volume from all previous steps This is not user editable 7 13 Table 7 4 continued Load Inject Sample Edit Load Inject Sample Load Inject Sample Fill Before Inject Rinse After Inject Enable Fill Before Flow Direction Sample gt C D ai Sample 2 Sample 3 Sample 4 This operation will occur BEFORE injecting sample on column Volume ml 1 0 Flow ml min 1 00 Step 4 Volume 1 00 ml OK Cancel SYSTEM OPERATION Edit Load Inject Sample Load Inject Sample Fill Before Inject Rinse After Inject
159. VR7 3 ASIE INJECT 7 5 VALVE MIXER 24 3 COLUMN WAST WORKSTATION INSTRUMENT BUS ee e UNE UV DETECTOR OR KENNEN INJECT QUADTEC DETECTOR PORT B RINSE MAXIMIZER CONDUCTIVITY MONITOR 6 G HE 7 m mi BACKPRESSURE REGULATOR USB BITBUS D A2 Communicator 3 A4 pH MONITOR USB BUS SEL IL Q DELL CONTROLLER WASTE COLLECT TO MODEL 2110 FRACTION COLLECTOR OR COLLECT TO BIOFRAC Figure 4 3 System Plumbing with Maximizer 4 3 SYSTEM PLUMBING SYSTEM INSTALLATION AND SETUP Plumbing the Maximizer The Maximizer Tubing Kit provides colored FEP PTFE 1 8 OD 0 062 ID prefitted tubing lengths Plumbing the Buffer Reservoirs to the Inlets on Maximizer Valves A and B a From the Maximizer Tubing Kit identify the following The red tubing labeled Inlet A1 connects the buffer container to the Maximizer valve port A1 The blue tubing labeled Inlet A2 connects the buffer container to the Maximizer valve port A2 The yellow tubing labeled Inlet B1 connects the buffer container to the Maximizer valve port B1 The green tubing labeled Inlet B2 connects the buffer container to the Maximizer valve port B2 For complete discussion of Maximizer tubing installation refer to the Maximizer Tubing Kit diagram b Screw the tubing into the inlet connectors on the sides of the valves Ensure a firm connection but do not over tighten Plumbing the Maximizer
160. YSTEM SETUP The ICM translates the signal from the QuadTec detector and transmits it to the DuoFlow Workstation DEVICE INSTR BUS NUMBER INSTR BUS COMM 19 99 S 9 25V 0 3A MAX FRONT VIEW REAR VIEW Figure 3 10 Instrument Control Module ICM front and rear views for use when the Maximizer is not to be used The ICM contains the following Front View of ICM Module required if Maximizer is not in use Address setting dial should always be set to position 1 Instrument bus connector System Cable 17 connects to the Workstation and the BioFrac fraction collector if used Refer to Figures 3 4 and 3 5 Rear View Serial Comm connector System Cable 25 QuadTec RS232 cable connects to the QuadTec detector Power connector System Cable 26 connects to the DuoFlow Workstation DC outlet The QuadTec detector is equipped with a universal power supply which operates with supply voltages from 90 to 260 Volts AC A manual setting of the supply voltage is not required CAUTION Make sure to use a properly grounded power outlet and the power cable provided with the system To connect the QuadTec to the Workstation via the ICM module 1 Use System Cable 25 to connect from the RS232 connector on the back of the QuadTec to the COMM connector on the ICM module 2 Use System Cable 26 the ICM power cord to connect to the back of the Workstation 3 Use System C
161. aLoop s sample end connector through port 2 on the AVR7 3 valve and may be loaded with a syringe or auxiliary pumps such as the Bio Rad Econo Gradient Pump or EP 1 pump The loading of the sample pushes the DynaLoop s sliding seal towards the buffer end connector As solution flows into the buffer end of the DynaLoop it pushes the sample into the system SAMPLE END PORT 6 ON AVR 7 3 RES BUFR END s 95 ym LS LEES SLIDING SEAL ASSEMBLY Figure 2 22 DynaLoop 2 40 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS WORKSTATION PUMP AVR7 3 eo 1 Pd LOOP OVERFILL TO WASTE SYRINGE OR AUXILIARY LOAD PUMP Figure 2 24 Plumbing the DynaLoop for use with an Inject Valve When plumbed directly to the AVR7 3 inject valve the DynaLoop functions just like a static sample loop The sample end of the DynaLoop should be plumbed to port 3 and the buffer end to port 6 of the inject valve The valve s operation and sample loading are controlled automatically simplifying the sample injection process and insuring precise sample loading and gradient formation The DynaLoop can be filled using a syringe or auxiliary pump Use a 1 4x28 to female Luer adapter between the sample load syringe and the AVR7 3 valve port 2 makes filling the loop much easier Consult the DynaLoop user s manual for additional information 2 41 DESCRIPTION OF SY
162. able 17 to connect between an instrument bus connector on the ICM and an available instrument bus connector on the Workstation 4 f you are using a Bio Rad device with an instrument bus connector such as the BioFrac fraction collector use another System Cable 17 to connect between the instrument bus connectors on the BioFrac and the ICM 3 11 SYSTEM SETUP SYSTEM INSTALLATION AND SETUP 3 6 3 pH Monitor The pH monitor available as an option from Bio Rad refer to section 2 5 4 for more information may be connected to the DuoFlow system in one of two ways To a Workstation connect the Signal Import Module SIM included with the pH Monitor The SIM connects to the DuoFlow Workstation through the bus communication cables System Cables 17 18 19 21 or 30 e To a Maximizer if in use connect to the rear BNC connector labeled pH SIGNAL CABLE pH ELECTRODE pH FLOW CELL Figure 3 11 pH Monitor To connect the Bio Rad pH electrode to the SIM 1 Connect the output cable from the pH electrode to the connector labeled pH Connector on the SIM 2 Connect the SIM to the USB Bitbus Communicator by using an Instrument Bus cable System Cables 17 18 19 21 or 30 as shown below 3 Connect an external power source to the USB Bitbus device see Section 2 1 2 CONTROLLER j USB BITBUS COMMUNICATOR WORKSTATION SYSTEM CABLE 20 pM CHART RECORDER BARE WIRE TO BANANA PLUGS FOR CH
163. ac accommodates 12 20 mm and 30 mm tube diameters microtubes and scintillation vials in a variety of diameters The optional ice bath microplate rack allows collection into 13 mm diameter tubes microplates and microtiter tubes that meet SBS standards required for automated microplate systems The drop head height can be adjusted to accomodate tubes up to 150 mm The following racks are available Table 2 12 BioFrac Racks Available F1 12 13 mm diameter tube 2 racks 6x15 180 tubes 150 mm height 15 16 mm diameter tube 2 racks 5x12 120 tubes 150 mm height F3 18 20 mm diameter tube 2 racks 4x10 80 tubes 150 mm height 1 5 2 0 ml capless microtubes 4 racks 6x7 168 tubes 0 5 ml capless microtubes 4 racks 7x9 120 tubes 16 mm scintillation vials 4 racks 5x6 120 tubes 30 mm scintillation vials 4 racks 2x3 24 vials H4 High 30 mm tubes 4 racks 2x3 24 tubes Ice Bath Ice bath for 13 mm diameter 1 rack 10x12 120 tubes Microplate rack tubes standard microplate or 4 plates 12x8 96 wells microtiter tubes SBS standard 4 plates 8x6 48 wells format 4 plates 4x6 24 wells 4 plates 3x4 12 wells Prep 20 Preparative rack bottle size fractions 20 funnels The BioFrac communicates with the DuoFlow Controller via the Instrument Bus To connect the BioFrac to the bitbus use either of the instrument bus connectors on the rear of the fraction collector The BioFrac fraction collector supports collection schem
164. ail the automatic validation done prior to the run The flow rate of the DuoFlow pump is not critical so you may set a low flow rate e g 0 1 ml min to minimize buffer waste It is important that either the time or volume length of this first step is of sufficient duration to allow the auxiliary pump to fill the DynaLoop In the Protocol screen select Load Inject Sample to program the DuoFlow system to automatically fill and inject the DynaLoop sample From the Load Inject Sample window a Select the Dynamic Loop as the type of loop to be used SAMPLE LOADING ADVANCED SYSTEM APPLICATIONS 5 Select Fill Before Inject This instructs the auxiliary pump to load the sample into the DynaLoop Note that the auxiliary pump flow rate is not under DuoFlow control The flow rate is used by the system when validating the protocol before the run Because the DuoFlow system only starts stops the auxiliary pump the correct flow rate must be set at the auxiliary pump The flow rate of the Econo pump is set from the pump and is recorded in the yellow data entry boxes of the Fill Sample section of the Load Inject Sample dialog box Note that the rinse function is not available when the DynaLoop is being used In the Fill Sample Loop area of the window select the sample to be loaded and enter its volume and flow rate of the auxiliary pump In the Inject Sample area of the window select the Injection Buffers the buffer composition the flow rate of the W
165. alve AVR9 8 the system can load up to 7 samples sequentially into a loop One of the Aux pump inlet valve ports must be assigned to a rinse solution Similarly an SVT3 2 or SV5 4 Aux pump inlet valve may be used to load 1 samples or 3 sample respectively System Setup 1 Connect an auxiliary pump to the system as described in Section 3 9 1 EP 1 or non Bio Rad pump or 3 9 2 Econo Gradient Pump 2 Plumb the Aux Pump Inlet valve and Aux Pump to port 2 of the AVR7 3 Sample Inject valve as shown in Figure 8 1 3 In the device setup screen add an Aux Pump to the setup and define it as a load pump Define non Bio Rad pumps as an EP 1 load pump 4 n the device setup screen add an AVR9 8 or SV5 4 or SVT3 2 valve Define the valve as an Aux Pump Inlet valve and name each position One position usually position 1 should be defined as a Rinse solution The rinse solution is used to clean the sample loop and sample inlet lines between injections Note that some sample will be lost in the process of pulling sample from a remote beaker Be sure to factor in the solution volume required to fill all the buffer lines leading up to the top of the column when programming the injection step 5 When using a low pressure column such as an Econo Pac cartridge remove the 40 psi backpressure regulator from the post column position and place it between the Workstation pump and the mixer SAMPLE LOADING ADVANCED SYSTEM APPLICATIONS AVR7 3 W
166. am and Rack and Tube fraction numbering 7 38 SYSTEM OPERATION MODES OF OPERATION BioLogic Duo Flow user name project name method name run name File Edit View Utilities Options Help Edi News Z 38 Haca ethod Run _ Browser Report Manual Setup Protocol Values at Cursor Run Time 00 03 38 4 Gradient 21 Buffer B QuadTec 1 280 nm 0 0345 AU ettings BAI PostRun New lethod s Nu Print Activity QuadTec 2 260 nm QuadTec 3 214 nm QuadTec 4 405 nm Conductivity GP pressure No Trace 0 0228 AU 0 4762 AU 0 0021 AU 8 47 mS cm 375 53 psi GP pressure Current Tagging Trace QuadTec 3 214 nm QuadTec 3 214 nm v Rack Pos Ds B Grid 1F 2F 12 13F14F 0 100 100 Buffer B L o o o o a Litt Min Tenth WL1 280nm WL2 260nm 214nm 0 40 0 15 2 00 Gradient Pump F 40 UV 1 00mV min 0 B 438 psi 0 03775 AU WL4 405nm 0 00 Flow Rate 0 00 ml min EGP Split 0 B 0 Conductivity SIM1 SIG Y SIM4 pH Y 359
167. an Econo 3 17 3 16 Example of Multiple Sample Loading using an Econo 3 17 3 17 Example of Large Sample Loading using an Econo Gradient Pump EGP 3 18 3 18 Example of Multiple Sample Loading using an Econo Gradient Pump 3 18 3 19 BioLogic Configuration Utility Software Screen sssssseeen 3 20 4 1 System Plumbing using Pre cut Tubing from Fittings Kit 4 1 4 2 Making 1 4 28 Flat Bottom Fittings eene 4 2 4 3 System Plumbing with een enn een nnne 4 3 4 4 Maximizer Plurbifig s Lacie tone E a rete act etu eet Goel ee ee ordei 4 4 vii TABLE OF CONTENTS 4 5 4 T 5 1 6 2 6 3 6 4 6 5 6 6 6 8 6 9 7 2 7 3 7 4 7 5 7 6 7 8 7 9 7 10 7 11 7 12 7 13 7 14 7 15 7 16 8 1 8 2 8 4 8 5 8 6 9 1 9 2 9 3 10 1 10 2 11 1 11 2 11 3 11 4 11 5 11 6 11 7 11 8 11 9 viii Plumbing Connections to the Workstation Pump 4 5 Inject Valve Plumbing for an 7 3 4 6 Backpressue DEVICE zi Hte te intpp e cede hehe rat ie Hae dert o e eadera ege etes 4 7 Layout of the Screen Display showing the Manual S
168. and DuoFlow Pathfinder 80 systems that have F40 pumps F10 and F40 pumphead kits are available to easily convert an F10 Workstation to F40 and vise versa See Section 2 9 7 Table 2 4 F10 and F40 Pumphead Flow Rates Pumphead Flow Rate Flow Rate Flow Rate Isocratic and with Maximizer with Maximizer Gradient Mode High Flow Non blending Buffer Blending Mode F10 0 01 10 ml min 0 02 20 ml min 0 5 20 ml min 3500 psi 3500 psi 3500 psi 233 bar 23 MPa 233 bar 23 MPa 233 bar 23 MPa F40 0 5 40 ml min 1 0 80 ml min 1 0 80 ml min 1000 psi 1000 psi 1000 psi 66 bar 6 6 MPa 66 bar 6 6 MPa 66 bar 6 6 MPa The Workstation houses the control circuitry for the Workstation pumps MX 1 mixer UV detector Conductivity monitor and system valves low pressure solenoid and automated high pressure inject and select valves Connectors on the rear panel provide inputs for valves and detectors as well as output to a chart recorder for UV and conductivity data at 1 V Full Scale and pen up down start stop control If a Maximizer is being used connect devices to it to connect the mixer Conductivity monitor and pH monitor to it rather than to the Workstation An AUX combicon connector used for a controlling fraction advances of the Model 2110 and generic fraction collectors b receiving an open closed signal from a device such as a manual inject valve and c starting and stopping the Model EP 1 Econo pump for samp
169. ation Diagnostics Information Gradient Pump Calibration Conductivity Flow Cell Constant Calibration pH Probe Calibration EGP Calibration The Utilities menu selections relate to system options and remain the same in each displayed screen The Utilities menu consists of the following Validate Method Verifies that the devices required by the method Protocol have been defined or selected in the Setup screen Validation is automatically run at the start of a new run Check Hardware connections Checks that all devices listed in the Setup screen are electrically connected to the system The mixer is always assumed to be connected so it does not appear in the Setup screen and its connection is not checked System Information Displays the current system configuration including each instrument in the system and its firmware version number the Windows version number available hard disk space and the number of methods and runs in the database Diagnostics Information Displays information for service diagnostic purposes only Gradient Pump Calibration Available only from the Manual screen and it is typically used only after servicing of the pump It allows the user to calibrate the gradient pump flow rate and zero the system pressure gauge Note You must exit the Calibration screen in order for the system to accept the calibration values Conductivity Flow Cell Constant Calibration Allows the user to calibrate the conductiv
170. atogram information 7 39 description iter pte tee 7 38 Export Chromatogram Image File WIDdOW irat ether hoe ce 7 44 Export Data Setup window 7 43 Post Run Tags window 7 40 resizing nter teri ete 7 39 Pressure conversion table B 1 IN 4 Priming the system flushing the system through to the fraction collector 4 8 Priming the Workstation pump 4 8 io ox ccrte dv cr reet e 2 53 Protocol screen Alarm DUttONn cccccccceeeeeessseeeeeeeeeeeeees 7 19 Change Valve button 7 16 Change Valve position in e epe 7 16 MED 7 16 Step Time or Volume 7 16 Chart Recorder button 7 19 Column Switching button Cantel 7 17 Cure 7 17 amp hector tenendo 7 17 Step Time or Volume 7 17 description eren 7 9 Editing toolbar buttons COPY iiie be des 7 29 QUE se deve cra del des TR 7 29 Delete testet bes 7 29 teet ettet 7 29 Paste aite Do tous 7 29 Fraction Collection button Collect All End Tube 7 20 Fraction Size 7 20 Start Tube uus 7 20 Tubes Required 7 20 Collection Windows Fraction Si
171. aximizer Gradient Pump F10 UV Conductivity SIM1 SIG Y SIM1 pH 1 00ml min 0 B2 438 psi 1 003 AU 1 23 mS cm 0 548 Volt 7 00 pH QuadTec METHODS IN QUEUE Figure 6 6 Queues Displayed in the Browser Window To create a queue 1 In the Browser screen select a User or enter a new User name 2 Select a Project or enter a new Project name 3 Select the NEW icon from the left screen sidebar and from the displayed menu select New Queue Enter a name and description for your Queue and click OK Your queue name will be listed under your User and Project name and the Queue tab will appear at the bottom of the screen See Figure 6 6 above INTRODUCTION TO THE BROWSER SCHEEN SYSTEM OPERATION 4 To place methods in a Queue highlight the method in the Browser and click the Queue icon on the sidebar Repeat until all desired methods are in the project Queue This places each method into your Project Queue and the Queue Tab window at the bottom of the screen e Whenever you highlight the Queue icon under the Project Queue the list of methods will appear in the Tab window e Multiple methods may be selected in the Browser by either shift click or control click e Methods will run in the sequence they are listed in the Tab window The run order of the methods can be changed by dragging and dropping met
172. ays the run chromatograms side by side e Overlay Chromatogram Places Trace Compare in overlay view e All Chromatograms Places Trace Compare in Tiled view and displays all chromatograms including the overlay view side by side e Cascade Chromatograms Displays chromatogram windows in cascase view e Legend Displays the legend for the the current chromatogram e Full View Expands the currently active chromatogram e Full View for All Chromatograms Expands all zoomed chromatograms including the Overlay chromatogram e Volume based Chromatograms Time based Chromatograms Table 6 5 Tools Drop down Menu BioLogic Duo Flow lt user name gt lt project name gt File Options View Tools Window Help Shift Up Shift Down The Window menu consists of the following e Shift Up Shifts chromatogram traces relative to each other in an upward direction e Shift Down Shifts chromatogram traces relative to each other a downward direction Table 6 6 Window Drop down Menu trace compare name File Options View Tools Ma Help The Window menu consists of the following e BioLogic Closes Trace Compare and returns to the Browser screen 6 16 SYSTEM OPERATION INTRODUCTION TO THE BROWSER SCREEN 6 5 4 Active Traces and Values at Cursor The Active traces and values in the at cursor window located in the upper left of the post run screen contain two drop down menus that control whi
173. be Amanual event mark from the Run screen Pressing the Event Mark button on the Run screen allows you to mark events as they happen Non Bio Rad UV detectors If the Model 1327 chart recorder is used in conjunction with a SIM and a third party detector to replace the DuoFlow UV Detector System Cable 20 should be used to control the recorder Channel 1 signals should be sent directly from the third party detector using a bare wires to banana plug cable In this case the appropriate input voltage range must be selected on the recorder Refer to the documentation for your non Bio Rad UV detector 2 9 9 Generic Chart Recorders A non Bio Rad chart recorder may be used as an integral part of the DuoFlow system If the DuoFlow system s UV detector is used a mini DIN to breakout cable System Cable 7 must connect the Workstation s UV Chart connector to the chart recorder Pen Up Down and Start Stop functions are available providing the control polarity of the generic recorder is compatible See the pin out information in your chart recorder manual Conductivity signals require a Bio Rad mini DIN to banana plug cable System Cable 4 Set the chart recorder s input signal voltage to 1 V for both BioLogic DuoFlow UV detector and Conductivity monitor signals Chart speed is set at the recorder itself it is not controlled by the DuoFlow system 2 52 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS 2 9 10 Uninterruptible Power Supply UPS
174. be sure to check this setting Remove the column and run the pumps to determine if the high backpressure is due to the column or not If the column is suspect the column may need cleaning along with a frit replacement Consult the column s instruction manual for cleaning procedure Alternatively the system tubing may have an obstruction so inspect the tubing path To isolate the blockage start by loosening connection fittings at the detector and work backwards towards the column and pumphead Prevent or minimize high backpressure problems by filtering buffers and samples and by changing the pump seals before they deteriorate completely MAINTENANCE AND TROUBLESHOOTING TROUBLESHOOTING 12 3 TROUBLESHOOTING THE UV DETECTOR AND UV TRACE Problem Possible Cause Solution UV baseline is unstable or noisy UV baseline shows a reproducible zig zag or saw tooth trace Air bubbles trapped in the analytical 5 mm Z cell are the most common cause of noisy UV baselines Note Always degas buffers before use Degas buffers by stirring vigorously under vacuum for approximately 20 minutes Use a heavy wall side arm Erlenmeyer flask as standard flasks may implode under vacuum Plumb the Z cell with the column outlet connected to the bottom of the flow cell This will force air bubbles to rise to the top of the flow cell and dissipate Use of the 40 psi backpressure regulator supplied with the DuoFlow sy
175. be used with the system see lower half of Figure 3 3 a Connect the USB Bitbus Communicator power adapter catalog number 760 2034 b Setthe Power Select switch to External DELL CONTROLLER PWR CON SELECT INSTRUMENT BUS SYSTEM CABLE iN FA X EXT INT 7 s 6 ences N REAR VIEW USB BITBUS COMMUNICATOR FRONT VIEW REAR VIEW SYSTEM CABLE 31 USB BUS 17 18 19 21 OR 30 CONNECTION TO THE FOLLOWING DEVICES LISTED IN SEQUENCE MAXIMIZER IF AVAILABLE WORKSTATION DELL CONTROLLER PWR SELECT N pa n i text int Ne P Ml SIGNAL IMPORT MODULE REAR VIEW REAR VIEW REAR VIEW SYSTEM CABLE 17 18 19 21 OR 30 INSTRUMENT BUS USB BITBUS COMMUNICATOR FRONT VIEW Eg SYSTEM CABLE 31 USB BUS m Figure 3 3 USB Bitbus Communicator Cabling 26 CONNECTION TO THE FOLLOWING DEVICES LISTED IN SEQUENCE SIM IF AVAILABLE OR MAXIMIZER IF AVAILABLE WORKSTATION WALL AC POWER UNIVERSAL AC DC INLINE ADAPTOR SYSTEM SETUP SYSTEM INSTALLATION AND SETUP 3 3 WORKSTATION CABLE CONNECTIO
176. ble 7 16 SYSTEM OPERATION MODES OF OPERATION Table 7 7 Column Switching Edit Switch Columns Switch Columns Position Column 2 Column 3 Column 4 Column 5 Column 6 Y Step 8 Volume 5 00 ml OK Cance The DuoFlow system supports the use of more than one column during a run Chapter 9 provides two examples of column switching during a run Use this button to specify which column to load sample onto Column switching between as many as eight columns can be done with two AVR9 8 valves A column switch valve is defined by pressing Column Switching in the device setup and assigning two AVR9 8 valves to the column switching valve This identifies the two valves as installed and synchronizes the switching of the valves without further user input Column switching permits identification of the electrical connection port of each valve and user naming for each column Installation of the AVR9 8 valve in setup automatically activates Column Switching in the Protocol screen e Position Select the column to switch to in a run e Adds the step to the protocol This is the same as pressing the Enter key on the keyboard e Cancel Does not add the step to the protocol This is the same as pressing the Esc key on the keyboard e Step Time or Volume Identifies current step number and calculates the elapsed time or volume from all previous steps This is not user editable
177. c Size Thresh Non Peak Frac Size Close ml ml ml AU ml 1 0 0 4 4 0 4 Waste EDITMODE 4 T Tubes Save Changes 2 7 00 8 00 1 00 0 010 Waste Delete Window Cancel Editing Hl Threshold and Collection Windows Each Collection Window discussed on page 7 22 can also contain a threshold value to further discriminate when eluant can be collected For each collection window non peak eluent can be directed to waste or to the fraction collector The Threshold and Time windows can be combined so the system collects fractions only during the programmed Time windows and within the Time windows only when the detector signal is above or below the threshold When using the Collection Windows functions the BioFrac and Model 2128 will skip a tube between each window collection For example if the first window ends collection at 20 minutes and the second window starts collection starting at 21 minutes then the fraction collector will leave an empty tube for the period between minutes 20 and 21 7 23 MODES OF OPERATION SYSTEM OPERATION Table 7 10 continued Fraction Collection Description Threshold and Collection Windows continued e Fraction Size Start Tube End Tube Start Rack and End Rack See description for Collect All on page 7 20 e Above below Threshold Used to select whether fractions are collected when the detector signal is above or below a designat
178. ch trace axis is displayed on the left and right sides of the active chromatogram The scroll bars on the left and right sides of the information box control the scaling of the trace associated with the left or right drop down menu When the vertical crosshatch bar is dragged across a chromatogram the values relating to the baseline or peak underneath the crosshatch is reflected in the upper left corner of the screen The value for each trace at the cursor position is shown in the dialog 6 5 5 Chromatogram Settings Tab The Chromatogram Settings window contains the following tabs to manipulate the display of the chromatogram Visibility In Tile mode each trace device type is displayed along with a check box legend and a run name Checking or unchecking the boxes causes the traces to be displayed or hidden In Overlay view only the traces selected in Options Select Overlay Traces or by the Select button are shown in the chromatogram settings dialog The currently active trace is highlighted in blue Y Axis Allows you to set the Baseline and Axis Max values for the currently active trace The active trace is selected in the Visibility tab X Axis Allows you to set the Start Time and End Time for the currently active chromatogram 6 17 SYSTEM OPERATION MODES OF OPERATION 7 0 MODES OF OPERATION There are two primary modes of system operation the user can operate the system manually from the Manual screen or through the use
179. ck 18 20mm BioFrac Rack set H1 4 x flatpack 1 5 ml microtubes BioFrac Rack set H2 4 x flatpack 0 5 ml microtubes BioFrac Rack set H3 4 x flatpack 16 mm scintillation vials BioFrac Rack set H4 4 x flatpack 30 mm large scintillation vials BioFrac Ice Bath amp Microplate Rack BioFrac Prep 20 Preparative Rack 741 ORDERING INFORMATION APPENDIX D 731 8238 731 8122 731 8120 760 0410 731 8130 731 8135 731 8136 731 9001 731 9002 731 8140 731 8142 731 8250 731 8253 731 8254 731 8255 750 0650 750 0651 750 0652 750 0655 760 1309 760 1307 760 1321 760 2004 760 2032 731 8262 731 8264 731 8265 731 8267 750 0653 760 0135 720 0001 760 0001 Model 2128 Diverter Valve Model 2110 Fraction Collector 110 V Model 2110 Fraction Collector 220 V Model 2110 Diverter Valve SVT3 2 Model 2110 Carousel 80 tube capacity Model 2110 Micro Tube Adapter Model 2110 Carousel Dust Cover Pumps Econo Gradient Pump 100 120 V Includes Tubing and Fittings kit Econo Gradient Pump 220 240 V Includes Tubing and Fittings kit Model EP 1 Econo Pump 100 120 V Model EP 1 Econo Pump 220 240 V Chart Recorder Model 1327 Chart Recorder with USA Canada Japan Mexico Taiwan and Latin America power adapter Model 1327 Chart Recorder with UK Commonwealth power adapter Model 1327 Chart Recorder with Australia New Zealand power adapter Model 1327 Chart Recorder with European power adapter
180. creen 5 1 The BFOWSGESCEIGGLI aieo cerei tt gies bates daa hA ER Ka bio Tea Ev RR 6 1 TRE Window trier eere ene dc sede deed N dee nt Ex e E me 6 4 Copyln with Information in Browser Tab Window sssem m 6 5 Set Browser Options 6 6 New Method Dialog Showing Method Templates sss e 6 7 Queues Displayed in the Browser Window sseenennm eene 6 9 Compare Displayed in the Browser Window 6 11 Trace Compare Window Tiled VieW ccccceceeeeeeeeeeeeeceaeeeeeeeeeeeeeeeeeeececaeaeeeeeeeeeeeteeeeee 6 12 Trace Compare Window Overlay 6 13 Relationships between Modes of Operation 7 1 Manual Screen for the BioLogic DuoFlow System Connected to a Maximizer BioFrac Fraction Collector QuadTec Detector Econo Gradient Pump and Four Valves 7 2 SOUP so LER 7 4 Protocol SChECM LS 7 10 Run Screen showing Run in Progress cccceessceeeeeeeeeeeeeeeeneeeeeseeneeeeeeeenaeeeeseeeieeeeeeeenaeeees 7 30 Run Screen s Abort Pause and Hold 7 33 Editing during aRUn s icio rdi tle cen dete
181. cting large volume samples Collects up to 8 samples The name specified for each position will appear in the Protocol screen s Change Valve dialog box When used for purposes other than described above The name specified for each position will appear in the Protocol screen s Change Valve dialog box Refer to Chapters 8 and 9 Allows you to install up to 8 columns and assign valve numbers and names to each column Requires two AVR9 8 valves one is an inlet valve and the other functions as an outlet valve The Buffer Editor is used to create new Buffer Blending buffer systems from user supplied information This information is used by the Maximizer to determine the amount of acid base water and salt required to produce a buffer solution at a desired pH and salt composition Each buffer system can include up to three buffers and each buffer can have up to three pKa s The Buffer Editor also generates a recipe describing how to make each Buffer Blending solution The Buffer Editor requires information about the buffer pKa s temperature coefficient s charge state molecular weight and concentration and the salt charge state molecular weight and concentration This information can also be supplemented with user determined 1 or 2 point pH correction for each buffer in a buffer system pH corrections are entered from the Buffer Blending setup dialog 7 8 SYSTEM OPERATION MODES OF OPERATION Table 7 2 Buffer Editor Buffer
182. ction keys Hold until Keypress To start a method that is on Hold during a run when a method includes a Hold until Keypress step Help Displays the Help menu for the currently displayed screen Esc Functions as an alternative to the Cancel selection in a Dialog box Alt Some system commands can be executed either by selecting them from a drop down menu or by holding down the Alt key and then pressing the appropriate character key These connect to the USB Bitbus Communicator which in turn connects to the instrument bus and allows components of the BioLogic DuoFlow system to communicate with the Controller Components connect to the instrument bus in a daisy chain and are recognized when the system is switched on Even when one component is switched off other components daisy chained to the system can be controlled by the Controller DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW Table 2 2 Rear View of the Dell PC Computer as the DuoFlow Controller MONITOR MONITOR POWER CONNECTOR DELL CONTROLLER SIGNAL CABLE ETHERNET CONNECTOR MOUSE COLOR MONITOR CONNECTOR CONNECTOR KEYBOARD CONNECTOR USB BUS CONNECTORS PARALLEL CONTROLLER CONNECTOR POWER CONNECTOR USB connectors These connect to the USB Bitbus Communicator which in turn connects to the instrument bus and allows components of the BioLogic DuoFlow system to communicate wi
183. d and base of a buffer to obtain a solution with a user defined pH and salt concentration One valve delivers an acid and base and the other valve delivers a salt and water Pre defined Buffer Blending buffer systems are provided for virtually all common buffers used in chromatographic applications Additional user defined buffers may be created using the BioLogic software Buffer Editor feature The Maximizer uses the buffer system information to determine the amount of acid base water and salt to add to obtained the desired buffer composition and pH When the Maximizer is set to local mode in the software Manual screen it will not be under DuoFlow system control In local mode the Maximizer front panel controls are accessible and can be used to prime the system calibrate the pH and conductivity monitor observe the status of each device and control the position of each Maximizer valve The Maximizer is designed to operate under normal laboratory and coldroom conditions 4 40 C with all commonly used aqueous chromatographic buffers The Maximizer includes the following hardware and circuitry e Front panel on off switches two 3 port two position proportioning valves for automated buffer blending an LCD screen and membrane switches for controlling valve positions and calibrating the pH probe and conductivity monitor Rear panel connectors that with the exception of the UV detector duplicate the connectors on the Workstation
184. de NaOH water methanol and water through the cell Dry the interior of the cell with a stream of high purity nitrogen gas never use house compressed air as it may contain oil droplets Check for leakage of liquid from the top of the optics unit onto the outside quartz windows of the flow cell Clean the windows with a soft non abrasive damp cloth Condensation may occur on the exterior windows of the flow cell when moving the optics unit from the lab to a cold room and vice versa Always allow an equilibration period to compensate for such temperature effects Drifting baseline Non homogeneous eluant perhaps due to poor mixing or flow rate variation Check flow rates of both pumps recalibrate if needed by selecting Gradient Pump Calibration from the Utilities drop down menu Slow column equilibration Certain ion exchangers are slow to re equilibrate especially if just sanitized Allow a longer equilibration period If drift is due to a higher absorbance suspect that UV absorbing material may be leaching from the column To confirm this remove the column and run deionizied DI water or an queous non absorbing buffer If the baseline is stable then the column is suspect Consult the column s user manual for cleaning procedures Negative peaks 12 8 The sample is applied to the column in a different buffer from that used to equilibrate and elute the column Refractive index changes
185. del 2128 fraction collector e Start Rack and End Rack Identifies the first and last BioFrac rack to receive fractions 7 20 SYSTEM OPERATION Table 7 10 continued Fraction Collection Description Threshold MODES OF OPERATION Fraction Collection Scheme BioFrac Rack Type F1 12 13 mm tubes Threshold Detector Threshold 0 100 Q Threshold and Collection Windows Collect All UV Detector Threshold Fraction Size Rack Tube Collection Windows 1 00 Start _A Yu B 90 Y Row Col 1F 1A Q Below Threshold Non peak Parameters Destination Waste Tubes Fraction Size 100 4m a AU 8 Above Threshold Close Threshold This method allows the flow to be sent to the collector if the detector signal of the eluant is above or below a certain signal level Note You can select any detection source to initiate threshold based fraction collection Non peak eluant can be sent to waste or to the fraction collector Fraction Size Start Tube End Tube Start Rack and End Rack See description for Collect All on page 7 20 e Threshold Enter the threshold value Fractions will be collected whenever the detector output goes above or below the threshold level e Above below Threshold Used to select
186. ding affinity chromatofocusing hydroxyapatite hydrophobic interaction ion exchange and gel filtration chromatography pH Monitor The BioLogic pH monitor allows direct pH monitoring during a run It is included as part of the DuoFlow Maximizer and Pathfinder systems and is available as an option for all other system configurations The pH monitor consists of a Calomel Tris compatible electrode in a PEEK biocompatible flow cell Flow Rate Flexibility The DuoFlow Workstation has two pump head options F10 and F40 that permit a wide range of flow rates 0 01 ml min up to 80 ml min Detection Flexibility e UV detector with fixed 254 nm and 280 nm filters long life mercury lamp and additional drop in expansion filters available e UV detector can be expanded to 214 nm filter with zinc lamp e QuadTec UV Vis detector analyzes samples simultaneously at 4 different wavelengths from 190 370 nm with a deuterium lamp or 370 790 with a halogen lamp SYSTEM OVERVIEW INTRODUCTION e Third party detectors such as refractive index or fluorescence may be utilized with the DuoFlow systems via a Signal Import Module SIM or Maximizer e Conductivity Monitor Monitors salt concentration to assure reliable gradient formation and pump function e Fraction Collection The DuoFlow system supports a wide variety of fraction collection options including Collect All Threshold Collection Collection Windows and Threshold amp Collection Windows Bo
187. e Chromatography with a Maximizer offers three major benefits First is its Buffer Blending feature that allows the composition of the pump effluent to be defined in terms of pH and salt concentration from a single set of reagents Buffers at any pH within the pH range of the buffer typically pKa 1 can be obtained Second solution preparation is simplified since buffer salts need to only be weighed out and diluted to the appropriate concentration There is no need to adjust the pH of the solutions The third benefit of using a Maximizer is that it doubles the accessible flow rates This occurs since gradients are no longer made by the pumps but by the Maximizer proportioning valves This allows both pumps to run at their full speed 10 1 DOUBLED FLOW RATE CAPACITY USING A MAXIMIZER The Maximizer valve system doubles the DuoFlow system flow rate capacity from 10 ml min F10 pump head and 40 ml min F40 pump head to 20 ml min and 80 ml min respectively This is accomplished since the Maximizer proportioning valves rather than the pumps are used to change buffer composition percent Buffer A and Buffer B This doubled flow rate capacity is available only when Buffer Blending is defined in the Device Setup The Buffer Blender feature can be used in two different ways 1 Buffer Blending where water salt and the conjugate acid and base of a buffer are combined to produce a solution with a specific salt concentration and pH or 2 Non Blending
188. e AVR7 3 Inject Valve a WASTE WASTE 4 6 The inject port assembly and needle are included with each AVR7 3 inject valve Screw this assembly into port 2 of the valve until it is secure Connect the sample loop to ports 3 and 6 Plumb the inject valve according to Figure 4 6 Use the tubes labeled waste or make fittings using 1 16 1 6 mm OD Tefzel tubing and 1 4 28 fittings for waste lines 1 and 7 Use the tube labeled 3 or make a fitting using 1 16 1 6 mm OD Tefzel tubing and 1 4 28 fitting to go from port 4 to the column WORKSTATION WORKSTATION WORKSTATION PUMP PUMP PUMP WASTE WASTE COLUMN SAMPLE WASTE SAMPLE WASTE SAMPLE LOOP LOOP LOOP SAMPLE E SAMPLE SAMPLE INJECT INJECT INJECT LL Lh Figure 4 6 Inject Valve Plumbing for an AVR7 3 Plumbing the UV Detector and Conductivity Monitor a Connect a piece of 1 16 1 6 mm OD orange PEEK tubing tube 4 in the tubing kit between the column outlet and the bottom inlet of the UV detector flow cell using 1 4 28 fittings If you are using the QuadTec detector connect it to the flow cell which is bidirectional using 10 32 fittings provided with the QuadTec Use the tube labeled 5 in the tubing kit or cCut approximately 8 cm of 1 16 1 6 mm OD orange PEEK tubing and attach 1 4 28 fittings Connect one end into the top of the UV detector flow cell Connect the other end into th
189. e Conductivity monitor flow cell which is bi directional If you are using a QuadTec detector connect tubing from its flow cell outlet using a 10 32 fitting to either port of the Conductivity monitor using 1 4 28 fittings Place the Conductivity flow cell into the notch of the optics bench This is a gentle push fit There is a tag with a number attached to the conductivity cable This number is the flow cell constant and must be entered in the software before beginning a run Refer to Table 5 6 page 5 10 Utilities drop down Menu Conductivity Flow Cell Constant Calibration For flow rates below 10 ml min insert the 40 psi backpressure regulator after the conductivity monitor Plumb the backpressure regulator following the direction of the arrow SYSTEM INSTALLATION AND SETUP SYSTEM PLUMBING Se gt FLOW DIRECTION gt Figure 4 7 Backpressure Regulator The backpressure regulator is required to help eliminate bubbles from becoming trapped in the detector flow cell When using low pressure columns such as an Econo Pac cartridge or Econo column plumb the 40 psi backpressure regulator at the Workstation pump outlet This aids in seating the check valves preventing permanent damage to the cartridge or column Refer to section 2 9 5 for more information 8 Connection to a Fraction Collector Connection to a BioFrac Fraction Collector a Use the tube labeled 6 in the fittings kit or make a fitting using 1 16 1 6
190. e closed with 1 4 28 plugs When loading samples directly through the Workstation pump first filter the sample through a 0 45 um filter Flush the valve pump and lines with a sanitizing solution to remove protein residues after the sample has been loaded protein residues might otherwise reduce pump piston seal lifetime Connect the SV5 4 valve cable to any of the available solenoid valve connectors on the back of the Workstation ports 1 2 or 3 If a Maximizer is in use connect to ports 4 5 or 6 before those on the Workstation From the Manual screen you may manually operate each inlet port on the SV5 4 Manual valve control is useful for the following When priming the tubing and the valves with buffer and or sample prior to starting a method e For purging all tubing lines for cleaning purposes Prior to programming a method in the Protocol screen you may name each of the valves positions in the Setup screen The name that you apply in the Setup screen will appear in the method Protocol screen when you program an Isocratic Flow Change Valve or Linear Gradient step 2 31 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW SV5 4 FOR BUFFER AND SAMPLE LOADING TO COLUMN WORKSTATION RUNNING RUNNING RUNNING BUFFER SAMPLE BUFFER BUFFER SANITIZING SOLUTION TORAGE SOLUTION E LE 1M SODI
191. e distinguished as follows Online The BioLogic DuoFlow icon in the upper left of the screen is green and the title bar does not indicate online Offline The BioLogic DuoFlow icon in the upper left of the screen is yellow and the title bar indicates offline To change between the online and offline windows you can use any of the following 7 34 Windows drop down menu The online and offline windows are listed Task bar The Windows taskbar lists the BioLogic DuoFlow windows Alt Tab Simultaneously hold down these two keys to list the open windows SYSTEM OPERATION MODES OF OPERATION 7 4 3 Editing A Method During a Run During a run you may find you want to change some of the run parameters The figure below shows how this is handled by the system and some of the restrictions that apply Method Name setup Steps Edit prior Edit during to run the first run Edit during or after any run after the first run Figure 7 7 Editing during a Run e During the first run of a method you can pause the method and return to the Protocol screen to edit steps that have not already been started The method name will not change e Any run after the first can be paused and then edited except runs in queue or scout which cannot be edited You will first be asked to rename the method or accept the default new name when the Edit Protocol button is clicked This is to ensure the integrity of the database in terms of
192. e of the following solvents dilute sodium dodecyl sulfate SDS 1M hydrochloric acid HCI 1M sodium hydroxide NaOH ethanol or acetone Run the solution through the flow cell using a syringe and leave for no more than 5 minutes Rinse extensively with water and then blow dry using a gentle stream of pure nitrogen Never dry with compressed air from a house line as this will contain microdroplets of oil that will coat the cell When the detector is not in use disconnect the flow cell and use a syringe filled with distilled water to clean out traces of salts and protein Before storing the flow cell inject a dilute solution 1096 to 2596 of ethanol or isopropanol into the cell to prevent microbial growth Use the plugs provided with the flow cell to seal the flow cell s inlet and outlet lines Conductivity flow cell requires little maintenance other than rinsing routinely with deionized water MAINTENANCE AND TROUBLESHOOTING MAINTENANCE 11 4 2 Replacing the Lamp in the UV Detector The following information covers the replacement procedures both for a new mercury lamp unit and a new zinc lamp unit Note that the zinc lamp assembly has cooling fins and is larger than the mercury lamp assembly Indications that a lamp replacement is necessary include an unstable baseline and a decreased response to a standard concentration of a test chromophore In this latter case it is advisable to ensure that the flow cell is clean
193. e optimal pH for a chromatography method e Buffer A Used to automatically run an experiment using a variety of load wash or elution buffer conditions through Inlet A e Buffer B Used to automatically run an experiment using a variety of load wash or elution buffer conditions through Inlet B e 9eB Used to systematical alter the salt composition of the buffer mobile phase during isocratic flow and Load Inject steps e Duration Used to systematically vary the duration of isocratic flow load inject and linear gradient steps 7 25 MODES OF OPERATION SYSTEM OPERATION Table 7 11 continued Scouting B Gradient Initial B Used to systematically vary the initial buffer composition of a gradient step B Gradient Final B Used to systematically vary the final buffer composition of a gradient step Flow Rate Used to optimize the flow rate for adsorption and elution steps Sample Name Used to inject samples that have been prepared under a variety of conditions Sample Volume Used to find the optimal sample load volume Column Used to automatically test up to eight column types when an AVR9 8 column switching valve is used Alternatively this method can be used similar to the Buffer A or Buffer B options above to simultaneously change Buffer A and Buffer B using the column switching valves Scouting Wizard Step 2 This dialog is used to select the protocol steps to be scouted Steps that are incompatible with the curr
194. e prompted for a new method name or the methods will automatically be numbered consecutively New Run Creates and names a new run for the current method and opens the Run screen Pressing Start in the Run screen starts the run e Open Opens the Browser screen Refer to Chapter 6 Browser Screen e Close Closes the current method and or run e Select User and Project Allows you to select the user and project for the system e Change Name Author Allows you to change the method run name author and description This function is inactive once a run has been run e Save setup Allows you to save and name different instrument setup configurations in the Setup screen and to set one as the default Load setup Recalls and loads a saved setup 5 5 INTRODUCTION TO THE SYSTEM SOFTWARE SYSTEM OPERATION Table 5 3 File Drop down Menu continued Print Report Allows you to print a report for the currently open method including its setup and run data run results and the run log report Data Management Displays the Browser screen from which you can copy and move run data Refer to Chapter 6 Browser Screen Export Data This feature is used to set data export parameters and export data text files This feature is available from the PostRun screen Export Chromatogram Image This feature exports a chromatogram image in a Windows Meta File WMF format This feature is available the PostRun screen Integratio
195. e reference chromatogram in the upper right window In the upper reference chromatogram use the vertical scrollbar to adjust the scale BioLogic Duo Flow user name project name method name run name File Edit View Utilities Options Help New Edit New E 4 KN 2 g a K Method Run Browser Report a A Su REN S FANS Print Del Tag Activit Values at Cursor Run Time 00 03 38 4 Gradient 2196 Buffer B QuadTec 1 280 nm 0 0345 AU QuadTec 2 260 nm 0 0228 AU QuadTec 3 214 0 4762 AU QuadTec 4 405 nm 0 0021 AU Conductivity 8 47 mS cm GP pressure 375 53 psi No Trace Current Tagging Trace QuadTec 3 214 nm GP pressure QuadTec 3 214 nm Rack Pos 1 13 14 0 100 7 100 Buffer B Milliliters WL1 280nm WL2 260 214nm WL4 405nm Econo Gradient Flow Rate EGP B Split QuadTec 0 15 2 00 0 00 Pump 0 00 ml min 0 B 0 0 40 Gradient Pump F 40 UV Conductivity SIM1 SIG v SIM4 pH Y 1 0 953 Volt 0 00 pH i 00ml min 0 B 438 psi 0 03775 AU 359 mS cm Figure 7 11a Post Run Screen showing a Volume based Chromatogr
196. e system bus and to set the rack type collection pattern BioFrac only and whether or not the maximum number of racks may be exceeded If you choose to exceed the maximum number of available racks the run will pause when the rack s are full and prompt you to add new racks The BioFrac or Model 2128 fraction collector must be selected in the BioLogic Configuration Utility see Chapter 3 11 3 before starting the BioLogic software The Model 2110 and generic options allow you to control these fraction collectors over the Aux port on the rear of the BioLogic Workstation or Maximizer If a Maximizer is connected to your system then the fraction collector must be connected to it and not the Workstation Note a SVT3 2 valve must be connected to your system and defined as a diverter valve in the device setup if threshold or windows collection is desired Buffer Blender Used to place the DuoFlow system in Buffer Blending mode select a pre defined Bio Rad or user generated buffer system set pH corrections and view solution preparation instructions pH range and buffer temperature coefficients The Buffer Editor feature is used to generate user defined buffers for Buffer Blending see Chapter 7 2 3 Detectors Used to define the detectors types used by a method Up to six detector traces may be defined including UV Detector QuadTec up to four traces Conductivity SIM pH and SIM signal up to two external detector traces such as refractive index or f
197. ead and no fittings are required SYSTEM PLUMBING SYSTEM INSTALLATION AND SETUP 4 3 PRIMING THE SYSTEM Before a method can be run the system must filled with buffer and all air bubbles purged from the system The following procedure should be used to prime the system for the first time and when changing buffers 1 Priming the Workstation pump a Immerse the Workstation pump A and B or Maximizer A1 A2 B1 and B2 inlet lines into a container of HPLC grade filtered degassed or other high quality water Place the 10 ml luer syringe supplied with the fittings kit in the priming port of pumphead A If a Maximizer is connected select Inlet A1 Turn the priming port counter clockwise to open the port and gently draw water into the syringe from the pumphead Repeat this operation until no air bubbles are visible in the inlet tubing Disconnect the pumphead outlet tube and hold a beaker up to the port With the syringe full of water inject water into the priming port using several short pulses to dislodge any trapped air bubbles Once all the bubbles have been dislodge close priming port and reconnect the pumphead outlet tube Repeat this priming procedure for the pump B inlet or inlets A2 B1 and B2 if a Maximizer is connected 2 Flush the System Through to the Fraction Collector a b 4 8 Take the column out of line if it has been connected From the Manual screen place the AVR7 3 injection valve into the Purge posi
198. eans it can be placed elsewhere by using the Paste button e To copy the currently highlighted step s in the protocol To paste the cut or copied step s before the currently highlighted step Delete To delete the currently highlighted step s from the protocol 7 29 MODES OF OPERATION SYSTEM OPERATION 7 4 RUN SCREEN The Run screen displays a run in progress All data associated with the run are automatically saved An example of a run in progress is shown in Figure 7 5 a and b below Table 7 13 discusses the buttons which may be used to control the run In addition if the EZLogic Integration software is installed the Integ toolbar button replaces the Log button PULL DOWN MENU PULE DOWNMENU TO SELECT LEFT TRACE TO SELECT RIGHT TRACE BioLogic Duo Flow user name project name method name run el ix File Edit View Utilities Options Window it 5 i c BA Kn HM _ A Bio Rad al Setup Protocol Run Notes Pos Settings Abort Pause Hold Full View Web Frac Collector Conductivity Y M i i Fractions Advance RackPos Divert Valve Tube 1 2 9 Collect 0 100 00 0 Buffer B Waste High psi 1000 H Low psi 0 Set Chart Recorder UV Range 0 01 Y Cond Range 500 Y
199. eb Maximizer Gradient Pump F10 Fraction Collector BioFrac QuadTec Detector Y Econo Gradient Pump 1 Y Mode System Local Mode System Local Y Flowrate ml min Rack F1 12 13 mm tubes Y Mode System Local Mode System Local pH Inlet B Rack Tube A Y 1 End B Y 90 Grid 1F n Y n Y High finit Low limit 1 00 X Zero Baseline ON OFF Flowrate 1 000 ml min Lamp Type Deuterium Range 190 370 nm EGP B 0 Split Wavelength Selection Fraction size X 280 t nm nm Tube Vol left Advance 7 Flow Direction 260 214 405 nm nm Set START STOP Set e ser sre Workstation Valves o SVT3 2 at port 1 SV5 4 at port 2 4O O2 AVR7 3 at port 4 AVR9 8 at port 5 70 1 6 902 eia Rack Pos Tube
200. eck that the O ring seal at the lamp housing aperture is present and undamaged If there is any indication of damage or if it is missing be sure to replace it using the O ring supplied with the kit 5 Reconnect the UV Lamp cable to the Workstation 6 Turn on electrical power to the Workstation and at the DuoFlow Controller go to the Manual screen and turn on the lamp Allow at least 30 minutes for the mercury lamp to warm up and at least several hours for the zinc lamp 11 5 MIXERS The Maximizer mixer and the MX 1 mixer may be disassembled for cleaning or to change the mixer capacity to accommodate the flow rate Cleaning may be required if there is erratic gradient performance Both mixers consist of the mixer base and barrel magnetic stir bar screws mixer top and sealing O rings Note If the system has been used make sure that any hazardous material has been flushed from the system the pumps are not running and any residual pressure has been bled from the system Drain fluid from the mixer disconnect any plumbing connections disconnect the cable from the workstation and then remove the mixer from the system rack 1 Use the hex key provided or any standard 5 32 hex key to remove the four assembly screws from the top of the mixer 2 Remove the mixer top and turn it upside down to remove the O ring If the O ring does not easily dislodge then use your fingers to remove it Note the position of the O rings refer to Figure
201. ect Fractions during entire method Chart Recorder Turn ON 25mM Tris 100 Time 2 0 min 25mM Tris Salt 0 Flow 2 0 ml min Static Loop Auto Inject Valve Edit Method i Load Sample Load standard Volume 1 0 ml Flow 2 0 ml min i 25mM Tris 100 Volume 4 0 min posteo 25mM Tris Salt 0 Flow 2 0 ml min Vi Onl Li di 25mM Tris 100 gt 55 Volume 16 0 min MG ny inear Gradient 25mM Tris Salt 0 gt 45 Flow 2 0 ml min 99 i i 25mM Tris 55 gt 100 Volume 2 0 min Linear Gradiant 25mM Tris Salt 45 gt 0 Flow 2 0 ml min 25mM Tris 100 Volume 3 0 min 25mM Tris Salt 0 Flow 2 0 ml min Isocratic Flow Isocratic Flow End of Protocol Fraction Collection WL1 280nm WL2 260nm WL3 214nm WL4 405nm Econo Gradient Flow Rate EGP 96 Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 B 0 Maximizer Gradient Pump F10 UV Conductivity SIM1 SIG Y SIM1 pH Y 1 00ml min 0 B2 438 psi 1 003 AU 1 23 mS cm 0 548 Volt 7 00 pH QuadTec Figure 7 8 Protocol Screen during a Run 7 36 SYSTEM OPERATION MODES OF OPERATION 7 4 A Run Notebook Screen Use the Run Notebook screen to maintain any information you want regarding the run It contains fields for description of the sample the column
202. ector a refractive index detector or a fluorescence detector The SIM digitizes the analog signal and transmits it to the BioLogic DuoFlow Controller External SIM modules are discussed later in this chapter Cond Flow cell To connect the Conductivity flow cell The Conductivity monitor flow cell must be connected to the Maximizer to supply temperature information for pH compensation SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM Table 2 8 continued Maximizer Rear Panel Connectors Instrument Bus The RJ 45 modular phone connectors and their bus communication cables connect the Maximizer to the Controller and the Workstation via the USB Bitbus Communicator The Instrument Bus handles all communications between the Controller and each of the components in the system Components can be connected in any order in the system Com 1 To connect the QuadTec UV Vis detector Com 2 Reserved for future use Mixer To connect the mixer Aux The 9 pin AUX port connects a variety of peripheral modules that cannot communicate with the DuoFlow Controller over the instrument bus Pin Description 1 Inject A contact closure between pins 1 and 9 GND satisfies a Hold for inject command which has been programmed in a method protocol n c No connection n c No connection n c No connection FC Adv Model 2110 and generic fraction collector Advance output AUX Pump A Stop Start command is sent to a pump e g B
203. ed Threshold e Start ml End ml Fraction Size ml Thresh AU and Non Peak Frac Size ml Each window is defined by these parameters Use the scroll bar to the right of the window display to scroll through the list of windows Enter the fraction size as volume ml e Add Mode Enter the desired values for Start End Frac Size and Thresh Select the Save Window button and the values will appear in line 1 To enter the values for the next window select Add Window and enter the values Select Save Window and the values will appear in line 2 When the collection scheme is complete select Finished Adding and the Select mode will appear e Select Mode This mode shows the following two buttons Add Window Inserts a window after the highlighted window Delete Window Deletes that window e Non peak parameters Destination and Fraction Size Enter the non peak parameters When collecting non peak material you must enter a fraction size 7 24 SYSTEM OPERATION MODES OF OPERATION Table 7 11 Scouting Scouting Setup a scout experiment for method optimization Scouting is a procedure used to systematically optimize the purification of a specific target molecule i e protein Molecules differ from one another in their charge hydrophobicity solubility reactivity substrate specificity and in their intermolecular interactions A purification protocol that is satisfactory for one type of mo
204. ee Figures 6 8 and 6 9 trace compare name File Options View Tools Window Aw AIN EA Tiled Run Cascade Overlays BioLogic Overlay View Q Queue Method 3 Run Q Queue Method 3 Fractions 176 177 18 19 2 21 2 233 24 23 27 28 429 1 E 0 400 T 0 400 0 100 F 0 090 100 0 Buffer B 0 350 l 0 350 E E 0 080 0 300 E E 0 070 0 250 0 250 E 0 060 0 200 0 200 F 0 050 0 150 0 150 4 F 0 040 t 0 035 0 100 4 0 100 F 0 020 F 0 010 0 000 4 i 0 000 4 70007 0 050 0 050 3 0 050 3 0 050 F O 0 010 00 00 00 00 02 00 00 04 00 00 06 00 00 00 00 00 02 00 00 04 00 au HrMin See A HrMin Sec Q Queue Method 2 Run Q Queue Method 2 Q Queue Method 4 Run Q Queue Method 4 Fractions Fractions 32 3 3 4 42 44 14024 8344 5 j6 9 n 12 14 0 100 0 400 n F 0 090 0 400 100 0 Buffer B 100 0 Buffer B 0350 F 0 080 E 0 300 F 0 070 E E 0 060 0 250 E 0 050 0200 550 E 0 040 0150 i 0 035 X 0 020 F 0 010 0 000 0 010 0 050 i 0 0 050 3 0 000 00 02 00 00 04 00 00 06 00 00 00 00 00 02 00 AU Hr Min Sec Active t
205. een collection schemes such as Collect All Threshold Collection Windows and Threshold Collection Windows all with Delay volume if required automatically become available in the Protocol screen The starting and ending tube numbers also may be specified The Model 2128 s optional diverter valve mounts on the fraction collector s arm and minimizes liquid spills during fraction advances If this valve is used it is automatically sensed by the system and hence is not configured by the user in the Setup screen This instrument is described in detail in its separate documentation 2 7 4 Generic Fraction Collectors A non Bio Rad collector may be used as an integral part of the DuoFlow system providing its tube advance function can be initiated by an active low TTL signal gt 100 ms Electrical connection from the collector is to pin 5 Frac Advance and pin 9 ground of the Workstation s AUX connector See the pin out information in your collector manual With an optional SVT3 2 diverter valve configured as a fraction collector diverter valve in the Setup screen the DuoFlow Controller will control all collection parameters including advanced features such as collection by Threshold and or Collection Windows If the optional valve is not used then only Collect All is available Fraction advance marks to a Bio Rad chart recorder are embedded on the UV signal from the Workstation 2 39 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW 2 8
206. emes are discussed below IMPORTANT NOTE The DuoFlow system is designed to control the BioFrac and the Model 2128 fraction collectors which are the only collectors that provide a choice of racks and the ability to overlay fractions from consecutive runs The DuoFlow system will also accommodate the Bio Rad Model 2110 fraction collector as well as generic collectors To use a fraction collection scheme other than Collect All with these collectors it is essential to assign a SVT3 2 valve as a Fraction Collector Diverter in the Setup screen Rack choice and overlay features are not available with these collectors Fraction Collection Scheme BioFrac Rack Type F1 12 13 mm tubes Threshold Q Collection Windows Fraction Size Start Rack Tube A 1 Q Threshold and Collection Windows 1 00 ml End B 90 Y Row Col 1F 1A Close Collect All The eluant from the entire run will be collected by the fraction collector e Fraction Size Enter the fraction size as volume ml e Tubes Required and Number of Racks The displayed value is calculated based on the total volume divided by fraction size e Start Tube and End Tube Identifies the first and last tubes to receive fractions This applies only to the BioFrac and the Mo
207. ent Scout type are grayed out Steps to be scouted are highlighted in green Steps that have not been selected are highlighted in white Steps to be concurrently scouted must all have the same initial value for the scouted parameter 7 26 Select All Highlights the first step that is compatible with the selected scout type along with all steps that can be concurrently scouted with it Concurrently scouted steps must each have the same initial value for the scout parameter Deselect All Deselects all protocol steps in scout setup Description Parameters Displays the currently defined protocol and is used to select or deselect the steps to be scouted with a mouse SYSTEM OPERATION MODES OF OPERATION Scouting Wizard Step 3 Scouting Wizard Step 3 Pune Gradient initial Run Name 96 Run 1 Scout Gradient Intial B 0 00 0 00 Run2 Scout B Gradient Intial B 5 00 5 00 Run 3 Scout B Gradient Intial B 10 00 10 00 Run 4 Scout Gradient Intial B 15 00 15 00 Auto naming Run 5 Scout B Gradient Intial B 20 00 20 00 Auto increment Increment Value 5 Back Next Finish Cancel This dialog is used to set the number of runs to be performed name each run and set the parameters for the scouted steps e Number of runs Sets the number of runs to be performed as part of the scout experiment e Auto
208. er 1 000 psi 66 bar 6 6 MPa from 0 5 to 40 0 ml min in 0 01 ml min increments With Maximizer 1 000 psi 66 bar 6 6 MPa from 1 0 to 80 0 ml min in 0 02 ml min increments Flow accuracy 0 5 to 40 0 ml min 296 e Compositional accuracy 1 0 e High pressure dynamic mixing MX 1 mixer volume without barrel 263 ul mixer without mixer barrel extender 750 ul mixer with mixer barrel extender 2 0 ml Maximizer mixer volume without barrel 750 yl mixer with mixer barrel extender 5 75 ml mixer with expansion barrel 12 75 ml Built in pressure transducer e Gradient by software control 1 increments A 1 SPECIFICATIONS APPENDIX A USB Bitbus Communicator Size 4 25 x 2 75 x 1 125 11 x 7 x 3 cm Lx W x H e Weight 6 oz Electrical 5 VDC output General System fittings Flangeless 1 4 28 flat bottom fittings fingertight nuts and ferrules e System Tubing for flow rates up to 15 ml min 1 16 1 6 mm OD 0 020 0 51 mm ID orange PEEK tubing The approximate volume of 1 cm of tubing is 2 pl System Tubing for flow rates of 15 ml min and greater 1 16 1 6 mm OD 0 030 0 76 mm ID green PEEK tubing The approximate volume of 1 cm of tubing is 4 5 ul Pump Inlet Tubing 1 8 3 2 mm OD 0 062 1 6 mm ID PTFE tubing Wetted materials 100 bio compatible e Solvent compatibility 1 0 M sodium hydroxide hydrochloric acid and organic acids 1 0 M sulfuric acid 7M urea and Guanidine hydrochloric acid 1009
209. er system 1 2 or 3 and then select the buffer s and specify their 2X concentration Press Next and to to the next step Select a salt from the salt selection screen and specify its 2X concentration Press Next and go to the next step Select the batch size for the solutions that will be prepared Note that this will automatically name each solution and generate the recipe text Change the solution names and recipe text as desired and press Next and to go the next step From the Save Buffer System screen enter the required information and then press Save The following information will be helpful in setting the pH range and reference temperature pH Range The View Table feature be used to estimate the buffer pH range The usable pH range is generally between 10 and 90 96 A2 but also depends on the ionic strength of the buffer The table will include user defined pH corrections if you are editing an existing buffer system that is also currently defined in the method setup and uses pH corrections User defined pH corrections are stored in the method setup and not in the Buffer Editor e Reference Temperature This is the temperature at which the Buffer Blending tables are calculated usually set to 25 except as noted below The Maximizer uses the buffer temperature coefficient to correct the Buffer Blending tables from the reference temperature to the run temperature In the case of multiple component buffers buffers wit
210. er barrel is to be used assure the O ring groove faces up 5 Place an O ring in each O ring groove If you are using only the mixer body only one O ring is required If the mixer barrel is used two O rings are required H SCREWS lt SCREWS lt ee Leg OUTLET PORT MIXER M TOP de OUTLET PORT TOP CO orm 12 ml MIXER O RING GROOVE O RING BARREL O RING GROOVE 750 ul MIXER 5 ml MIXER BARREL EXTENDER BARREL EXTENDER ORG O RING GROOVE O RING GROOVE MAGNETIC MAGNETIC STIR BAR NER STIR BAR MIXER INLET PORT INLET PORT BODY ASSEMBLY OF THE MAXIMIZER MIXER ASSEMBLY OF THE MX 1 MIXER Figure 2 4 Assembly of Mixers 2 20 SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM 2 5 DETECTION SYSTEMS The DuoFlow system supports the following detection and monitoring devices e UV Detector e Conductivity Monitor e QuadTec UV VIS Detector pH Monitor 2 5 1 UV Detector The UV detector is a single beam fixed wavelength UV absorbance detector specifically designed for high resolution protein chromatography Available in several configurations e Rack mountable and portable which enables it to be positioned close to a column outlet for better resolution and decreased peak band broadening e Optional 214 nm filter and zinc lamp available for sensitive peptide analysis UV FLOW CELL FILTER TRAY WITH 254 nm AND 280 nm UV FILTER MERCURY
211. es are available in 1 ml and 5 ml formats to accommodate most sample loads They are recommended for method scouting and for first step purification of crude samples They are based on 50 um Macro Prep supports Refer to bulletin 1946 Macro Prep High Q Support This is a strong anion exchanger containing quaternary amine functional groups with a 50 uim particle size It is ideal for rapid purification of acidic and neutral proteins and peptides Refer to bulletins 1840 A 100 1917 and 1985 2 10 2 Cation Exchange S Strong Cation Exchange The S strong cation exchanger chemistry is available in the following formats 2 54 UNO S Biochromatography Columns These columns are designed to handle separations at high flow rates with low back pressure Instead of a traditional bed of packed beads or particles each column contains an advanced polymer matrix called the Continuous Bed matrix which is nonporous and homogeneous The matrix is designed to maximize resolution binding capacity and speed Refer to bulletins 2116 and 1946 Bio Scale S Pre packed Medium Pressure Columns These columns are designed for high resolution separations of proteins peptides and polynucleotides in analytical to semipreparative medium pressure applications They are available in four column sizes Methods developed on the Bio Scale columns can be transferred to production scale using the Macro Prep 50 um supports Refer to bulletins 1881 1946 and 2079
212. es such as Collect All Threshold Collection Windows and Threshold Collection Windows all with Delay volume if required The BioFrac diverter valve can be mounted on either side of the fraction collector s upright supports See Section 3 8 1 for more information on connecting the BioFrac to the DuoFlow system The BioFrac is plumbed to the DuoFlow system using 0 020 diameter PEEK tubing and 1 4 28 fittings for flow rates up to 20 ml min or with 0 030 diameter PEEK tubing and 1 4 28 fittings for flow rates up to 100 ml min This instrument is described in detail in its separate documentation 2 36 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS 2 7 2 Model 2110 Fraction Collector The Model 2110 is programmed in the DuoFlow software and can function as a stand alone fraction collector with non Bio Rad systems It uses a stationary drop dispensing head and collects up to 80 fractions in a motor driven carousel It also uses standard 13 x 100 test tubes An optional adapter is available for use with 1 5 ml microcentrifuge test tubes The Model 2110 includes the following features n stand alone mode the Model 2110 accepts small chromatography columns that can mount directly to the drop former to minimize dead volume e collect from 1 drop 50 to 9 ml fraction in the 13 x 100 test tubes or 1 5 ml micro test tubes e Itis coldroom compatible
213. fer Systems resisaad iade AETA Nakra RAKERA nemen nnne nennen 10 2 B f Press re ConversiOon nuit ende toe cosa ced e ae eun Hoo oe enano B 1 SYSTEM OVERVIEW INTRODUCTION 1 0 INTRODUCTION 1 1 OVERVIEW The BioLogic DuoFlow chromatography system is specifically designed for the high resolution purification of proteins peptides and other biomolecules where recovery of biological activity is of primary concern The DuoFlow F10 pumphead operates at up to 20 ml min and 3500 psi 233 bar 23 MPa when used with the Maximizer The DuoFlow F40 pumphead operates at up to 80 ml min and 1000 psi 66 bar 6 6 MPa when used with the Maximizer The BioLogic DuoFlow system software provides an easy to use graphic interface and menu driven software for manual operation system setup method editing and run operations The system software may be run on any PC running Microsoft Windows 2000 The flexible control architecture allows the seamless integration of a wide variety of configurations with other Bio Rad and non Bio Rad components to meet your purification requirements UNO COLUMN AVR7 3 SAMPLE INJECT VALVE QUADTEC DETECTOR MAXIMIZER MIXER pH MONITOR i USB BITBUS COMMUNICATOR CONDUCTIVITY MONITOR WORKSTATION 4 v o o gt Hs
214. fer system list This high flow non blending method uses inlet valve A to load Buffer A and inlet valve B to control the percent Buffer A and B in the effluent In this case Buffer A is plumbed to inlets A1 and B1 Buffer B is plumbed to inlet B2 and must be prepare at 1X in buffer and 2X in salt Inlet A2 is not used and should be plugged 10 1 MULTIPLE COLUMNS 10 2 BUFFER BLENDING WITH THE MAXIMIZER ADVANCED SYSTEM APPLICATIONS The BioLogic DuoFlow software provides 26 predefined buffer systems for use in Buffer Blending experiments see Table 10 1 These buffer systems cover a wide pH range and include most of the commonly used chromatography buffers Each buffer system includes recipe text that describes how to prepare the required Buffer Blending solutions In addition the Buffer Editor can be used to create user defined buffer systems The Buffer Editor is a simple tool used to edit or modify predefined buffer Systems or to create new buffer systems Buffer Blending solutions should always be made from reagent grade materials and then filtered and degassed before use Filtering the solutions will increase the life of the DuoFlow check valves as well as the column Degassing solutions will help prevent air bubbles from forming in the DuoFlow pumps and the detector Table 10 1 Buffer Blending Buffer Systems Buffer System pH Range 1 Acetate 20 mM 3 7 to 5 7 2 Acetate 50 mM 3 6 to 5 6 3 Bis Tris 20 mM 5 6 to 7 6 4 Bis
215. fittings if needed 2 Immerse the Workstation pump A and B or Maximizer A1 A2 B1 and B2 inlet lines into filtered degassed buffer 3 Remove air from the inlet buffer lines and the pumps a Place the 10 ml luer syringe supplied with the fittings kit in the priming port of pumphead A If a Maximizer is connected select Inlet A1 b Turn the priming port counter clockwise to open the port and gently draw buffer into the syringe from the pumphead c Repeat this operation until no air bubbles are visible in the inlet tubing 4 Remove air trapped behind the pump heads This procedure should also be done as part of the instruments daily maintenance and anytime there are pressure fluctuations greater than 10 a Disconnect the pumphead outlet tube and hold an empty beaker up to the outlet port b With the syringe connected as described in step 3 inject buffer into the priming port using Several short pulses to dislodge any trapped air and to push the air out of the pump head c Once all the bubbles have been dislodge close the priming port and reconnect the pumphead outlet tube 5 Repeat this priming procedure for the pump B inlet or inlets A2 B1 and B2 if a Maximizer is connected Pumping 200 ml of 10096 methanol MeOH through each pumphead at 5 ml min can greatly reduce air bubbles Immerse inlet lines from both pumps into the methanol and pump at 50 B with the 40 psi backpressure regulator in place 11 3 2 Daily
216. g and 1 4 28 fittings From the Manual screen you can manually switch inlet ports on the SVT3 2 Manual valve control is useful for the following When priming the tubing and the valves with buffer prior to starting a method For purging or rinsing all tubing lines during cleaning process In the Setup screen you can name each of the two valve positions For example when the SVT3 2 is placed before the Workstation pump you can identify the valve by the name or composition of the buffer The name that you apply in the Setup screen will appear in the method Protocol screen when you program an Isocratic Flow step a Change Valve step or a Linear Gradient step When used as a diverter valve with a Model 2110 or generic fraction collector this valve should be named as such in the Setup screen Position 1 of this valve is Waste position 2 Collect 2 33 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW WORKSTATION UV MONITOR CONDUCTIVITY FLOW CELL DIVERTER VALVE SAMPLE SELECT VALVE VALVE SAMPLE WATER WASTE COLLECT TO MODEL 2110 RINSE OR GENERIC FRACTION COLLECTOR Figure 2 18 Two Configurations of the SVT3 2 Valve When used as a diverter valve with a Model 2110 or generic fraction collector this valve should be named as such from the Setup screen Position 1 of this valve is Waste position 2 Collect 2 34 SYSTEM OVERVIEW
217. g or damaged contact Bio Rad Laboratories immediately Bio Rad ships DuoFlow systems in a number of different configurations each with its own catalog number These systems are described in the following table Because of its modular design any of these systems can be upgraded any time simply by adding system options 1 3 INTRODUCTION SYSTEM OVERVIEW 1 4 SYSTEM CONFIGURATIONS The BioLogic DuoFlow is available in the following system configurations Each system configuration is identified by its name its standard components and devices and optional components and devices that may be ordered separately for use with the system BioLogic DuoFlow System Guide S Standard Catalog Number Fraction Collector QuadTec UV Vis PERG BioFrac c EKA He a N F10 Workstation o Wut a c c EU c PH Monitor 760 0037 DuoFlow Basic System 100 120 V 760 0036 DuoFlow Basic System Japan and Korea 760 0038 DuoFlow Basic System 220 240 V 10 ml min flow rate to 3500 psi 254 280 nm detection 760 0047 DuoFlow Standard System 100 120 V 760 0046 DuoFlow Standard System Japan Korea 760 0048 DuoFlow Standard System 220 240 V 10 ml min flow rate to 3500 psi 254 280 nm detection Fraction collection 760 1137 DuoFlow QuadTec Basic System 100 120 V 760 1136 DuoFlow QuadTec Basic System Japan Korea 760 1148 DuoFlow QuadTec Basic System 220 240 V 10 ml min flow rate to 3500 psi UV Vis detec
218. gure 7 3 is used to select instruments and devices for use in a user defined method name alias the Workstation and Maximizer inlets and create Buffer Blending buffer systems Buffer Editor Instruments and devices may include fraction collector BioFrac Model 2128 Model 2110 or generic non Bio Rad detector Standard UV QuadTec UV Vis pH Conductivity SIM Valves SVT3 2 SV5 4 AVR7 3 AVR9 8 auxiliary pump Econo Gradient Econo EP 1 or a generic non Bio Rad pump and a Buffer Blender When a new method is created the devices defined in the default setup are automatically loaded into the Devices in setup list For this reason devices that are used routinely should be saved as part of the default setup with the File Save Setup function Furthermore the Method Template feature determines the type of detector Standard UV or QuadTec that should be included in each template from the default setup Devices are added to the setup using the Available Devices buttons shown in Figure 7 3 When a device is selected a dialog is displayed that allows device specific parameters to be entered When the maximum allowed number of a device has been added to the Devices in setup list the corresponding Available Devices button becomes grayed out Devices may be removed from the Devices in setup list by pressing the toolbar Delete button Double clicking a device in the Devices in setup list opens a dialog used to edit the device settings
219. h as the BioFrac fraction collector the QuadTec UV Vis detector the Model EP 1 Econo pump the Econo Gradient Pump EGP and Signal Import Modules SIM 2 1 1 Controller The Controller runs the BioLogic DuoFlow software version 4 0 or higher on a Windows 2000 operating system From the Controller you can set up and run methods perform simple data analysis and store method and run data The following tables show the key features on the Dell computer provided by Bio Rad Table 2 1 Front View of the Dell PC Computer as the DuoFlow Controller COLOR DISPLAY MONITOR POWER SWITCH CD ROM DRIVE USB CONNECTORS 2 BEHIND COVER PANEL FLOPPY DISK DRIVE Description Turns on off the Controller and monitor Floppy To backup methods to and restore methods from a floppy disk Press the button Disk Drive next to the drive slot to manually eject a floppy disk from the drive SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM Table 2 1 continued Front View of a Dell PC Computer as the DuoFlow Controller fme Pel CD ROM Drive Keyboard Mouse amp Function Keys USB Connectors To load updates of the BioLogic DuoFlow operating software To open the drive press the button on the front of the drive The keyboard and mouse interface devices control the system They are standard PC compatible input devices The keyboard includes the following special fun
220. h more that one type of buffer an average temperature coefficient is used to correct the tables In this case the Reference Temperature should be set near the run temperature to obtain the best pH accuracy MULTIPLE COLUMNS ADVANCED SYSTEM APPLICATIONS 10 3 PH MEASUREMENT AND CORRECTIONS Buffer pH plays an important role in chromatography of biomolecules since it affects both their stability and their chromatographic properties Both solution temperature and ionic strength affect the pH of a buffer The Maximizer is designed to compensate for both of these effects during an experiment A sensor in the conductivity flow cell monitors temperature and so must always be connected to the Maximizer Changes in pH due to changes in ionic strength are compensated for by information stored with each Buffer System Changes in pH due to sample injection and or adsorption or desorption of ions from a column cannot be compensated for and may cause the pH to deviate from the specified run pH For this reason it is advisable to run ion exchange experiments at a pH near the middle of the buffers pH range where the buffer capacity is greatest Accurate pH measurements require that samples be of uniform composition and temperature These conditions do not generally occur when monitoring pH during a chromatographic run For this reason pH s measured during a run should always be considered approximate Furthermore some pH accuracy is sacrificed in order to make pH p
221. h the stator body when it is lifted off 11 11 MAINTENANCE MAINTENANCE AND TROUBLESHOOTING 3 Remove the stator face assembly with your fingers and set it aside 4 Remove the rotor seal by placing a small flat blade screwdriver into the slot on the side of the stator ring Gently pry up the rotor seal which will pop off 5 Inspect the components from the disassembled valve Make sure there are no scratches or foreign material on the sealing surfaces of the valve Clean as necessary Soak or sonicate in a bath containing a mild detergent To reassemble the valve 1 Place the new rotor seal slot side facing the stator body onto the three seal pins of the stator ring It will only fit one way Press the rotor seal so that it fits firmly 2 Place the new stator face assembly onto the stator body so that the pins slip into the mating holes in the stator body It will only fit one way 3 Replace the stator body and stator face assembly so that the stator ring enters the mating hole on the stator body 4 Using the three screws re assemble the valve Tighten each screw equally H o H STATOR SCREWS 3 E STATOR BODY STATOR FACE C 7 ASSEMBLY ROTOR SEAL ACCESS TO b ROTOR SEAL STATOR RING Figure 11 8 AVR7 3 and AVR9 8 Valve Assembly 11 12 MAINTENANCE AND TROUBLESHOOTING MAINTENANCE 11 7 MAXIMIZER VALVES Over time the Maximizer valves may become worn and require replacement
222. hat does not leak FITTINGS TIGHTENER PEEK FERRULE STAINLESS STEEL COMPRESSION RING CUT THE TUBING TUBING i Oc Figure 4 2 Making 1 4 28 flat bottom fittings 4 2 SYSTEM INSTALLATION AND SETUP SYSTEM PLUMBING 2 Slide the nut stainless steel compression ring and the ferrule in that order onto the tubing as shown in Figure 4 2 The flattened end of the compression ring should face towards the nut with the tapered end facing the tapered end of the brown ferrule The stainless steel lock ring is not in the fluid path so biocompatibility is maintained 3 Allow the tubing to extend slightly beyond the end of the ferrule 4 Place the fitting and tubing into the green fittings tightener Do not allow the tubing to slip out of the ferrule 5 Tighten with your fingers to seat the ferrule onto the tubing but do not over tighten The ferrule should be flush with the tubing 6 Once the fitting is made the compression ring and ferrule should adhere to the end of the tubing while the nut will be moveable 4 2 PLUMBING DUOFLOW SYSTEM This section discusses how to create your own plumbing arrangement of a DuoFlow system The plumbing for each of the DuoFlow valves is discussed later in the chapter The Fittings Kit includes an F10 tubing kit that includes all tubing required for a basic installation Refer to Figure 4 1 A
223. he Instrument Bus handles all communications between the Controller and each of the components in the system For example the Instrument Bus connects the Workstation to the USB Bitbus Communicator Maximizer BioFrac fraction collector or Econo Gradient Pump Components can be connected to the system in any order Aux The 9 pin AUX PORT connects a variety of peripheral modules that cannot communicate with the DuoFlow Controller over the Instrument Bus If the Maximizer is being used use its AUX connector before the connector on the Workstation Pin Description 1 Inject A contact closure between pins 1 and 9 GND satisfies a Hold command which has been programmed in a method protocol n c No connection n c No connection n c No connection FC Adv Model 2110 and generic fraction collector Advance output AUX Pump A Stop Start command is sent to a pump e g Bio Rad EP 1 n c No connection n c No connection GND Ground Reserved for internal Bio Rad use SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM 2 3 BIOLOGIC MAXIMIZER VALVE SYSTEM The Maximizer enables buffer blending applications doubles the accessible pump flow rate and doubles valving capacity to 6 low pressure valves and 6 high pressure valves The Maximizer includes a separate Maximizer mixer see Section 2 4 2 and pH monitor see Section 2 5 4 Proportioning valves on the Maximizer blend water salt and the conjugate aci
224. her and a Method and its runs can be moved from one project to another Individual runs cannot be moved Not that the selected project method and run have to be closed before they are moved To move an item a Highlight the item in the database tree you want to move You may highlight a Project or a Method b Click once on the Move button on the Browser toolbar The items to be moved will appear in the MOVE List in the Browser tab window From the MOVE List select the items you want to move From the database tree select the new destination for the items you are moving e Again click on the Move button The items will automatically move to the destination Hint Alternatively highlight the destination icon right mouse click and select the Move Selected message with a left mouse click f Once you finish the move procedure use the Clear List button on the Browser toolbar to delete the remaining methods and runs in the MOVE List Places methods in a queue Highlight the method s you wish to place in a queue and select Queue The methods will appear in the tab screen Refer to Section 6 3 for detailed description of queuing 6 5 INTRODUCTION TO THE BROWSER SCHEEN SYSTEM OPERATION Compare Places runs in a list for overlay comparison Highlight the Run s you wish to compare and select Compare The run will appear in the tab screen See Section 6 4 for detailed description of trace compare Reset Updates and refreshes the Br
225. hod QuadTec 1 280 nm Run Queue Method Run Time 1 91 min Visibility Y Axis X Axis Chromatogram Settings Visibility Trace Run QuadTec 1 280 nm QuadTec 1 280 nm QuadTec 1 280 nm QuadTec 1 280 nm Run Q Queue Meth Run Q Queue Meth Run Q Queue Meth Run Q Queue Meth 6 5 1 Chromatogram Display Screen Figure 6 9 Trace Compare Window Overlay View The Chromatogram Display Screen consists of one or more windows containing a chromatogram Only one window is active at a time The window appearance is controlled by the mouse as well as the Toolbar Buttons Drop down menus Active Traces and Values at Cursor control and Chromatogram Settings control as described below Windows may be resized by doing a click and drag on the side of the window with the mouse Double clicking on the window title bar makes the window full screen The window chromatograms can be zoomed by doing a click and drag with the mouse 6 13 INTRODUCTION TO THE BROWSER SCHEEN SYSTEM OPERATION 6 5 2 Toolbar Buttons The function of each toolbar button is provided in Table 6 1 Table 6 1 Toolbar Buttons Pei Ti dd Tiled Run Displays Trace Compare chromatograms in in tile view lle un EJ Cascade Displays Trace Compare chromatograms in in cascade view ascaae RA Overlays Displays Trace Compare chromatog
226. hods in the queue in the Tab window 5 After all methods are listed in the project queue click the Verify Queue button in the right sidebar of the Tab window to confirm that all methods meet the necessary criteria to be run e If the methods meet the necessary run criteria to run correctly Methods were successfully verified will appear f methods do not meet the necessary run criteria a list of errors will appear Errors will appear if methods are not compatible to run in sequence or the hardware Setup screens are not identical e To run your queue click the Run Queue icon in the right sidebar of the Tab window Description of the Queue Icons S Open Method M Delete Method X Edit Method 2 Verify Queue Run Queue 09 Edit Queue 6 10 Open Method Opens a highlighted method for editing or viewing in the Protocol Screen Delete Method Deletes a highlighted method from the queue This is displayed only prior to the queue running Edit Method Copies the highlighted method allows you to Rename it and then opens it for editing in the Protocol screen This is displayed only when the Queue has completed its run Queued methods cannot be edited after the run has started Verify Queue Checks methods in a queue to assure there is not a conflict in the method Protocol and Setups to prevent running If there is a conflict a list of errors will appear If methods are compatible the message Me
227. ift Solution temperature is changing Allow all solutions to come to thermal equilibrium Fluctuations observed in pH readings during run Changes in salt concentrations during a run may cause errors in the observed pH The actual pH is may not be in error but only the observed pH Collect fractions and measure the pH of the solution using a suitable bench top pH monitor during a run Ensure that the mixer size is appropriate for the flow rate The observed buffer pH does not match the desired pH The software pH correction may have been entered incorrectly in the Buffer Blending Setup Check the pH correction setting and change it if necessary The pH electrode is not calibrated Recalibrate the pH electrode using standards that span the pH range of the buffer being used Buffers may be incorrectly prepared The pH elecrode will not calibrate 12 10 The electrode may be dirty or damaged Clean the pH electrode as described in the instructions that came with the probe Replace the pH electrode if necessary MAINTENANCE AND TROUBLESHOOTING TROUBLESHOOTING 12 6 TROUBLESHOOTING OTHER BIO RAD INSTRUMENTS AND DEVICES For troubleshooting information about other Bio Rad instruments and devices such as the BioFrac fraction collector and the QuadTec detector refer to their separate documentation 12 11 TROUBLESHOOTING MAINTENANCE AND TROUBLESHOOTING 12 12 APPENDIX A SPECIFICATI
228. ii User 3 08 04 2001 11 55 42AM 1 Y RS USER User1 n BROWSER METHOD Compare 09 10 2001 10 07 25AM TAB SUMMARY WINDOW ICON Information MOVE List QUEUES COMPARE COPYIN List Error List BROWSER TABS QuadTec WL1 280 WL2 260nm 214nm WL4 405nm Econo Gradient Flow Rate Split 0 40 0 15 2 00 0 00 Pump 0 00 ml min 0 B 0 Maximizer Gradient Pump F10 UV Conductivity SIM1 SIG SIM1 pH 1 00ml min 0 2 438 psi 1 003 AU 1 23 mS cm 0 548 Volt 7 00 pH Figure 6 1 The Browser Screen 6 1 OVERVIEW The Browser screen displays the following information and controls e Collapsing expanding the tree hierarchy Click on this icon in the database tree to collapse the listing for a User Project Method or Run folder Click on this icon in the database tree to expand the listing for a User Project Method Queue Scout or Run folder Updates and refreshes the Browser screen by collapsing all folders to the top Users icon INTRODUCTION TO THE BROWSER SCHEEN SYSTEM OPERATION Icon colors in the database tree indicate the following Green Currently open in the active window The active window may be the online window or the off line window For more detailed information about online and offline refer to the discussion in Section 7 4 2 Working Offline Red Currently open in the window that is no
229. ilable from the Del Tag button in the toolbar Delete all tags Deletes all tags in the chromatogram Not available from the toolbar Trace Legend Displays different line formats for each of the chromatogram traces This is useful for distinguishing traces when printing to a black and white printer SYSTEM OPERATION INTRODUCTION TO THE SYSTEM SOFTWARE Table 5 5 View Drop down Menu BioLogic Duo Flow user name lt project name gt File Utlilties Options Window Help Manual Setup Protocol Run Post Run Run Notes Run Log TraceCompare Bio Rad webpage BioFrac tube format Rack and Tube BioFrac tube format Rack and Grid Volume based Chromatogram The contents of View menu remain the same in each displayed screen Manual Displays the Manual screen for individual control of installed instruments and devices in the system Setup Displays the Setup screen which allows you to specify the components required for your method Protocol Displays the Protocol screen for creating and editing chromatography steps in the method Run Displays the Run screen from which you can initiate a sample run of the open method Post Run Displays the Post Run screen from which you can view the chromatogram and apply tags to UV Conductivity and or B traces Run Notes Displays the run notes screen used to store information such as sample description column type operator buffer s flow rate
230. ile wmf or enhanced metafile emf Copy Overlay Image to Clipboard Allows you to copy the overlay view to the clipboard for pasting into other applications Table 6 3 Options Drop down Menu lt trace compare name gt File View Tools Window Help Select Overlay Traces Autocolor overlay traces User Preferences Time as HH MM SS Grid for BioFrac Tube The Options menu consists of the following Select Overlay Traces Allows you to select which traces will appear in overlay view AutoColor Overlay Traces Toggles between coloring each trace a different color or coloring each trace by its trace type User Preferences Allows you to set the default time and BioFrac tube numbering format Time as HH MM SS Time as Minute tenth Toggles time format between HH MM SS and Minute tenth Grid for BioFrac Tube Tube for BioFrac Tube Toggles tube number format for the BioFrac between Grid numbering and Tube number 6 15 INTRODUCTION TO THE BROWSER SCHEEN Table 6 4 View Drop down Menu trace compare name Tiled Run Chromatograms Overlay Chromatogram All Chromatograms Cascade Chromatograms Legend Full View Full View for All Chromatograms Volume based chromatograms File Options Tools X Window S SYSTEM OPERATION The View menu consists of the following e Tiled Run Chromatograms Places Trace Compare in Tiled view and displ
231. in peak 1 00 01 41 6 7 0 08617 7 999 N A Conalbumin peak 2 00 01 48 7 2 0 05921 9 025 N A Ovalbumin 00 02 44 11 0 0 03799 16 472 N A Soybean Trypsin Inhibitor 00 03 30 14 0 0 06844 22 308 N A To change a tag name in the Post Run screen chromatogram move the cursor over the tag and note that the cursor changes Double click on the tag and in the window that appears enter the tag name To deselect a tag highlight the undesired tag and press DelTag from the button bar To remove all tags select Figure 7 12 Post Run Tags for UV Detector Note Additional Traces will be shown when the QuadTec Detector is being used Delete all tags from the Edit drop down menu 7 40 SYSTEM OPERATION MODES OF OPERATION 7 5 4 Entering Activity Data The Activity Trace Editor permits data collected by a separate offline method to be included with data collected by the DuoFlow method For example if fractions collected by the DuoFlow are also analyzed by an ELISA method or for radioactivity CPM or DPM the data collected by these assays can be entered into the Activity Trace Editor and a trace will appear on screen reflecting the activity for each fraction 1 To enter activity data Select the run for which you wish to enter the post
232. indow The Browser tabs at the bottom of the Browser screen controls the information to be displayed in the Browser Tab window The following tabs are available Information tab Displays two panels in the Browser Tab window The left panel shows details about the specific item selected the right panel displays a chromatogram when a run is selected MOVE List tab Displays the list of all items selected using the Move button to be moved from one place to another in the database list Items selected to be moved are indicated by a red checkmark in the database tree Projects can be moved from one user to another methods can be moved from one project to another When a project is moved it takes all methods and runs associated with it when a method is moved it takes all runs associated with it The procedure for moving an item is on page 6 5 Queue tab Displays the methods in the Browser that have been placed in a Queue The sequence of methods in a queue can be changed by dragging and dropping Refer to Section 6 3 for running a queued method Compare tab Displays the runs selected or highlighted in the Browser for comparison Refer to Section 6 4 for running Trace Compare COPYIN List tab Displays the list of all items selected to be copied in restored to the database from an archived data file or disk The procedure for copying in items is discussed on page 6 4 Error List tab Displays setup discrepancies that exist between methods in a
233. io Rad EP 1 pump n c No connection n c No connection GND Ground Power Cord The grounded 3 prong connector inputs power to the Workstation and outputs power to any unit connected to the Workstation The Workstation s input power cord should be plugged into a 3 prong grounded power outlet 2 15 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW Table 2 9 Maximizer Screens mE Function and Description on Maximizer Faceplate in Local Mode Inlet Selection Arrow buttons switch between Inlets A1 0 and A2 1 BLEND A POS 0 PREV changes to previous screen PREV NEXT NEXT changes to next screen Arrow buttons switch between Inlets B1 0 and B2 1 BLEND B POS 1 PREV NEXT Valve Control Arrow buttons select a port Port on valve Valve Up to six valves lt Valve gt lt Port gt POS 0 may be displayed including three motorized AVR7 3 AVR9 8 and three PREV NEXT solenoid SVT3 2 SV5 4 valves ENTER accepts the change and moves the valve to the new position SIM Calibration SIM 0 000 Volts Displays the current Maximizer SIM voltage PREV NEXT CALSIM ENT SET Used in conjunction with a 1 Volt calibration source PREV NEXT Pressing ENTER sets the current voltage reading to 1 V Pressing the UP arrow resets the calibration to the factory setting CAL SIM ENT SET After ENTER is pressed the display responds with SET to show that the PREV set NEXT calibration was successful CALSIM ENT SET After the
234. ioLogic Configuration Utility Software Screen SYSTEM INSTALLATION AND SETUP SYSTEM PLUMBING 4 0 SYSTEM PLUMBING This chapter discusses recommended plumbing practices and provides general guidelines for system setup The system will work more efficiently if tubing lengths are as short as possible Bio Rad provides precut and 1 4 28 fitted labeled tubing in the Fittings Kit The illustration below shows where the tubing is designed to be connected Discussion of how to create your own tubing can be found in the following section Use 1 16 1 6 mm OD PEEK tubing and 1 4 28 fittings Use orange PEEK 0 020 0 51 mm ID tubing for the F10 pumps and green PEEK 0 030 0 76 mm ID tubing for the 40 pumps Premade tubing kits are provided with your Fittings kit AVRT 3 INJECT VALVE AVR7 3 SAMPLE INJECT VALVE ed DXX 00008 UV DETECTOR WET PORT COLUMN s CONDUCTIVITY MONITOR QUADTEC DETECTOR COLUMN e TO FRACTION 5 COLLECTOR MIXER CONDUCTIVITY MONITOR Figure 4 1 System Plumbing using Tubing Kit from Fittings Kit 4 1 SYSTEM PLUMBING SYSTEM INSTALLATION AND SETUP 4 4 GENERAL GUIDELINES FOR CREATING YOUR OWN TUBING CONNECTIONS The DuoFlow system uses three types of tubing Use the following table to select the appropriate tubing The Fittings Kit c
235. ion and the protocol can be programmed In the Protocol screen select Load Inject Sample In the Load Inject Sample window note that the AVRT 3 valve will automatically move to the Inject position at the start of the step and to the Load position at the end of the step a In the Load Inject Sample window specify Dynamic Loop b In the Injection Buffer area of the window select the buffer s and percent 96 composition to be used to inject the sample onto the column c In the Volume ml field enter the sample volume In the Flow ml min enter the flow rate of the Workstation pump that will be used to inject the sample onto the column Continue writing the separation protocol The following steps should be programmed within the method protocol to automatically inject a sample using an auxiliary pump 1 Connect the Aux pump The EGP pump connects to the Instrument bus If you are using the EP 1 Econo pump consult Section 8 2 In the Setup screen select Aux Load Pump and AVRT 3 valve in addition to the other device and instruments you have connected to the system In the Protocol screen program an Isocratic Flow step that is long enough for the auxiliary pump to load the desired quantity of sample into the DynaLoop For example if you are loading 25 ml of sample at an auxiliary pump flow rate of 5 ml min you will need at least a 5 minute step prior to the sample loading step Otherwise the protocol will f
236. irm that the UV flow cell capacity volume is appropriate for the flow rate Complete discussion of the UV detector and conductivity monitor is provided in Section 2 5 including the procedure for changing the UV flow cell Using the rod clamps attach the UV detector to a vertical or horizontal bar close to the column outlet Connect the power cable square connector from the UV detector lamp into the connector marked UV Lamp on the rear of the Workstation Refer to Figures 3 4 and 3 5 Connect the UV detector signal cable mini DIN connector to the connector marked UV Optics on the rear of the Workstation Refer to Figures 3 4 and 3 5 Connect the Conductivity monitor s combined power and signal cable mini DIN connector to the connector marked Cond Flow cell on the rear of the Maximizer if available Otherwise connect it to the Workstation Refer to Figures 3 4 and 3 5 The Conductivity monitor is designed to be held in the circular notch of the optics bench but may be placed anywhere in the fluid path SYSTEM SETUP SYSTEM INSTALLATION AND SETUP 3 6 2 QuadTec UV Vis Detector The QuadTec detector is shipped with a dummy flow cell installed Before operating the detector you need to install the biocompatible 3mm PEEK flow cell For complete discussion of the QuadTec detector including the procedure for installing the flow cell refer to the QuadTec Instruction Manual 1 2 3 10 Make sure the detector power is OF
237. its pH calibration mode CANCEL aborts the calibration Conductivity Calibration Displays the current conductivity reading mS cm ENTER causes the Maximizer to enter calibration mode CURSOR moves the cursor to the next digit of the calibration constant ENTER accepts the conductivity cell constant calibrates the conductivity meter and exits calibration mode CANCEL aborts the calibration Beeper ENTER causes the Maximizer to enter volume adjustment mode The UP and DOWN arrows adjust the volume of the alarm Firmware Version Displays the current version of the Maximizer firmware 2 17 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW 2 4 MIXERS Bio Rad s DuoFlow mixers improve gradient quality by mixing the output from the DuoFlow Workstation pumps There are two PEEK biocompatible mixers for DuoFlow systems e MX 1 mixer A low volume mixer for use with a DuoFlow system not equipped with the Maximizer Maximizer mixer A large volume mixer for use with the Maximizer The mixer cable plugs into the connector labeled Mixer on the rear of the Maximizer or Workstation If you will be using the MX 1 mixer connect it to the Workstation if you will be using the Maximizer mixer connect it to the Maximizer The mixers have two inlet ports seal the unused port with the plug provided 2 41 MX 1 Mixer The Model MX 1 mixer is used when the DuoFlow system is not equipped with the Maximizer The mixer may
238. ity flow cell The conductivity flow cell constant is printed on a tag attached to the flow cell cable When a new conductivity flow cell is installed use this value to enter the flow cell constant Note You must exit the Calibration screen in order for the system to accept the calibration values pH Probe Calibration This utility allows the user to calibrate the pH monitor It is typically used at the start of each day s use of the system Note You must exit the Calibration screen in order for the system to accept the calibration values EGP Calibration This is informational only It reminds the user to calibrate the pump through the pump software Refer to the EGP Instruction Manual 5 10 SYSTEM OPERATION INTRODUCTION TO THE SYSTEM SOFTWARE Table 5 7 Options Drop down Menu Options menu Manual Screen Options menu Browser Setup Protocol Post Run Screen e BioLogic Duo Flow user name project name method e BioLogic Duo Flow user name project name method File Edit View Utilities Window Help File Edit View Utilities Window Help Edit User Preferences Edit User Preferences Chromatogram Settings Chromatogram Settings Status lines Status lines Browser Settings Browser Settings Manual Setup Use Time min Buffer Blender Setup Use Volume ml Time and Volume are displayed The contents of the Options menu depends up the disp
239. k space The copy out function copies the User Project and Method names associated with each Initiating a CopyOut to floppy disk completely erases the contents of the floppy disk To copy out highlight the runs you wish to copy out Press the CopyOut button on the Browser toolbar A dialog box appears that allows you to select the destination of the ZIB file either the hard drive or a floppy disk Choose the destination and press OK 6 3 INTRODUCTION TO THE BROWSER SCHEEN SYSTEM OPERATION BioLogic Duo Flow user name project name gt method name gt run name gt File View Utilities Options Window Help Z A A Settings CE Default 08 04 2001 11 55AM 1 Default 08 04 2001 11 55AM 0 Edit Delete Select Copy In File Print File name Folders biologic zib CopyOut biologic zib z a Cancel Copyln Help K Move List Files of type Drives Biologic Archive ZIB a Network Queue Ef Select All Compare SS Clear List Reset e w BK V Information MOVE List QUEUES COMPARE COPYIN List Error List WL1 280nm
240. kpressure device See Section 11 3 for additional information SYSTEM OPERATION INTRODUCTION TO THE SYSTEM SOFTWARE 5 0 INTRODUCTION TO THE SYSTEM SOFTWARE The BioLogic DuoFlow system software is run on computers running the Microsoft Windows 2000 operating system This chapter discusses the DuoFlow system software version 5 0 5 1 SYSTEM INTERFACE The Manual screen Figure 5 1 is the first screen displayed when the BioLogic software is started This screen like all DuoFlow screens is grouped into the system menus the control window and the status bar The use of the Control window functions is discussed in Chapters 6 and 7 and in the separate documentation for the Econo Gradient Pump EGP and the QuadTec UV VIS detector Note that many menus and tool bar functions are grayed out and inacessible until you select or enter a User name in the Browser see chapter 6 Browser screen Chapter 6 Manual screen Chapter 7 section 7 1 Setup screen Chapter 7 section 7 2 Protocol screen Chapter 7 section 7 3 Run screen Chapter 7 section 7 4 Post Run screen Chapter 7 section 7 5
241. lable in your laboratory press Done and go back to the main screen If the buffer is in the list but is made of reagents that are not available in your laboratory press Copy rename the buffer and change the buffer information as necessary Save the buffer press Done and go back to the main screen If the desired buffer is not in the list press New and enter the required buffer information Save the buffer press Done and go back to the main screen The Use Activity Correction option is used to correct for the pKa dependence on buffer ionic strength In practice this option should be checked for all pKa s where the charge of one of the associated buffer ions is greater than 0 or less then 1 2 Define a Salt From the Buffer Editor main screen press Edit Salt List Use the Salt Name pull down list to select and view the desired salt If the salt is in the list and is made from reagents available in your laboratory press Done and then back to the main screen If the desired salt is not in the list Copy an existing Salt or Press New to create a new salt Enter the required information save the salt and press Done 3 Create or Edit a Buffer System a From the Buffer Editor main screen press Create New Buffer System to create a new buffer system or press Edit Current Buffer System to copy and edit an existing Read Only buffer system From the Buffer selection screen select the number of buffers that will be included in the buff
242. layed screen as indicated above These selections are used to change screen options Edit User Preferences Lets you specify the following Run Options Allows you to elect to have the the valves automatically return to position 1 at the end of a run Protocol Editor Mode This determines whether the default protocol is to be based on time or volume Time Format Allows you to select the time format HH MM SS or min tenth used for the chromatogram X axis BioFrac Tube Number Format Allows you to select how the tube numbers are displayed on the Manual Run and Post Run chromatograms when a BioFrac fraction collector is used In Rack and Tube mode tubes are identified by the rack that they are located in and by the order that the tubes were filled In Rack and Grid mode the tubes are identified by the rack they are located in and by their rack grid location This feature is particularly useful for locating samples in microplates Chromatogram settings Allows you to select which instrument traces will be visible on the chromatogram Up to 8 instrument traces are selectable from among the following standard UV detector conductivity monitor pH system backpressure theoretical B concentration four QuadTec wavelengths and detector traces acquired via the Signal Import Module SIM For the Manual screen the X axis time and the Y axis AU ranges can be set For the Run and Post Run screens only the Y axis range can be set Status Line
243. le 7 9 Miscellaneous Description To repeat the highlighted step s a specified number of times To highlight more than one step hold down the Ctrl key while selecting steps with the mouse To pause the method at a specific step during the run Pause stops the progression of the method and time holds the B composition and stops the pumps Time Out Req d permits a pause time to be entered The step will be paused for the specified length of time after which it will automatically resume An audible alarm may be programmed to sound when the step is paused and when it automatically resumes To sound a 10 second audible alarm at the programmed time The progress of the run will not be affected To zero the detector baseline at a programmed time At system start up the default condition is Lamp ON The lamp may be set to turn off at the end of a run If the lamp is OFF it takes about 20 minutes for the lamp to warm up prior to use To start stop an Econo Gradient Pump at the programmed time for a user defined task To turn the chart recorder on off at the programmed time Inserts a step that tells the chart recorder to start stop the paper feed and lower lift the pen To display additional steps so that they can be added to the method 7 19 MODES OF OPERATION Button Fraction Collection Table 7 10 Fraction Collection Description Collect All SYSTEM OPERATION A To set up fraction collection Fraction collection sch
244. le loading If a Maximizer is being used connect these devices to its AUX connector rather than to the Workstation Power supply for the Workstation electronics as well as all devices connected to and controlled by the Workstation Output power connectors to the UV lamp and a Model 1327 chart recorder Two instrument bus phone type connectors SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM Table 2 5 Workstation Front Panel Controls PUMPHEAD WASHOUT INLET PORTS quu PRIMING PORT A PRESSURE TRANSDUCER PRESSURE BioLogic DuoFlow Workstation OUTLET PORT MR PUMPHEAD OUTLET PORTS PAUSE PRIMING PORT B ALERT LEDs POWER PURGE INLET PORTSA amp B Been Power Button Turns power on off to the Workstation and to the components connected to it Pause Button Stops the Workstation pumps and pauses a running method When it is pressed the status LEDs for pumps A and B change from green to flashing red To resume the run Press the button again to restarts the pumps and the method or Press the Continue button in the Run screen or Press the START button in the Manual screen WASHOUT OUTLET Purge A amp B Before you press either purge button switch the AVR7 3 inject valve to the Purge Buttons position in the Manual screen so that the column is not exposed to high pressures and flow rates The purge buttons flush the tubing lines with the solutio
245. lecule may not work for a different molecule type Several factors influence the quality of a purification procedure Some of these factors include buffer composition pH ionic strength co solutes elution type gradient slope and gradient duration flow rate column chemistry and sample composition In principle each of these can be adjusted to produce the most efficient and effective purification strategy for a molecule In practice only a few of these are generally tested due to time and cost considerations By performing a series of automated scout runs the time and resources required for protocol optimization can be significantly reduced Any method can be turned into a Scout Method using the Protocol Editor s Scouting Wizard Methods used in scout experiments can be copied from an existing method created from scratch or loaded from the Bio Rad method templates Before starting the Scouting Wizard make sure the method setup and protocol are correct and that the step parameters are set to the values desired for the first scout run Scouting Wizard Step 1 Scouting Wizard Step 1 Ix Type of Scout pH Buffer A Buffer B Duration B Gradient Initial B B Gradient Final B Flow Rate Sample Name Sample Volume Column Back Next gt gt Finish Cancel This dialog is used to select the type of scout to be carried ran e pH Used in Buffer Blending mode to find th
246. lie flush against each other with no space in between them Place the two larger screws removed in step 3 above through the valve assembly Set this aside Set the actuator with its open end facing up Place the spring in the opening at the center of the actuator so that it fits into the deepest part of the opening This may take a couple of tries Place the valve body assembly onto the actuator the orientation is not specific then tighten the two Screws Attach the clamp to the valve body with the remaining screws Apply the valve label Make sure that the hole in the label matches with the Pump Common port and does not overlap it 11 6 2 AVR7 3 and AVR9 8 Valves The AVR7 3 and AVR9 8 valves are disassembled and cleaned in the same way The replaceable parts in the valve are the rotor seal and the stator face assembly these items are included in the AVR7 3 Rebuild kit catalog 760 0401 and the AVR9 8 Rebuild kit catalog 760 0403 Indications of damage include valve leaks and valve switching problems Safety Note Before disassembling the valve be sure to flush any hazardous material from the valve and to disconnect it from the Workstation Wear protective clothing as appropriate To disassemble the valve follow the procedure below 1 2 With the hex key that is provided remove the three stator screws Lift the stator body and stator face assembly from the stator ring The stator face assembly usually stays wit
247. ll has a swept volume of approximately 80 ul when the pH electrode is inserted The flow cell mounts to the BioLogic rack using an attached mounting bracket which can also be used to attach the UV detector and Conductivity monitor The flow cell should be positioned so that the flow path is angled upward to promote bubble clearance Plumb the flow cell with the inlet tube attached to the lower port and the outlet tube attached to the upper port The inlet and outlet ports are threaded for use with 1 16 1 6 mm OD tubing and 1 4 28 flat bottom fittings The pH monitor is designed for flow rates up to 80 ml min and flow cell pressure less than 75 psi and therefore should be plumbed downstream of the backpressure regulator pH electrode The pH probe is a sealed Calomel type electrode consisting of a pH and a reference electrode built into the same body The sealed reference design eliminates the need to add electrolyte solutions and minimizes reference dryout The Calomel electrode is fully compatible with buffers such as Tris that may not be compatible with the Ag AgCl silver silver chloride containing electrodes Signal Cable To connect the pH probe to the BNC connector of a Signal Import Module or Maximizer SIGNAL CABLE pH ELECTRODE pH FLOW CELL Figure 2 9 pH Monitor The pH electrode and flow cell are described in detail in their separate documentation 2 5 5 Other Detectors Detectors other than those offered by Bio Rad
248. lt the system assumes that the current user and project shown in the window title bar is the path for the new method Name a New Run in a previously defined Method To start a new run from the Browser a Select a Method or a Run in the Method where you want the new Run to be located b Click once on the New button c Select New Run Enter the Run name and press OK The Run screen appears Pressing the Start button in the toolbar will begin your sample run Hints 1 Alternatively highlight the Method icon right mouse click and select New Run with a left mouse click 2 You can also use the New Run button in the toolbar By default the system assumes that the current User Project and method shown in the window title bar is the path for the new Run SYSTEM OPERATION INTRODUCTION TO THE BROWSER SCREEN 6 3 CREATING AND RUNNING A QUEUE Queue allows you to run multiple methods in sequence Methods placed in a queue must have identical hardware setups or the system will not permit them to run BioLogic Duo Flow user name project name method name run name File Edit View Utilities Options Window Help Date 6 USERS 4 ChemLab 03 02 2001 10 04 56AM i0 LP Project for ChemLab 01 07 2001 07 08 34PM Open f Mary Jones 01 13 2001 07 34 43AM
249. lumn Econo Columns have two male luer lock fittings Catalog 750 0565 Low Pressure To connect to the system use female luer to 1 4 Econo Column to Columns and flow 28 adapters BioLogic System Fittings Kit adapters If a flow adapter with flexible tubing is to be used attach the barbed to male luer fitting included in this kit then connect the female luer to 1 4 28 adapter If the column adapter uses PTFE tubing attach This kit includes all the parts the small length of 1 16 1 6 mm ID Tygon needed to connect Econo tubing and insert the barbed male luer fitting into Columns and flow adapters open end Five cm of Tygon tubing is included in to the BioLogic DuoFlow this kit Connect as above system FPLC Column Pharmacia s FPLC columns use M 6 fittings To Catalog 750 0561 connect to the system use 1 4 28 to M6 adapter Union 1 4 28 to M6 union Two of these are supplied with the Fittings j TE 2 58 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS Table 2 15 continued Columns and Column Fittings HPLC Column or These columns accept 10 32 nuts To connect this Catalog 750 0564 Pharmacia s type of column to the system use two 1 4 28 to HPLC Column to BioLogic RESOURCE 10 32 adapters System Adapters Column Catalog 42750 0567 MG Fittings Kit UNO Column to This kit includes two nuts and four ferrules to FPLC system connect an UNO column to an FPLC system UNO Column to This kit i
250. luorescence detectors The external detectors are connected through a SIM HR and or a Maximizer SIM see Chapter 2 9 6 and Table 2 7 respectively Valves SVT3 2 SV5 4 AVR7 3 and AVR9 8 Used to define valve functionality for 3 solenoid and 3 motorized valves 6 solenoid and 6 motorized valves if a Maximizer is installed with your system The valve setup dialogs are used to define the valve name and function specify which port the valve is connected to and to assign a name function to each valve position See Table 7 1 for valve function options 7 5 MODES OF OPERATION SYSTEM OPERATION 7 2 2 Inlet and Valve Naming One of the DuoFlow Software s most powerful features is the ability to define a valves function and to name inlet and valve positions This greatly simplifies programming individual steps in the Protocol Editor Each DuoFlow inlet A B or A1 A2 B1 B2 on a system configured with a Maximizer can be assigned to a specific valve Inlet Valve or be assigned to a specific buffer The Gradient Pump and Maximizer Gradient Pump section of the Setup screen is used to assign Inlets A and B A1 A2 B1 and B20n a system with a Maximizer to a specific buffer Alternatively the SVT3 2 SV5 4 and AVR9 8 valves can be assigned as Inlet valves and each port of the inlet valves assigned to a specific buffer SYSTEM OPERATION Valve Type SVT3 2 Low pressure solenoid valve Valve Name Function Fraction Collector Diver
251. mS cm 0 953 Volt 0 00 pH n s Figure 7 11b Post Run Screen showing a Time based Chromatogram and Rack and Grid fraction numbering Econo Gradient Pump QuadTec 7 5 1 Resizing The main chromatogram shows the current Zoom region To resize the image or zoom in to a particular region of the chromatogram either change the axes scale in the reference chromatogram or use the rubber band controls on either chromatogram To use the rubber band controls click and hold the left mouse button Drag it across the chromatogram region Release the mouse button and the main chromatogram will be resized Click on the Full View button in the toolbar to return to normal view 7 5 2 Chromatogram Information Values at Cursor The top left corner of the PostRun screen contains information about the chromatogram The Values at Cursor window in the upper left corner will list up to eight detector traces depending on the devices selected in the runs device setup The position of the vertical bar within the main chromatogram dictates the displayed information Run time absorbance AU conductivity mS cm gradient progression and the activity trace values are displayed for each trace at the position of the vertical bar To view the vertical bar place the cursor inside the main chromatogram 7 39 MODES OF OPERATION 7 5 3
252. mables Duo gro ut The foregoing obligations are in lieu of all other obligations and liabilities including negligence and all warranties of merchantability fitness for a particular purpose or otherwise expressed or implied in fact or by law and state Bio Rad s entire and exclusive liability and Buyer s exclusive remedy for any claims or damages in connection with the furnishing of goods or parts their design suitability for use installation or operation Bio Rad will in no event be liable for any special incidental or consequential damages whatsoever and Bio Rad s liability under no circumstances will exceed the contract price for the goods for which liability is claimed Under this warranty Bio Rad s obligation with respect to transportation expenses is limited to the cost of shipping the repaired or replacement instrument to the buyer provided that such repair or replacement comes within the terms of this warranty For additional help contact your local Bio Rad representative In the United States call Technical Service at 1 800 4BIORAD WARRANTY APPENDIX C APPENDIX D ORDERING INFORMATION APPENDIX D ORDERING INFORMATION 760 0180 760 0110 760 0164 760 0161 760 0162 760 0184 760 0172 750 0162 750 0111 760 2030 760 0170 760 0171 100 1627 920 8529 760 2010 760 2005 760 2012 760 1300 760 1330 760 1331 760 1332 760 1306 760 1311 760 1406 760 1408 760 1301 750 0200 750 0214 750 02
253. mate Acetate Phosphate pH Range at 25 Celcius 3 10 to 6 80 Desired pH pH at0 B pH at 100 Sulis Molarity at 100 B 1 0 M 476 4 62 440 Acetate Temperature Compensation Coefficient 0 0002 3 75 1 60 3 29 Formate 7 20 7 04 6 71 Phosphate Figure 10 2 Buffer Blender Setup Dialog for a Multi Component Buffer Two point correction best for gradient experiments 1 From the manual screen Press the Buffer Blender Setup button and select the desired buffer system Make sure the pH correction box is not checked Set the pH and flow rate Set the salt composition to 0 B Press run After the system has equilibrated collect some effluent Set the salt composition to the maximum B that will be used in the experiment Press run BO d After the system has equilibrated collect some effluent in a second tube 10 5 MULTIPLE COLUMNS ADVANCED SYSTEM APPLICATIONS 10 11 Measure the pH of the low salt and then the high salt solutions using a high quality pH probe Be sure to correct the reading for any difference between the sample temperature and run temperature Note that if you are using a multi component buffer system and want to use a buffer specific pH correction you will need to repeat steps 2 through 6 for each buffer component Load your experimental method from the Browser dialog and press Setup Double click on the Buffer Blending icon and select the desired buffer The
254. material has been flushed from the system and the pumps are not running Drain fluid from the UV detector and disconnect its plumbing and cables Bop 2 22 To remove the UV flow cell loosen the flow cell thumbscrews and lift the flow cell out To insert a flow cell place it into the UV detector and tighten the thumbscrews To change the UV filter loosen the flow cell thumbscrews and lift the filter tray out Rotate the filter tray to use the correct filter and then place it into the UV detector and tighten the thumbscrews SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM 2 5 2 Conductivity Monitor The Conductivity monitor included with all DuoFlow systems measures fluid conductivity to track the accuracy of a salt gradient This data is useful for optimizing purification protocols and column cleaning procedures Conductivity monitor sensitivity ranges from 0 to 500 mS cm The Conductivity monitor consists of the following Flow cell with Inlet Outlet ports To connect the tubing use 1 4 28 flat bottom fittings The flow cell allows flow in either direction The flow cell can be plumbed immediately after the UV flow cell or at any other point in the flowpath It is designed to fit into a receptacle on the UV detector see Figure 2 7 The volume in the cell is a nominal 6 e Signal cable To connect to the back of the Workstation Electrical power for the Conductivity monitor is drawn from the signal cable If a Maxi
255. may be responsible for the negative peaks The elution buffer may have a higher UV absorbance than the sample components Apply the sample in the same buffer used to equilibrate the column Check the elution buffer s UV absorbance MAINTENANCE AND TROUBLESHOOTING Possible Cause False or ghost The injection valve injection peaks port and loop were not rinsed out between sample injections 1 TROUBLESHOOTING Solution It is a good idea to allow a larger volume of injection buffer to pass through the inject valve and loop For example if the sample loop contains 100 ul then program a load injection volume of 200 yl to completely flush the valve and loop Always clean large volume dynamic loops such as the Bio Rad DynaLoop between use to avoid cross contamination Strongly retained sample components may still be eluting from a previous run Use stringent elution conditions or consult the column s user manual for an appropriate wash step to remove such components between runs 12 4 TROUBLESHOOTING THE CONDUCTIVITY FLOW CELL AND TRACE Problem Possible Cause No values for There may be a loose or buffer conductivity incorrect cable connection are displayed on the Controller Solution Make sure the cable from the Conductivity flow cell is plugged into the correct connector on the rear of the Workstation and that the pins are not bent status bar Conductivity monitor may
256. may be used when connected to the Signal Import Module SIM Refer to Section 2 9 6 2 25 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW 2 6 VALVES BioRad offers an extensive variety of valves that enable greater flexibility for multiple sample and advanced application options The DuoFlow system controls 3 AVR high pressure valves and 3 SV low pressure valves DuoFlow systems with a Maximizer double the number of valves to 6 AVR high pressure and 6 SV low pressure valves Methods that include automated injection buffer selection column switching and large volume fraction collection are easily performed using various valve configurations For further discussion of valving applications and protocols refer to Chapters 8 9 2 6 1 AVR7 3 Sample Inject Valve The AVR7 3 sample inject valve is a 7 port 3 position valve for injecting samples and is an essential component of all DuoFlow systems It is rated to 3500 psi 233 bar and is designed with non metallic wetted parts and minimal internal dead volume Some features of this valve include e Patented make before break design MBBTM prevents pressure spikes when the valve rotates from one port to another eliminating baseline interferences and prevents pump shutdowns due to transient over pressure situations This is especially beneficial when using more fragile low pressure columns or flow sensitive detectors e g refractive index Applications include sample injection reve
257. mizer is in use connect to its COND port CONDUCTIVITY MONITOR FLOW CELL SIGNAL CABLE TO CONDUCTIVITY FLOW CELL CONNECTOR ON REAR OF WORKSTATION Figure 2 7 Conductivity Monitor The Conductivity monitor can be calibrated by either entering the cell constant or by calibrating the cell against a known standard The Conductivity Flow Cell Constant Calibration feature is found under the Utilities drop down menu The cell constant can be found on the tag attached to the cable The Conductivity sensitivity range for the chart recorder is set in the system software Manual and Run screens When setting the range choose one that will accommodate the maximum conductivity expected during the chromatography run Keep in mind that although the chart recorder range can be manipulated during a chromatography run the data will be stored unattenuated allowing for re scaling at a later time 2 5 3 QuadTec UV VIS Detector The BioLogic QuadTec UV Vis detector enables four wavelengths to be monitored simultaneously and displayed on the DuoFlow Controller The QuadTec detector has a wavelength range of 190 370 nm using the deuterium lamp and 370 740 nm with the halogen lamp The deuterium lamp is standard and is required for the detection of peptides proteins and nucleic acids Both lamps are pre aligned calibrated and user serviceable Wavelengths are selected through the use of a moveable grating monochromator with an accuracy of 1 nm in 1
258. mm OD orange PEEK tubing and 1 4 28 fittings Connect the fitting between the Conductivity flow cell or the backpressure regulator or pH monitor flow cell if they are installed and the BioFrac For complete instructions refer to the documentation for the BioFrac fraction collector Connection to a Model 2128 Fraction Collector a Use the tube labeled 6 in the fittings kit or make a fitting using 1 16 1 6 mm OD orange PEEK tubing and 1 4 28 fittings Assure that the tubing length allows unrestricted movement of the fraction collector arm Connect the fitting between the Conductivity flow cell or the backpressure regulator or pH monitor flow cell if they are installed and the drop head of the Model 2128 or the Model 2128 diverter valve For complete instructions refer to the documentation for the Model 2128 fraction collector Connection to a Model 2110 Fraction Collector and a SVT3 2 Fraction Collector Diverter valve a Connect a piece of 1 16 OD orange PEEK tubing between the Conductivity flow cell or the backpressure regulator if that was installed and the SVT3 2 valve common port labeled Common using the 1 4 28 fittings Connect a piece of 1 16 OD Tefzel tubing from port 1 of the SVT3 2 valve see Figure 4 3 for use as a waste line Connect a piece of 1 16 OD Tefzel tubing from port 2 of the SVT3 2 valve see Figure 4 3 to the Model 2110 fraction collector drop head This tubing sits inside the drop h
259. n 2 9 5 Backpressure Regulator The 40 psi backpressure regulator is used with flow rates below 10 ml min Plumb the backpressure regulator following the direction of the arrow The Backpressure Regulator helps eliminate bubble formation within the detector As a solution is pumped through a column the column exerts a backpressure that serves to keep any air bubbles in solution Solution exiting the column returns to atmospheric pressure and air bubbles may form As the bubbles pass through or lodge in the detector flow cell they may cause artifacts on the baseline chromatogram that appear as spikes This outgassing may be minimized by thoroughly degassing buffers and by placing a backpressure regulator after the Conductivity monitor The backpressure from the regulator helps to keep the bubbles in solution When using low pressure columns such as an Econo Pac cartridge plumb the 40 psi backpressure regulator between the Workstation pump outlet and the mixer This aids in seating the check valves preventing permanent damage to the cartridge or column ex Figure 2 28 Backpressure Regulator 2 49 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW 2 9 6 Signal Import Module SIM The Signal Import Module digitizes an analog signal and transmits it to the DuoFlow Controller It is used to import signals from non Bio Rad detectors into the DuoFlow software SIM allows for connection of any pH probe and detector
260. n Launches the Bio Rad EZLogic integration software Contact Bio Rad Technical Support at 1 800 4 BIORAD in the USA or your local Bio Rad representative for more information on EZLogic Buffer Editor Opens the Buffer Editor that is used to create new Buffer Blending buffer systems Exit Exits the BioLogic system software and returns to the Windows desktop 1 through 4 Display the last four methods and runs used SYSTEM OPERATION INTRODUCTION TO THE SYSTEM SOFTWARE Table 5 4 Edit Drop down Menu Edit menu Setup Screen Edit menu Protocol Screen e BioLogic Duo Flow user name project name method name e BioLogic Duo Flow user name project name gt method name gt File d View Utilities Options Window Help N File EI View Utilities Options Window Help 4 Edit Edit Delete Cut Copy Select All Paste Delete Select All Edit menu Run Screen Edit menu Post Run Screen e BioLogic Duo Flow user name project name method name e BioLogic Duo Flow user name project name method name 4 File Bel View Utilities Options Window Help N File Bel View Utilities Options Window Help Full view in Run Chromatogram Edit Activity Trace Multiple runs Tag and Trace Options Start run Full view in zoom Chromatogram Pause buffer pump Print zoom Chromatogram Hold gradient Trace Legend Copy
261. n connected to pumps A and B When the buttons are pressed the green LEDs for pumps A and B flash To stop the flow press the purge buttons again and the LEDs go off These buttons do not operate when the system is running a method When the purge buttons are pressed the pumpheads run at their default maximum flow F10 purge rate is 10 0 ml min or 20 ml min when used with the Maximizer 40 purge rate is 40 ml min or 80 ml min when used with the Maximizer To alter the default purge flow rate select Manual Setup from the Options drop down menu Eight This LED serves two functions a Constant red Indicates that the pump has shut down due to a mechanical or electrical problem This includes a shutdown due to a high or low pressure limit being exceeded Pressure limits are set in the Manual and Run screens Flashing red Occurs during a method run and indicates that a pre programmed ALARM step has been reached The system operator must respond before the method continues Also the Controller emits an audible tone Constant green Indicates the pump is running DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW Table 2 5 continued Workstation Front Panel Controls O OES Plumbing Ports on the front of the Workstation Connections a Pumphead Inlet ports Buffer inlet lines attach to the bottom of each pumphead using standard 1 4 28 flat bottom fittings The pumphead inlet tubing is 1 8
262. ncludes two nuts and four ferrules to Catalog 14750 0568 HPLC system connect an UNO column to an HPLC system 10 32 Fittings Kit 2 59 SYSTEM INSTALLATION AND SETUP SYSTEM SETUP 3 0 SYSTEM SETUP The modular design of BioLogic DuoFlow systems permits you to arrange the system to best meet application and space requirements Each of the following subsections discuss how to set up the different components Setup of each component is the same regardless of the system you have The Workstation and the Maximizer have many rear panel connectors that are identical The conductivity monitor must be connected to the Maximizer UNO Q1 COLUMN AVR7 3 SAMPLE INJECT VALVE eere MAXIMIZER MIXER AT QUADTEC DETECTOR pH MONITOR JT USB BITBUS COMMUNICATOR CONDUCTIVITY MONITOR WORKSTATION BIOFRAC KEYBOARD Figure 3 1 Example of a DuoFlow Pathfinder System Configuration SYSTEM SETUP SYSTEM INSTALLATION AND SETUP The compact design of the Workstation and rack are ideal for laboratories with limited space or for systems that must go into a cold box or cold room For these instances the Controller can be placed away from the Workstation see Figure 3 2 bel
263. nde A ARa 3 6 2 HH 3 7 3 6 Detection System Connections 3 9 3 6 1 UV Detector and Conductivity 3 9 3 6 2 QuadTec UV Vis 3 10 3 6 3 gt pH MOnltor eoe a ecc trees etie Lo re ele eaten 3 12 3 6 4 Non Bio Rad Detectors ssssssssssseeeeeeeeenn mener nnns 3 13 3 Valve Connections e ace eee ende le e e dee eade Ive e DV fe 3 14 3 8 Fraction Collector Connections 3 15 3 8 1 BioFrac Fraction 1 a 3 15 3 8 2 Model 2110 Fraction Collector essssee 3 15 3 8 3 Model 2128 Fraction Collector 3 15 3 9 PUMP Gonnections iid ettet e iac fot D e da Eo 3 16 3 9 1 Model EP 1 Econo Pump ssssssssssssssseeereeneeene nennen 3 16 3 9 2 Econo Gradient Pump EGP ssssssssseeeeenenm ees 3 17 3 10 Model 1327 Chart Recorder Connections 3 19 3 11 Completing System Setup sssssssssssseeeeeneeenmeeeenen nemen nnne nennen nnn 3 19 3 11 1 DuoFlow System Network 3 19 3 11 2 System Power Up
264. ng Fraction Collector Scheme CollectionWindows Start End Frac Size Close ml ml ml 1 4 00 5 00 0 60 SELECT MODE Add Window 2 7 00 8 00 1 00 Collection Windows This method specifies collection during specified parts of the run Time or Volume Each collection window can have a unique fraction size ml Note Collection windows programming should be done after all other steps have been written When using Collection Windows the BioFrac and Model 2128 will skip a tube between each window collection For example if the first window ends collection at 20 minutes and the second window starts collection starting at 21 minutes then the fraction collector will leave an empty tube for the period between minutes 20 and 21 e Fraction Size Tubes Required Number of Racks Start Tube End Tube Start Rack and End Rack See description for Collect All on page 7 20 Add Mode Enter the desired values for Start End and Frac Size a Select the Save Window button and the values will appear in line 1 b Enter the values for the next window and select Save Window The values will appear in line 2 c When the collection scheme is complete select Finished Adding and the Select mode will appear e Select Mode This mode not shown in illustration above shows the following two buttons Add Window Inserts a window after the highlighted window Delete Window Deletes that window 7 22 SYSTEM
265. nnected to the Workstation using an Instrument Bus communication cable System Cables 17 18 19 21 or 30 Connect its power cable to an available outlet Be sure to use the appropriate fuse Refer to the separate documentation for the EGP for fuse specifications The EGP is connected to the DuoFlow Workstation with Bus communication cable System cables 17 18 19 or 21 When the DuoFlow assumes control of the EGP the EGP is automatically set to Remote mode 3 17 SYSTEM SETUP SYSTEM INSTALLATION AND SETUP In Remote mode the EGP keys provide limited control allowing only basic observation of EGP operating parameters For a complete discussion of the EGP refer to its separate documentation The illustration below shows the Aux pump connected to port 3 of the inject valve WASTE WORKSTATION PUMP WASTE 6 5 LOW PRESSURE COLUMN WASTE TON EGP PUMP ECONO PURGE GRADIENT PUMP Figure 3 17 Example of Direct Inject Sample Loading using an Econo Gradient Pump EGP The Econo Gradient Pump EGP can be used to load up to 7 samples sequentially when used with an AVR9 8 at the pump s inlet valve as shown below See Chapter 8 for more details WORKSTATION PUMP LOW PRESSURE LOOP OVERFILL COLUMN TO WASTE ECONO GRADIENT PUMP EGP 8 SAMPLE 2 SAMPLE 3 Figure 3 18 Example of Multiple Sample Loading using an Econo Gradient Pump EGP 3 18 SYSTEM INSTALLATION AND SETUP SYSTEM SETUP
266. nt The rack should be assembled before placing it on the Workstation as described below see Figure 2 26 Keep in mind that the illustration is only an example Alternative rack arrangements include mounting the column clamps on the side of the rack to hold tall columns or setting up the rack to use only one or two trays 1 Fit the tapered collars into the ringed grooves on the rods The collars are tapered so that when they are attached to the rods and the rods are fitted into the holes at each end of the tray the rods and collars serve as the legs of the tray With this in mind note the following guidelines For a2 or 3 tray rack you will need four long rods for a 1 tray rack you will need just two long rods The long rods have three grooves one at each end and one in the middle Attach the collars to one of the end grooves It doesn t matter which of the end grooves you use Attach the collars so that they flare out toward the end of the rod that will be placed at the bottom of the rack e For a one tray rack use the two short rods which have only one groove Fit the collars onto the grooves so that the collars flare out toward the end of the rod that will be placed at the bottom of the rack 2 45 DESCRIPTION OF SYSTEM COMPONENTS SYSTEM OVERVIEW 2 46 Insert the rods into the holes at each corner of a tray Insert the rods from underneath the tray such that they produce a firm fit in the holes For a 2 or 3 tray rack attach
267. nual screen and the status bar at the bottom of the screen show which devices and instruments are connected and communicating with the system The system is ready for operation 3 19 SYSTEM SETUP SYSTEM INSTALLATION AND SETUP 3 11 3 BioLogic Configuration Utility Software The BioLogic Configuration utility is used anytime the pumpheads are changed or a Maximizer is installed It is also used to choose between Bio Rad s BioFrac and Model 2128 fraction collectors To run this software 1 2 3 3 20 Exit the BioLogic DuoFlow software by selecting Exit from the File drop down menu Double click on the BioLogic Configuration icon In the BioLogic Configuration Utility window indicate the pump that will be used with the system whether or not the Maximizer will be used with the system and the default Bio Rad fraction collector either the BioFrac or the Model 2128 Exit the BioLogic Configuration Utility and double click on the BioLogic DuoFlow icon to start the software BioLogic Configuration Utility Workstation Configuration OK v Maximizer is installed Cancel Help r Fraction Collector Model 2128 BioFrac Pump r Pump Head F10 F40 r Spec Flow Rate Range 0 02 20 00 ml min Max Pressure 3500 psi r Setting Purge Flow Rate 10 00 ml min High Pressure Limit 3500 psi Low Pressure Limit 0 psi Figure 3 19a B
268. of a user defined method Each of these modes of operation is discussed below as well as the screens involved in setting up defining and running a method Operating the system using the Manual screen This allows you to individually control the devices and instruments connected to the Workstation Examples of its use include purging the pumps and equilibrating the columns For further discussion refer to Section 7 1 Operating the system using a user defined method This involves the following New Method Allows you to create a method from scratch or load a Method Template Setup screen Allows you to define the instruments and devices to be used for any method Protocol screen To define each of the steps to be run during a method Most steps are programmed using a dialog box to enter the parameter values Note that the number of the step being programmed is displayed in the upper left corner of the dialog box Run screen To run a method or view a run in progress Start Starts a selected run Note You may move through the four screens by activating buttons on the system toolbar as long as a run is not in progress Those functions available during a run are discussed in section 7 4 Run Screen The relationship between the the two modes of operation as well as the screens involved in defining and running a method is illustrated in Figure 7 1 below To list the devices needed by the To list the steps in the run method
269. of the Workstation Pump a Cleaning Sonicate check valves in a warm detergent solution rinse with deionizied DI water and re install Replacing Replace badly clogged or damaged check valves Recalibrate the pumps after cleaning replacing check valves Select Gradient Pump Calibration from the Utilities drop down menu The backpressure displayed on the Controller status bar drops to zero or is much lower than expected Flow rate may have been changed Confirm that the flow rate setting has not been changed There may be a tubing or fitting leak Re inspect all tubing connections especially at the pump inlet Pump seals may need replacement Replace pump seals A build up of crystallized buffer salts on the rear of the pumphead is a clear sign for seal replacement see Section 11 3 3 Routine Maintenance of the Workstation Pumps Check valves may need cleaning or replacement Clean or replace check valves see Section 11 3 3 Routine Maintenance of the Workstation Pumps 12 5 TROUBLESHOOTING Problem No pressure psi reading on Controller status bar or psi value always reads zero Possible Cause Pump may not be running or inlet lines are not primed or there is a problem in the buffer flow MAINTENANCE AND TROUBLESHOOTING Solution Check that the pumps are running and that the inlet lines are primed and not pulled out of the buffer reservoirs Check f
270. ollection 7 20 connecting to system 3 15 nne 2 37 Model 2128 fraction collector collection 7 20 connecting to system 3 15 description eee 2 38 racks available 2 38 Model EP 1 Econo pump connecting to Workstation 3 16 descriptiOri ziii dte te 2 42 examples of sample loading using 8 1 8 2 Modes of operation using a User Defined Method 7 1 using the Manual screen 7 1 Multiple columns Column switching AVR7 3 Two column switching 9 1 AVR9 8 eight column switching 9 2 Multi dimensional chromatography 9 5 MX 1 mixer attaching to 3 8 changing mixer capacity 2 20 cleaning rm ae heel acest 11 8 description 2 18 mixer barrels and mixer capacity 2 18 N Non Bio Rad detectors connecting through SIM e apse oe 3 13 New features 1 2 P pH monitor connecting to Workstation with Maximlzer oe ctetu 3 12 connecting to Workstation without Maxirnlzer icta 3 12 description 2 25 Post Run screen Activity Trace Editor window 7 41 chrom
271. olume i pH Range 7 20 to 9 20 pH Volume ml Buffer A1 Y A 100 1 0 Y Solution A1 Tris HCI 50 mM 7 00 1 0 Solution A2 Tris 50 mM i Flow mimin Solution B1 Degassed Water B Flow ml min Buffer B1 Y 0 100 Solution B2 Sodium Chloride 2 00M 0 1 00 Molarity at 10096B 1 0 M Y Step 2 Volume 0 00 OK Cance Step 2 Volume 1 00 ml OK Cancel Isocratic Flow Screen Isocratic Flow Screen with Maximizer in Use l ti The Edit Isocratic Flow dialog is used to define the isocratic step buffer composition The buffer composition is determined by the percentage of total buffer delivered through pumps A 96A and B e Buffers Displays all the available buffers The names displayed for Buffer A and Buffer B are the names assigned to the pump inlets or inlet valves in the device setup see Chapter 7 2 2 e Composition Allows you to set the buffer composition for the step e g 75 A and 25 B The Edit Isocratic Flow pH View dialog is used to define the isocratic step buffer composition when a Maximizer is connected to the system and Buffer Blending is turned on The dialog box on the left lists the current buffer system its pH range and the solutions assigned to each Maximizer valve inlet pH Allows you to enter the pH for the current step e 9eB Allows you to set the buffer composition for the step as a percentage of the 1x salt concentration The following selections are available in both the Edit Isoc
272. omes with fittings tubing and an F10 Tubing Kit with the necessary tubing cut and fitted for each system connection see Figure 4 1 Complete description of each of these items is provided with the Fittings kit Table 4 1 Tubing Guidelines Pre pump 1 8 OD x 0 062 ID clear PTFE Super flangeless fittings Workstation 3 2 mm OD x 1 6 mm ID for 1 8 3 2 mm OD tubing and Aux pumps Post pump 1 16 OD x 0 02 ID clear Tefzel Super flangeless fittings 1 6 mm OD x 0 5 mm ID for 1 16 1 6 mm OD tubing Post pump 1 16 OD x 0 020 ID orange PEEK 1 4 28 Super flangeless fittings 2 ul with F10 1 6 mm OD x 0 5 mm ID for 1 16 1 6 mm OD tubing pumphead Post pump 1 16 OD x 0 030 ID green PEEK 1 4 28 Super flangeless fittings 4 5 pl with 40 1 6 mm OD x 0 76 mm ID for 1 16 1 6 mm OD tubing pumphead Pre pump refers to all tubing leading up to pump inlet port Pumps include Workstation pumps EP 1 pump and Econo Gradient Pump Maximizer uses pre pump tubing Post pump refers to all tubing following the pump outlet port When plumbing the system be sure to keep tubing lengths to a minimum All fittings should be finger tight Do not over tighten as you risk damaging the connection Ferrule Installation 1 8 3 2 mm OD and 1 16 1 6 mm OD tubing 1 the tubing with the tubing cutter provided in the Fittings Kit This will give a flat clean cut to the tubing this is important in making a fitting t
273. oolbar buttons are located on the top left side of the protocol screen and are used to create new methods edit a copy of the current method or to select an existing method see Chapter 6 0 for more detail ADD STEP BUTTONS 4 COLLECTOR BUTTON SCOUTING BUTTON BioLogic DuoFlow user name project name method name run name mE File Edit View Utilities Options Window Help Tas New Edit New tea E I D DI BID BID H Bio Rad Method Method Run Browser Report Manual Setup Protocol Run N tes PostRun agi Settings Edit Cut Copy Paste Delete Web Add Steps Isocratic 2 0 0 Collect Fractions of size 1 00 ml during entire method Flow 2 0 0 Chart Recorder Turn ON oad Inject o i 25mM Tris 100 Time 2 0 min Sample 3 080 Ieocratic Flow 25 Tris Salt 0 Flow 2 0 ml min tatic Loop Auto Inject Valve Ee ci 4 30 Load Sample Load standard Volume 1 0 ml Flow 2 0 ml min i 25mM Tris 100 Volume 4 0 min Change 3 70 Iocratie Flow 25mM Tris Salt 0 Flow 2 0 m min Val e 230 PER 25mM Tris 100 55 Volume 16 0 min Inear Gradient 25mM Tris Salt 0 45 Flow 2 0 Column gm i
274. or inlet valves in the device set up see Chapter 7 2 2 e 9 Composition Allows you to choose initial and final values for the linear binary gradient Notice that changing one value in the Initial column affects the other initial value The Final column behaves identically The Edit Linear Gradient pH View screen appears when you are using a Maximizer in Buffer Blending mode This box sets the pH initial and final salt concentration and the period over which the change in composition is to occur There is no limit to the number of gradient steps in a protocol The following selections are available e pH Allows you to enter the pH for the current step The dialog box on the left hand side lists the currently defined buffer system and its pH range as well as the solutions present at each inlet Initial and Final B Allows you to set the initial and final salt concentration as a percentage of the maximium allowable concentration for a linear binary gradient The following selections are available in both screens e Volume Allows you to choose the duration of this step e Flow Allows you to choose the flow rate of this step e OK Adds the step to the protocol This is the same as pressing the Enter key on the keyboard e Cancel Does not add the step to the protocol This is the same as pressing the Esc key e Step Time or Volume Identifies current step number and calculates the elapsed time or volume from all previous
275. or leaking fittings Check that the buffer flow is going to the column and not to waste via the injection valve purge position The zero pump pressure calibration routine may have been used while the system was under pressure Stop the pump and undo the line from the pump to the valve such that the pressure transducer is definitely at zero psi Select Gradient Pump Calibration from the Utilities drop down menu and perform a Zero psi procedure Incorrect backpressure readings for a given column Flow rate may have been changed Column may need cleaning and or a frit replacement The pressure transducer calibration may be incorrect Check that the pump is set to deliver the correct flow rate Consult the column s instruction manual for cleaning procedure Alternatively the system tubing may have an obstruction so inspect the tubing path Stop the pump momentarily loosen the pump outlet fitting to release pressure Select Gradient Pump Calibration from the Utilities drop down menu and perform a Zero psi procedure High backpressure is shutting down the pumps 12 6 Maximum pressure for the pumps is F10 3500 psi F40 1000 psi The pressure limit settings in the Manual screen see Figure 7 2 have been set too low Pressure increases are due to a build up of particulate matter in the system causing a resistance to flow An increase in flow rate also increases the pressure so
276. orkstation pumps and the sample volume to be injected onto the column The DuoFlow system will now automatically control the loading and injection of the sample Note that the rinse function is not available when the DynaLoop is being used Continue writing your desired separation protocol During the run the valve functions as follows e Load While in this position the valve connects ports 5 and 4 for equilibration of the column and for sample elution In this position sample loop is loaded to the desired volume via port 2 and buffer is expelled from the dynamic loop through port 1 Inject While in this position the valve connects ports 5 and 6 and ports 3 and 4 for applying the sample onto the column The flow from the Workstation pump forces the sliding piston to expel the sample onto the column of WASTE WASTE Purge While in this position the valve connects ports 5 and 7 and allows purging or buffer changes the Workstation pump without the need to remove the column from the system DYNAMIC WORKSTATION DYNAMIC WORKSTATION DYNAMIC WORKSTATION MP MP UMP LOOP PU LOOP PU LOOP P WASTE WASTE aa DNE cb mm i j WASTE j 4 WASTE DYNAMIC OR AUXILIARY DYNAMIC OR AUXILIARY DYNAMIC OR AUXILIARY LOOP LOADING LOAD PUMP LOOP LOADING LOAD PUMP LOOP LOADING L
277. ot modify the method The event is recorded in the Run Log To set the pressure limits of the Workstation pumps This is not part of a protocol it is a system setting from the Manual screen The method pauses and the pumps stop when the pressure goes above or below the set values To set the UV Detector range for the chart recorder output The settings are 0 001 0 002 0 005 0 01 0 02 0 05 0 1 0 2 0 5 1 0 2 0 AUFS To set the Conductivity range on the chart recorder output The settings are 500 200 100 50 20 10 5 mS cm To set an Event Mark on the chart recorder UV trace output only It will not record event marks on the screen Use the Zero Baseline button to reset the UV or QuadTec detector absorbance value to zero Note Use care during a collection scheme which uses a Threshold value These drop down menus located on either side of the chromatogram are used to select the active data trace to be scaled during or after a run As each trace scale is selected the scroll bars can be used to adjust the scale setting To work offline while a run is in progress Refer to section 7 4 2 Working Offline Notes displays the Run Notebook screen which allows you to enter notes regarding the run Log displays the Run Log screen which provides information about the run and is non editable Accessed from the view drop down menu if EZ Logic has been installed To stop pause or hold a method in progress Refer to Figure
278. ough data is collected at a rate of five points per second it can be exported at a user defined rate Data is exported in an ASCII TXT file format The file can be imported into Excel as comma delimited data Export Data Setup Export Run Time Window Min 10 5 Min Cancel Export Parameters Export Rate UV 5 0 points sec Conductivity Calculate GP Pressure 135 KB 3150 Gradient B File Size Number of Records Fraction Figure 7 15 Export Data Setup Screen 7 43 MODES OF OPERATION SYSTEM OPERATION 7 5 6 Exporting Chromatogram Images Chromatographic images can be exported into other applications such as word processing programs Before transferring an image modify the chromatogram by selecting the desired traces and set the scale and zoom region in the PostRun screen This should be done now since changes cannot be made to the file once it is transferred out of the DuoFlow program The image displayed in the main chromatogram will be exported Select Export Chromatogram Image from the File drop down menu see Figure 7 16 The image is exported in a Windows Meta File wmf format which can be transferd into most Windows based applications Alternatively a chromatogram can be copied to
279. ow Bus communication cables of lengths up to 100 feet can be purchased from Bio Rad and USB cables can be purchased from any computer supply store LABORATORY COLD ROOM USB BITBUS COMMUNICATOR USB BUS INSTRUMENT BUS EX NY Figure 3 2 DuoFlow Pathfinder Setup in Non Condensing Environment 3 1 CONTROLLER CABLE CONNECTIONS The Controller consists of a monitor computer keyboard and mouse Bio Rad provides a Dell computer for use as a Controller Detailed instructions for setting up your Controller are provided in the separate documentation provided with your Controller 1 Place the Controller so that the back side is facing you Connect the monitor keyboard and mouse to the computer 2 Connect the power cables to the computer and monitor If the computer has a second power outlet use it to plug in the monitor power cable Do not turn on power to these devices yet 3 2 SYSTEM INSTALLATION AND SETUP 3 2 USB BITBUS COMMUNICATOR CABLE CONNECTIONS SYSTEM SETUP The USB Bitbus Communicator allows communication between the Controller and the rest of the DuoFlow system To connect the USB Bitbus Communicator 1 Connect System Cable 31 from the USB Bitbus Communicator to the computer USB connector Refer to the illustration below 2 Ifa Signal Import Module SIM is to
280. ow direction Note that if an EP 1 or non Bio Rad pump is used the flow rate must be set both in this dialog and at the pump If an Aux Pump Inlet valve is connected select the sample to load If the tubing is to be rinsed between sample injections add a second Load Inject step select the rinse buffer and set the rinse volume flow direction and flow rate SAMPLE LOADING ADVANCED SYSTEM APPLICATIONS When doing Aux Pump Direct Injection the AVRT 3 injection valve operates as follows Load In this position the valve connects ports 4 and 5 and is used for column equilibration and sample elution Inject In this position the valve connects ports 3 and 4 and is used to inject sample onto the column e Purge In this position the valve connects ports 5 and 7 and is used to take the column out of line for purging or buffer changes WASTE WASTE WASTE WORKSTATION PUMP WASTE WORKSTATION PUMP WASTE LOW LOW LOW PRESSURE PRESSURE PRESSURE COLUMN COLUMN COLUMN AUXILIARY AUXILIARY AUXILIARY AUXILIARY LOAD PUMP AUXILIARY AUXILIARY LOAD PUMP PUMP PUMP PUMP PURGE PURGE PURGE Figure 8 3 AVR7 3 Valve Positions During a Run with Direct Sample Loading and Injection 8 3 GRADIENT PUMP DIRECT INJECTION Large volume samples can be directly loaded through the Workstation pumps Up to 7 samples can be sequentially loaded if an inlet valve AVR9 8 is added to the setup One of the inlet valve ports must be as
281. owser screen by collapsing all folders to the single user icon There are several options available for how information is displayed in the Browser screen Select Browser Settings from the Options menu discussed in Table 5 7 Options Drop down Menu The Set Browser Options window provides the following options e Enable Projects A summary of all Projects regardless of User will be listed in the Browser e Enable Methods A summary of all Methods regardless of User or Projects will be listed in the Browser Enable Runs A summary of all Runs will be listed in the Browser Enable User Methods A summary of all Methods for the specified User will be listed in the Browser Enable Project Runs A summary of all Project Runs will be listed in the Browser Set Browser Options Starting Browser Selections Enable Projects Enable Methods Enable Runs Enable User Methods Enable Project Runs OK Cancel Figure 6 4 Set Browser Options Window SYSTEM OPERATION INTRODUCTION TO THE BROWSER SCREEN 6 2 METHOD TEMPLATES The BioLogic software includes Method Templates to simplify the method creation process These templates can be used as is or modified to fit experimental requirements The templates that are available depend on the system configuration BioLogic DuoFlow BioLogic DuoFlow QuadTec BioLogic DuoFlow Maximizer and BioLogic DuoFlow Pathfinde
282. portant to wash out the Maximizer valves and the SVT3 2 and SV5 4 valves Failure to do so may result in valve failure 3 Use deionizied DI water and the 10 ml syringe provided in the Fittings kit to wash behind each pumphead with at least 10 ml of water This applies to both the F10 and 40 pumps 4 Fill the entire system with either 2096 ethanol EtOH or 0 05 sodium azide to inhibit bacterial growth If necessary fit the pump inlets UV detectors and conductivity monitors and all solenoid valves with 1 4 28 plugs which are provided in the Fittings kit 11 1 MAINTENANCE MAINTENANCE AND TROUBLESHOOTING 11 3 CARE AND MAINTENANCE OF THE WORKSTATION PUMPS The following three sections describe the procedures for system priming and periodic maintenance of the Workstation pumps 11 3 1 Priming the Workstation Pumps and Removing Trapped Air Bubbles Warning Do not run the Workstation pumps dry without buffers in line as this may result in damage to the pumpheads Care should be taken when setting up the DuoFlow system to ensure air does not get trapped in the pumps Air trapped in the pumps can lead to erratic flow rates and poor gradient performance To reduce the likelihood of bubbles getting into the pumps all buffers should be thoroughly degassed 1 Ensure that all inlet tube fittings are securely fastened to the Workstation Maximizer and valve inlets Use the fittings tightener supplied with the fittings kit to tighten the
283. r a New User Enter a new User to allow you to define your Methods and to group Projects Methods and Runs within the Browser a Click once on the Users icon in the Browser b Click once on the New button and select New User You will be prompted for a new User Name Enter the name you would like to use c Press OK to accept the new name If you define a new User you must also assign a Project to the User before writing a new Method Hint Alternatively highlight a User icon right mouse click and select New User with a left mouse click Define a New Project The Browser is further segmented into Projects within a User s domain To define a new project a With the new User Name highlighted click once on the New button b Select New Project Enter the project name and description c Press OK to accept the new name Hint Alternatively highlight the User icon right mouse click and select New Project with a left mouse click Write a New Method To begin writing a new method from the Browser a Select the Project folder or a Method in the Project folder where you want the new method to be located Click once on the New button Select New Method Enter the method name and press OK This transfers you to the hardware Setup screen Hints 1 Alternatively highlight the Project icon right mouse click and select New Method with a left mouse click 2 You can also use the New Method button in the toolbar By defau
284. r ds 7 37 7 5 Post R ti SCEreeh uc is ee tree fete teen pete Be ERR Tene o de det eR Ee ERR E 7 38 Lu EL labem 7 39 7 5 2 Chromatogram Information Values at 7 39 7 5 3 Annotating tagging the Chromatogram sse 7 40 7 5 4 Entering Activity Data eene nnns 7 41 7 5 5 Exporting Chromatogram Data to other Software Applications 7 43 7 5 6 Exporting Chromatogram 7 44 SECTION 4 SAMPLE LOADING APPLICATIONS Chapter 8 0 Sample Loading 8 1 8 1 Automatic Loop Fill and Rinse cccccccceeeeeeeeeeceeeneeeeeeeeeeeeeeeeeeeccnceeeeeeeeeeeeeteeeeees 8 2 8 2 AuxPump Direct Inject 22 etc decer aeneo reine re iaces v ere 8 3 8 3 Gradient Pump Direct Injection sssn eme 8 4 8 4 DynaLoop Sample Injection sssseeneneeenn eee nenne 8 6 Chapter 9 0 Column and Buffer Flow Switching 9 1 9 1 Col mn SWItChing Aae tee ine ctt etes te RR CER ERE BERI es 9 1 9 1 1 AVRT 3 Two Column Switching e 9 1 9 1 2 AVR9 8 Eight Column Switching sseee e 9 2 9 2 Reverse Flow Chromatography sse
285. r systems Before using the Method Template feature the system must be configured in the BioLogic Configuration utility and a QuadTec or UV detector should be defined in the default setup see Section 7 2 New Method Method Identification OK The select method template will be used to create the method Cancel User Name Phoenix Y Project Name Project A1 Y Method Name lon Exchange with Auto Inject Y Method Description Y Method Author James R Y Template Selection Experiment Type Template Description Affinity METHOD DESCRIPTION Size Exclusion with Auto Inject C 1 This Method is used to separate molecules on the basis of molecular size Loaded samples are Chromatofocusing injected automatically with an AVR7 3 sample inject valve Hydrophobic Interaction COLUMN SAMPLE This method was written for a 490 to 530 ml gel filtration column 2 5 x 100 cm Hydroxyapatite to 2 6 x 100 cm but can be scaled to work with columns of different sizes Sample volumes should lon Exchange typically be 1 to 326 of the column volume Consult your column instructions for specific sample Gel Filtration size and flow rate information REQUIRED DEVICES BioFrac fraction collector AVR7 3 sample inject valve conductivity Templates monitor standard UV detector and a sample loop up to 20 ml Size Exclusion wi
286. race Compare eoe E Pete Doe terre eee en 6 12 6 5 1 Chromatogram Display 6 13 6 5 2 Toolbar Buttons Ti eicere doves toa erases 6 14 6 5 3 Drop down MenUSi nnne nennen 6 15 6 5 4 Active Traces and Valves at 6 17 6 5 5 Chromatogram Settings 6 17 Chapter 7 0 Modes of 7 1 7 1 Maniu al S6reen ntt 7 2 7 2 Setup SCreen uno eie e onte eec ae ies a ee ovi tuber ee eese eas 7 4 7 27 Device Selection 7 5 7 2 2 Inlet and Valve Naming sssseeeeeeeeneeenm menn 7 6 7 2 9 v Buffer Editor etc teet eese ia MR reete rede ee 7 8 7 3 Protocol Screen niece edna Sie een eee 7 10 TA RUM SGFeem c nc ete d die utes ear t em dati opu todd e ea depen Mns 7 30 7 4 4 Pausing Stopping a Method in 7 33 7 4 2 Working Offline During a mem 7 34 7 4 3 Editing a Method During a Run seessseeeemeemm 7 35 7 4 A Notebook 7 37 TAS Run Log Screens 2 ico eee dete lv era dee deepe
287. races and values at cursor Chromatogram Settings QuadTec 1 214 Run Queue Method 4 Q Y QuadTec 1 214 nm Queue Method 3 QuadTec 1 280 nm Run Q Queue Method 22 0 0150 AU Buffer B Run Queue Method 2 Queue Burer B Visibility Trace Color Run QuadTec 2 260 nm Run Q Queue Method 2 aegis Run Time 00 00 50 8 y QuadTec t 280 nm Run Q Queue Meth QuadTec 3 214 nm Run Q Queue Method 2 0 0048 AU QuadTec 1 280 Run Q Queue Meth QuadTec 4 405 nm Run 0 Queue Method 2 1 insem V QuadTec 1 280 nm Run 0 Queue Meth Conductivity Run Queue Method 2 Que Visibility Y Axis X Axis Figure 6 8 Trace Compare Window Tiled View 6 12 SYSTEM OPERATION INTRODUCTION TO THE BROWSER SCREEN trace compare name gt File Options View Tools Window Help 2 55 RN EIE Select Active traces and values at cursor Min Tenth QuadTec 3 280 nm Run Q Queue Method 4 Q Y QuadTec 3 280 nm Run Queue Method 3 Q QuadTec 1 280 nm Run Queue Method QuadTec 1 280 nm Run Queue Method QuadTec 1 280 nm Run Queue Met
288. rams in in overlay view Overlays iim Select Allows you to select which traces will appear in overlay view elect Color Toggles between two trace coloring schemes Color by device type or color Colors each trace uniquely Full View Expands the currently active chromatogram to full scale Report Allows you to preview and print a report of the currently open compare including an Overlay Report Trace Compare Report Chromatograms Trace Compare Report Legends and Trace Compare Report Summary BioLogic Closes Trace Compare and returns to the browser Shift Up Shifts chromatogram traces relative to each other in an upward direction Shift Down Shifts chromatogram traces relative to each other in a downward direction Make sure that vertical placement of text is consistent throughout the Table 6 14 SYSTEM OPERATION INTRODUCTION TO THE BROWSER SCHEEN 6 5 3 Drop down Menus Table 6 2 File Drop down Menu trace compare name Options View Window Help Report Export Overlay Image Copy Overlay Image to Clipboard LAN The File menu consists of the following Report Allows you to preview and print a report of the currently open compare including an Overlay Report Trace Compare Report Chromatograms Trace Compare Report Legends and Trace Compare Report Summary Export Overlay Image Allows you to export the overlay veiw to a windows metaf
289. raphy methods that are then ran sequentially as part of a queued run Once written each one dimensional method can be combined with other methods to make the desired experiment For example a single affinity protocol could be could be combined with either a desalting method or size exclusion method to create one of the two dimensional experiment shown in Table 1 On the other hand an affinity method could be combined with both a desalting method and an ion exchange method to create the three dimensional experiment shown in Table 1 When writing a multi dimensional method care should be taken to prevent reintroduction of impurities removed in previous dimensions Cleaning and equilibration steps should be included at the start of each dimension to wash any impurities from the tubing and to fill the system with the appropriate buffers 9 6 ADVANCED SYSTEM APPLICATIONS MULTIPLE COLUMNS 10 0 BUFFER BLENDING The Maximizer is a component of the DuoFlow system that dynamically Blends water salt and the conjugate acid and base of a buffer to produce a solution with a specific salt concentration and pH Buffer Blending is a powerful chromatographic tool that in conjunction with the scouting feature of the DuoFlow software can be used to optimize salt concentration gradient slope and pH in a set of unattended experiment The results of these experiments can then be easily compared and analyzed using the Trace Compare feature of the DuoFlow softwar
290. ratic Flow screen and the Edit Isocratic Flow pH View screen e Volume Time Allows you to choose the duration of the step e Flow rate ml min Allows you to choose the flow rate of the step e OK Adds the step to the protocol This is the same as pressing the Enter key on the keyboard e Cancel Cancels all input This is the same as pressing the Esc key on the keyboard e Step Time or Volume Identifies current step number and calculates the elapsed time or volume from all previous steps This is not user editable 7 11 MODES OF OPERATION SYSTEM OPERATION Table 7 4 Load Inject Sample Edit Load Inject Sample Load Inject Sample LFill Before Inject Rinse After Inject Injection Buffers 96 Comp Volume ml e Siatic Loop Buffer A1 A 100 10 Dynamic Loop Flow ml min A C Direct inject Buffer B1 Lo 100 Load Inject Sample Screen Auto Inject Valve will move to INJECT at start of step and return to LOAD at end of step Step 3 Volume 1 00 ml OK Cancel Edit Load Inject Sample Load Inject Sample LEill Before Inject Rinse After Inject r Type 7 Buffer System Tris 25 mM tatic Loop pH Range 7 20 to 9 20 pH Volume ml Solution A1 Tris HCI
291. recorder is available Connect the red line to the positive terminal and the black line to the negative or ground terminal of channel 2 CH2 The chart recorder should be set to 1 V full scale If you are using the Model 1327 chart recorder move all switches to the green settings DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYSTEM OVERVIEW Table 2 6 continued Workstation Rear Panel Connectors 2 10 UV Chart For UV signal output to a single or dual pen chart recorder When the Bio Rad Model 1327 is used chart recorder Pen Up Down Stop Start commands and event marks are sent from this connector The Bio Rad Model 1327 dual pen recorder needs an 8 pin mini DIN to standard DIN cable System Cable 2 and a mini DIN to banana plugs cable System Cable 4 Generic chart recorders require an 8 pin mini DIN to breakout cable System Cable 7 available from Bio Rad When a Signal Import Module signal replaces the standard BioLogic UV Detector use System Cable 20 to control a Bio Rad Model 1327 dual pen chart recorder The chart recorder should be set to 1V Power Cord The grounded 3 prong connector inputs power to the Workstation and outputs power to any unit connected to the Workstation The Workstation s input power cord should be plugged into a 3 prong grounded power outlet Instr Bus The RJ 45 modular phone connectors and the bus communication cables connect the Workstation to the other components in the system T
292. robes that are able to operate at the flow rates and pressures used in chromatography applications If highly accurate pH measurements are required collect fractions and measure their pH using a high quality pH probe such as an Orion Ross electrode or other Tris compatible electrode connected to a desk top pH meter The pH electrode supplied with the Maximizer is designed to give optimum performance during the inline measurement of pH It can be used at flow rates up to 80 ml min and pressures up to 75 psi Its use provides useful information about the Maximizer performance and buffer composition during a run However itis not required for accurate pH delivery by the Maximizer Since the pH probe has an approximately 80 ul flow cell volume it should be taken out of line when collecting small fractions sizes i e when collecting in microplates Prior to use the pH electrode should be calibrated using two standard solutions that span the pH range over which the experiment will be run for example pH 7 amp 4 pH 7 amp 10 or pH 4 and 10 Calibration is performed from the Utilities DH Probe Calibration menu option Although the Maximizer produces buffer with good pH accuracy the accuracy can be improved by applying a 1 or 2 point correction using the DuoFlow software as described below Single point correction best for isocratic experiments 1 From the manual screen press the Buffer Blender Setup button and select the desired buffer system
293. rse flow chromatography and two column switching e Avalve rebuild kit is available Selection Figure 2 10 AVR7 3 Sample Inject Valve 2 26 SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM The three valve positions are Load position 1 Inject position 2 and Purge position 3 See Figure 2 11 Load is the default position when the system is powered up or at the end of a method run unless configured differently from the Edit User Preferences window available from the Options menu in the system software WORKSTATION WORKSTATION WORKSTATION PUMP PUMP PUMP WASTE WASTE WASTE 2 SAMPLE LOOP SAMPLE E SAMPLE B SAMPLE E INJECT INJECT INJECT Figure 2 11 Sample Load Positions The valve uses 1 16 1 6 mm OD tubing and 1 4 28 fittings Sample loop sizes are available from 50 ul loop in the Starter kit included with all systems to a maximum of 5 ml PEEK loop Larger volume injections may be obtained using Dynaloops additional valves and or an auxiliary pump Connect the AVR7 3 valve signal cable to any of the available Automated Valve connectors on the back of the Workstation ports 4 5 or 6 If a Maximizer is in use connect to ports 7 8 or 9 before those on the Workstation If more than 3 valves are desired you may connect additional valves to ports 4 5 and 6 on the Workstation All valves will be active 2 27 DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM SYST
294. run activity data from the Browser screen 2 Press the Activity toolbar button A box will appear listing each fraction collected in the opened run See Figure 7 13 3 Enter activity values for each fraction and enter the desired units Check the Show Activity Trace box to display the data in the Post Run screen 4 The activity values entered will appear in the chromatogram information screen and as a trace in the chromatogram See Figure 7 14 BioLogic Duo Flow user name project name method name run name Oe File _ Edit View Utilities Options Window Edit New RA Method Method Run Browser Report Manual Setup PO Values at Cursor Run Time 00 03 38 4 ie Gradient 21 Buffer B i QuadTec 1 280 nm 0 0345 AU QuadTec 2 260 nm 0 0228 AU QuadTec 3 214 nm 0 4762 AU QuadTec 4 405 nm 0 0021 AU Conductivity 8 47 mS cm GP pressure E Activity Trace Editor oP Activity Fraction Fraction Fraction Activity Activity units QuadTec 3 214 nm start time end time value GP pressure A 1F 00 00 00 00 00 30 0 00 dPm 2 E Bio Rad Settings Stans Print Del Te Activity Web 100 0 90 0 80 0 70 0 60 0 50 0 40 0 30 0 20 0 0 1007
295. s Allows the status information at the bottom of the screen to be toggled on or off Browser settings This enables you to select a heirarchy of how the Browser is to display users projects methods runs For example select Projects to display a top level folder called Projects which contains all projects in the database Use Time Use Volume These are available only from the Protocol screen when defining a new protocol The following selections are available from the Manual screen Manual Setup This enables you to set the pump purge flow rate Additionally you can set an EGP split time period select a QuadTec time constant and set the SIM parameters For the F10 pumps you can specify a maximum purge rate of 10 ml min for the F40 pumps you can specify a maximum purge rate of 40 ml min Buffer Blender Setup This is available only when the Maximizer is used with the system This allows you to switch between buffer blending and non buffer blending modes In buffer blending mode a window listing buffer systems available for use is displayed From this window a user can enter one or two point pH corrections if needed 5 11 INTRODUCTION TO THE SYSTEM SOFTWARE SYSTEM OPERATION Table 5 8 Window Drop down Menu e BioLogic Duo Flow user name project name method name File Edit View Utilities Options Help 1 BioLogic Online method name gt 2 BioLogic Offline method name gt
296. s gradients chart speed fraction size and general notes These notes are printed with the report Run Log Displays a log of all events occurring during the run This information cannot be edited The Run Log may be disabled by selecting Edit User Preferences from the Option drop down menu It is recommended however that the Run Log be active for assistance in troubleshooting Note If the Bio Rad EzLogic Integration software option is installed the Log toolbar button is replaced by the Integ toolbar button To display the Log window select Run Log from the View drop down menu TraceCompare Allows you to compare the trace results from different runs Bio Rad webpage If your Controller has internet access you can use this button to access the Bio Rad web page BioFrac tube format Rack and Tube Rack and Grid Toggles the chromatogram fraction collection trace between Rack and Tube and Rack and Grid mode see Figures 7 5a 7 5b 7 11a and 7 11b for examples Volume Based Chromatogram Time Based Chromatogram Toggles the chromatogram horizontal axis between time and volume mode see Figures 7 5a 7 5b 7 11a and 7 11b for examples 5 9 INTRODUCTION TO THE SYSTEM SOFTWARE SYSTEM OPERATION Table 5 6 Utilities Drop down Menu e BioLogic Duo Flow user name project name method name File Edit Options Window Validate Method Check Hardware connections System Inform
297. s done on a routine basis Multi dimensional experiments usually involve some type of affinity purification step followed by a high resolution step such as ion exchange hydroxyapatite or size exclusion chromatography Table 9 1 shows a few common multi dimensional chromatography experiments types Table 9 1 Common Multidimensional Chromatography Experiments Experiment Type Dimension 1 Dimension 2 Dimension Affinity Desalt Affinity Size Exclusion Affinity Desalt lon Exchange Most of the common protein purification techniques can be incorporated into multidimensional chromatography experiments Generally purification techniques that are sample independent provide the greatest benefit when included as part of a multidimensional experiment Examples include affinity chromatography of proteins tagged with His tags glutathione s transferase and maltose binding protein size exclusion chromatography and ion exchange chromatography Both two and three dimensional chromatography experiments can be performed using the BioLogic DuoFlow system The type and complexity of experiments that you can run will depend on the hardware installed on your system Maximizer and Pathfinder systems with their increased valve capacity can run the most complex experiments System Setup When setting up a multi dimensional chromatography protocol it is important to consider a few essential factors First of all sequential purification pro
298. s of use they should be replaced following the information in the maintenance section Ater re assembly of the pumpheads pump 100 ml of 100 methanol MeOH through each pumphead at 5 ml min with the 40 psi backpressure regulator in place 12 4 MAINTENANCE AND TROUBLESHOOTING Problem Delivery of liquid is erratic and the pressure readout on the Controller status bar is fluctuating more than 2096 Possible Cause Severe fluctuation in the back pressure readout indicates an air bubble trapped in one or both of the pumpheads Note Always degas buffers before use TROUBLESHOOTING Solution Put the AVR7 3 valve into the purge position and purge the system by pressing the Purge buttons on the front panel of the Workstation Stop or Pause the pumps and re prime Loosen the top fitting on the pumphead and force any trapped air out of the pumphead by squirting buffer through the priming port using a syringe Refer to the maintenance section on how to prime the Workstation pump and remove trapped air bubbles Always use degassed buffers To minimize this problem use degassed buffers and solutions Degas buffers by stirring vigorously under vacuum for approximately 20 minutes Use a heavy wall side arm Erlenmeyer flask as standard flasks may implode under vacuum The pump s check valves may need cleaning or replacement Clean or replace the check valves see section 11 3 3 Routine Maintenance
299. setup Refer to Section 7 2 for more information about the Device Setup screen Start the Protocol Editor using the tool bar Protocol Editor button Use the protocol screen Add Step tools to create a new method as illustrated with below for an ion exchange protocol See Chapter 7 for more information a Add an Isocratic Flow step to equilibrate the column and enter the required flow rate step size and B The parameters entered here will automatically appear in the next step but can be changed at any time b Add a Zero Baseline step to zero the selected detector prior to sample injection c Add a Load Inject Sample step and then enter the sample inject volume and flow rate If a static injection loop is used with an AVR7 3 Sample Inject Valve select static loop For information about the other injection options see Chapter 8 d Add an Isocratic Flow step to wash the column and enter the required flow rate step size and B e Add a Linear Gradient step to elute the column and enter the required flow rate step size initial B and final B f Add Isocratic Flow step to clean the column and enter the required flow rate step size and usually 100 This ensures that the entire sample is removed from the column g Add an Flow step to re equilibrate the column and enter the required flow rate step size and B see step a above h Adda Fraction Collection step to specify how fractions are to be collected
300. signed to the running buffer Similarly an SVT3 2 or SV5 4 Aux pump inlet valve may be used to load 1 sample or 3 samples respectively Samples loaded through the pumps should be filtered through a 0 45 um filter Furthermore the pump should be washed with a sanitization solution afterwards to remove residual protein contamination that could reduce the piston seal lifetime Consult the DuoFlow online help for additional information System Setup 1 Connect a valve SVT3 2 SV5 4 or AVR9 8 to Inlet A or B as shown in Figure 8 4 For systems configured with a Maximizer valves can be connected at Inlets A1 A2 B1 and B2 2 n the device setup screen define a connected valve SVT3 2 SV5 4 or AVR9 8 as an Inlet A or Inlet B valve and name each valve position As shown in Figure 8 4 one position usually position 1 should be defined as the running buffer To automate system cleaning ports may also be assigned to sanitization and storage solutions 3 To prevent air from entering the system unused valve ports should be plugged with the 74 28 plugs supplied in the DuoFlow fittings kit 8 4 ADVANCED SYSTEM APPLICATIONS SAMPLE LOADING TO COLUMN WORKSTATION RUNNING BUFFER SAMEUE SANITIZING SOLUTION LE 20 ETHANOL HH SODIUM ELUTION SV5 4 HYDROXIDE BUFFER Figure 8 4 Sample Loading Through the Workstation Pump Writing the Gradient Pump Direct
301. ssing this button initiates the process of editing the method s protocol You may edit the method s protocol using any of the Add Step buttons and the Cut Copy Paste and Delete buttons Continue To continue the method run AN Run After editing the method s protocol return to the Run screen You then can Continue or Abort the run Figure 7 6 Run Screen s Abort Pause and Hold Buttons 7 33 MODES OF OPERATION SYSTEM OPERATION 7 4 2 Working Offline During a Run The DuoFlow system software allows you to work offline creating a new method or editing an existing method while a run is in progress The following actions can be performed Write a new method Edit an existing method View results from a completed run Print a report from a completed run Integrate run data from a completed run using the optional EZLogic software Perform PostRun analysis on a completed run Export Data or Export Chromatogram Image of a completed run Access the HELP screen These functions are not available while a run is in progress PostRun analysis of the run in progress Integration of the data from the run in progress Copy Out data Copy In data Initiation of a new run Utilities functions including calibrating pH probe gradient pumps conductivity Manual mode functions During a run the browser toolbar button is used to activate the offline window The online and offline windows ar
302. stem will remove most air bubbles Place it directly after the conductivity flow cell and run buffer for several minutes If the column being used cannot be run with the backpressure regulator and air bubbles are a continuing problem for example during some affinity chromatography steps use the 2mm path length cuvette cell The straight line flow of the cuvette cell makes bubble removal easier Excessive pump pulsations exhibited as regular noise on the baseline Air bubbles trapped in the pumpheads can produce exaggerated pulsations which appear as a noisy baseline Purge the pumpheads to remove the bubble Refer to Section 11 3 1 Priming the Workstation Pump and Removing Trapped Air Bubbles Baseline noise continues when pumps are turned off Noise spikes can also be caused by external environmental influences If the spikes occur at regular intervals for example once every 20 30 sec check for the presence of heating baths drying ovens or other heating devices on the same electrical circuit or in close proximity to the DuoFlow system Turn off these devices to see if the problem goes away 12 7 TROUBLESHOOTING Problem UV trace will not zero Possible Cause If no air bubbles are present in the flow cell check that the cell itself is clean both internally and externally MAINTENANCE AND TROUBLESHOOTING Solution Clean the interior of the cell by passing 1M sodium hydroxi
303. steps This is not user editable 7 15 MODES OF OPERATION SYSTEM OPERATION Table 7 6 Change Valve Edit Change Valve Change Valve Valve Position SVT3 2 Valve Inlet A Port 1 Y A Buffe SVT3 2 Valve Inlet A Port 1 A Buffer 2 SVT3 2 Valve Inlet B Port 2 AVR7 3 Valve User Assigned Name Port 4 Step 8 Volume 2 00 ml OK Cancel Change Valve To select any valve and change its position e Change Valve name and position These drop down menus allow you to choose a valve and to make a change in valve position Note that certain valve functions defined in the Setup screen such as Fraction Collector Diverter Aux Pump Inlet Sample Inject Inlet A Inlet B are tied to other protocol Steps so valve position changes will be made automatically An example would be an AVR7 3 valve defined as a Sample Inject valve in the Setup screen which is then tied to the Load Inject Sample step in the Protocol screen Valves assigned a User defined name during Setup require the Change Valve step at the desired point in the protocol e OK Adds the step to the protocol This is the same as pressing the Enter key on the keyboard e Cancel Does not add the step to the protocol This is the same as pressing the Esc key on the keyboard e Step Time or Volume Identifies current step number and calculates the elapsed time or volume from all previous steps This is not user edita
304. t active This condition is displayed during off line use it applies to the method and run data for the window that is not active online or offline Items with a red check mark indicate that item is in the Move List or is marked for deletion Blue Currently not open in the Online or Offline window Bolded text in the database tree applies to the following Top level folders Typically this applies to the USERS top level folder which lists all Users in the system By selecting Browser Settings from the Options menu you can use Starting Browser Selections to display projects methods and runs on the top level of the tree hierarchy where they will be bolded Summary information The total number of methods are listed for each user and the total number of runs are provided for each user project folder You cannot delete summary information Browser screen controls and information e Browser toolbar Located down the left side of the screen controls the different Browser functions They are discussed in greater detail on the following page Name and Date bars Toggle buttons located across the top of the database tree that allow you to sort the tree alphabetically or chronologically For example you can click on the Name button to sort from a to z and then click again to sort from z to a If you click on the Date button to sort from the latest listing then clicking again sorts from the earliest listing Browser Tabs and Browser Tab w
305. t in the Protocol Editor Method for editing The new method is assigned a default name method name version number that can be changed by the user Creates and names a new run for the current method and opens the Run screen Pressing Start in the Run screen starts the run Opens the Browser and displays all the users projects methods and runs associated with the database Many of the menu and toolbar options will remain Browser grayed out until a user name is selected See Chapter 6 for more detail on the Browser screen Opens the Print Report dialog that is used to specify the type of run reports to Report print Opens the Manual screen that is used for manual control and monitoring of the Manual devices connected to the Workstation and instrument bus Opens the Device Setup screen that is used to specify the devices required for the current method The Device Setup screen is used to name the pump inlet ports add valves and name the valve positions add detectors add a fraction collector add auxiliary pumps and add Buffer Blending Maximizer systems only for the method This screen is automatically opened after the New Method button is selected Setup 5 3 INTRODUCTION TO THE SYSTEM SOFTWARE SYSTEM OPERATION Table 5 2 Toolbar Buttons continued 5 4 PostRun
306. t recorder as well as to monitor the status of the two signal import modules SIM 1 and SIM 2 if they are connected to the they system see Chapter 2 9 6 for more information The UV detector panel is used to turn the Standard UV detector lamp on and off and to zero the baseline The Chart Recorder panel is used to start stop the chart recorder and to set the conductivity and UV range Event marks can be added to the chart recorder output by pressing the Event mark button The down arrow in the upper right corner of the panel is used to toggle between the QuadTec and UV detector control panels QuadTec Detector This Panel is used to control the QuadTec detector From this panel the lamp type and wavelength range is viewed the QuadTec detector can be turned on or off up to four wavelengths can be selected and the UV Vis baseline can be zeroed For best performance the QuadTec wavelengths should be entered in numerical order starting in the upper most field The Set button is used to accept any changes made in the Wavelength Selection boxes The down arrow in the upper right corner of the panel is used to toggle between the QuadTec and UV detector control panels The System Local option buttons are used to toggle the QuadTec between System and Local mode Econo Gradient Pump This panel allows you to control an Econo Gradient Pump EGP if it is connected to the system bit bus From this panel you can control the flow rate split see Chapter
307. te Note if an EP 1 or non Bio Rad pump is used the flow rate must be set both in this dialog and at the pump 8 2 AUX PUMP DIRECT INJECT With the addition of an Econo Gradient Pump EP 1 Econo pump or other compatible pump to the DuoFlow system samples can be loaded directly onto low pressure columns Up to 7 samples can be loaded sequentially if an Aux pump inlet valve AVR9 8 is connected to the system and defined in the setup One of the Aux pump inlet valve ports must be assigned to a rinse solution Similarly an SVT3 2 or SV5 4 Aux 8 2 ADVANCED SYSTEM APPLICATIONS SAMPLE LOADING pump inlet valve may be used to load 1 samples or 3 samples respectively System Setup 1 Connect an auxiliary pump to the system as described in Section 3 9 1 EP 1 or non Bio Rad pump or 3 9 2 Econo Gradient Pump Plumb the Aux Pump to port of the AVR7 3 Sample Inject valve as shown in Figure 8 2 If an auxiliary pump inlet valve is connected to the system it should be plumbed between the load pump and the AVR7 3 Sample Inject valve In the device setup screen add an Aux Pump to the setup and define it as a load pump Define non Bio Rad pumps as an EP 1 load pump Optional If an Aux Pump Inlet valve is used add the valve to the device setup define the valve as an Aux Pump Inlet valve and name each position One position usually position 1 should be defined as a Rinse solution The rinse solution is used to clean the sample loop bet
308. ter Inlet A Inlet B Aux Pump Inlet User Assigned Name MODES OF OPERATION Table 7 1 Valve Setup Information Position Names 1 Waste 2 Oollect Named by user Named by user Named by user Named by user Functions as a fraction collection diverter determined by the actual fraction collection parameters chosen When used before the inlet to Pump A or B the valve enables buffer selection The buffer name specified for each position will appear in the Protocol screen s Isocratic Flow Linear Gradient and Change Valve dialog box Refer to Chapter 8 Sample Loading for examples Used for auxiliary pump load selection to select one of two solutions Refer to Chapter 8 Sample Loading for examples When used for a purpose other than described above The name specified for each position will appear in the Protocol screen s Change Valve dialog box SV5 4 Low pressure solenoid valve Inlet A Inlet B Aux Pump Inlet User Assigned Name Named by user Named by user Named by user Named by user When used before the inlet to Pump A or B the valve provides preparative sample loading or buffer selection The buffer or sample name specified for each position will appear in the Protocol screen s Isocratic Flow Linear Gradient and Change Valve dialog box Refer to Chapter 8 Sample Loading for examples Used for auxiliary pump load selection to select one of four solu
309. th Above Threshold and Below Threshold collection are supported The software also supports tube numbering by Rack amp Tube and Rack amp Grid e Multiple Valve Capabilities e Workstation provides connection for low pressure and 3 high pressure valves e Workstation with the addition of the Maximizer doubles the capacity to 6 low pressure and 6 high pressure valves e Starter Kit and UNO Q1 Anion Exchange Column Includes the necessary reagents protein sample and columns for running an anion exchange chromatography experiment The kit includes easy to follow tutorial style instructions for the first time user e Q OQ Protocols Validation protocols are available or can be performed by certified Bio Rad service engineers 1 3 UNPACKING When you receive the BioLogic DuoFlow system carefully inspect the shipping containers for any damage which may have occurred in shipping Severe damage to a container may indicate damage to its contents If you suspect damage to the contents immediately file a claim with the carrier in accordance with their instructions before contacting Bio Rad Laboratories A Caution Lift items from the bottom as you remove them from their containers Open each of the shipping cartons and lift the contents out of its packing Check the contents of each box against the supplied packing list Remove the plastic bag from each unit and inspect the unit for external damage If any part is missin
310. th Auto Inject A A METHOD PARAMETERS Flow Rate 2 5 ml min Size Exclusion with Auto Inject B Sample Size Rinse 20 ml Size Exclusion with Auto Inject C Fraction Size 2 5 ml Size Exclusion with Auto Loop Fill Rinse A Fraction Collector Rack F1 12 13 mm tubes y Size Exclusion with Auto Loop Fill _Rinse B Size Exclusion with Auto Loop Fill Rinse C y View Template Help Figure 6 5 New Method Dialog Showing Method Templates To load a Method Template 1 Select New New Method from the Browser toolbar or the New Method button on the system tool bar 2 In the New Method dialog place a check in the Use Method Templates box see Figure 6 5 Select the Experiment Type Select the Method Template A brief description of each method template is given in the Template Description box or a more detailed description can be viewed from the online help by pressing View Template Help 5 After entering the method name press OK to load the template 6 If your system includes a Model 2128 fraction collector you will need to change the Method Template Setup To this go to the Setup Screen and delete the BioFrac fraction collector and replace it with a Model 2128 In the protocol screen open the fraction collection step and make sure the correct number of tubes is defined INTRODUCTION TO THE BROWSER SCHEEN SYSTEM OPERATION Procedures for creating a run in the Browser 1 6 8 Ente
311. th the Controller Components connect to the instrument bus in a daisy chain and are recognized when the system is switched on Even when one component is switched off other components daisy chained to the system can be controlled by the Controller Monitor connector To connect the color monitor to the Controller Keyboard connector To connect the keyboard to the Controller Mouse connector To connect the mouse to the Controller Parallel connector Devices designed for connection to the parallel port include printers and external storage devices Refer to Windows 2000 and or the device documentation for installation instructions Some printer drivers are pre installed on the Controller Power connectors To connect the power cable 2 4 SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM 2 1 2 USB Bitbus Communicator The USB Bitbus Communicator is used to connect the Controller to DuoFlow system instrument bus and to supply power to the instrument bus when a Signal Import Module SIM is used Table 2 3 USB Bitbus Communicator PWR BUS INSTR BUS SELECT PWR mn Ea EXT m FRONT VIEW REAR VIEW Description om Used to connect to the Controller USB port by way of a USB cable catalog number 760 2032 ON LED Indicates that there is power to the USB Bitbus Communicator Is used to supply power to the USB Bitbus Communicator when a Signal Import
312. that outputs an analog signal between 2 5 Volts to 2 5 Volts Instruments that may be connected in this way could include a variable wavelength UV detector a refractive index detector or a fluorescence detector e Two SIMs may be connected to a DuoFlow system IN IN GND INSTRUMENT BUS DEVICE POWER S eeg Be O xj Figure 2 29 Signal Import Module front and rear views The output from a pH electrode is connected to the pH connector a BNC connector on the SIM the output from any other detector is connected at the 3 pin connector gnd of the SIM The Automatic Temperature Compensation ATC connector is reserved for future upgrades and enhancements The instrument bus cable connects the SIM to the USB Bitbus Communicator Refer to Figures 2 29 and 2 30 If a second SIM is to be used one of the instrument bus cables is used to connect the two SIMs The SIM is described in detail in its separate documentation When a SIM is connected to the instrument bus the USB Bitbus device must be connected to an external powersource see Section 2 1 2 CONNECTING A SINGLE SIM INSTRUMENT BUS DEVICE POWER NUMBER STATUS ES TO USB BITBUS 2 CONNECTION TO THE COMMUNICATOR FOLLOWING DEVICES LISTED IN SEQUENCE MAXIMIZER IF AVAILABLE WORKSTATI
313. the Workstation All valves will be active AVR7 3 INJECT VALVE FOR SAMPLE APPLICATION WORKSTATION WASTE TWO AVR9 8 VALVES FOR COLUMN SWITCHING COLUMN COLUMN INLET 3 WASTE SAMPLE 4 7 AVR9 8 AS COLUMN LOOP 3 SWITCHING VALVE 8 TO RECEIVE SAMPLE UP TO8 DIFFERENT COLUMNS 5 5 6 6 COLUMN OUTLET 7 SECOND AVR9 8 AS COLUMN SWITCHING VALVE TO DIRECT ELUENT TO MONITORS AND FRACTION COLLECTOR RINSE SAMPLE 1 SAMPLE 2 SAMPLE 3 UV MONITOR amp FRACTION COLLECTOR AVR9 8 FOR LOADING MULTIPLE SAMPLES Figure 2 14 Two Examples Using AVR9 8 Valves 2 30 SYSTEM OVERVIEW DESCRIPTION OF BIOLOGIC DUOFLOW SYSTEM 2 6 3 SV5 4 Buffer Select and Automated Sample Loading Valve The SV5 4 valve is a low pressure 5 port 4 position valve used for automatic buffer selection and sample loading The SV5 4 valve may be used for e Buffer and or sample selection when placed before the Workstation pumps or an auxilary load pump SV5 4 valve connected to a Workstation pump inlet enables access to four separate solutions Fraction collection when placed after the column e Purging or rinsing all tubing lines for cleaning purposes Figure 2 15 SV5 4 Buffer Select Valve Use 1 8 3 2 mm OD PTFE tubing and 1 4 28 fittings when plumbing the SV5 4 valve to the Workstation pump inlet To prevent air from entering the system unused ports should b
314. the operator the buffer s flow rates gradients chart speed fraction size and any other information you might want to enter Run Notebook Method Name Method 1 Method Description Date Run Name 1 OK Sample Column Operator Buffer A Buffer B Flow Rate Gradient Chart Speed Fraction Size Run Description Run Notes Figure 7 9 Run Notebook Screen 7 4 5 Run Log Screen The Run Log screen details the time and order of the execution of each step error and or event of the run This information cannot be edited It is possible although not recommended to turn off the Run Log by deselecting its checkbox in the Edit User Preferences window available from the Options menu Event Log for run name Close Description Author Date lt lt Time and Date gt gt User Name lt lt name gt gt Project Name lt lt name gt gt Method Name lt lt name gt gt Run Name lt lt name gt gt Run Operator name run description Figure 7 10 Run Log Screen 7 37 MODES OF OPERATION SYSTEM OPERATION 7 5 POST RUN Screen The Post Run screen enables peak annotation tagging and data export To enter the Post Run screen open a run file in the Browser screen or when a run is finished choose PostRun from the toolbar Two chromatograms are viewed the main chromatogram in the lower large window and th
315. thods were successfully verified appears Run Queue Starts a run queue Edit Queue Used to change the name and description of a Queue that has not been run or to make a copy of a Queue that has been run SYSTEM OPERATION INTRODUCTION TO THE BROWSER SCREEN 6 4 CREATING AND VIEWING A COMPARE Trace Compare allows you to view and compare an unlimited number of chromatogram runs simultaneously in either tile or overlay mode BioLogic Duo Flow user name project name gt method name gt run name gt File View Utilities Options Window Help New Edit Method Method New Wash Run mei PostRun a Z a Browser Report lt 8 Manual Setup Protoco Settings Date New Edit Delete 8 6 o Print CopyOut Copyln f USERS eo jemLab Project for ChemLab Mary Jones inm Project for Mary Jones Joe Smith Project for Joe Smith Default mo Chromatagraphy E Project for Demo Chromatagraphy lll Demonstration Queue iji Demonstration Queue 1 Run Q Queue Method 3 WU Run Q
316. ties drop down menu Liquid is continu The pump s piston seals are 1 Replace the piston seals on both pumpheads ously leaking from worn or damaged Clean pistons and piston guides with water the pumphead followed by methanol as a precautionary washout drain maintenance step trough 2 Recalibrate the pumps after changing seals by selecting Gradient Pump Calibration from the Utilities drop down menu _ Erratic or reduced Columns packed with soft gels Insert the backpressure regulator between the flow rate when or large particles typically pro pump and the injection valve pumping onto duce very low back pressures columns which when run at low flow rates CAUTION Low pressure columns may burst produce low back if the backpressure regulator is placed after pressures the column eg Econo Pac cartridges or small agarose gel affinity columns Loud or unusual Residue buildup behind 1 The pistons should be rinsed on a nightly basis noise coming from piston seals This may easily be done automatically by the front of the programming a water rinse step at the end of workstation each protocol This may also be done manually by washing the pistons with the syringe provided in the Fittings kit Sticky pistons 2 Follow the maintenance procedure to carefully remove and disassemble the pumpheads Wash the pistons thoroughly with water to remov debris or salt crystals If the piston seals have many hour
317. tion The AVR7 3 valve control panel is labeled with the valve type and the Workstation port where the valve is connected For example if the valve is connected at Workstation port 4 the panel will be named AVRT 3 at port 4 The AVR7 3 valve the panel shows three valve positions Load L Inject 1 and Purge P Press the Purge buttons A and B on the front of the Workstation The Workstation pump will run and the indicator lights will flash green The default purge flow rate is 10 ml min for F10 pumps and 40 ml min for F40 pumps The default purge flow rate can be changed from the Options Manual Setup menu option on the Manual screen Run both pumps for about 2 minutes If a Maximizer is connected to the system place the valve inlets at position A2 and B2 for about a minute and then at A1 and B1 for an additional minute Press the Purge buttons again to stop the pump From the Manual screen place the AVRT 3 injection valve into the Inject 1 position Set the pump flow rate to 1 0 ml min and start the pump Water will now flow through the sample loop the UV Conductivity flow cells and ultimately to the fraction collector After the system is filled change the AVR7 3 injection valve to Load and monitor the pressure displayed on the status bar for a few minutes If the pressure variation exceeds 10 96 repeat step 1e until the pressure is stable It is best to monitor the pressure with some backpressure on the pumps such as the 40 psi bac
318. tion key and the down arrow key on the front panel of the EP 1 Econo pump If the Econo pump does not have the correct firmware version contact BioRad for information on how to upgrade the pump The EP 1 Econo pump receives start stop signals only The flow rate of the EP 1 Econo pump is set from the pump and the flow rate is recorded in the yellow data entry boxes of the Load Inject Sample dialog box The DuoFlow system starts stops the Aux pump and cannot control the flow of the EP 1 Econo pump The correct flow rate must be set at the EP 1 Econo pump Consult the EP 1 Econo pump user s manual for additional information 2 42 SYSTEM OVERVIEW DESCRIPTION OF SYSTEM COMPONENTS 2 8 3 EGP Econo Gradient Pump The Econo Gradient Pump EGP is a two channel bi directional variable speed peristaltic pump for low pressure chromatography and general laboratory use The EGP has the following features The EGP can be programmed to run a gradient The EGP controls a splitter valve for stream splitting The EGP may be used with most flexible tubing having an inner diameter less than or equal to 3 2 mm 1 8 and a wall thickness of 1 0 mm or less including Silicone Tygon and PharMed Flow rates are 0 01 20 ml min per channel depending upon tubing ID It has start stop control of fraction collectors and chart recorders It is coldroom compatible Platen ECONO GRADIENT PUMP E Foe e we LCD Display an Soft Keys
319. tion with 4 simultaneous wavelengths 760 1147 DuoFlow QuadTec Standard System 100 120 V 760 1146 DuoFlow QuadTec Standard System Japan Korea 760 1148 DuoFlow QuadTec Standard System 220 240 V 10 ml min flow rate to 3500 psi UV Vis detection with 4 simultaneous wavelengths Fraction collection 760 2237 DuoFlow Maximizer 20 System 100 120 V 760 2236 DuoFlow Maximizer 20 System Japan Korea 760 2238 DuoFlow Maximizer 20 System 220 240 V 20 ml min flow rate to 3500 psi 254 280 nm detection Buffer blending Fraction collection 760 2247 DuoFlow Maximizer 80 System 100 120V 760 2246 DuoFlow Maximizer 80 System Japan Korea 760 2248 DuoFlow Maximizer 80 System 220 240 V 80 ml min flow rate to 1000 psi 254 280 nm detector Buffer blending pH monitoring Fraction collection 760 2257 DuoFlow Pathfinder 20 System 100 120 V 760 2256 DuoFlow Pathfinder 20 System Japan Korea 760 2258 DuoFlow Pathfinder 20 System 220 240 V 20 ml min flow rate to 3500 psi UV Vis detection with 4 simultaneous wavelengths Buffer blending pH monitoring Fraction collection 760 2267 DuoFlow Pathfinder 80 System 100 120 V 760 2266 DuoFlow Pathfinder 80 System Japan Korea 760 2268 DuoFlow Pathfinder 80 System 220 240 V 80 ml min flow rate to 1000 psi UV Vis detection with 4 simultaneous wavelengths Buffer blending pH monitoring Fraction collection SYSTEM OVERVIEW INTRODUCTION 1 5 QUICK START PROCEDURE The general proced
320. tions Refer to Chapter 8 Sample Loading for examples When used for a purpose other than that described above The name specified for each position will appear in the Protocol screen s Change Valve dialog box Sample Inject User Assigned Name 1 Load Sample 2 Inject Sample 3 Purge Named by user For automatically loading a sample When used for a purpose other than described above The name specified for each position will appear in the Protocol screen s Change Valve dialog box Refer to Section 4 Advanced System Applications Chapters 8 through 10 MODES OF OPERATION Valve Type Valve Name Function Aux Pump Inlet Inlet A Inlet B Fraction Collector User Assigned Name Column Switching 7 2 3 Buffer Editor SYSTEM OPERATION Table 7 1 continued Valve Setup Information Position Names Named by user Named by user Named by user 1 Waste 2 Collect 3 8 Named by user Named by user Named by user Used for auxiliary pump load selection to select from up to eight samples buffers or rinse solution When used before the inlet to Pump A or B the valve provides preparative sample loading or buffer selection The buffer or sample name specified for each position will appear in the Protocol screen s Isocratic Flow Linear Gradient and Change Valve dialog box Refer to Section 4 Advanced System Applications Chapters 8 and 9 Useful for colle
321. to port 4 and outlet to port 3 Column 2 should have its inlet connected to port 6 and outlet to port 1 2 Plumb port 5 of the column switching valve to port 4 of the upstream sample inject valve Plumb port 2 of the column switching valve to your detector 4 n the device setup configure the AVR7 3 valve as a user define valve and name the valve Column Switcher Name valve positions 1 2 and 3 as Column 1 Column 2 and Purge respectively SAMPLE LOADING ADVANCED SYSTEM APPLICATIONS Writing the Column Switching Protocol When using multiple columns care should be taken to prevent cross contamination Purge steps should be included before placing a column inline to ensure that the tubing is clean and filled with the appropriate buffer The following three steps should be included as part of a column switching step 1 Add a Change Valve step to your protocol and place the Column Switcher valve in the Purge position 2 Add an isocratic flow step to wash the tubing and mixer with 3 5 volumes of buffer Add a Change Valve step to your protocol and select the desired column on the Column Switcher valve Column switching steps are usually placed at the beginning of a protocol or at the start of each dimension in a multi dimensional chromatography experiment 9 1 2 AVR9 8 Eight Column Switching AVR7 3 INJECT VALVE FOR SAMPLE APPLICATION WORKSTATION PUMP WASTE COLUMN f 3 COLUMN INLET WASTE SAMP
322. tocols must be compatible In other words the material collected in one dimension must be suitable for injection in the next dimension in terms of composition pH concentration etc Another factor to consider is the number of fractions to be collected and the capacity of your fraction collector Although fractions can be collected during the entire experiment it is usually best to only collect fractions during the final dimension in order to avoid running out of tubes This is particularly important if you are purifying multiple samples The setup of a multidimensional experiment can be complex and requires that care be taken in plumbing the valves and equilibrating the system The placement of valves pumps and detectors should be planned so as to minimize tube lengths Prior to connecting columns to the system make sure that all the tubing has been filled with buffer A few plumbing diagrams have been supplied in the BioLogic software online help that show how to plumb both two and three dimensional chromatography experiments Method templates designed for these plumbing diagrams are available in a file named Multi D Templates zib located in the BioLogic directory MULTIPLE COLUMNS ADVANCED SYSTEM APPLICATIONS Writing the Protocol The powerful queuing feature of the BioLogic software makes writing multi dimensional chromatography experiments simple Multi dimensional experiment can be written as a series of individual one dimensional chromatog
323. uential method steps Step 4 Run to inject sample and view the real time chromatogram e Two easy steps if you want to run a sample using a current Setup and Protocol Step 1 Browser to select a user and along with a method or Method Template Step 2 Run to inject sample and view real time chromatogram On screen Help Includes detailed information and a troubleshooting guide USB Bitbus Communicator The USB Bitbus communicator allows the BioLogic DuoFlow system to be controlled from any computer running Windows 2000 and BioLogic software 4 0 or above over a USB port Buffer Blending Buffer Blending is a feature of the BioLogic DuoFlow Maximizer and Pathfinder systems that dynamically Blends the conjugate acid and base of a buffer with water and salt to produce a solution with a specific salt concentration and pH Buffer Editor The Buffer Editor is a feature used to create buffer systems for use in Buffer Blending experiments Both single and multiple component buffers can be created Scouting This feature facilitates the optimization of a chromatography protocol for a specific target molecule Scouting systematically increments a user selected variable and then performs a chromatography run at each increment Variables that can be Scouted include pH B step duration column buffer flow rate sample and sample volume Method Templates Includes ready to run chromatography protocols for a variety of experiment types inclu
324. uggested solutions for the different instruments in the system You can obtain more information about your system by registering for Consult Bio Rad at www biorad com where an online technical support service offers an extensive database of frequently asked questions FAQs 12 1 TROUBLESHOOTING THE DUOFLOW CONTROLLER AND SOFTWARE Problem Software response is slow Possible Cause Database size may need to be reduced Solution Archive unnecessary methods and runs a Open the Browser window b Use COPY OUT to archive seldom used methods and runs You may archive to a CD ROM the BioLogic C drive or any other drive selectable from the Drives drop down menu Use the Windows 2000 Disk Defragmenter utility Minimize the Duo Flow application and then from the Windows Start button select Programs Accessories System Tools gt Disk Defragmenter Follow the online instructions to defragment files on your disk A method or run you expect is not displayed in the Browser There may be no apparent cause for this Use the Reset button in the Browser toolbar From the Browser toolbar click on the Reset button and then check the database tree for your method or run Note Using Reset collapses the database tree While in offline mode the offline window is not displayed The Windows 2000 minimize button may have been selected Use any of the following three means of activating the offline window a
325. uld be used with the backpressure regulator see page 2 52 Connect the backpressure regulator after the Conductivity monitor in the system plumbing Preparative 2 mm flow cell This flow cell is recommended for most applications which demand less sensitivity for flow rates greater than 10 ml min or when working with high protein concentrations It has a 2 mm path length a volume of 30 and is rated to 750 psi The UV detector receives power via the Workstation to which it is connected by the UV lamp cable The UV detector communicates with the system via the UV optics cable which plugs into the UV optics connector on the back of the Workstation The UV detector sensitivity ranges from 0 to 2 0 AUFS Absorbance Units Full Scale The UV sensitivity range for the chart recorder is set in the system software in either the Manual or Run screens A Zero Baseline button is available in the Manual and Run screens and a programmable Zero Baseline command is available as part of a method protocol Refer to the discussion of the chart recorder for setting the chart recorder range page 2 55 To replace an expired lamp refer to Chapter 11 Maintenance or to the instruction sheet for the replacement lamp UV FLOW CELL FILTER TRAY WITH 214 nm UV FILTER Figure 2 6 UV Detector with Zinc Lamp 214 nm Filter and Conductivity Flow Cell To change the UV flow cell or UV filter If the system has been used make sure that any hazardous
326. ume Sample Injection 2 40 2 8 2 Model EP 1 Econo P MP ee ea ateen aaaea e eaa aenea Ea aa a a aaa kreadi 2 42 2 8 3 EGP Econo Gradient Pump 2 43 2 8 4 Other Gradient 2 44 system Peripherals eiie tacere IB ue ee ethene aval dee en aep denen ee 2 45 29 1 System Rack sien Sasi eden cet ede ende te beetles eet eme dus 2 45 2 9 2 Starter Kit Eee e ea Net ee al eae eh os 2 A7 2 9 3 Fittings Kit including Tubing 2 47 2 9 4 Fittings Tightener tet reete eR denne a iain stre en Lue de 2 48 2 9 5 Regulator ssssssssssseeeeeem eem 2 49 2 9 6 Signal Import Module SIM ssseeeeeenn mmm 2 50 2 937 PUMP KIsz o eae eo t tarder esee meet aet eet 2 51 TABLE OF CONTENTS 2 9 8 Model 1327 Charn Recorder rosiorii R 2 52 2 9 9 Generic Chart Recorders sssssssssseeeeeeeeee nennen nenne 2 52 2 9 10 Uninterruptible Power 2 53 LETTRE iic 2 53 2 10 Columns and Column Fittings seseeneeeenn emen 2 54 2 10 1 Anion Exchange Q Strong Anion Exchange 2 54 2 10 2 Cation Exchange S Strong Cation
327. ure used to create and run a chromatography experiment on a DuoFlow system is described below 1 Install the required devices and instruments on the system see Figures 3 4 and 5 5 for cable connections and 4 1 and 4 3 for plumbing connections Flush all plumbing with DDI H20 to ensure that the system is clean and free of air and then prime the pumps with starting buffer See Chapter 4 for more detail Attach a column and set the pressure limits in the Manual screen The high limit should be less than or equal to the pressure limit for the column Equilibrate the column and system with starting buffer System equilibration is controlled from the Manual screen see Section 7 1 Create a new method in the Browser a Start the Browser using the Browser button on the tool bar and then select or create a user and project Refer to Chapter 6 for more information on the Browser screen b Use the New New New User option in the Browser tools on the left side of the screen to create a new user and enter a username c Use the New and New Method option in the Browser tools to create and name a new method Click OK to proceed to the hardware Setup screen Alternatively check the Use Method Templates box select a method template and press OK In the Device Setup screen select the devices that are connected to the system Select the File Save Setup menu item and save the device setup check the Default Setup box if this is the default
328. utomatically by programming a water rinse step at the end of each protocol It is also important to rinse behind the piston seals by injecting water through the washout port located at the top of the pumphead A standard syringe may be used for this purpose SHORT SCREWS 8 32 X 1 0 WASHOUT PORT OUTLET BLOCK O RING CHECK VALVES CENTER BLOCK FLOW RATE IDENTIFICATION 10 or 40 O RING CHECK VALVES INLET BLOCK PUMP HEAD ASSEMBLY LONG SCREWS 8 32 X 1 5 NOTE FOR BOTH SHORT AND LONG SCREWS USE PUMPHEAD DISASSEMBLY ALLEN WRENCH OR STANDARD 9 64 ALLEN WRENCH Figure 11 3 Pumphead Assembly MAINTENANCE MAINTENANCE AND TROUBLESHOOTING 11 4 MAINTENANCE OF THE UV DETECTOR AND THE CONDUCTIVITY FLOW CELL The following two sections discuss the cleaning of the UV detector and the Conductivity flow cell and replacement of the lamp in the UV detector 11 4 1 Cleaning the UV Detector and the Conductivity Flow Cell Cleaning of the UV flow cell is indicated when the baseline is very noisy or unstable yet air bubbles have been removed from the flow cell However problems with UV detection during medium pressure chromatography are often solved by a few simple precautions Air bubbles trapped in the flow cells give rise to a sawtooth signal pattern that will never stabilize We recommend always running with a backpressure regulator after the detector if using low pressure columns
329. valve 11 4 Toolbar daily maintenance 11 2 about PERSE EIAS MEAN HO VUE E BUNT EA 5 2 priming and removing air bubbles 11 2 4 8 File buttons replacing a piston 11 3 BrOWSer esee 5 3 troubleshooting ecce 12 3 Edit 5 3 New 5 3 Workstation New rome 5 3 10 25V 0 3A Max connector 2 9 Report NONE ANE 5 3 alert hibet 2 7 View buttons automated valves connector 2 9 Buffer Blending 5 4 aux connector Sgen eee SS 2 10 Bio Rad eme 5 4 cable connections with Maximizer eee 3 5 RES 5 4 Workstation cable connections D LLLI 5 4 without 3 4 Manualet soot ot ae rar ot 5 3 cond chart connector 2 9 NOTES 5 4 cond flowcell 2 9 Peet tease 5 4 description ii a til 2 6 IN 6 instr bus 2 10 mixer connector essen 2 9 pause button 2 7 plumbing connections 2 8 power button 2 2 7 power cord connector
330. ve mount the valve to a vertical bar on the system rack and then connect its cable to any of the connectors labeled Solenoid Valves 7 8 or 9 on the rear of the Maximizer if available Otherwise connect it to connectors 1 2 or 3 on the Workstation e Refer to section 2 6 for more detailed information about each valve HIGH PRESSURE LOW PRESSURE Figure 3 13 DuoFlow Valves 3 14 SYSTEM INSTALLATION AND SETUP SYSTEM SETUP 3 8 FRACTION COLLECTOR CONNECTIONS This section discusses the connections for the following instruments and devices BioFrac fraction collector e Model 2110 fraction collector Model 2128 fraction collector Refer to section 2 7 for more detailed information about the fraction collectors 3 8 1 BioFrac Fraction Collector The BioFrac fraction collector is controlled by the DuoFlow software version 4 0 or greater via the Instrument Bus The BioFrac fraction collector is connected to the system as discussed below 1 Connect the USB Bitbus to the Controller as discussed in Section 3 2 2 Use either of the instrument bus connectors on the rear of the fraction collector to connect the BioFrac fraction collector to the Instrument bus see Section 3 3 3 Select BioFrac in the BioLogic Configuration Utility 3 8 2 Model 2110 Fraction Collector The Model 2110 fraction collector may be connected to the Workstation as discussed below 1 Connect the DB 9 connector on System Cable 5 to the Model
331. w of the Dell PC Computer as the DuoFlow 2 4 2 3 USB Bitbus Communicator seisin eeraa e ia a ea e iaaa Eana Eaa 2 5 2 4 F10 and F40 Pumphead Flow enne nnne 2 6 2 5 Workstation Front Panel 2 7 2 6 Workstation Rear Panel Connectors sse eren 2 9 2 7 Maximizer Front Panel 2 12 2 8 Maximizer Rear Panel Connectors 2 14 2 9 Maximizer SCreens dieere dt ede de I aad ere ee Medi Pee e eed ke 2 16 2 10 Mixer Barrels and Mixer Capacity for the MX 1 2 18 2 11 Mixer Barrels and Mixer Capacity for the Maximizer 2 19 2 12 BioFrac Racks Available 2 36 2 13 Model 2128 Racks Available ssssssssssssssssssssssssseeeee nennen nennen nnne nennen 2 38 2 14 Workstation Pump Configuration Flow Rates 2 51 2 15 Columns and Column menn nnm 2 58 3 14 MixerEloW Rates tete tette te ER EL EORR eo ede aaa
332. ware control of an auxiliary pump for multiple sample loop fills and large volume dynamic loop filling Valve Control Up to three low pressure automated solenoid valves SV5 4 and SVT3 2 for stream selection Up to three high pressure automated valves AVR7 3 and AVR9 8 for sample inject column switching and stream selection A 2 APPENDIX A SPECIFICATIONS Fraction Collection BioFrac fraction collector collection by Collect All Threshold Collection Windows Collection Windows Threshold Model 2110 fraction collector collection by Collect All when used with optional SVT3 2 Diverter valve it also offers collection by Threshold Collection Windows Collection Windows Threshold Chart Recorder Control Model 1327 Dual pen recorder Paper feed Start Stop Pen Up Down Signal Import Module e Up to two Signal Import Modules for importing analog signals from peripheral chromatography detectors A 3 SPECIFICATIONS APPENDIX A APPENDIX B PRESSURE CONVERSION TABLE APPENDIX B PRESSURE CONVERSION TABLE Use this table to convert pressure units for the pump Table B 1 Pressure Conversion Multiply the units in the left column by the conversion factors listed Kg cm Torr 51 713 6 8948 0 06895 Inches Hg 2 0359 14 696 1 760 101 32 1 0133 29 921 14 223 0 9678 1 735 56 98 06 0 9806 28 958 0 0193 0 00132 0 00136 1 0 1330 0 00133 0 0394 0 1450 0 00987 0 0102 7 52 1 0
333. ween injections Note that some sample will be lost in the process of pulling sample from a remote beaker Be sure to factor in the solution volume required to fill all the buffer lines leading up to the top of the column when programming the injection step Remove the 40 psi backpressure regulator from the post column position and place it between the Workstation pump and the mixer This allows low pressure peristaltic pumps to push sample through the column while at the same time will help the check valve to seat properly and ensure pump flow performance In addition low pressure columns may burst if the backpressure regulator is placed after the column Plumb the Aux Pump to port 3 of the AVR7 3 Sample Inject valve as shown in Figure 8 2 Optional If an Aux Pump Inlet valve is used connect it to the Aux pump inlet WASTE WASTE 5 WORKSTATION PUMP LOW PRESSURE COLUMN AUXILIARY AUXILIARY LOAD PUMP LOAD PUMP PURGE Figure 8 2 Plumbing an AVRT 3 Inject Valve with an Auxiliary Load Pump Writing the Aux Pump Direct Inject Protocol Several methods that include the Aux Pump Direct Inject feature have been included in the BioLogic DuoFlow Method Templates see Section 6 2 and the online help for more information This feature may be added to any protocol 1 2 In the protocol load inject step select Direct Inject as the injection type Select the Aux Load Pump option and set the sample volume flow rate and fl
334. ween the two modes 7 10 Add Step buttons These buttons are used to add steps to the protocol They are located in a scroll box on the left side of the protocol screen To insert a step highlight the step below where the new step is to be added and then press the appropriate button to define the new step see Tables 7 3 through 7 9 for more information The buttons that are active depend on which devices have been defined in the device setup Fraction Collector button This button is used to define the fraction collection scheme used by the protocol It is located below the Add Step buttons on the protocol screen see Table 7 10 for more details This button is active if a fraction collector has been defined in the device setup Scout Button This button is used to convert the currently defined method into a scout method Note that the method cannot be edited once a scout has been defined unless the scout is deleted see Table 7 11 Edit buttons These toolbar buttons are located at the top right side of the protocol screen and are used to edit cut copy paste and delete method steps To cut copy or delete a step select the step with the mouse for multiple step selection do a click drag or Cirl click and press the appropriate button To paste steps into a method select the step below the place where the new step s is to be added and press paste see Table 7 12 for more detail File buttons The New Method Edit Method and Browser t
335. whether fractions are collected when the detector signal is above or below a designated Threshold e Non peak parameters Destination and Fraction Size Enter the non peak parameters If the buffer stream is diverted to Waste there is no need to enter a fraction size But if you want to collect non peak material you must enter a fraction size 7 21 MODES OF OPERATION SYSTEM OPERATION Table 7 10 continued Fraction Collection Description Collection Windows Fraction Collection Scheme BioFrac Rack Type F1 12 13 mm tubes Collect All EN Threshold m Rack Row Col Start A Yj 1 Y 90 v 1A Threshold and Collection Windows O Close Fraction Collector Scheme CollectionWindows Start End Frac Size Close ml ml ml ADD MODE oo m o H o e0 Rl Save Window 1 4 00 5 00 0 60 2 7 00 8 00 1 00 Finished Adding Fraction Collector Scheme CollectionWindows Start End Frac Size Close ml ml ml 1 4 00 1 5 00 4 0 60 2 EDITMODE x M Save Changes 2 7 00 8 00 1 00 Delete Window Cancel Editi
336. ze 7 22 Tubes Required 7 22 Start Tube 7 22 End Tube 7 22 Add Mode 7 22 Select Mode 7 22 Threshold and Collection Windows Above below threshold 7 24 Fraction size 7 24 Start T be 7 24 End Tube 7 24 Start 7 24 End 7 24 Threshold AU 7 24 Non Peak Frac Size ml 7 24 Add Mode 7 24 Select Mode hes 7 24 Non peak parameters 7 24 Threshold Above below threshold 7 20 End 7 20 Fraction Size 7 20 Non peak parameters Destination 7 20 Non peak parameters fraction ir MER 7 20 Start Tube 7 20 7 20 Tubes Required 7 20 Hold button Above Threshold 7 18 Below Threshold 7 18 piese rte fete 7 18 End of 7 18 Key Pressed ees 7 18 MEER 7 18 Sound Alarm 7 18 Start of

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