Bio-Rad BioLogic DuoFlow Pathfinder 80 System DuoFlow Chromatography System Starter Kit User Manual

BioLogic DuoFlow

F3 F4 F5

1 2 3

4 5 6

7 8 9

F1 F2

A B

VALVE A VALVE B

BioFrac Franction Collector

™

Chromatography System

Starter Kit

Instruction Manual

Catalog # 760-0135

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

1.1 Prime the Workstation Pumps.....................................................5

1.2 Move the AVR7-3 Inject Valve to the Purge Position....................5

1.3 Purge the Workstation Pumps ....................................................5

1.4 Manual Control of the Workstation Pumps ..................................6

1.5 Flush the System Through to the Fraction Collector ....................6

BioFrac Fraction Collector

1.6 Turn on the UV Lamp ..................................................................6

DuoFlow UV Detector QuadTec UV/Vis Detector

1.7 Manual Screen Chromatogram Window......................................7

1.8 Status Bar...................................................................................7

Section 2. Anion Exchange Separation of Protein Standards........8

2.1 Overview of the Procedure..........................................................8

2.2 Prepare Buffers...........................................................................9

2.3 Prepare Sample..........................................................................9

2.4 Install UNO Q1 Column ............................................................10

2.5 Prime the Pumps and Equilibrate the UNO Q1 Column ............10

2.6 Create a New Method...............................................................10

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 ...................................................21

3.5 Flush the System Through to the Fraction Collector ..................21

BioFrac Fraction Collection

3.6 Turn on the UV Lamp................................................................22

DuoFlow UV Detector QuadTec UV/Vis Detector

3.7 pH Electrode Calibration ...........................................................23

3.8 Manual Screen Chromatogram Window ...................................23

3.9 Status Bar ................................................................................24

Section 4. Anion Exchange Separation of Protein Standards ......24

4.1 Overview of the Procedure........................................................24

4.2 Prepare Solutions .....................................................................25

4.3 Prepare Sample........................................................................26

4.4 Install the UNO Q1 Column.......................................................26

4.5 Prime the Pumps and Equilibrate the UNO Q1 Column.............27

4.6 Create a New Method...............................................................29

Section 5. Ordering Information.....................................................36

Introduction

This instruction manual and starter kit contents may be used for the BioLogic DuoFlow system and the BioLogic DuoFlow Maximizer 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 8.1 (10x concentrate)

• 50 ml of buffer B, 250 mM Tris-HCI buffer, pH 8.1, plus 5.0 M NaCl (10x concentrate)

• 50 ml of Maximizer solution A1, 500 mM Tris-HCl (10x concentrate)

• 50 ml of Maximizer solution A2, 500 mM Tris base (10x concentrate)

• 50 ml of Maximizer solution B2, 5.0 M NaCl (2.5 x concentrate)

• One vial of anion exchange protein standard (catalog #125-0561)

• One 1 ml disposable sample injection syringe

• One 50 µl sample loop

The chromatographic separation for this kit requires approximately 6 minutes.

2. Materials You Will Need

In order to prepare the starter kit buffer solutions you will need the following materials:

• Filtered high-quality water (i.e., HPLC grade water)

™

and Pathfinder

™

• One 500 ml graduated cylinder

• One 1 L side-arm flask

• Stirbar and stirplate

• Vacuum source for degassing

• Two 500 ml bottles

• Fraction collection tubes, 13 x 100 mm (at least 14 tubes)

• 100 ml beaker

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 1.5 or 2 ml micro tubes

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. The arrow button in the upper right corner of the detector control panel toggles between the UV and QuadTec detector control panels. Only those instruments connected to the system will be displayed.

BioLogic Duo-Flow - - - no method -

File View Utilities Options Window Help

New

Method

Flowrate

Inlet A

Inlet B

High limit

Low limit

SVT5-4 at port 1 SVT3-2 at port 3SV5-4 at port 2

UV Conductivity

Zero baseline for the UV detector

New

Edit

Method

Run Browser Manual Setup Setup

Gradient Pump: F10

1.00

700

START

QuadTec

Gradient Pump: F10 UV Conductivity

0.00 ml/min

50 %B

Report

Fraction Collector: BioFrac

Mode: System

ml/min

psi.

Set

STOP

1.50

1.00

0.50

0.00

F1 (12-13 mm tubes)

Rack:

Start Tube:

End Tube:

Fraction size:

Tube number:

Volume left:

Advance

100% Buffer B

02 46 8

1 psi 0.2583 AU 0.000 mS/cm

Fig. 1. Manual Control Screen with UV detector

wash

1 2

load

Protocol

1.00

START STOP

Workstation Valves

Run

Local

Notes

Conductivity range (mS/cm):

UV range (AU):

L21

Fractions

Minutes

PostRun

UV Detector

Chart Recorder

AVR7-3 at port 4

Econo Gradient

Log

Settings

Zero Baseline

ON OFF

1.0

Event Mark

Pump

Econo Gradient Pump 1

Mode: System

Flowrate

EGP %B

% Split

Flow Direction

OFFON

AVR9-8 at port 5

400.0

300.0

200.0

100.0

0.0

mS/cm

Flow Rate EGP %B

1.000

0.00

START STOP

AVR9-8 at port 6

0 %B0.00 ml/min 0%

SIM1/pHSIM1/SIG

™

Bio-Rad

Web

ml/min

Set

Resize

Clear

Traces

% Split

Local

BioLogic Duo-Flow - - - no method -

Fig. 2. Manual Control Screen with QuadTec detector

File View Utilities Options Window Help

New

Method

Flowrate

Inlet A

Inlet B

High limit

Low limit

New

Edit

Method

Run Browser Manual Setup Setup

Gradient Pump: F10

1.00

700

START

ml/min

psi.

Set

STOP

Report

Fraction Collector: BioFrac

Mode: System

F1 (12-13 mm tubes)

Rack:

Start Tube:

End Tube:

Fraction size:

Tube number:

Volume left:

Advance

wash

1 2

load

Protocol

1.00

START STOP

Notes

Run

Local Mode: System Local

Mode: System Local

Lamp Type

Range

Log

PostRun

Settings

QuadTec Detector

Zero Baseline

ON OFF

Deuterium

190 - 370 nm

Wavelength Selection

280

260

214

405

Set

Econo Gradient Pump 1

Flowrate

1.000

EGP %B

% Split

0.00

Flow Direction

START STOP

Workstation Valves

SVT5-4 at port 1 SVT3-2 at port 3SV5-4 at port 2

UV Conductivity

1.50

1.00

0.50

0.00

QuadTec

0.00 ml/min

Sets fraction collector to system mode

WL1 - 280 nm

0.2232 AU

Gradient Pump: F10

50 %B

100% Buffer B

02 46 8

WL2 - 260nm WL3 - 214nm WL4 - 405nm

0.15 AU 1.15 AU

2 psi 0.2583 AU 0.000 mS/cm

AVR7-3 at port 4

L21

Fractions

Minutes

0.30 AU

Econo Gradient

Pump

Conductivity

AVR9-8 at port 5

400.0

300.0

200.0

100.0

0.0

mS/cm

Flow Rate EGP %B

0 %B0.00 ml/min 0%

AVR9-8 at port 6

SIM1/pHSIM1/SIG

Bio-Rad

Web

ml/min

Set

Resize

Clear

Traces

% Split

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. 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 B.

1.2 Move the AVR7-3 Inject Valve to the Purge Position

Prior to purging the pumps at 10 ml/min it is essential to place the AVR7-3 valve in the purge position. This directs the flow to waste and not to the column and detector.

To change the position of the AVR7-3 inject valve, select P from the Manual screen valve control panel for the AVR7-3 valve. If you plugged the AVR7-3 inject valve into port 4 on the workstation rear panel, you will see a valve box designated AVR7-3 at port 4. The three 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.

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.

1.3 Purge the Workstation Pumps

a. Make sure that the AVR7-3 inject valve is in the Purge position.

b. Press the Purge buttons A then B on the front of the workstation. The

workstation pumps will run at a default flow rate of 10 ml/min and the indicator light will flash green.

c. Run each pump for 2 minutes. Press the purge buttons again to stop the pump.

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. To change the pump parameters while the pump is running, enter the new value and then click on the Set button.

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. If you are using an UNO

™

Q1 column, set the high limit to 700 psi and the low limit to 20 psi.

1.5 Flush the System Through to the Fraction Collector

With the gradient pumps stopped, move the AVR7-3 valve back to position L (Load) by clicking L (AVR7-3) on the Manual screen.

From the gradient pump control panel on the Manual screen, set the pump flow rate to 1.0 ml/min and start the pump. Water will flow through the UV or QuadTec and conductivity flow cells to the fraction collector, as described below.

BioFrac

™

Fraction Collector

The BioFrac fraction collector has two operating modes:

• System—Controlled by the DuoFlow system

• Local—Controlled from its own faceplate in stand-alone mode

Ensure that the System button is selected.

When in System mode, the fraction collector control panel will show fields for Rack type, Start tube, End tube, Fraction size, Tube number, Volume left, a toggle button for Start and Stop, and a button for Advance (see Figures 1 and 2).

1.6 Turn on the UV lamp

DuoFlow UV detector

a. The UV lamp automatically turns on when you turn on power to the

workstation. The UV lamp can be turned on and off by clicking the On and Off buttons from the UV detector control panel on the Manual screen (see Figure 1). Check that the lamp is on; the mercury lamp requires approximately 30 minutes to warm up.

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. When connection is completed, “SLAVE” appears in the corner of QuadTec faceplate. The QuadTec appears in its own control panel as shown in Figure 2.

b. From the QuadTec detector control panel on the Manual screen (Figure 2),

set the four wavelengths of the QuadTec detector to 280, 260, 214, and 405 nm. Select Set. The active wavelengths will appear in the lower screen status bar.

c. Click the Zero Baseline button to zero the four UV/Vis signals. 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.

1.7 Manual Screen Chromatogram Window

A feature of the Manual screen is its ability to display up to eight traces of a chromatogram; including UV/Vis, pH, conductivity, %Buffer B, and pressure traces, over a 10-minute interval. This is useful during column equilibration. The chromatogram window is displayed at the bottom of the screen, under the valve control panel (See Figures 1 and 2). Features of the chromatogram window include:

• The time axis is reset automatically at the end of 10 minutes or reset manually by clicking the Clear Traces button

• The chromatogram window can be enlarged by pressing the Resize button.

• A chromatogram trace may be selected for scaling by using the drop-down menus on the upper right and left of the display

• The Y-axis scale can be changed using the scroll bars on the right or left of the display

• The maximum and minimum axis settings can be changed by pressing Settings on the manual screen toolbar.

1.8 Status Bar

At the bottom of the Manual screen is a status bar that is continually updated with system parameters.

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. 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, 0.5 M NaCl

• Flow rate 4.00 ml/min

• Sample volume 50 µl

• UV detection 0.1 AUFS

• QuadTec detection 0.1 AUFS (λ = 280 nm), 0.1 AUFS (λ = 260 nm),

1.0 AUFS (λ = 214 nm), and 0.4 AUFS (λ = 304 nm)

• Conductivity 100 mS/cm

General Procedure

Step 1 Prepare buffer

Step 2 Prepare sample

Step 3 Install the UNO Q1 column

Step 4 Prime the workstation pumps and equilibrate the column

Step 5 Write a method

a. Program the instrument Setup

b. Program the method Protocol

c. Load sample into 50 µl loop

d. Select Run

e. Select Start

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. Place the contents of the graduated cylinder 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 buffer for approximately 15 minutes with gentle stirring.

c. When degassing is complete, pour the buffer into a bottle and label it “Buffer A,

25 mM Tris-HCI, pH 8.1”.

Buffer B

Prepare buffer B by following the same procedure for preparation of buffer A. Label the buffer as “Buffer B = 25 mM Tris-HCl, pH 8.1, 0.5 M NaCl”.

Conversion of Maximizer Solutions to Buffers A and B

The starter kit contains solutions for use with the DuoFlow Maximizer or Pathfinder systems. These can be converted to buffer A, 25 mM Tris-HCI, pH 8.1, and buffer B = 25 mM Tris-HCl, pH 8.1, 0.5 M NaCl as follows:

a. Dilute Maximizer solutions A1 and A2 to 500 ml each with filtered water.

b. Combine 150 ml of diluted A1, 100 ml of diluted A2 and the entire contents

of solution B2. Dilute the mixture to 500 ml. Check the pH and adjust to pH 8.1, if necessary. Degas the solution and label it as “Buffer B, 25 mM Tris-HCI, pH 8.1, 0.5 M NaCl“.

c. Combine the remaining diluted solutions A1 and A2 with water in a 1:1:2

ratio (i.e., 250 ml each of diluted A1 and A2 with 500 ml water). Check the pH and adjust it to pH 8.1, if necessary. Degas the solution and label it as “Buffer A, 25 mM Tris-HCI, pH 8.1”.

2.3 Prepare Sample

a. Remove the aluminum cap from the anion exchange standard vial. Slowly

remove the rubber plug from the vial (the contents may be under vacuum).

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