BIO RAD BioLogic Duo-Flow Manual

BioLogic
Chromatography System
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)
AB
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Table of Contents
Introduction .......................................................................1
1. Starter Kit Components ................................................................1
2. Other Materials You Will Need ....................................................2
Part 1 BioLogic Duo-Flow Chromatography System
Section 1 Preparation for Use of the System ...................................3
Section 2 Anion Exchange Separation of Protein Standards ........7
2.1 Overview of the Procedure ...........................................................7
2.2 Buffer Preparation ........................................................................8
2.3 Sample Preparation.......................................................................8
2.4 Installation of the UNO™ Q1 column..........................................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 Pumps and
Equilibrating the UNO Q1 Column............................................24
4.6 Creating a Method for the Manual
BioLogic HR System..................................................................24
4.7 Creating a Method for the Automated
BioLogic HR system ..................................................................31
4006147a.qxd 12/22/2000 1:29 PM Page i

Introduction
This instruction manual and starter kit contents may be used for both
the BioLogic Duo-Flow and BioLogic HR Chromatography Systems.
To use the starter kit with the BioLogic Duo-Flow System, refer to
Section 1. To use the starter kit with the BioLogic HR System, refer to
Section 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 number 720-0001)
The chromatographic separation for this kit requires approximately
6 minutes.
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2. Other Materials You Will Need
• Filtered high quality water (i.e.,HPLCgradewater)
• 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
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Section 1
Preparation for Use of the
Duo-Flow System
When the BioLogic Duo-Flow System is turned on, the Manual Control
Screen is displayed. (see Figure 1) This screen consists of a series of
boxes containing instrument faceplates which provide direct control of
the gradient pumps, a fraction collector, the UV and conductivity detector
range settings and valves.
1. Priming the BioLogic Duo-Flow 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 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 displayed on the controller monitor.
If you plugged an inject valve into port 4, you will see a valve box
designated AVR7-3 at port 4. The three radio buttons of this box
correspond to valve positions as follows: L= Load position, I= Inject
position, P= Purge position.
To move the AVR7-3 valve to Purge position, click button P.
Note: The default position at power up and at the end of a programmed
method for the AVR7-3 is L. For all other automated valves the default
is position 1.
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3. Purging the BioLogic Duo-Flow Pumps.
a. Make sure that the inject valve is in the Purge position.
b. Press the Purge buttons A and B on the front of the workstation.
The gradient pumps will run and the indicator lights will flash
green.
c. Run both pumps for 2 minutes and then press the purge buttons
again to stop the pump.
Fig. 1. Manual Control Screen.
4. Control of the BioLogic Duo-Flow Pumps.
The gradient 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 spin arrows. You can set the flow rate between 0.1 to
10 ml/min and the gradient composition between 0% and 100% B.
To start the pump, click the Start button. Note the running man icon.
To change the pump 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
limit:
0
1000
Low
limit: psi
AVR7-3 at port 4SVT3-2 at port 1 SV5-4 at port 2
PL21
21
34
STOP START STOP
Set
psi
Tube number:
ml
Volume left:
Report PostRun Log ON
Window Help
Mode: System Local
Start Tube:
End Tube:
Fraction size:
1
1
128
1.00
Rack: Rack 1 (128 tubes)
I
AVR9-8 at port 5
37
82
654
I
Conductivity
47.5 mS/cm 0.089 Volt 7.00 pH
SIM2/pHSIM1/SIG
Chart Recorder
Signal Import Module
SIM 1 and SIM 2 connected
UV Detector
Conductivity
range(mS/cm): 500
UV range (AU): 0.1
ON OFF
Event mark
ON OFF
Zero Baseline
Flowrate of the gradient pump
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Pressure limits can be adjusted to match the pressure limits of a
column. If the pressure limit is exceeded, the pump will stop and an
alarm will sound. For now, set the high limit to 700 psi and the low limit
to 20 psi.
5. Flushing the System through to the fraction collector.
With the gradient pumps stopped, move the inject valve back to
position L (Load) by clicking radio button L (AVR7-3) 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.
Using a Model 2128 Fraction Collector
Connect the Model 2128 Fraction Collector by inserting the gray bus
cable between it and the workstation. When it is connected, it will
appear 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:
Flow rate: ml/min
%
%
High
limit:
0
1000
Low
limit: psi
AVR7-3 at port 4SVT3-2 at port 1 SV5-4 at port 2
PL21
21
34
STOP START STOP
Set
psi
Tube number:
ml
Volume left:
Report PostRun Log ON
Window Help
Mode: System Local
Start Tube:
End Tube:
Fraction size:
1
1
128
1.00
Rack: Rack 1 (128 tubes)
I
AVR9-8 at port 5
37
82
654
I
Conductivity
47.5 mS/cm 0.089 Volt 7.00 pH
SIM2/pHSIM1/SIG
Chart Recorder
Signal Import Module
SIM 1 and SIM 2 connected
UV Detector
Conductivity
range(mS/cm): 500
UV range (AU): 0.1
ON OFF
Event mark
ON OFF
Zero Baseline
Flowrate of the gradient pump
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The Model 2128 Fraction Collector has two operating modes.
• System: Controlled by the BioLogic Duo-Flow System.
• Local: Controlled from its own faceplate.
For now, insure that the system button is selected.
When in System mode, the fraction collector operates as follows.
• Control of the Model 2128 is identical whether or not the optional
on-arm diverter valve is used. Without a valve, the drop-head returns
to the waste trough during divert operations.
• The fraction collector 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 2).
6. Turning on the UV lamp.
a. The UV lamp automatically turns on when you turn on power to
the workstation. (The UV lamp is turned off and on by clicking
the On and Off buttons in the Manual 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 column equilibration.
• To access this feature, simply click the Chromatogram Window On
button, at the far right of the toolbar (See Figure 2). The chromatogram
window will be displayed at the bottom of the screen, over the valve
position indicators. The time axis is reset automatically at the end of
10 minutes or reset manually by clicking the Clear Traces button.
Use the scroll bars to change the scaling of the axes.
• To close the Chromatogram Window, again click the toolbar button.
The valve status indicators will reappear.
8. The Status Bar.
At the bottom of the Manual Screen is a status bar which is continually
updated with system parameters.
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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
• BufferA=25mMTris-HCl, pH 8.1
• BufferB=25mMTris-HCl,pH8.1,0.5MNaCl
• 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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2.2 Buffer Preparation
Buffer A
1. Empty the contents of the bottle labeled buffer A into a 500 ml
graduated cylinder and add filtered, high-quality water to 500 ml
volume.
2. Place the contents of the graduated cylinder intoa1Lside-arm flask
and drop in a stir bar. Cap the side arm flask, place it on a stir plate and
connect it to a vacuum source. Degas the buffer for approximately
15 minutes with gentle stirring.
3 When degassing is complete, pour the buffer into a bottle and label
appropriately.
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 standards.
2.4 Installation of the UNO Q1 column
Remove end caps from the UNO Q1 column. Keeping tubing lengths
to a minimum, connect 1/16" tubing from port 4 of the inject valve to the
column inlet. Connect the column outlet to the bottom of the UV flow
cell. Secure the column in a vertical position.
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2.5 Priming the Gradient Pumps and Equilibrating
the UNO Q1 Column
Be sure the gradient pumps are stopped and the inject valve is in
the purge position. Re-prime and purge pumps A and B as described in
Section 2, step 1 of this manual.
Set the inject valve to position L (Load). Set the flow rate to
2.0 ml/min. Set the UV range to 0.1 AUFS and the conductivity range to
100 mS/cm.
1. Wash the column with 6.5 ml (5 column volumes) of buffer B at
2 ml/min to remove the column storage solution.
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.
2.6 Creating a Method
From the Manual Control screen, click the New Method toolbar
button. 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 buttons grouped in the left hand box
(Available Devices) shows all the devices that could theoretically be
connected to the BioLogic Duo-Flow System.
The list of items in the right hand box (Devices 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 Duo-Flow System. The default devices
in Setup can be changed by choosing Save setup as default from the
Data pull-down menu.
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1. 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 Model 2128. Click on
Model 2128 and click the OK button.
You will see 2128 Fraction Collector in the Devices in Setup box.
2. Next, click on the AVR7-3 Valve button in the Available Devices box.
You will see a dialog box with a pull-down menu under Valve
Name/Function. You should see Sample Inject already selected.
Other functions are available from the pull-down menu. This action
is called valve function aliasing. You will see that the valve position
names are Load for position 1, Inject for position 2, and Purge for
position 3.
Fig. 3. Setup editor.
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 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
Conductivity Monitor
SVT3-2 Valve - Fraction Collector Diverter Port 1
Available Devices
Gradient Pump: F-10
SIM1
pH
SVT3-2
Valve
AVR7-3
Valve
SV5-4
Valve
AVR9-8
Valve
1
SIM1
Signal
1
SIM2
pH
SIM2
Signal
22
UV
Detector
Conductivity
Monitor
Fraction
Collector
SV3
INJECT
P
U
M
P
Aux Load
Pump
Delete
Edit
Method
New
Method
New
Run Manual Setup Protocol
wash
load
1
2
Run Notes
Report PostRun Log
Gradient Pump: F-10 UV
Inlet A:
Inlet B:
0.00ml/min 0 %B 438 psi -0.00284 AU
Conductivity
59.0 mS/cm
SIM1/pHSIM1/SIG
File
Browser
0.004 Volt 7.00 pH
25 mM Tris
25 mM Tris + 0.5 M NaCl
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4. The Setup Editor is now complete and you are now ready to program
the separation steps. To see the protocol editor, click on the Protocol
Editor toolbar menu button.
Programming the Protocol Editor
1. From the Options pull-down menu, insure that Use Volume (ml) is
selected, so that the programming base is Volume.
Fig. 4. Protocol editor.
2. Program the separation method listed below and in Figure 4.
• From the left side of the screen, press the Fraction collection
button. In the pop-up window that appears, choose Collect All
with a fraction size of 2.00 ml and a delay of 0. Make sure that you
choose the correct rack number for the rack you will be using
with the Model 2128.
• Program the remaining steps using the add step icons from the
left-hand side of the screen.
Cut Copy Paste Delete
Isocratic
Flow
Add Step
Linear
Gradient
Load/Inject
Sample
Change
Valve
Hold
Alarm
Zero
Baseline
UV Lamp
Chart
Recorder
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
41.0 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
100%
0%
Volumn Description Parameters
100%
0%
Volume: 0.8 ml
Flow: 4.00 ml/min
Volume: 8.0 ml
Flow: 4.00 ml/min
25mM tris
25mM tris, 0.5M NaCl,
100% --> 50%
0% --> 50%
25mM tris
25mM tris, 0.5M NaCl,
25mM tris
25mM tris, 0.5M NaCl,
25mM tris
25mM tris, 0.5M NaCl,
Volume: 1.0 ml
Volume: 0.5 ml
Flow: 4.00 ml/min
Volume: 13.0 ml
Flow: 4.00 ml/min
End of Protocol
Gradient Pump: F-10 UV
0.00ml/min 0 %B 1 psi 0.00375 AU
Conductivity
3.59 mS/cm
SIM1/pHSIM1/SIG
-0.003 Volt
File
Browser
Enter volume of sample to load
BioLogic Duo-Flow - Dr. J. Smith - Starter Kit - UNO Q1 -
11
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Step Start
Number (ml) Step
1. 0.0 Collect fractions of size 2.00 ml during entire run
2. 0.0 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
1.0 ml
3. 1.0 Set UV baseline to 0.0
4. 1.0 Static loop: Inject 0.5 ml sample at 4.00 ml/min for
0.1 min
5. 1.5 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 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 on the
toolbar button RUN. You will see a dialog box asking you to name the
run. For now, type in Run 1 and click the OK button. You will now see
the Run Control Screen (see Figure 5).
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The Run Control Screen
1. Use the toolbar buttons at the left-hand side of the screen to check
that the screen display ranges for UV (0.1 AUFS) and conductivity
(100 mS/cm) are correctly set and that the gradient pump pressure
limits are appropriate (set to 700 psi High and 20 psi Low).
2. If you were equilibrating the column while writing the method, you
will notice that the Status Bar is displaying the flow-rate and values
for UV 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 automatically move to tube 1
when the Run is started.
2. Insure that the AVR7-3 valve is in the LOAD position (position L). If
it is not, return to the Manual Screen by clicking the toolbar Manual
button and click on valve position L.
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Fig. 5. Run control screen.
3. Insure that the 50 µl sample loop is connected to ports 3 and 6 of the
inject valve. Completely fill the loop with sample protein standard
via port 5 and a syringe and needle. Do not remove the syringe from
the injection port after filling the loop or the sample will siphon to
waste.
4. To launch the Run, click on the green Start toolbar button. The sample
will be loaded automatically.
5. Clicking the Hold toolbar button will hold the gradient pumps at the
current %B value and will not advance the programmed method until
the Continue toolbar button is pressed. Clicking the Pause toolbar
button will stop the pumps completely. Clicking the Continue toolbar
button will re-start the pumps at exactly the point where the program
was paused.
6. When this run is finished, the pumps automatically stop and a run
finished 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% 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 Info
0 psi 0.00003 AU -0.014 mS/cm
Hr:Min:Sec
AU mS/cm
Collect
Waste
View Utilities Options
Edit
Method
New
Method
New
Run Manual Setup Protocol
wash
load
1
2
Run Notes FullView
Report PostRun Log
Help
Grad. Pump
Chart Recorder
1000
High psi
0
Low psi
0.01
0.080
0.070
0.060
0.050
0.040
0.030
0.020
0.010
0.000
-0.010
50.0
0.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
-5.0
Fractions
1 23 456 78 910111213
Fraction Vol. Left
SIM1/pHSIM1/SIG
File
Browser
ConductivityUV
Window
BioLogic Duo-Flow - Carole Smith - Uno Q1 data - Uno Q1 - Standards
14
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Section 3
Preparation for Use of the
BioLogic HR System
The procedures in this document are written for two configurations,
which are distinguished by the following.
• Manual: Uses a V7-3 Manual Inject Valve, a Model 2110 Fraction
Collector, and an SV3-2 Diverter Valve.
• Automated: Uses an AV7-3 Automatic Inject Valve and a Model
2128 Fraction Collector.
When you turn on the BioLogic HR System, the Manual Control
Screen is displayed (see Figure 6). This screen consists of a series of
boxes containing instrument faceplates which provide direct control of
the gradient pumps, a fraction collector, a chart recorder (apart from chart
speed, which is set on the recorder itself), the UV and conductivity detector
range settings and solenoid and automated valves.
1. Priming the BioLogic 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 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.
• For a manual inject valve V7-3, move the arm to the Purge position
(left, then up, then left). The figure in the valve position window
displays P.
Flow from the gradient pumps will now go directly to waste,
bypassing any column or detector downstream of the valve.
• To change the status of an automated inject valve AV7-3, go to the
Manual Screen displayed on the controller monitor.
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If you plugged an inject valve into port 4, you will see a valve
box designated AV7-3 at port 4. The three radio buttons of this
box correspond to valve positions as follows: L= Load position,
I= Inject position, P= Purge position.
To move the AV7-3 valve to Purge position, click button P.
Note: The default position at power up and at the end of a programmed
method for the AV7-3 is L and for all other automated valves is
position 1.
3. Purging the BioLogic HR Pumps.
With the inject valve in position P(Purge):
a. Press the Purge buttons A and B on the front of the Workstation.
The gradient pumps will run and the indicator lights will flash
green. The default purge flow rate is 7 ml/min 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
Gradient Pump UV
0.00 ml/min 0 %B 1 psi 0.00889 AU
START
Inlet A:
4.00
100
0
Inlet B:
Flow rate: ml/min
%
%
High
limit:
0
1000
Low
limit: psi
SV3-2 at port 1
21
STOP START STOP
Set
psi
Tube number:
ml
Volume left:
Report PostRun Log ON
Window Help
Fraction size: 1.00
Conductivity
3,57 mS/cm
SIM2/pHSIM1/SIG
Chart Recorder
UV Detector
Conductivity
range(mS/cm): 100
UV range (AU): 2.0
ON OFF
Event mark
ON OFF
Zero Baseline
Flowrate of the gradient pump
Fraction Collector
Gradient pump
17
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4. Control of the BioLogic HR Pumps.
The gradient 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 spin arrows. You can alter the flow rate from
0.1 to 10 ml/min for any given proportion of pumps A and B. Note that
the total flow rate can not exceed 10 ml/min (i.e. with a set flow-rate
of 10 ml/min at 50% B, both pump heads A and B are delivering at
5 ml/min each).
To start the pump, click the Start button. Note the running man icon.
To change the pump parameters while the pump is running, enter the
new value and then click on the Set button.
The maximum pressure capability of the BioLogic HR gradient pumps
is 1000 psi. Pressure limits can be adjusted to match the pressure
limits of a column. If the pressure limit is exceeded, the pump will stop
and an alarm will sound. For now, set the high limit to 700 psi and the
low limit to 20 psi.
5. Flushing the System through to the fraction collector.
With the gradient pumps 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 valve as
a diverter valve.
If you plugged the SV3-2 valve into Solenoid valve port 1, you will
see a valve box on the Manual Screen designated SV3-2 at port 1.
(see Figure 2) The two radio buttons of this box correspond to
valve positions; 1 = Waste, 2 = Collect. Select position 1 (waste)
to divert the flow to waste. The fraction collector box on the
manual screen (refer to Figure 2) will show fields for fraction
size–ml,Tube number,Volume left, a toggle button for Start and
Stop and a button for Advance. Note that tube number is a display
field only.
• Place tubes in the fraction collector rack and set a fraction size of
1 ml. Click on the Start button and select valve position 2 of the
SV3-2 valve box. Flow will now be diverted to the fraction
collector tubes. Click the Advance button to test its function if
desired.
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• Continue to pump water through the system for at least 10 minutes.
You may wish to continue fraction collection or return the
SV3-2 valve to position 1 and then stop fraction collection.
Note:Inmanual operation, starting or stopping fraction collection
does not change the SV3-2 diverter valve position. You must always
manually set the SV3-2 valve to the waste or collect position.
b. Using a Model 2128 Fraction Collector.
The Model 2128 Fraction Collector has two operating modes:
• System: Controlled by the BioLogic HR System.
• Local: Controlled from its own faceplate.
For now, insure that the System button is selected.
When in System mode, operation of the fraction collector is as follows.
• Control of the Model 2128 is identical regardless of whether or not
the optional on-arm diverter valve is used. Without a valve, the
drop-head returns to the waste trough during divert operations.
• The fraction collector 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
Advance
Gradient Pump UV
0.00 ml/min 0 %B 438 psi -0.00181 AU
Fraction Collector
START
Gradient pump
Inlet A:
1.00
100
0
Inlet B:
Flow rate: ml/min
%
%
High
limit:
0
1000
Low
limit: psi
AV7-3 at port 4SV3-2 at port 1 SV5-4 at port 2
PL21
21
34
STOP START STOP
Set
psi
Tube number:
ml
Volume left:
Report PostRun Log ON
Window Help
Mode: System Local
Start Tube:
End Tube:
Fraction size:
1
1
128
1.00
Rack: Rack 1 (128 tubes)
I
AV9-8 at port 5
37
82
654
I
Conductivity
47.5 mS/cm 0.089 Volt 7.00 pH
SIM2/pHSIM1/SIG
Chart Recorder
Signal Import Module
SIM 1 and SIM 2 connected
UV Detector
Conductivity
range(mS/cm): 500
UV range (AU): 0.1
ON OFF
Event mark
ON OFF
Zero Baseline
Flowrate of the gradient pump
19
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