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4006208B.qxd 6/28/2004 11:03 AM Page 3 Table of Contents Introduction .......................................................................................1 1. Starter Kit Components ..............................................................1 2. Materials You Will Need...............................................................1 I. BioLogic DuoFlow System ....................................................3 Section 1. DuoFlow System Preparation .........................................3 1.
4006208B.qxd 6/28/2004 11:03 AM Page 4 II. DuoFlow Maximizer and Pathfinder Systems.....................18 Section 3. System Preparation.......................................................18 3.1 Prime the Workstation Pumps ..................................................20 3.2 Move the AVR7-3 Inject Valve to the Purge Position..................20 3.3 Purge the Workstation Pumps ..................................................21 3.4 Manual Control of the Pumps .....................................
006208B.qxd 6/28/2004 11:03 AM Page 6 Introduction This instruction manual and starter kit contents may be used for the BioLogic DuoFlow system and the BioLogic DuoFlow Maximizer™ and Pathfinder™ chromatography systems. The use of the starter kit with these systems is described in Sections 1 and 3, respectively. 1. Starter Kit Components This starter kit contains the following items for running a separation: • 50 ml of buffer A, 250 mM Tris-HCI buffer, pH 8.
4006208B.qxd 6/28/2004 11:03 AM Page 7 If you are using the DuoFlow Maximizer or Pathfinder systems you will also need the following materials: • pH 7.00 and pH 10.00 standard buffer • One 200 ml graduated cylinder (optional) • Two additional 500 ml bottles • Fraction collection 1.
4006208B.qxd 6/28/2004 11:03 AM Page 8 I. BioLogic DuoFlow System Section 1. DuoFlow System Preparation When the DuoFlow system is turned on, the Manual screen is displayed (see Figures 1 and 2). This screen displays instrument control panels that provide direct control of the pumps, valves, fraction collector, UV detector, QuadTec™ UV/Vis detector, and Econo™ Gradient Pump.
4006208B.qxd 6/28/2004 11:03 AM BioLogic Duo-Flow - - - no method File View Utilities Options Window New Help 1 2 New Edit Method Method Run Browser Report Mode: ml/min 1.00 wash load Manual Setup Protocol Run Notes PostRun Log Fraction Collector: BioFrac Gradient Pump: F10 Flowrate Page 9 Mode: System Rack: F1 (12-13 mm tubes) Inlet A 50 % Start Tube: 1 Inlet B 50 % End Tube: 20 Fraction size: High limit 700 psi. Low limit 0 psi. ON 1.
4006208B.qxd 6/28/2004 11:03 AM Page 10 1.1 Prime the Workstation Pumps a. Immerse the workstation pump A and B inlet lines in a container of HPLC grade (filtered, degassed) or other high quality water. b. Connect the syringe (supplied with the fittings kit) to the priming port of pump A. c. Turn the priming port counter-clockwise one full turn to open the seal. Gently withdraw the syringe plunger to draw water into the pump head. d.
4006208B.qxd 6/28/2004 11:03 AM Page 11 1.4 Manual Control of the Workstation Pumps The workstation pump parameters are set from the Manual screen either by clicking in the appropriate field and entering a value from the keyboard or by using the arrows. You can set the flow rate between 0.01 to 10 ml/min and the gradient composition between 0 and 100% B. To start the pump, click the Start button. Note that the running man icon will start running.
4006208B.qxd 6/28/2004 11:03 AM Page 12 b. Click the Zero Baseline button to zero the UV signal. The Status bar along the bottom of the screen provides AU output for the detector. Ensure that it goes to zero when you select the zero baseline option. QuadTec UV/Vis detector a. The QuadTec detector should be powered On before starting the BioLogic software. If the QuadTec detector is not powered up, exit the software, power up the QuadTec detector and restart the software.
4006208B.qxd 6/28/2004 11:03 AM Page 13 Section 2. Anion Exchange Separation of Protein Standards The starter kit enables you to learn to use the DuoFlow system by 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 (catalog #720-0001). Equine myoglobin is not retained on the UNO Q1 column and elutes in the void volume.
4006208B.qxd 6/28/2004 11:03 AM Page 14 2.2 Prepare Buffers During solution preparation, wear appropriate laboratory protective clothing including, eye protection, and gloves. Avoid skin and eye contact with starter kit solutions. In case solutions come in contact with eyes, rinse immediately with plenty of water and get medical advice. Buffer A a. Empty the contents of the bottle labeled buffer A into a 500 ml graduated cylinder and add filtered, high-quality water to a 500 ml volume. b.
4006208B.qxd 6/28/2004 11:03 AM Page 15 b. Add 1.0 ml of prepared buffer A to the vial. c. Replace the rubber stopper and gently invert the vial to solubilize the protein standards. 2.4 Install the UNO Q1 column Remove the end caps from the UNO Q1 column. Keeping tubing lengths to a minimum, connect 1/16" tubing from port 4 of the AVR7-3 inject valve to the column inlet. Connect the column outlet to the bottom of the UV flow cell or to the QuadTec flow cell. Secure the column in a vertical position.
4006208B.qxd 6/28/2004 11:03 AM Page 16 BioLogic Duo-Flow - - - - File Edit View Utilities Options Window Help New Edit 1 wash 2 load New Method Method Run Browser Report Manual Setup Protocol Run Notes PostRun Log Available Devices Settings Bio-Rad Web Delete Devices in setup BioFrac Fraction Collector, Rack: F1 (12-13 mm tubes) Aux Load Pump Fraction Collector UV Detector Signal Import Module 2 pH Range: 0.00 to 14.
4006208B.qxd 6/28/2004 11:03 AM Page 17 standard with the BioLogic DuoFlow system. The DuoFlow QuadTec system includes a QuadTec UV/Vis detector in place of a UV detector. a. Click on the Fraction Collector button in the Available Devices box. A dialog box will appear asking you to choose the type of collector; i.e., a generic collector, a Model 2110, or a BioFrac. Click on BioFrac and click the OK button. You will now see BioFrac fraction collector in the Devices in Setup box.
4006208B.qxd 6/28/2004 11:03 AM Page 18 BioLogic DuoFlow - - - - File Edit View Utilities Options Window Help New 1 wash 2 load New Edit Method Method Run Browser Report Manual Setup Protocol Run Notes PostRun Log Settings Edit Cut Copy Paste Delete Add Step V Isocratic Flow Load/Inject Sample Linear Gradient Change Valve Column Switching Repeat Steps 1 0.00 Collection Fractions of size 2.00 ml during entire run 2 0.
4006208B.qxd 6/28/2004 Step Number 11:03 AM Start (ml) Page 19 Step 1. 0.0 Collect fractions of size 2.00 ml during entire run 2. 0.0 Isocratic flow with 100% 25 mM Tris-HCI, pH 8.1, 0% 25 mM Tris-HCI, 0.5 M NaCl, pH 8.1 at 4.00 ml/min for 1.0 ml 3. 1.0 Zero Baseline to set UV baseline to 0.0. Select either UV detector or QuadTec detector. 4. 1.0 Load inject sample, static loop: Inject 0.5 ml sample at 4.00 ml/min. You will be injecting the loop size of 50 µl. 5. 1.
4006208B.qxd 6/28/2004 11:03 AM Page 20 zero the UV or QuadTec UV/Vis trace by clicking on the Zero baseline button in the appropriate box. This button may be selected at any time. c. To scale the on-screen chromatogram trace display axes, use the scroll bars located on the left and right axes of the chromatogram window. d. To enlarge the view select the Resize button to the right of the chromatogram display. Start the Run a.
4006208B.qxd 6/28/2004 11:03 AM Page 21 BioLogic Duo-Flow - Demo Chromatography - Standard UV Detector - Sample Run File Edit View Utilities Options Window Help New 1 2 New Edit wash load Method Method Run Browser Report Manual Setup Protocol Run Notes PostRun Log Frac. Collector Advance Divert Valve Bio-Rad Web Full View Settings UV Conductivity 0.100 1 2 3 100.0% Buffer B 4 5 Fractions 6 7 8 9 10 11 12 13 14 50.0 Collect Waste Grad. Pump High psi 700 Low psi 0 40.0 0.075 30.
4006208B.qxd 6/28/2004 11:03 AM Page 22 BioLogic Duo-Flow - Demo Chromatography - Standard UV Detector - Sample Run File Edit View Utilities Options Window Help New Edit 1 2 New wash load Method Method Run Browser Report Manual Setup Protocol Run Notes PostRun Log Frac. Collector Advance Divert Valve 0.100 Bio-Rad Web Full View Settings QuadTec (280 nm) Conductivity 1 2 3 100.0% Buffer B 4 5 Fractions 6 7 8 9 10 11 12 13 14 50.0 Collect Waste Grad.
4006208B.qxd 6/28/2004 11:03 AM Page 23 II. DuoFlow Maximizer and Pathfinder Systems Section 3. System Preparation When the DuoFlow Maximizer or Pathfinder system is turned on, the Manual screen is displayed in either Buffer Blending (Figure 7) or Non-Buffer Blending mode (Figure 8). This screen displays instrument control panels that provide direct control of the pumps, valves, fraction collector, UV detector, QuadTec UV/Vis detector, and Econo gradient pump.
4006208B.qxd 6/28/2004 11:03 AM BioLogic Duo-Flow - - - no method File View Utilities Options Window New 1 2 Browser Report Mode: System Mode: Local 1.00 Inlet Valves Inl A 50 % A1 A2 Inl B 50 % B1 B2 700 psi. 0 psi. System Local Mode: 1 End Tube: 20 Fraction size: System Local System 1.000 Local ml/min OFF Deuterium EGP %B 0 % Range 190 - 370 nm % Split 0.
4006208B.qxd 6/28/2004 11:03 AM Page 25 3.1 Prime the Workstation Pumps a. Immerse Maximizer inlet lines A1, A2, B1, and B2 in a container of HPLC grade (filtered, degassed) or other high-quality water. b. From the Manual screen place the Maximizer in Local mode and use the Valve Port Select button, under the A1/A2 Maximizer valve inlet, to select inlet port A1. c. Connect the syringe (supplied with the fittings kit) to the priming port of pump A. d.
4006208B.qxd 6/28/2004 11:03 AM Page 26 3.3 Purge the Workstation Pumps a. Make sure that the inject valve is in the Purge position. b. Select solutions A2 and B2 from the Valve Port Select buttons on the Maximizer faceplate. c. Press the Purge buttons A then B on the front of the workstation. The workstation pumps will run at 10 ml/min and the indicator light will flash green. d.
4006208B.qxd 6/28/2004 11:03 AM Page 27 BioFrac Fraction Collector The BioFrac fraction collector has two operating modes: • System—Controlled by the BioLogic DuoFlow system • Local—Controlled from its own faceplate in stand-alone mode Ensure that the System button is selected.
4006208B.qxd 6/28/2004 11:03 AM Page 28 3.7 pH Electrode Calibration Prior to starting a run the pH electrode should be calibrated as follows: a. Remove the electrode from the flow cell and rinse it with deionized water. b. Place the electrode in pH 7 standard buffer. c. From the Utilities drop-down menu, select “pH probe calibration”. d. Enter the temperature and reference pH (at the current temperature) for the first buffer. Press Set.
4006208B.qxd 6/28/2004 11:03 AM Page 29 3.9 Status Bar At the bottom of the Manual screen is a status bar that is continually updated with system parameters. Section 4. Anion Exchange Separation of Protein Standards The starter kit enables you to learn to use the DuoFlow Maximizer or Pathfinder systems by programming and running separation of a premixed anion exchange standard containing equine myoglobin, conalbumin, chicken ovalbumin, and soybean trypsin inhibitor, using a 1.
4006208B.qxd 6/28/2004 11:03 AM Page 30 Step 4 Prime the workstation pumps and inlet valves, and equilibrate the column Step 5 Write a method a. Program in the instrument Setup b. Program the method Protocol c. Load sample into 50 µl loop d. Select Run e. Select Start 4.2 Prepare Solutions During solution preparation, wear appropriate laboratory protective clothing, including eye protection and gloves. Avoid skin and eye contact with starter kit solutions.
4006208B.qxd c. 6/28/2004 11:03 AM Page 31 When degassing is complete, pour the buffer into a bottle and label “Solution A2, 50 mM Tris base”. Solution B1 Place 1 L of water into a 1 L side-arm flask and drop in a stirbar. Cap the side arm flask, place it on a stirplate and connect it to a vacuum source. Degas the solution for approximately 15 minutes with gentle stirring. Label solution as “Solution B1, water”. Solution B2 a.
4006208B.qxd 6/28/2004 11:03 AM Page 32 4.5 Prime the Pumps and Equilibrate the UNO Q1 Column Ensure the gradient pumps are stopped and the inject valve is in the purge position. Place the tubing from inlets A1, A2, B1 and B2 into solutions A1 (TrisHCl), A2 (Tris base), B1 (water), and B2 (NaCl), respectively. Re-prime and purge the pumps and inlets A1, A2, B1, and B2 as described in Section 3.1 of this manual. Set the inject valve to position L (Load). Set the flow rate to 2.0 ml/min.
4006208B.qxd 6/28/2004 11:03 AM Page 33 Two-Point Correction (best for gradient applications) a. In the manual screen, set the pH to 8.1, the salt composition to 0 %B and the flow rate to 2.0 ml/min. b. Take the column out of line, if it has been connected, and start the pump. c. When the pH has stabilized, read the pH from the status bar or, alternatively, collect the effluent and measure the pH using a high quality Tris compatible pH probe. d.
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4006208B.qxd 6/28/2004 11:04 AM Page 35 Program the Instrument Setup In the Setup screen, select the instruments and devices to be used for the Starter Kit method. The icons grouped on the left hand side of the screen (refer to Figure 3, Available Devices), show all the instruments and devices that can be connected to the BioLogic DuoFlow systems. The list of devices in the right box (Devices in Setup) identifies those devices selected for use with a specific method.
4006208B.qxd 6/28/2004 11:04 AM Page 36 BioLogic DuoFlow - - - - File Edit View Utilities Options Window Help New 1 2 New Edit Method Method Run Browser Report Add Step V Isocratic Flow Load/Inject Sample Linear Gradient Change Valve Column Switching Repeat Steps Volume wash load Settings Edit Cut Copy Paste Delete Manual Setup Protocol Run Notes PostRun Log Description Bio-Rad Web Parameters 1 0.00 Collection Fractions of size 2.
4006208B.qxd 6/28/2004 11:04 AM Page 37 Program the Method Protocol a. From the Options pull-down menu, ensure that Use Volume (ml) is selected, so that the programming base is Volume. b. Program the separation method listed below and in Figure 10. • From the left side of the screen, press the fraction collection icon. In the pop-up window that appears, choose Collect All with a fraction size of 2.00 ml and a delay of 0.0. Make sure the correct rack type is displayed.
4006208B.qxd 6/28/2004 11:04 AM Page 38 The Run Screen a. The toolbar buttons on the left side of the screen enable you to check that the screen display ranges for UV, QuadTec UV/Vis, pH, and conductivity are correctly set (see page 24) and that the workstation pump pressure limits are appropriate (700 psi high and 20 psi low limit) for the UNO Q1 column. b.
4006208B.qxd 6/28/2004 11:04 AM Page 39 BioLogic Duo-Flow - Demo Chromatography with Buffe... - Standard UV Detector with Buffer Blending - Sample Run File Edit View Utilities Options Window Help New 1 2 New Edit wash load Method Method Run Browser Report Manual Setup Protocol Run Notes PostRun Log Frac. Collector Advance Divert Valve Bio-Rad Web Full View Settings UV Conductivity 0.100 1 2 3 100.0% Buffer B 4 5 Fractions 6 7 8 9 10 11 12 13 14 100.0 Collect Waste Grad.
4006208B.qxd 6/28/2004 11:04 AM Page 40 BioLogic Duo-Flow - Demo Chromatography with Buffe... - Standard UV Detector with Buffer Blending - Sample Run File Edit View Utilities Options Window Help New 1 2 New Edit wash load Method Method Run Browser Report Manual Setup Protocol Run Notes PostRun Log Frac. Collector Advance Conductivity 0.100 Divert Valve Bio-Rad Web Full View Settings UV 1 2 3 100.0% Buffer B 4 5 Fractions 6 7 8 9 10 11 12 13 14 100.0 Collect Waste 75.0 0.
4006208B.qxd 6/28/2004 11:04 AM Page 41 Section 5.
06208B.qxd 6/28/2004 11:03 AM Page 1 For technical service, call your local Bio-Rad office, or in the U.S., call 1-800-4BIORAD (1-800-424-6723) Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr., Hercules, CA 94547 USA 510-741-1000 1-800-424-6723 4006208 Rev.
