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Section 2
Anion Exchange Separation of Protein
Standards

The starter kit allows you to learn how to use the BioLogic Duo-Flow

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

*

.

Equine myoglobin is not retained on the UNO Q1 Column and elutes in
the void volume. Conalbumin, chicken ovalbumin and soybean trypsin
inhibitor bind to the column and require increased salt concentrations for
elution. Separation requires approximately 6 minutes.

2.1 Overview of the Procedure

Run Conditions

•

Buffer A = 25 mM Tris-HCl, pH 8.1

•

Buffer B = 25 mM Tris-HCl, pH 8.1, 0.5M NaCl

•

Flow rate

4.00 ml/min

•

Sample volume

50 µl

•

UV detection

0.1 AUFS

•

Conductivity

100 mS/cm

General Procedure

Step 1

Buffer preparation

Step 2

Sample preparation

Step 3

Installation of the UNO Q1 column

Step 4

Priming the gradient pumps and equilibrating the column

Step 5

Writing a method

a) Programming the Setup Editor

b) Programming the Protocol Editor

c) The Run Control Screen

d) Starting a Run

* UNO Q1 Column: catalog number 720-0001

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Содержание 750-0135

Страница 1: ...em Starter Kit Instructions for the First Time User of the BioLogic Duo Flow and BioLogic HR Systems Catalog Number 750 0135 For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723 A B ...

Страница 2: ...umn 8 2 5 Priming the Gradient Pumps and Equilibrating the UNO Q1 Column 9 2 6 Creating a Method 9 Part 2 BioLogic HR Chromatography System Section 3 Preparation for Use of the System 16 Section 4 Anion Exchange Separation of Protein Standards 22 4 1 Overview of the Procedure 22 4 2 Buffer Preparation 23 4 3 Sample Preparation 23 4 4 Installation of the UNO Q1 Column 23 4 5 Priming the Gradient Pu...

Страница 3: ...3 1 Starter Kit Components This starter kit contains the following items for running a separation 50 ml of buffer A 250 mM Tris buffer pH 8 1 10x concentrate 50 ml of buffer B 250 mM Tris buffer pH 8 1 plus 5 0 M NaCl 10x concentrate One vial of anion exchange protein standards catalog number 125 0561 One 1 ml disposable sample injection syringe One 50 µl sample loop One UNO Q1 column catalog numb...

Страница 4: ...red high quality water i e HPLC grade water One 500 ml graduated cylinder One 1 L sidearm flask Stir bar and stir plate Vacuum source for degassing Two 500 ml bottles Fraction collection tubes 13 x 100 mm at least 14 tubes 100 ml beaker 2 ...

Страница 5: ... the seal Gently withdraw the syringe plunger to draw water into the pump head d Repeat this operation several times until no air bubbles are visible in the inlet tubing e Tighten the priming port by turning it clockwise f Repeat this priming procedure for pump head B 2 Moving the Inject Valve to Purge Position To change the status of an automated inject valve AVR7 3 go to the Manual Screen displa...

Страница 6: ... parameters while the pump is running enter the new value and then click on the set button BioLogic Duo Flow Default no method File View Utilities Options Edit Method New Method New Run Browser Manual Setup Protocol wash load 1 2 Run Notes Advance Gradient Pump F 10 UV 0 00 ml min 0 B 438 psi 0 00181 AU Fraction Collector START Gradient pump F 10 Inlet A 1 00 100 0 Inlet B Flow rate ml min High li...

Страница 7: ...pear in the Manual Screen as shown in Figure 2 Fig 2 Manual Control Screen showing Model 2128 Fraction Collector BioLogic Duo Flow Default no method File View Utilities Options Edit Method New Method New Run Browser Manual Setup Protocol wash load 1 2 Run Notes Advance Gradient Pump F 10 UV 0 00 ml min 0 B 438 psi 0 00181 AU Fraction Collector START Gradient pump F 10 Inlet A 1 00 100 0 Inlet B Fl...

Страница 8: ...nual Screen see Figure 2 You should now check that the lamp is on the mercury lamp requires approximately 30 minutes to warm up b Clicking the Zero Baseline button will zero the UV signal 7 The Manual Control Screen Chromatogram Window A feature of the Manual Screen is the ability to display a chromatogram window showing UV and conductivity values over a 10 minute interval This is useful during co...

Страница 9: ...or bind to the column and require increased salt concentrations for elution Separation requires approximately 6 minutes 2 1 Overview of the Procedure Run Conditions Buffer A 25 mM Tris HCl pH 8 1 Buffer B 25 mM Tris HCl pH 8 1 0 5M NaCl Flow rate 4 00 ml min Sample volume 50 µl UV detection 0 1 AUFS Conductivity 100 mS cm General Procedure Step 1 Buffer preparation Step 2 Sample preparation Step 3...

Страница 10: ...iately Buffer B Prepare buffer B by following the same procedure for preparation of buffer A 2 3 Sample Preparation 1 Remove the aluminum cap from the anion exchange standards vial Slowly remove the rubber plug from the standards vial the contents may be under vacuum 2 Add 1 0 ml of prepared buffer A to the vial 3 Replace the rubber stopper and gently invert the vial to solubilize the protein stan...

Страница 11: ...tton You will see a dialog box asking for a method name input Starter Kit Separation Auto Use the keyboard tab key to go to the Method Description and Method Author fields Make an entry in these fields if desired and click the OK button You will now see the Setup Editor Programming the Setup Editor This information entered in the Setup Editor is important for the subsequent separation method The b...

Страница 12: ...ump Use the tab key or click and drag on the field buffer A to highlight it and type in 25 mM Tris pH 8 1 Use the tab key to highlight buffer B and type 25 mM Tris 0 5 M NaCl pH 8 1 This operation is called aliasing the gradient pump inlets BioLogic Duo Flow Jennifer System training Starter Kit 2128 Fraction Collector Edit View Utilities Options Window Help Devices in setup UV Detector Conductivit...

Страница 13: ...er Repeat Steps Fraction Collection Edit View Utilities Options Edit Method New Method New Run Manual Setup Protocol wash load 1 2 Run Notes Report PostRun Log Window Help 1 2 3 6 5 0 0 0 0 0 0 1 5 Collect Fractions of size 2 00 ml during entire run Chart Recorder 1 0 Turn ON 7 Isocratic Flow 4 1 0 Set Zero Baseline Load Inject Sample UV Detector 2 3 Linear Gradient 8 9 15 3 18 1 Isocratic Flow Is...

Страница 14: ...for 0 8 ml 6 2 3 Linear gradient with 0 to 50 25 mM tris 0 5 M NaCl pH 8 1 at 4 00 ml min for 13 0 ml 7 15 3 Isocratic flow with 0 25 mM tris pH 8 1 100 25 mM tris 0 5 M NaCl pH 8 1 at 4 00 ml min for 2 8 ml 8 18 1 Isocratic flow with 100 25 mM tris pH 8 1 0 25 mM tris 0 5 M NaCl pH 8 1 at 4 00 ml min for 8 0 ml 9 26 1 End of protocol 3 When you have finished programming the Protocol Editor click ...

Страница 15: ...V and conductivity If necessary you may wish to zero the UV trace by clicking on the Zero baseline button This button may be clicked at any time 3 To scale the on screen chromatogram display axes use the scroll bars located on the left and right axes of the chromatogram window Starting the Run 1 Insure that sufficient tubes are in the fraction collector rack approximately 14 The drophead will auto...

Страница 16: ...ed message appears in the bottom right of the status bar 7 Figure 5 shows a typical run screen and chromatogram for this separation Advance Frac Collector Divert Valve 0 090 0 100 50 0 100 Buffer B Run Time Run Volume 0 0 ml 0 24 9 0 00 0 0 00ml min 0 B Gradient Pump F 10 UV Conductivity UV Detector Set UV Range Zero Baseline Event mark Cond Range 500 Protocol 1 0 00 0 Isocratic Flow with 100 Buff...

Страница 17: ... HR Gradient Pumps a Immerse the gradient 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 head A c Turn the priming port counter clockwise to open the seal Gently withdraw the syringe plunger to draw water into the pump head d Repeat this operation several times unti...

Страница 18: ...in per pumphead although this may be changed using the options manual setup pull down menu b Run both pumps for 2 minutes and then press the Purge buttons again to stop the pump Fig 6 Manual control screen for a Manual BioLogic HR System BioLogicHR Dr J Smith Starter Kit UNO Q1 File View Utilities Options Edit Method New Method New Run Browser Manual Setup Protocol wash load 1 2 Run Notes Advance ...

Страница 19: ...ps stopped move the inject valve back to position L Load by either moving the lever to the right V7 3 or clicking radio button L AV7 3 on the Manual Screen Using the Manual Screen set the pump flow rate at 1 0 ml min and start the pump Water will flow through the UV and conductivity flow cells and to the fraction collector as described below a Using the Model 2110 Fraction Collector and SV3 2 valv...

Страница 20: ...ector box on the manual screen will show fields for Rack type Start tube End tube Fraction size ml Tube number Volume left a toggle button for Start and Stop and a button for Advance see Figure 7 Fig 7 Manual Control Screen for the Automated BioLogic System BioLogicHR Default no method File View Utilities Options Edit Method New Method New Run Browser Manual Setup Protocol wash load 1 2 Run Notes ...

Страница 21: ...ivity monitor range is set by clicking on a value from 0 10 to 500 0 mS cm from the drop down menu Note that this range of settings applies only to the output to a chart recorder On screen displays of the conductivity range are controlled by the scroll bars and have no effect on the output to a chart recorder For now set the scale to 100 mS cm 8 The Manual Control Screen Chromatogram Window A feat...

Страница 22: ...top start functions are controlled by the ON OFF toggle switch which is displayed in the Manual screen Chart speed is set at the chart recorder Clicking the Event Mark button will send an event mark to the recorder 10 The Status Bar At the bottom of the Manual Screen is a status bar which is continually updated with system parameters 21 ...

Страница 23: ...utes 4 1 Overview of the Procedure Run Conditions Buffer A 25 mM Tris HCl pH 8 1 Buffer B 25 mM Tris HCl pH 8 1 0 5M NaCl Flow rate 4 00 ml min Sample volume 50 µl UV detection 0 1 AUFS Conductivity 100 mS cm Chart speed 60 cm hr General Procedure Step 1 Buffer Preparation Step 2 Sample Preparation Step 3 Installation of the UNO Q1 Column Step 4 Priming the gradient pumps and equilibrating the col...

Страница 24: ...ately Buffer B Prepare buffer B by following the same procedure for preparation of buffer A 4 3 Sample Preparation 1 Remove the aluminum cap from the anion exchange standards vial Slowly remove the rubber plug from the standards vial the contents may be under vacuum 2 Add 1 0 ml of prepared buffer A to the vial 3 Replace the rubber stopper and gently invert the vial to solubilize the protein stand...

Страница 25: ...ion 2 Equilibrate the column with13 ml 10 column volumes of 100 buffer A The conductivity monitor on the status bar should now read approximately 3 mS cm 4 6 Creating a Method for the Manual BioLogic HR System From the Manual Control Screen click on the New Method toolbar button In the pop up window which appears input the method name Starter Kit Separation Manual Use the keyboard s tab key to ste...

Страница 26: ...dure is for a Manual BioLogic HR System Refer to Section 4 7 for the Automated BioLogic HR System setup procedure Fig 8 Setup Editor for a Manual BioLogic HR System BioLogicHR Jennifer System training Starter Kit 2110 Fraction Collector Edit View Utilities Options Window Help Devices in setup UV Detector Conductivity Monitor SV3 2 Valve Fraction Collector Diverter Port 1 Available Devices Gradient...

Страница 27: ...1 and Collect for position 2 Note that the functioning of this particular valve is now automatically tied to the programmed operation of the fraction collector during a Method but does not affect the manual screen operation 3 Next go to the box labeled Gradient Pump Use the tab key or click and drag on the field buffer A to highlight it and type in 25 mM Tris pH 8 1 Use the tab key to highlight bu...

Страница 28: ... Help 1 2 3 6 5 0 0 0 0 0 0 1 5 Collect Fractions of size 2 00 ml during entire run Chart Recorder 1 0 Turn ON 7 Isocratic Flow 4 1 0 Set Zero Baseline Load Inject Sample UV Detector 2 3 Linear Gradient 8 9 15 3 18 1 Isocratic Flow Isocratic Flow Isocratic Flow 26 1 100 100 0 0 Load Sample Static Loop 25mM tris 25mM tris 0 5M NaCl Manual Inject Valve Flow 4 00 ml min Volume 2 8 ml Flow 4 00 ml min...

Страница 29: ...1 at 4 00 ml min for 0 8 ml 7 2 3 Linear gradient with 0 to 50 25 mM tris 0 5 M NaCl pH 8 1 at 4 00 ml min for 13 0 ml 8 15 3 Isocratic flow with 0 25 mM tris pH 8 1 100 25 mM tris 0 5 M NaCl pH 8 1 at 4 00 ml min for 2 8 ml 9 18 1 Isocratic flow with 100 25 mM tris pH 8 1 0 25 mM tris 0 5 M NaCl pH 8 1 at 4 00 ml min for 8 0 ml 10 26 1 End of protocol 3 When you have finished with the protocol ed...

Страница 30: ...completed run on a Manual BioLogic HR System Advance Frac Collector Divert Valve 0 090 0 100 50 0 100 Buffer B Run Time Run Volume 0 0 ml 0 24 9 0 00 0 0 00ml min 0 B Gradient Pump UV Conductivity UV Detector Set UV Range Zero Baseline Event mark Cond Range 500 Protocol 1 0 00 0 Isocratic Flow with 100 Buffer B at 1 00 ml min for 25 0 min 00 00 00 00 02 00 00 04 00 00 06 00 StepTime Left Valve Inf...

Страница 31: ...ft The sample will be loaded onto the column and the method will be advanced You may choose to leave the valve in the inject position for the duration of the run or return it to load position after all the sample has been loaded 5 To Hold Pause or Abort a run click on the appropriate toolbar button Clicking the Hold toolbar button will hold the gradient pumps at the current B value and will not ad...

Страница 32: ...evices in Setup identifies those devices you wish to use in the subsequent method The initial default Devices in Setup are a UV detector and a conductivity monitor as these come as standard with the BioLogic HR System The default devices in Setup can be changed by choosing Save setup as default from the Data pull down menu 1 Click on the fraction collector button in the available devices box A dia...

Страница 33: ...training Starter Kit 2128 Fraction Collector Edit View Utilities Options Window Help Devices in setup UV Detector Conductivity Monitor AV7 3 Valve Sample Inject Port 4 Available Devices Gradient Pump SIM1 pH SV3 2 Valve AV7 3 Valve SV5 4 Valve AV9 8 Valve 1 SIM1 Signal 1 SIM2 pH SIM2 Signal 2 2 UV Detector Conductivity Monitor Fraction Collector SV3 INJECT P U M P Aux Load Pump Delete Edit Method ...

Страница 34: ...s Edit Method New Method New Run Manual Setup Protocol wash load 1 2 Run Notes Report PostRun Log Window Help 1 2 3 6 5 0 0 0 0 0 0 1 5 Collect Fractions of size 2 00 ml during entire run Chart Recorder 1 0 Turn ON 7 Isocratic Flow 4 1 0 Set Zero Baseline Load Inject Sample UV Detector 2 3 Linear Gradient 8 9 15 3 18 1 Isocratic Flow Isocratic Flow Isocratic Flow 26 1 100 100 0 0 Load Sample Stati...

Страница 35: ... 4 00 ml min for 0 8 ml 7 2 3 Linear gradient with 0 to 50 25 mM tris 0 5 M NaCl pH 8 1 at 4 00 ml min for 13 0 ml 8 15 3 Isocratic flow with 0 25 mM tris pH 8 1 100 25 mM tris 0 5 M NaCl pH 8 1 at 4 00 ml min for 2 8 ml 9 18 1 Isocratic flow with 100 25 mM tris pH 8 1 0 25 mM tris 0 5 M NaCl pH 8 1 at 4 00 ml min for 8 0 ml 10 26 1 End of protocol 3 When you have finished programming the Protocol...

Страница 36: ...and conductivity If necessary you may wish to zero the UV trace by clicking on the Zero baseline button This button may be clicked at any time 3 To scale the on screen chromatogram display axes use the scroll bars located on the left and right axes of the chromatogram window Starting the Run 1 Insure that sufficient tubes are in the fraction collector rack approximately 14 The drophead will automa...

Страница 37: ... automatically stop and a run finished message appears in the bottom right of the status bar 7 Figure 13 shows a typical run screen and chromatogram for this separation Advance Frac Collector Divert Valve 0 090 0 100 50 0 100 Buffer B Run Time Run Volume 0 0 ml 0 24 9 0 00 0 0 00ml min 0 B Gradient Pump UV Conductivity UV Detector Set UV Range Zero Baseline Event mark Cond Range 500 Protocol 1 0 0...

Страница 38: ...and 358 0 9 804 2200 France 01 43 90 46 90 Germany 089 318 84 0 Hong Kong 852 2789 3300 India 91 11 461 0103 Israel 03 951 4127 Italy 02 21609 1 Japan 03 5811 6270 Korea 82 2 3473 4460 The Netherlands 31 318 540666 New Zealand 64 9 4152280 Singapore 65 2729877 Spain 91 661 70 85 Sweden 46 0 8 627 50 00 Switzerland 01 809 55 55 United Kingdom 0800 181134 000097 Sig 120197 Bio Rad Laboratories 40061...

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