Getting Started with - Amazon S3 Started with ÄKTAavant and UNIC ORN 6™ ™ ... UNICORN....

GE Healthcare

Getting Started with

ÄKTAavant and UNIC ORN 6™ ™

Table of Contents51 About this guide ...................................................................................................61.1 Get started ...............................................................................................................................................

101.2 Regulatory information ......................................................................................................................

122 Safety instructions ...............................................................................................132.1 Safety precautions ...............................................................................................................................202.2 Labels .........................................................................................................................................................232.3 Emergency procedures ......................................................................................................................

263 Start up .................................................................................................................273.1 Start the system and log on to UNICORN ..................................................................................

314 The instrument and the software .....................................................................324.1 ÄKTAavant instrument overview ...................................................................................................364.2 UNICORN software overview ..........................................................................................................394.3 UNICORN software modules ...........................................................................................................404.3.1 Method Editor module ......................................................................................................................434.3.2 System Control module ...................................................................................................................454.3.3 Evaluation module .............................................................................................................................

475 Files and folders in UNICORN .............................................................................485.1 Navigator overview .............................................................................................................................515.2 Handling files and folders .................................................................................................................

556 Create a method ..................................................................................................566.1 Guide to method creation .................................................................................................................576.1.1 Create and edit methods ................................................................................................................656.1.2 Predefined methods ..........................................................................................................................676.1.3 Predefined phases .............................................................................................................................686.2 Print a method .......................................................................................................................................

707 Prepare the system for a run .............................................................................717.1 Before you prepare the system ......................................................................................................727.2 Prepare the flow path .........................................................................................................................787.3 Prime buffer inlets and purge System pumps .........................................................................837.4 Connect a column ................................................................................................................................887.5 Calibrate the pH monitor ..................................................................................................................907.6 Prepare the Fraction collector ........................................................................................................

958 Run a method ........................................................................................................968.1 Before you start .....................................................................................................................................988.2 Applying the sample ............................................................................................................................

1018.3 Start a method run ..............................................................................................................................

Getting Started with ÄKTAavant and UNICORN 6 28-9440-69 AB 3

Table of Contents

1068.4 Monitor the run ......................................................................................................................................1118.5 After run procedures ...........................................................................................................................

1159 Evaluate and print the results ...........................................................................1169.1 View the results .....................................................................................................................................1209.2 Peak integration ....................................................................................................................................1259.3 Print the results ......................................................................................................................................

13010 Maintenance .........................................................................................................13110.1 Maintenance program .......................................................................................................................

4 Getting Started with ÄKTAavant and UNICORN 6 28-9440-69 AB

Table of Contents

1 About this guide

IntroductionThis chapter describes the purpose of the guide and provides regulatory informationrelevant for ÄKTAavant system.

ContentsThis chapter contains the following sections:

See pageSection

61.1 Get started

101.2 Regulatory information


1 About this guide

1.1 Get started

IntroductionRead this section for an understanding of the purpose and conventions of this guide, aswell as the requirements that you must fulfill before using the ÄKTAavant system.

Purpose of Getting StartedThe purpose of this guide is to present a quick and easy guide to the system for a userwith limited or no experience of UNICORN software and ÄKTAavant instrument. Theworkflow is presented as practical instructions on how to operate the software and theinstrument. The instructions forma basic framework that you can expand on by readingselected parts in the other manuals. This Getting Started guide includes the followingtopics:

• Basic features of ÄKTAavant and UNICORN

• Create methods

• Prepare the system for runs

• Perform runs

• Make simple evaluations

• Print reports

For best results, follow the guide from page to page in front of the system.

PrerequisitesIn order to follow this guide and use the system in themanner it is intended, it is importantthat:

• you have a general understanding of how the computer and Windows™ work.

• you understand the concepts of liquid chromatography.

• you have read and understood the Safety instructions chapter in the userdocumentation.

• the instrument and software are installed, configured and calibrated according toÄKTAavant and UNICORN 6 Installation Guide.

• a user account has been created according to UNICORN 6 Administration andTechnical Manual.


1 About this guide1.1 Get started

Pushing the limits beyondGetting Started

Users who are familiar with the instrument and software and want to learn more aboutthe system’s advanced features should refer to the list below.

please read..To find out moreabout...

ÄKTAavant and UNICORN 6 Installation Guideinstallation

UNICORN 6 Administration and Technical Manualadministration ofdatabases

ÄKTAavant and UNICORN 6 User Manualinstrumentmodulesandfunctions

ÄKTAavant and UNICORN 6 User Manualcalibration

UNICORN 6 Method Manualpredefined methods

UNICORN 6 Method Manualcolumn handling

UNICORN 6 Method Manualmanual method editing

UNICORN 6 Method ManualBufferPro–automaticbuffer preparation

UNICORN 6 Method Manualscouting

UNICORN 6 Method ManualDesign of Experiment

UNICORN 6 Evaluation Manualevaluation

UNICORN 6 Evaluation Manualpeak integration

UNICORN 6 Evaluation Manualcustomizedprint format



Intended useÄKTAavant is a liquid chromatography system intended for method development. Thesystem can be used to screen for optimal choice of columns, media and runningparameters to purify selected proteins.

The ÄKTAavant system is intended for research use only, and shall not be used in anyclinical procedures, or for diagnostic purposes.

Safety noticesThis user documentation contains WARNINGS, CAUTIONS and NOTICES concerning thesafe use of the product. See definitions below.

Warnings

WARNINGWARNING indicates a hazardous situation which, if not avoided,could result in death or serious injury. It is important not to proceeduntil all stated conditions are met and clearly understood.

Cautions

CAUTION

CAUTION indicates a hazardous situation which, if not avoided,could result in minor or moderate injury. It is important not toproceed until all stated conditions aremet and clearly understood.

Notices

NOTICE

NOTICE indicates instructions that must be followed to avoiddamage to the product or other equipment.



Typographical conventionsSoftware items are identified in the text by bold italic text. A colon separatesmenu levels,thus File:Open refers to the Open command in the Filemenu.

Hardware items are identified in the text by bold text (e.g., Power switch).

Text entries that UNICORN generates or that the user must type are represented by amonotype typeface (e.g., \Program Files\GEHealthcare\UNICORN\bin\UNICORN Instrument Server.exe.config).



1.2 Regulatory information

IntroductionThis section describes the directives and standards that are fulfilled by the ÄKTAavantsystem.

CE conformityThis product complies with the European directives listed in the table below, by fulfillingthe corresponding harmonized standards. A copy of the Declaration of Conformity isavailable on request.

TitleDirective

Machinery Directive2006/42/EC

Low Voltage Directive2006/95/EC

ElectroMagnetic Compatibility (EMC) Directive2004/108/EC

Radio Equipment and Telecommunications TerminalEquipment Directive

1999/5/EC

CE marking

The CEmarking and the corresponding declaration of conformity, is valid for theinstrument when it is:

• used as a stand-alone unit, or

• connected to other CEmarked GE Healthcare instruments, or

• connected to other products recommendedor described in the user documentation,and

• used in the same state as it was delivered fromGEHealthcare, except for alterationsdescribed in the user documentation.


1 About this guide1.2 Regulatory information

International standardsThis product fulfills the requirements of the following standards:

NotesDescriptionStandard

Safety requirements for electricalequipment for measurement,control, and laboratory use

EN 61010-1, IEC61010-1, UL 61010-1,CAN/CSA-C22.2No.61010-1

Harmonized with2004/108/EC

EMC emissions and immunityrequirements for electricalequipment for measurement,control and laboratory use.

EN 61326-1

CISPR 11 Group 1,Class B


Safety of machinery — Basicconcepts, general principles fordesign

EN-ISO 12100-1,12100-2


Safety of machinery — Principles ofrisk assessment

EN ISO 14121-1,14121-2

Software declaration ofconformity

UNICORN 6 is technically compatible with all relevant sections of FDA 21 CFR Part 11.

A part 11-system assessment checklist is available on request through the local GEHCrepresentative.

Regulatory compliance ofconnected equipment

Any equipment connected to theÄKTAavant systemshouldmeet the safety requirementsof EN 61010-1/IEC 61010-1, or other relevant harmonized standards. Within EU,connected equipment must be CE marked.


1 About this guide1.2 Regulatory information

2 Safety instructions

IntroductionThis chapter contains instructions of how to handle the ÄKTAavant instrument in a safeway.


See pageSection

132.1 Safety precautions

202.2 Labels

232.3 Emergency procedures


2 Safety instructions

2.1 Safety precautions

IntroductionThe ÄKTAavant instrument is powered bymains voltage and handles pressurized liquidsthatmay be hazardous. Before installing, operating ormaintaining the system, youmustbe aware of the hazards described in this manual. Follow the instructions provided toavoid personal injuries or damage to the equipment.

The safety precautions in this section are grouped into the following categories:

• General precautions

• Personal protection

• Installing and moving the instrument

• System operation

• Maintenance

General precautions

WARNINGAlways follow theseGeneral precautions to avoid injurywhenusingthe ÄKTAavant instrument.

• Do not operate the ÄKTAavant instrument in any other way than described in theÄKTAavant and UNICORN manuals.

• Operation and usermaintenance of the ÄKTAavant instrument should be performedby properly trained personnel only.

• Before connecting a column to the ÄKTAavant instrument, read the instructions foruse of the column. To avoid exposing the column to excessive pressure, make surethat the pressure limit is set to the specified maximum pressure of the column.

• Do not use any accessories not supplied or recommended by GE Healthcare.

• Do not use the ÄKTAavant instrument if it is not working properly, or if it has sufferedany damage, for example:

- damage to the power cord or its plug

- damage caused by dropping the equipment

- damage caused by splashing liquid onto it


2 Safety instructions2.1 Safety precautions

NOTICE

Avoid condensationby letting the instrument equilibrate to ambienttemperature.

Using flammable liquids

WARNINGWhen using flammable liquids with the ÄKTAavant instrument,follow these precautions to avoid any risk of fire or explosion.

• Fire Hazard. Before starting the system, make sure that there are no unintentionalleakage in the instrument or tubing.

• Explosion hazard. To avoid building up an explosive atmosphere when usingflammable liquids, make sure that the room ventilation meets the requirementsspecified in ÄKTAavant and UNICORN 6 Installation Guide.

• Fraction collector. Donot fractionate flammable liquids.When runningRPCmethods,or other procedures using solvent based buffers, collect fractions through the outletvalve.

Personal protectionWarnings

WARNINGTo avoid hazardous situations when working with the ÄKTAavantsystem, take the following measures for personal protection.

• Always use appropriate personal protective equipment during operation andmaintenance of ÄKTAavant system.

• Hazardous substances.When using hazardous chemical and biological agents,take all suitable protectivemeasures, such aswearing protective glasses and glovesresistant to the substances used. Follow local and/or national regulations for safeoperation and maintenance of the system.



• Spread of biological agents. The operator has to take all necessary actions to avoidspreading hazardous biological agents in the vicinity of the instrument. The facilityshould comply with the national code of practice for biosafety.

• High pressure. The ÄKTAavant instrument operates under high pressure. Wearprotective glasses at all times.

Cautions

CAUTION

To avoid hazardous situations when working with the ÄKTAavantsystem, take the following measures for personal protection.

• Closedoors. Tominimize the risk of exposure to hazardous chemicals andpressurizedliquids, always close the Foldable door and the Pump cover before starting a run.

• Cut injuries. The tubing cutter is very sharp andmust be handled with care to avoidinjuries.

Installing and moving theinstrument

Warnings

WARNINGTo avoid personal injury when installing or moving the ÄKTAavantinstrument, follow the instructions below.

• Heavy object. The ÄKTAavant instrument weighs about 100 kg. Use proper liftingequipment, or use four or more persons whenmoving the instrument. All lifting andmoving must be performed in accordance with local regulations.

• Moving the instrument horizontally. Three persons are recommendedwhenmovingthe instrument horizontally.

• Supply voltage.Make sure that the supply voltage at the wall outlet corresponds tothe marking on the instrument, before connecting the power cord.

• Protective ground. The ÄKTAavant instrument must always be connected to agrounded power outlet.

• Power cord. Only use power cords delivered or approved by GE Healthcare.



• Access to power switch and power cord. Do not block the rear and side panel ofthe instrument. The Power switch must always be easy to access. The power cordmust always be easy to disconnect.

• Installing the computer. The computer should be installed and used according tothe instructions provided by the manufacturer of the computer.

Notices

NOTICE

To avoid damage to ÄKTAavant or other equipmentwhen installingor moving the instrument, follow the instructions below.

• Vents on the ÄKTAavant instrument. To ensure adequate ventilation, keep papersand other objects away from the vents of the instrument.

• Disconnect power. To prevent equipment damage, always switch off power to theÄKTAavant instrument before an instrument module is removed or installed, or acable is connected or disconnected.

• Misuse of UniNet-9 connectors. The UniNet-9 connectors at the rear panel shouldnot be mistaken for Firewire connectors. Do not connect any external equipment tothe UniNet-9 connectors. Do not disconnect or move the UniNet-9 bus cable.

System operationWarnings

WARNINGTo avoid personal injury when operating the ÄKTAavant system,follow the instructions below.

• Rotating the instrument.Make sure that there is always at least 20 cmof free spacearound the ÄKTAavant instrument to allow rotation on the swivel foot.When rotatingthe system, take care not to stretch or squeeze tubing or cables. A disconnectedcable may cause power interruption or network interruption. Stretched tubes maycause bottles to fall, resulting in liquid spillage and shattered glass. Squeezed tubingmay cause increase in pressure, or block liquid flow. To avoid the risk of turning overbottles, always place bottles on the buffer tray, and close the doors before rotatingthe instrument.



• Fasten sample bottles. Always fasten bottles and cassettes to the rails at the sampletray. Use appropriate holders for bottles. Shattered glass from falling bottles maycause injury. Spilled liquid may cause fire hazard and personal injury.

• Electrical shock hazard after spillage. If there is a risk that large volumes of spilledliquid may penetrate the casing of the ÄKTAavant instrument, immediately switchoff the instrument, disconnect the power cord, and contact an authorized serviceengineer.

• Moving parts in Fraction collector. Do not open the Fraction collector drawerwhenthe Fraction collector is active. If you need to access the Fraction collector, pressPause, and make sure that the movement has stopped before opening the drawer.

• Using a Superloop. After loading a Superloop, always plug the Syr port on theInjection valve with a Stop plug. With a Superloop connected to the valve, anover-pressure may be created during injection.

• Hazardous chemicals during run.When using hazardous chemicals, run SystemCIP and Column CIP to flush the entire system tubing with distilled water, beforeservice and maintenance.

• Hazardous biological agents during run.When using hazardous biological agents,run SystemCIP andColumnCIP to flush the entire system tubingwith bacteriostaticsolution (e.g., NaOH) followed by a neutral buffer and finally distilled water, beforeservice and maintenance.

• RPC runs with 100% acetonitrile. Always replace the green PEEK tubing betweenthe used system pump and the pressure monitor with orange PEEK tubing, i.d. 0.5mm, before running RPC with 100% acetonitrile. Set the System pressure alarm to10 MPa.



Cautions

CAUTION

To avoid hazardous situations when operating the ÄKTAavantsystem, follow the instructions below.

• Risk of breaking test vials. Do not use excessive force to press vials with incorrectdimensions into the Fraction collector cassettes. Glass vials may break and causeinjuries.

• Hazardous chemicals or biological agents in UV flow cell. Make sure that the entireflow cell has been flushed thoroughly with bacteriostatic solution (e.g., NaOH) anddistilled water, before service and maintenance.

• pH-electrode. Handle the pH-electrodewith care. The glass tipmay break and causeinjury.

• Max. weight on top tray. Do not place containers with a volume of more than 10liters on the top tray. The total allowed weight on the top tray is 40 kg.

• Max. size of bottles on front panel. Do not fasten bottles with a volume of morethan 1 liter in the front panel rails.

Notices

NOTICE

To avoid damage toÄKTAavant or other equipmentwhenoperatingthe instrument, follow the instructions below.

• Keep UV flow cell clean. Do not allow solutions containing dissolved salts, proteinsor other solid solutes to dry out in the flow cell. Do not allow particles to enter theflow cell, as damage to the flow cell may occur.

• Waste container height. The maximum level of the waste container content mustbe lower than 30 cm above the lab bench, for trouble-free use.



MaintenanceWarnings

WARNINGTo avoid personal injury when performing maintenance on theÄKTAavant instrument, follow the instructions below.

• Electrical shock hazard. All repairs should be done by service personnel authorizedby GE Healthcare. Do not open any covers or replace parts unless specifically statedin the user documentation.

• Disconnect power. Always switch off power to the instrument before replacing anycomponent on the instrument, unless stated otherwise in the user documentation.

• Hazardous chemicals during maintenance.When using hazardous chemicals forsystem or column cleaning, wash the system or columns with a neutral solution inthe last phase or step.

NOTICE

Cleaning. Keep the instrument dry and clean. Wipe regularly witha soft damp tissue and, if necessary, amild cleaning agent. Let theinstrument dry completely before use.



2.2 Labels

IntroductionThis section describes the safety labels and labels concerning hazardous substancesthat are attached to the ÄKTAavant instrument. For information about marking of thecomputer equipment, refer to the manufacturer’s instructions.

Labels on the ÄKTAavantinstrument

The illustrations below show the labels that are attached to the ÄKTAavant instrument.


2 Safety instructions2.2 Labels

Safety symbolsThe following safety symbols are used in the labels:

MeaningLabel

Warning!

Electrical shock hazard.All repairs should be done byservice personnel authorized byGEHealthcare. Do notopen any covers or replace parts unless specificallystated in the user documentation.

Supply voltage.Make sure that the supply voltage atthe wall outlet corresponds to the marking on theinstrument, before connecting the power cord.

Warning!Do NOT fractionateflammable liquids. Fraction collector. Do not fractionate flammable

liquids. When running RPC methods, or otherprocedures using solvent based buffers, collectfractions through the outlet valve.

The system complies with the requirements forelectromagnetic compliance (EMC) in Australia andNew Zealand.

The system complies with applicable Europeandirectives.



Labels concerning hazardoussubstances

The following symbols on the labels concern hazardous substances:

MeaningLabel

This symbol indicates that electrical and electronicequipmentmust not be disposedof as unsortedmunicipalwaste and must be collected separately. Please contactan authorized representative of the manufacturer forinformation concerning the decommissioning ofequipment.

This symbol indicates that the product contains hazardousmaterials in excess of the limits establishedby the Chinesestandard SJ/T11363-2006Requirements for ConcentrationLimits for Certain Hazardous Substances in ElectronicInformation Product.



2.3 Emergency procedures

IntroductionThis section describes how to do an emergency shutdown of the ÄKTAavant system.The section also describes the result in the event of power failure or network interruption.

Emergency shutdownIn an emergency situation, follow the steps below to stop the run:

ActionStep

Press the Pause button on the instrument display. This will stop all pumpsin the instrument.

1

To pause the run from UNICORN, click the Pause icon:


2 Safety instructions2.3 Emergency procedures

ActionStep

If required, switch off power to the instrument by pressing the Power switchto the O position. The run is interrupted immediately.

2

Power failureThe result of a power failure depends on which unit is affected.

will result in...Power failure to...

• The run is interrupted immediately

• The data collected up to the time of the powerfailure is available in UNICORN.

ÄKTAavant instrument



will result in...Power failure to...

• The UNICORN computer shuts down

• The instrument display shows status Notconnected

• The run is interrupted immediately

• Data generated up to 10 seconds before thepower failure can be recovered

Note: The UNICORN client mayclose down during atemporary overload of theprocessor. This may appearas a computer failure. Therun continues and you canrestart theUNICORN client toregain control.

Computer

Uninterruptible power supply(UPS)

A UPS can eliminate data loss after a power failure, and allow time for a controlledshut-down of the ÄKTAavant system.



3 Start up

IntroductionThis chapter describes how to start the system, both the software and the instrument,and also how to log off and exit UNICORN.

It is assumed that your user profile is already created. For information about how to setup and define users, user groups and access rights, please refer to UNICORN 6Administration and Technical Manual.


See pageSection

273.1 Start the system and log on to UNICORN


3 Start up

3.1 Start the system and log on to UNICORN

IntroductionThis section describes how to start UNICORN and the instrument, and how to connectto the system.

Start UNICORN and log onFollow the instruction below to start and log on to UNICORN.

ActionStep

To start the program, double-click the UNICORN icon on the desktop.1

Result: The Log On dialog opens.


3 Start up3.1 Start the system and log on to UNICORN

ActionStep

In the Log On dialog:2

• Select your User Name from the drop-down list.

• Enter your Password.

• Click the Options button.

Result: Checkboxes for each of the UNICORN modules are displayed inthe Log On dialog.

In the Log On dialog:3

• Select which UNICORN modules to start by clicking the correspondingcheckboxes.

• Click OK.

Result: The selected UNICORN modules open.



Start the instrumentandconnectto the system

Follow the instruction below to start the instrument and connect to the UNICORNsoftware.

ActionStep

Switch on the instrument by pressing the Power switch to the I position.1

Open the the System Controlmodule in UNICORN and click the Connect toSystems icon.

2

Result: The Connect to Systems dialog opens.



ActionStep

In the Connect to Systems dialog:3

• Select the instrument.

• Select Controlmode.

• Click OK.

Result: The selected instrument can now be controlled by the software.



4 The instrument and the software

IntroductionThis chapter gives an overview of the ÄKTAavant system: instrument, software andaccessories.

Illustration of the systemThe illustration below shows the ÄKTAavant instrumentwith UNICORN software installedon a computer.


See pageSection

324.1 ÄKTAavant instrument overview

364.2 UNICORN software overview

394.3 UNICORN software modules


4 The instrument and the software

4.1 ÄKTAavant instrument overview

IntroductionThis section provides an overview of the ÄKTAavant instrument. Technical details aboutthe instrument and the individual modules are found in ÄKTAavant and UNICORN 6 UserManual.

Exterior designÄKTAavant has a modular design, with all the liquid handling modules placed on theexterior of the instrument. Buffer vessels are placed on the Buffer tray on top of theinstrument. An Instrument display is placed on the front. From this side the built-inFraction collector is handled, as well as the sample. The rest of the modules are placedon the right-hand side of the instrument. This side can be covered by a foldable doorand a pump cover. By rotating the instrument using the swivel foot any side is easilyaccessed.

Operating rangesThe table below shows some of the instrument’s operational limits.

LimitsParameter

Up to 25 ml/minFlow rate

20 MPaMax. operating pressure

190 - 700 nmWavelength


4 The instrument and the software4.1 ÄKTAavant instrument overview

Illustration of the main parts ofthe instrument

The illustration below shows the location of the main parts of the instrument.

DescriptionPart

Fraction collector1

Buffer tray2

Instrument display3

Wet side4

Foldable door5

Pump cover6

Holder rails7

Swivel foot Lock/Unlock knob8

Swivel foot9

Swing out toolbox10

Power switch11



Illustration of the wet sidemodules of the instrument

The illustration below shows the modules of the wet side of the instrument.

DescriptionPart

Injection valve1

Column valve2

Flow restrictor3

pH valve4

UV monitor5

Conductivity monitor6

Inlet valve B7

Quaternary valve8

System pump B9

Pressure monitor of System pumps10

Inlet valve A11



DescriptionPart

System pump A12

Mixer13

Pressure monitor of Sample pump14

Sample pump15

Pump piston rinsing system tubes16

Sample inlet valve17

Outlet valve18

Holder rails19



4.2 UNICORN software overview

IntroductionThis section gives a brief overview of the UNICORN software—a complete package forcontrol, supervision and evaluation of chromatography instruments and purificationruns. It also describes how to access the help utility that is included in UNICORN.

UNICORN modules overviewUNICORN consists of fourmodules:Administration,Method Editor, SystemControl andEvaluation. The main functions of each module are described in the table below.

Main functionsModule

Performuser and system setup, system log anddatabaseadministration.

Administration

Create and edit methods.Method Editor

Start, view and control runs.System Control

Open results, evaluate runs and create reports.Evaluation

Enter a UNICORN moduleTo enter a module:

• click the Taskbar button of the module of interest,

or

• choose themodule of interest in the Toolsmenu in any of the other softwaremodules.

The illustration below shows the Toolsmenu of the Evaluationmodule.


4 The instrument and the software4.2 UNICORN software overview

Access the help utilityA comprehensive help utility is included in the UNICORN software. The table belowdescribes how to access the different parts of the help utility.

then...If you want to...

select Help:Help for... in the UNICORN module of interestfind information abouta UNICORN module

• press the F1 keywith the itemof interest selected andin focus

Note: To find information about a phase,make sure that the PhaseProperties tab is selected beforepressing the F1 key.

or

• click the Help icon in the open dialog

find information aboutthe item currentlyselected and in focus(e.g., a pane, a dialog, ora method phase)




• selectHelp:Help for... in any of theUNICORNmodules(see illustration above)

• in the TOC pane, expand the headings of interest tonavigate the content structure

• click the heading of interest to open a section

navigate the online help


• in the Search pane, enter the term of interest in theinput field

• click the Search button

search for a specificterm in the online help


• in the TOCpane, expand the headingUNICORNonlinedocumentation portal and select Onlinedocumentation overview

• in the PDFmanuals section, click one of the text links

• click the illustration or the text link of the manual ofinterest

access any of themanuals in PDF format

In theMethod Editormodule:find information abouta method instruction

• open a method

• select the instruction of interest in the Instruction boxin the Text instruction pane

• press the F1 key

In the System Controlmodule:

• selectManual:Execute Manual Instructions

• expandaheadingand select the instruction of interest

• click the Help icon in the dialog



4.3 UNICORN software modules

IntroductionThree of the four UNICORNmodules are used in this Getting Started guide and describedin this section:Method Editor, System Control and Evaluation. The Administrationmodule and its icons are described in UNICORN 6 Administration and Technical Manual.

ContentsThis section contains the following subsections:

See pageSection

404.3.1 Method Editor module

434.3.2 System Control module

454.3.3 Evaluation module


4 The instrument and the software4.3 UNICORN software modules


IntroductionIn UNICORN software, the instructions to control a chromatography run are defined ina method. TheMethod Editormodule is used to create or edit such methods. Thegraphical user interface combines default settings with the ability to change settings.This simplifies method creation and replaces the method wizard in earlier versions ofUNICORN.

Predefinedmethods and phasesA method is built up by a number of phases. Each phase represents a major processstep in themethod, for example, equilibration or elution. Predefinedmethods, that includeall the phases necessary to run the system, are available for different chromatographytechniques and also for system cleaning.


4 The instrument and the software4.3 UNICORN software modules4.3.1 Method Editor module

Method Editor panesAs illustrated below, four panes show in theMethod Editor by default. Available phasesare found in the Phase Library (1), and an overview of the phases included in the activemethod is displayed in aMethod Outline (2). Detailed information about the method ispresented in the upper right-hand pane (3), containing the two tabs Phase Propertiesand Text Instructions. The gradient used in the method is displayed in a Gradientillustration (4).

1 32

4




Method Editor toolbar iconsThe table below shows theMethod Editor toolbar icons that are referred to in this GettingStarted guide.

FunctionIconFunctionIcon

Open Method Navigator:Opens theMethodNavigator where availablemethods are listed.

New Method: Opens theNew Method dialog wheremethods can be created.

Print: Opens the Printdialog from where amethod can be printed.

Save: Saves the activemethod.

Paste: Pastes a copiedmethod or folder to a newlocation.

Copy: Copies the selectedmethod or folder.

Redo: Restores the methodto the state it was in beforethe Undo command wasused.

Undo: Restores themethodto the state it was in beforethe last change.


4 The instrument and the software4.3 UNICORN software modules4.3.1 Method Editor module


IntroductionThe System Controlmodule is used to start, view, and control a method run.

System Control panesAs illustrated below, four panes show in the System Control by default. The Run Datapane (1) presents current data in numerical values, while the Chromatogram pane (2)illustrates data as curves during the entire method run. Information about the methodprogression is presented in the Run Log (3) and the current flow path is illustrated in theFlow Scheme (4).

1

2

3 4




System Control toolbar iconsThe table below shows the System Control toolbar icons that are referred to in thisGetting Started guide.


Run: Starts a method run.Open Method Navigator:Opens theMethodNavigator where availablemethods are listed.

Pause: Suspends themethod run and stops allpumps.

Hold: Suspends themethodrun, while current flow rateand valve positions aresustained.

End: Permanently ends themethod run.

Continue: Resumes forexample a held or pausedmethod run.

Connect to Systems: Opensthe Connect to Systemsdialog where systems canbe connected, andcurrentlyconnected users aredisplayed.

Customize: Opens theCustomize dialog wherecurve settings, run datagroups and run logcontents can be set.


4 The instrument and the software4.3 UNICORN software modules4.3.2 System Control module


IntroductionThe Evaluationmodule is used to evaluate the results from chromatography runs.

Evaluation panesAs illustrated below, the Evaluationmodule contains three panes. When a result isopened from the Result Navigator (1) the Chromatogram pane (2) is displayed. After apeak integration has been performed, details about the peaks are shown in the lowerright-hand pane (3), in the tabs Peak data and Integration summary. In the Evaluationmodule it is also possible to view the complete documentation about the results, and togenerate reports.

1 2

3




Evaluation toolbar iconsThe table below shows the Evaluation toolbar icons that are referred to in this GettingStarted guide.


Save: Saves the changesmade to the current result.

Open Result Navigator:Opens theResultNavigatorwhere available results arelisted.

Copy: Copies the selectedresult or folder.

Print: Opens the Printchromatograms dialogfrom where achromatogram can beprinted.

Undo: Restores themethodto the state it was in beforethe last change.

Paste: Pastes a copiedresult or folder to theselected folder.

View Documentation:Opens the Documentationdialog that contains thecomplete documentationfor a method run.

Report: Opens the Createreport dialog where areport of the result can becreated.

Peak Integrate: Opens thePeak Integrate dialog fromwhere curves can beintegrated.

Customize: Opens theCustomize dialog wherecurve settings, peak tablecontent andheader contentcan be set.


4 The instrument and the software4.3 UNICORN software modules4.3.3 Evaluation module

5 Files and folders in UNICORN

IntroductionIn the UNICORN user interface,method and result data are represented by files. All usershave a designated home folder where all methods and result files created by the userwill automatically be located. The files and folders are displayed in Navigator panes.

This chapter briefly describes the Navigator panes available in the different modules. Italso describes how to handle the files and folders.


See pageSection

485.1 Navigator overview

515.2 Handling files and folders


5 Files and folders in UNICORN

5.1 Navigator overview

IntroductionThis section describes the navigator panes that are available in all UNICORN modules,with exception for the Administrationmodule. The navigator panes can be used tolocate and open methods and results, and to handle files and folders.

In the modulesMethod Editor and System Control, the navigator panes are calledMethod Navigator and are used primarily to access methods.

In the Evaluationmodule, the navigator pane is calledResult Navigator. In this navigatorpane three different panes can be used to access the results–Results, Recent Runs andFind Results.

Show the navigator paneTo open and display the navigator pane in any of the UNICORNmodules, click theOpen...toolbar icon.

The navigator panes may either be displayed statically in the position where they open,or theAutoHide function can be selected by clicking the pin symbol in the top right-handcorner of the pane (illustrated below). If Auto Hide is selected, the panes openautomaticallywhen themouse pointer is placedover the corresponding tab, and remainopen as long as themouse pointer remains over the pane. The pane closes automaticallywhen the pointer is moved outside.


5 Files and folders in UNICORN5.1 Navigator overview

Navigator toolbar iconsThe table below shows the common navigator toolbar icons.

FunctionIcon

Open: Opens the selected file.

New folder: Creates a new folder in the folder that is currently selected.

Refresh: Updates all items in the navigator pane to the current status.

Opens the ViewDetails droplist (see illustration below)where the followingoptional information may be selected for display in the navigator pane:

• System

• Created by (user name of the person who created the original file)

• Lastmodified (date and timewhen then file or folderwas lastmodified)



Filter settingsBy default, theMethod Navigator shows only methods, and the Result Navigator onlyresults. This is the Default Filter setting for the items that are displayed. However, eachnavigator may also show items that primarily are used in another UNICORN module.The options are:

• Default Filter: only items belonging to the active module show

- InMethod Editor and System Control: "Methods, Method Queues"

- In Evaluation: "Results, DoE Results"

• All: all items show, regardless of the active module

• All except Archived: only items that are not archived show

The illustration below shows the filter settings available in theMethod Navigator.

Close the navigator paneClick the small cross in the top right-hand corner of the pane to close the navigator pane.



5.2 Handling files and folders

IntroductionThe handling of files and folders in UNICORN is similar to the generalWindows functions.This section describes how to create new folders and how to copy, move, delete andrename both files and folders. It also describes the general search function that helpsyou find the files and folders that you are looking for.

Create a folderTo arrange your files for better overview, you can create sub-folders in any folder thatyou have access to. Follow the instruction below to create a new folder in theMethodNavigator or in the Result Navigator.

ActionStep

Select the folder in which you want to create the new sub-folder.1

Click the New Folder icon.2

Result: A sub-folder named "New Folder" is created in the selected folder.

Note: The new folder is placed after all other sub-folders, butbefore any files directly in the selected folder.

Type a name for the new folder.3

Result: The new folder is renamed.


5 Files and folders in UNICORN5.2 Handling files and folders

Copy and paste files and foldersFollow the instruction below to copy and paste files and folders in theMethodNavigatoror in the Result Navigator. If you copy a folder you will also copy all the files and foldersthat it contains.

ActionStep

Select one or several files and/or folders.1

Click the Copy toolbar icon.2

Result: The selected items are copied.

Select a target folder.3

Click the Paste toolbar icon.4

Result: The copied items are pasted into the selected folder.

TIP: You can select a range of items by pressing the keyboard Shift key orseveral individual items by pressing the keyboard Ctrl key

Move files and foldersFiles and folders may be moved by drag-and-drop within the folder structure that youhave access to. The destination must always be a folder and the contents will normallybe re-sorted automatically so that:

• folders are placed first, in alphabetical order, and

• individual files are placed in alphabetical order, after the folders.

Note: The sorting order may be changed by clicking the header of eachcolumn. You can also move folders and files by using Cut and Paste inthe same manner as Copy and Paste.



Delete files and foldersFollow the instruction below to delete files and folders in theMethod Navigator or inthe Result Navigator.

Note: Top level folders, for example home folders, cannot be deleted thisway.Refer to UNICORN 6 Administration and Technical Manual for moreinformation about home folders.

ActionStep

Select the item(s) that you want to delete.1

Choose Edit:Delete.2

Result: A dialog for confirmation of the delete action opens.

Press Yes to confirm the delete action in the confirmation dialog.3

Result: The selected items are deleted.

Note: If you have chosen to delete a folder that is not empty, a specialconfirmation dialog opens. This dialog displays all the items that willbe deleted as a consequence, both items in the active UNICORNmoduleand items in other UNICORN modules (see illustration below).

Rename files and foldersFollow the instruction below to rename files and folders in theMethod Navigator or inthe Result Navigator.

ActionStep

Select the item that you want to rename.1

Choose Edit:Rename.2

Result: The current name is selected.

Type a new name.3



ActionStep

Press Enter.4

Result: The selected item is renamed.

Find files and foldersFollow the instruction below to use the Find dialog to search for files or folders in theMethod Navigator or in the Result Navigator.

ActionStep

• Select the file or folder in the navigator pane from where you want toperform the search.

• Choose Edit:Find.

Result: The Find dialog opens.

1

• Type a name or a partial name in the Find what field.

• Select none, one, or both of the the matching levels described below.

- Match whole word only: Matches only whole words and disregardspartial matches.

- Match case: Matches the case of the Find what field entry.

• Select a Direction for the search.

- Up: The search proceeds upwards in the folder structure from theselected level.

- Down: The search proceeds downwards in the folder structure fromthe selected level.

2

Click Find Next.3

Result: The first locatedmatch is selected in the navigator pane. The searchcan be continued step by step until the whole folder structure has beensearched.



6 Create a method

IntroductionAmethod is created in theMethodEditormodule. This chapter describesmethodcreationbased on predefined methods, and also how to print a method.


See pageSection

566.1 Guide to method creation

686.2 Print a method


6 Create a method

6.1 Guide to method creation

IntroductionThis section describes how to create a newmethod based on a predefinedmethod, andhow to edit a method. The section also provides an overview of the predefinedmethodsandphases that are available. You cancreatebothpurificationmethodsandmaintenancemethods. You can also add a maintenance phase at the end of a purification method.For further information about method creation, see UNICORN 6 Method Manual.

ContentsThis sections contains the following subsections:

See pageSection

576.1.1 Create and edit methods

656.1.2 Predefined methods

676.1.3 Predefined phases


6 Create a method6.1 Guide to method creation

6.1.1 Create and edit methods

IntroductionMethod creation in UNICORN 6 is performed in theMethod Editormodule. Predefinedmethods are available for a number of different chromatography techniques, for exampleaffinity chromatography and gel filtration. There are also predefinedmethods for columnpreparation and system cleaning. A method is comprised of a number of phases. Eachphase represents a major process step in the method, for example, equilibration orelution. If desired, a predefined method can be edited by adding, deleting and/orrearranging phases, and also by editing the settings of each phase included in themethod.

Illustration of the Method Editoruser interface

The illustration and table below shows theMethod Editormodule and its panes.

1 32

4


6 Create a method6.1 Guide to method creation6.1.1 Create and edit methods

DescriptionPart

Phase Library: Displays all available phases.1

MethodOutline: Presents an overview of the phases included in the activemethod.

2

Phase Properties: Presents detailed settings for the selected phase.3

Gradient: Illustrates the gradient used in the active method.4

Create a methodFollow the instruction below to create and edit a chromatographic method based on apredefined method.

ActionStep

Open theMethod Editormodule and click the New Method icon.1

Result: The New Method dialog opens.

Select System and Predefined Method in the dialog. Click OK.2

Result: The phases included in the chosen method show in theMethodOutline pane, and the default settings for each of the phases show in thePhase Properties pane.



ActionStep

In the Phase Properties pane of theMethod Settings phase, edit generalsettings like Column type andMethod Base Unit. UNICORN automaticallycalculates correct settings for volume, flow rate, and pressure limits basedon the selected column type.

3

The illustration below shows theMethod Outline pane and the PhaseProperties pane of theMethod Settings phase.



ActionStep

In the Phase Properties pane of theMethod Settings phase, click theResultName & Location button to specify the name and location of the resultsfrom the method runs.

4

Result: The Result Name & Location dialog opens.

In the Result Name & Location dialog:

• Set Result location by clicking the Browse button and select a folder inwhich to save the results. By default, the results will be saved in yourhome folder.

• SelectResult name. With the default selectionMethod name the resultswill be named with the method name together with a serial number.

• Click OK to confirm and close the dialog.



ActionStep

In the Phase Properties pane of theMethod Settings phase, click the StartProtocol button if you wish to include a start protocol.

5

Result: The Start Protocol dialog opens.

In the Start Protocol dialog:

• Select items to display at method start. Result Name and Location isselected by default.

• Click OK to confirm and close the dialog.

Select the next phase in theMethod Outline pane by clicking it, and chooseappropriate parameter values in the Phase Properties pane.

6

Repeat step 6 until all phases are edited.7

TIP: If the instrument will not be used for a couple of days orlonger, add one or several System CIP phases to the endof the method as described below.



ActionStep

Click the Save the method icon.8

Result: The Save As dialog opens.

In the Save As dialog:9

• Select a target folder to enable the Save button.

• Type a Name for the method.

• Select a System from the list.

• Click the Save button.

Result: The created method is saved in the selected folder.



Add phases to a methodFollow the instruction below to add phases to a method.

ActionStep

Select the desired phase in the Phase Library pane.1

Drag-and-drop the phase to requested position in theMethodOutline pane.The phase can be placed at any postion below theMethod Settings phase.

2

Result: The phase is included in the method at the the requested position.

Delete phases from a methodFollow the instruction below to delete phases from a method.

ActionStep

Select the phase to delete in theMethod Outline pane.1

Note: TheMethod Settings phase cannot be deleted.

Click the Delete button located below theMethod Outline pane.2

Result: The selected phase is excluded from the method.

Rearrange phases within amethod

Follow the instruction below to rearrange phases within a method.

ActionStep

Select the phase to move in theMethod Outline pane.1

Note: TheMethod Settings phase cannot be moved.

Drag-and-drop the phase to requested position in theMethodOutline pane.2

Result: The selected phase is moved to the requested position.



Choose a maintenance methodA number of predefined methods for preparation and cleaning are available. Use thesemaintenance methods to prepare and clean the system and columns, and to fill thesystem and columns with storage solution. The table below gives suggestions for whatmethods and solutions to choose for the different purposes.

SolutionPhase/MethodIf you wish to...

suitable buffersSystem PreparationPrepare tubing and instrument modulesbefore a run

suitable buffersColumn PreparationPrepare column before a run

0.5-1 M NaOHSystem CIPClean tubing and modules

0.5-1 M NaOHColumn CIPClean column

20% EthanolSystem CIPLeave system in storage solution

20% EthanolColumn CIPLeave column in storage solution

TIP: CIP (Cleaning-In-Place) and preparation of columns and system maybe run either as separate predefined methods or as phases includedin chromatographic methods.

TIP: The System CIP and System Preparation phases are designed to useone cleaning solution each. The SystemPreparationmethod includestwo SystemPreparationphases and the SystemCIPmethod includesthree SystemCIP phases. To use additional solutions sequentially, addphases to the method.

Note: Usually 0.5-1 M NaOH is used for cleaning of the system and column.However, before choosing cleaning agent always consider the mediaand buffers used, and also the chemical resistance of the column.



6.1.2 Predefined methodsThe predefined methods that are available are described in the table below.

DescriptionMethod

After equilibration and sample application, the protein ofinterest is adsorbed to the column ligand. After a wash toremove unbound sample, elution is performed either by usinga buffer containing a competitor to displace the protein ofinterest, or by changing the pH or ionic strength. Finally, thecolumn is re-equilibrated with start buffer.

Affinitychromatography(AC)

After equilibrationand sample application, negatively chargedproteins are adsorbed to the column ligand. After a wash, toremoveunbound sample, elution is performedusingagradientof increasing salt concentration (of e.g., NaCl). Finally, thecolumn is washed and re-equilibrated with start buffer.

Anion ExchangeChromatography(AIEX)

After equilibration and sample application, positively chargedproteins are adsorbed to the column ligand. After a wash, toremoveunbound sample, elution is performedusingagradientof increasing salt concentration (of e.g., NaCl). Finally, thecolumn is washed and re-equilibrated with start buffer.

Cation ExchangeChromatography(CIEX)

After equilibrationand sample application, elution is performedusingapHgradient. Theproteins separateandelute accordingto their isoelectric points. Finally, the column is re-equilibrated.

Chromatofocusing(CF)

The column is filled with a cleaning solution. Select inletpositions. Enter the solution identity, volume, flow rate andincubation time. By adding steps, several cleaning solutionscan be used. Suggestions for cleaning steps are available fora number of column types.

Column CIP

After equilibration of the column, sample is injected via acapillary loopandeluted isocratically. A non-adsorbing samplelike acetone or salt should be used. After the run, calculatecolumn performance in the Evaluationmodule. The efficiencyof the column is determined in terms of height equivalent toa theoretical plate (HETP), and the peak asymmetry factor (As).The result is logged in the column logbook.

ColumnPerformance Test

The column is filled with buffer solution. Select inlet positions.Enter the solution identity, volume, flow rate and incubationtime. By adding steps, several preparation solutions can beused.

ColumnPreparation



6.1.2 Predefined methods

DescriptionMethod

After equilibration and sample application, the proteins areeluted isocratically. This technique is commonly used for bufferexchange.

Desalting (DS)

After equilibration and sample application, proteins separateand elute according to their size (largest first).

Gel filtration (GF)

After equilibration and sample application (use a buffercontaining a high salt concentration, for example 2 MAmmonium Sulphate) hydrophobic proteins are adsorbed tothe column ligand. After a wash to remove unbound sample,elution is performed using a gradient of decreasing saltconcentration. Finally, the column is washed andre-equilibrated with start buffer.

HydrophobicInteractionChromatography(HIC)

After equilibration and sample application, hydrophobicproteins adsorb to the column ligand. After a wash to removeunboundsample, elution is performedbygeneratingagradientof a non-polar, organic solvent such as Acetonitrile. Finally,the column is washed and re-equilibrated.

Reversed PhaseChromatography(RPC)

The system is filled with cleaning solution. Select for exampleinlets, outlets and column positions to be cleaned. ThreeSystem CIP phases are included in the method to facilitatethe use of three different cleaning solution. Additional SystemCIP phases can be added from the Phase Library if desired.

System CIP

The system is filled with preparation solution. Select forexample inlets, outlets and column positions to be prepared.Two System Preparation phases are included in the method.Additional System Preparation phases can be added fromthe Phase Library if desired.

SystemPreparation


6 Create a method6.1 Guide to method creation6.1.2 Predefined methods

6.1.3 Predefined phasesThe predefined phases that are available in the predefined methods and in the PhaseLibrary are described in the table below.

DescriptionPhase

The first, and mandatory, phase in any method. Definescommon parameters used in the subsequent phases.

Method Settings

Equilibrates the column before purification, or re-equilibratesthe column after purification.

Equilibration

Applies sample to the column. Defines the sample applicationtechnique, the sample volume, and the handling offlowthrough.

SampleApplication

Washes out unbound sample after sample application orremoves strongly bound proteins after elution.

ColumnWash

Elutes the sample from the column. Defines parameters forthe elution and fractionation settings.

Elution

Prepares the column before use by removing the storagesolution and equilibrating the column. By adding steps, severalpreparation solutions can be used sequentially.

ColumnPreparation

Cleans the columnafter purification runs by rinsing the columnwith a cleaning solution to remove unspecifically boundproteins. By adding steps, several cleaning solutions can beused sequentially.

Column CIP

Prepares the systembefore a runby removing storage solutionand filling the system and inlets with buffer solution. Onepreparation solution is used per phase.

SystemPreparation

Cleans the systemafter purification runs by rinsing the systemwith a cleaning solution. One cleaning solution is used perphase.

System CIP

Tests the efficiency of a packed column in terms of heightequivalent to a theoretical plate (HETP), and the peakasymmetry factor (As).

ColumnPerformance Test



6.1.3 Predefined phases

6.2 Print a method

IntroductionThis section describes how to print the text instructions and variables of a method.UNICORN uses the printers and printer settings that are installed on your computer.

How to print a methodFollow the instruction below to print a method.

ActionStep

Open theMethod Editormodule and click theOpenMethodNavigator iconin the toolbar.

1

Result: TheMethod Navigator pane opens.

Select themethod to print and click theOpenaMethod icon in the navigatortoolbar.

2

Result: The selected method opens.


6 Create a method6.2 Print a method

ActionStep

Click the Print icon.3

Result: The Print dialog opens.

In the Print dialog:4

• select Printer

• click OK

Result: The method is printed.


6 Create a method6.2 Print a method

7 Prepare the system for a run

IntroductionFollow this chapter to perform the necessary preparations of the system before startinga run.

WARNINGAlways use appropriate personal protective equipment duringoperation and maintenance of ÄKTAavant system.


See pageSection

717.1 Before you prepare the system

727.2 Prepare the flow path

787.3 Prime buffer inlets and purge System pumps

837.4 Connect a column

887.5 Calibrate the pH monitor

907.6 Prepare the Fraction collector


7 Prepare the system for a run

7.1 Before you prepare the system

IntroductionIt is important to prepare the system in accordance with the settings in the method tobe run. Before preparing the system, check the settings in theMethod Editor andmakesure that all accessories to be used are available.

ChecklistMake sure to prepare the system in accordance with the settings in the method to berun. Remember to check:

• which valve ports to use for inlets and outlets

• which cassettes with corresponding deep well plates and/or tubes to use in theFraction collector, if applicable

• which column type to use

• which column position to use

• which sample application technique to use

• that the pH electrode is connected, if applicable

• if it is a reversed phase chromatography (RPC) run

WARNINGRPC runs with 100% acetonitrile. Always replace the greenPEEK tubing between the used system pump and the pressuremonitor with orange PEEK tubing, i.d. 0.5 mm, before runningRPC with 100% acetonitrile. Set the System pressure alarm to10 MPa.

WARNINGFraction collector. Do not fractionate flammable liquids.Whenrunning RPCmethods, or other procedures using solvent basedbuffers, collect fractions through the outlet valve.


7 Prepare the system for a run7.1 Before you prepare the system

7.2 Prepare the flow path

IntroductionThe flowpath contains tubing, valves, pumpsandmonitors. This sectiongives anoverviewof the flow path and describes how to prepare the flow path before a run.

Illustration of the flow pathThe illustrationbelow shows the flow schemeas illustrated in the SystemControlmodule.The individual instrument modules are presented in the table below.

1

9

8

7

6

5

3

42

12

11

10

16

15 14

13


7 Prepare the system for a run7.2 Prepare the flow path

DescriptionPart

Sample inlet valve1

Sample pump2

Inlet valve A3

System pump A4

Quaternary valve5

Inlet valve B6

System pump B7

Injection valve8

Capillary loop or Superloop9

Column valve10

Column11

UV monitor12

Conductivity monitor13

pH valve with pH monitor and flow restrictor14

Outlet valve15

Fraction collector16



Tubing and connectorsThe table below showswhat types of tubing and connectors to use for different purposes.

PhotoConnectorTubingScope of use

Tubing connector, 1/8"+ Ferrule (yellow), 1/8"

Teflon™, o.d.1/8", i.d. 1.6mm

Inlet tubing

Fingertight connector,1/16"

PEEK, o.d.1/16", i.d. 0.50mm

Tubing toconnectcolumns

Fingertight connector,1/16"

Tefzel™, o.d.1/8", i.d. 1.0mm

Waste tubing

Inlet portsThe table below shows the inlet ports of Inlet valve A, Inlet valve B and Sample inlet valve.

IllustrationsValves and ports

Inlet valve A

Inlet ports:

• A1-A7




Inlet valve B

Inlet ports:

• B1-B7

Sample inlet valve

Inlet ports:

• S1-S7, Buff



Waste portsThe table below shows the waste ports of Injection valve, pH valve, and Outlet valve.


Injection valve

Waste ports:

• W1, W2

pH valve

Waste port:

• W3

Outlet valve

Waste port:

• W



Prepare inlets and outletsFollow the instruction below to prepare the inlets and outlets that are to be used in themethod run.

ActionStep

Connect inlet and outlet tubing to the ports that are to be used.1

Immerse all inlet tubing that is to be used during the method run in thecorrect buffers.

2

Immerse the waste tubing from the Injection valve, the pH valve, the Outletvalve, and the Fraction collector into a sufficiently largewaste vessel (typically2-10 liters).

3



7.3 Prime buffer inlets and purge System pumps

IntroductionBefore usage of the System pumps, it is important to:

1 Prime the inlets (fill the buffer inlets with liquid).

2 Purge the System pumps (remove air from the pumps).

Prime buffer inletsFollow the instruction below to prime the buffer inlets. Each buffer inlet that is to be usedduring the method run has to be primed.

ActionStep

Make sure that all buffer inlet tubing that is to be used during the methodrun is immersed in the correct buffers.

1

Make sure that the waste tube connected to Injection valve portW1 isimmersed in a waste vessel.

2

Open the System Controlmodule in UNICORN, and selectManual:ExecuteManual Instructions.

3

Result: TheManual instructions dialog opens.


7 Prepare the system for a run7.3 Prime buffer inlets and purge System pumps

ActionStep

TIP: If desired, the flow rate and volume of the pump washcan be edited before the pump wash is started. In theManual instructions dialog, select Pumps andpressures:Wash settingsand edit the settings for Systemflow rate and Systempumpwash volume. Click Execute.

4

In theManual instructions dialog:

• Select Pumps and pressures:Pump wash, and select the inlet to beprimed from the Inlet A or Inlet B drop-down list.

• If several inlets are to be used, click Insert to insert a selected inlet inthe Instruction execution list. Insert all inlets to be used in the Instructionexecution list.

• Click .

Result: A pumpwash of the first inlet in the Instruction execution list starts.



ActionStep

Connect a 25-30ml syringe to one of the purge valves of the selected systempump. Make sure that the syringe fits tightly into the purge connector.

5

Open the purge valve by turning it counterclockwise about 3/4 of a turn.Draw 5-10 ml of liquid slowly into the syringe.

6

Close the purge valve by turning it clockwise. Disconnect the syringe anddiscard its contents.

7

Click the Continue button on the Instrument display to start a pump washof the next inlet in the Instruction execution list.

8

Repeat steps 4-7 until all inlets in the Instruction execution list have beenprimed (filled with liquid).

9



Purge the system pumpsFollow the instruction below to purge System pump A and/or System pump B. Note thatboth pump heads of each system pump have to be purged. Also, note that both systempumps have to be purged after priming of buffer inlets.

TIP: Prime all buffer inlets and purge SystempumpBbefore finally purgingSystempumpAwith start buffer. The instrument is then ready for startof a method run.

ActionStep

In theManual instructions dialog:1

• Select Pumps and pressures:Pump wash, and select one inlet from theInlet A or Inlet B drop-down list. Preferably, choose the buffer inlet to beused in the beginning of the method run.

• Click .

Result: A pump wash of the selected inlet starts.



ActionStep

Connect a 25-30 ml syringe to the left purge valve of the selected systempump. Make sure that the syringe fits tightly into the purge connector.

2

Open the purge valve by turning it counterclockwise about 3/4 of a turn.Draw 5-10 ml of liquid slowly into the syringe with a rate of about 1 ml/s.

3

Close the purge valve by turning it clockwise. Disconnect the syringe anddiscard its contents.

4

Connect the syringe to the purge valve on the right pump head of theselected system pump, and repeat steps 3-4.

5



7.4 Connect a column

IntroductionThis section describes how to connect a column to the instrument, using a columnholderand without introducing air into the flow path. Several types of column holders areavailable for ÄKTAavant.

WARNINGBefore connecting a column to the ÄKTAavant instrument, readthe instructions for use of the column. To avoid exposing the columnto excessive pressure, make sure that the pressure limit is set tothe specified maximum pressure of the column.

Methods automatically include a pressure alarm based on the tolerance of the chosencolumn type. However, when running manual runs you have to set the pressure limitsyourself.

CAUTION

Donot overtightenwhenconnectingcolumns.Overtighteningmightrupture the connectors or squeeze the tubing and thereby resultin high backpressure.


7 Prepare the system for a run7.4 Connect a column

Attach a column holder andconnect a column to theinstrument

Follow the instruction below to connect a column to the instrument. Always use a columnholder. The column is connected to two opposite parts of the Column valve, usingappropriate tubing and connectors.

ActionStep

Choose a column according to the column selections made in the methodto be run.

1

Attach an appropriate column holder to the rail on the instrument.2

Attach the column to the column holder.3



ActionStep

Connect a suitable tubing to a Column valve port, for example port 2A ifcolumn position 2 was chosen in the method to be run.

4

Open the System Controlmodule and selectManual:Execute ManualInstructions.

5

Result: TheManual instructions dialog opens.


• Select Flow path:Column position.

• Choose the Position used for connection of the column, in this examplePosition 2.

• Choose the Flow Direction Down flow.

• Click .

Result: The Column valve switches to position 2.



ActionStep


• Select Pumps and pressure:System flow.

• Enter a low Flow rate (e.g., 0.2 ml/min).

• Click .

Result: A system flow of 0.2 ml/min starts.

When buffer leaves the tube on port 2A in a continuous mode and the toppart of the column is filled with buffer, connect the tube to the top of thecolumn.

8



ActionStep

Connect a tube to the bottom of the column.9

When buffer leaves the tube at the bottom of the column in a continuousmode, connect this tube to the Column valve. Use the port opposite to theone already connected to the column, in this example port 2B.

10

Click the End icon in the System Control toolbar to end the run.11



7.5 Calibrate the pH monitor

IntroductionIf pH will be measured during the chromatographic run, the pH monitor should becalibrated before the run is started. Use two pH calibration buffers with a difference ofat least one pH unit. Preferably use a pH standard buffer pH 4 or 7 as the first calibrationpoint, and a pH standard buffer close to the lowest or highest pH you need to measureas your second point. Allow the buffers to reach the operating temperature before use.

Note: Do not run a system flow during pH calibration.

Calibrate the pH monitorFollow the instruction below to perform the calibration.

ActionStep

Open the System Controlmodule and select System:Calibrate.1

Result: The Calibration dialog opens.

Set the pHmonitor as theMonitor to calibrate by selecting pH from the list.2

Click .3

Result: The pH valve switches to the calibration position.

Enter the pH of the first pH standard buffer in the pH for buffer 1 field.4


7 Prepare the system for a run7.5 Calibrate the pH monitor

ActionStep

Fill a syringe with approximately 10 ml of the first pH standard buffer.Connect the syringe to the Luer connector in pH valve port Cal, and injectthe buffer.

5

When the Current value is stable, click .6

Wash the pH flow cell by injecting water into pH valve port Cal using a newsyringe.

7

Enter the pH of the second pH standard buffer in the pH for buffer 2 field.8

Repeat steps 5-6 using the second pH standard buffer.9

Result: The calibration date and time are displayed in the dialog, and alsovalues for Calibrated electrode slope and Asymmetry potential at pH 7.

Is the Calibrated electrode slope ≥ 80% and the Asymmetry potential atpH 7 inside the interval ±60 mV?

10

• If Yes: Click to switch the pH valve back to the default position,and to close the Calibration dialog.

• If No: Clean the pH electrode, and repeat the calibration procedure. Ifthis does not help, replace the electrode. For information about cleaningand replacing the pH electrode, see User Manual Chapter Maintenance.

CAUTION

pH-electrode. Handle the pH-electrode with care. The glass tipmay break and cause injury.


7 Prepare the system for a run7.5 Calibrate the pH monitor

7.6 Prepare the Fraction collector

IntroductionThis section describes how to prepare the Fraction collector. For information regardingthe types of deep well plates, tubes and cassettes, see ÄKTAavant and UNICORN 6 UserManual.

WARNINGFraction collector. Do not fractionate flammable liquids. Whenrunning RPC methods, or other procedures using solvent basedbuffers, collect fractions through the outlet valve.

Prepare the Fraction collectorFollow the instruction below to prepare the Fraction collector before a run. Choose deepwell plates and/or tubes according to the fractionation settings in themethod to be run,and choose appropriate cassettes.

ActionStep

If youare to use cassetteswith theQuickRelease function, open the cassettes.1


7 Prepare the system for a run7.6 Prepare the Fraction collector

ActionStep

Place the tubes and deep well plates in the cassettes. Make sure that thedeep well plates are rotated so that the well marked A1 is positioned abovethe A1 marking on the cassette.

2

Close the cassettes that have the QuickRelease function.3



ActionStep

Place the cassettes on the Cassette tray. Make sure that the Cassette typecode (see illustration below) faces the front of the tray marked with the GElogo.

4

Open the Frac drawer by pressing the handle upwards, and pulling out thedrawer.

5



ActionStep

Place the Cassette tray on the Tray support of the Frac drawer. Make surethat the front of the tray (marked with the GE logo) faces the front of thedrawer and is hooked onto the two pins.

6

Close the Frac drawer. Make sure that it snaps into closed position.7

Cassette identificationAfter the Frac drawer has been closed, the Frac arm scans the Cassette type code ofeach cassette to identify the cassette types. If deep well plates are used, the instrumentalso identifies the types of deep well plates.

WARNINGMoving parts in Fraction collector. Do not open the Fractioncollector drawer when the Fraction collector is active. If you needto access the Fraction collector, press Pause, and make sure thatthe movement has stopped before opening the drawer.



View Fraction collector contentTo view the content of the Fraction collector, open the System controlmodule and selectView:Fraction Collector Content.



8 Run a method

IntroductionThis chapter describes how to start up and run a method, and also how to handle thesystem after the run.


See pageSection

968.1 Before you start

988.2 Applying the sample

1018.3 Start a method run

1068.4 Monitor the run

1118.5 After run procedures


8 Run a method

8.1 Before you start

IntroductionBefore starting a run, it is necessary to read and understand the information in thissection and to perform the checks listed below.

ChecklistMake sure that the system is correctly prepared. Check that:

• the system is prepared according to chapter 6.

• a suitable column has been selected for the application (consider target protein andpressure range).

• the buffer inlet tubing is immersed in correct buffer vessels (consider solution identityand volume).

• the waste tubing is immersed into appropriate waste vessels (consider vessel sizeand vessel material).

• no tubing is twisted and the flow path is free from leakage.

Hold, pause or stop the runAt the end of a method the run stops automatically. All pumps stop and the systemmakes an acoustic end signal and displays End in the Run Log.

To interrupt amethod during a run youmay use theHold, Pause or End icons in SystemControl. A held or paused method run can be resumed by using the Continue icon. Seethe instructions in the table below.


click the Hold icon.temporarily hold themethod,with currentflow rate and valve positions sustained

click the Pause icon.temporarily pause the method, and stopall pumps


8 Run a method8.1 Before you start


click the Continue icon.resume, for example, a held or pausedmethod run.

Note: An ended methodcannot be continued.

click the End icon.permanently end the run

Note: When ending amethod run in advance, it is possible to save the partialresult.

Warnings concerning use ofhazardous substances

WARNINGHazardous chemicals during run.When using hazardouschemicals, run System CIP and Column CIP to flush the entiresystem tubingwith distilledwater, before service andmaintenance.

WARNINGHazardous biological agents during run.When using hazardousbiological agents, run System CIP and Column CIP to flush theentire system tubing with bacteriostatic solution (e.g., NaOH)followedby a neutral buffer and finally distilledwater, before serviceand maintenance.


8 Run a method8.1 Before you start

8.2 Applying the sample

IntroductionA number of different sample application techniques are available. The sample can beapplied either directly onto the column using the Sample pump, or via a loop. A loop canbe filled either manually or by use of the Sample pump. This section describes sampleapplication using a syringe tomanually fill a capillary loop. The two stages of the sampleapplication are described in the table below. For detailed instructions and informationregarding the different sample applications techniques, see User Manual.

DescriptionStage

The capillary loop is filled with sample.Load

The sample is injected onto the column.Inject

Sample application through acapillary loop

A capillary loop is manually filled with sample using a syringe connected to the Injectionvalve port Syr. During the method run, the sample is automatically injected onto thecolumn. The loop is emptied and washed out using buffer from the system pumps. Thetotal buffer volume to be used for emptying and washing the capillary loop is set in thePhase Properties tab of the Sample Application phase at Empty loop with.


8 Run a method8.2 Applying the sample

TIP: Empty the capillary loopwith a buffer volume that exceeds the volumeof the loop. This will ensure that the loop is completely emptied.

How to fill a capillary loopFollow the instruction below to fill the capillary loop with sample.

ActionStep

Connect a suitable capillary loop to Injection valve ports LoopF (fill) andLoopE (empty).

1

Fill a syringe with sample.2

Connect the syringe to the Injection valve port Syr.3



ActionStep

Load sample into the capillary loop. To avoid sample loss due to siphoning,leave the syringe in the port until the sample has been injected onto thecolumn during the run.

4

TIP: It is recommended to overload the loop tomake sure thatthe loop is completely filled. Excess of sample will leavethe valve through portW1.



8.3 Start a method run

IntroductionThis section describes how to start a run using a previously created method. If ColumnLogbook was enabled during installation of the software, registration and selection ofindividual columns is possible at method start. The Column Logbook function includes,for example, logging of column history. For further information on column handling,please refer to UNICORN 6 Method Manual.

Choose and start a methodThe instruction below describes how to open a method and start a run.

ActionStep

Open theSystemControlmoduleandclick the iconOpenMethodNavigator.1

Result: TheMethod Navigator pane opens.

Select the method to run, and click the Run icon.2

Result: The Start Protocol dialog opens.

Step through the displayed pages in the Start Protocol, add requested inputand make appropriate changes if necessary. Click Next.

3


8 Run a method8.3 Start a method run

ActionStep

Click Finish on the last page of the Start Protocol.4

Result:

• If column logging was chosen at installation of UNICORN and a columntypewas selectedatmethod creation, the Select Columnsdialog opens.Continue with steps 5-9.

• If column logging was not chosen at installation of UNICORN and/or nocolumn type was selected at method creation, the run starts directly.

Is the column to be used already registered?5

• If No, continue to step 6.

• If Yes, continue to step 9.

In the Select Columns dialog, click New.6

Result: The first New Column dialog opens.



ActionStep

Register the column using the 2D Barcode scanner as follows:7

• Make sure that the pointer is placed in the first position of theCode field.

• Point the 2DBarcode scanner towards thedatamatrix tagon the column.

• Press and hold the trigger to create a beam.

• When the 2D Barcode scanner beeps, the column ID is registered anddisplayed in the dialog.

• Alternatively, manually enter the column ID, that you find on the columnlabel, in the dialog using your keyboard.

• Click Continue.

Result: The expanded New Column dialog opens.



ActionStep

In the expanded New Column dialog:8

• enter Alias (optional)

• select Technique and Column type from the drop-down lists.

• click OK.

TIP: Alias can be used for easy identification of a column.

Result: The entered information is saved and the dialog closes.



ActionStep

In the Select Columns dialog:9

• Select Enter ID.

• Use the 2D Barcode scanner (see step 7) to enter the column ID.

• Alternatively, choose Select ID and select the column individual to beused in the run from the drop-down list.

• Click OK.

Result: The run starts. All necessary actions occur automatically accordingto the method, including ending of the run.



8.4 Monitor the run

IntroductionYou may follow the on-going method run in the System Controlmodule. The currentsystem status is shown in the System state panel in the Run Data pane. For example,it may state Run,Wash or Hold. The same information is also shown on the Instrumentdisplay.

This section describes the data shown in System Control during a run, the layout of themodule and the procedure to customize the view of the different panes.

Illustrationof the SystemControluser interface

In the System Controlmodule, four panes show by default (see illustration and tablebelow).

1

2

3 4


8 Run a method8.4 Monitor the run

DescriptionPart

Run Data: Presents current run data values.1

Chromatogram: Illustrates data as curves.2

Run Log: Presents all registered actions.3

Flow Scheme: Illustrates the current flow path.4

Customize Run Data

TheRunDatapane shows real-timedatafrom available monitors during the run.

To change what parameters to displayin the pane:

• Click the Customize icon to open theCustomize dialog.

• Under the Run Data Groups tab,select the parameters to display.

• Click OK to close the dialog.

Customize ChromatogramThe Chromatogram pane displays a graphical presentation of registered data fromavailable monitors during the run.

To change the chromatogram properties:

• Click the Customize icon to open the Customize dialog.



• Make appropriate selections under the tabs described in the table below.

CustomizationTab

Select curves to be displayed.Curves

Customize the appearance of the displayed curves.Curve style and color

Select base unit (time or volume) and set X-axis scale.X-axis

Select which Y-axes to display and set Y-axis scale forthe different curves.

Y-axis


Note: These operations determine which curves are displayed and theirappearance in theChromatogram pane and on printouts. The originalraw data curves can never be modified or removed from the result.

View details in ChromatogramFollow the instructions below to view selected parts of the chromatogram.


position the mouse pointer over the curve of interest.identify a curve

Result: The curve name is displayed.

click the curve of interest.select which curve theY-axis scale refers to Result: The Y-axis scale changes to the unit applicable for

the curve.

select the area of interest by moving the pointer with theleft mouse button held down.

zoom in the curves

Result: The selected area is enlarged.

click the right mouse button and select Reset Zoom.zoom out again

Result: The original area is restored in one step.



Customize Run LogThe Run Log pane displays all registered actions during the run. Scroll up to see theentire log.

To change what items to display in the pane:


• Under the Run Log tab, select the items to display.


Note: These operations determinewhich data are shown in theRun Log paneduring a run. All original raw data are saved and can never bemodifiedor removed from the result.



Flow schemeThe Flow Scheme pane displays the current flow path during the run. Color indicationis applied, as shown in the table below. Real-time data frommonitors are also displayedin the flow scheme. See illustration below.

IndicationColor

Open flow pathGreen

Closed flow pathGrey

AlarmRed (not shown)



8.5 After run procedures

IntroductionThis section describes how to clean the instrument and columnsafter a chromatographicrun, and how to prepare the system for storage.

The instrument and the columns should be cleaned between the runs. This will prevent,for example, sample contamination, protein precipitation and column clogging. If theinstrument is not going to be used for a couple of days or longer, the instrument, columnsand the pH flow cell should be filled with storage solution. For further information aboutcleaning and maintenance procedures, see User Manual.

TIP: To clean and fill the instrument and columns with storage solution,use System CIP and Column CIP either as separate, predefinedmethods or as phases included in a chromatographic method.

WARNINGHazardouschemicalsduringmaintenance.Whenusinghazardouschemicals for system or column cleaning, wash the system orcolumns with a neutral solution in the last phase or step.

System cleaningAfter a method run is completed, perform the following:

• Rinse the instrument with one or several cleaning solution(s) (e.g., NaOH, buffersolution or distilled water) using System CIP.

• Empty the Fraction collector.

• Clean all spills on the instrument and on the bench using a moist tissue.

• Empty the waste vessel.

• Check that the pH electrode is in appropriate buffer.

System storageIf the instrument is not going to be used for a couple of days or longer, also perform thefollowing:

• Fill the system and inlets with storage solution (e.g., 20% ethanol) using System CIP.


8 Run a method8.5 After run procedures

Column cleaningAfter a method run is completed, perform the following:

• Clean the column with one or several cleaning solution(s) using Column CIP.

Column storageIf the column is not going to be used for a couple of days or longer, also perform thefollowing:

• Fill the column with storage solution (e.g., 20% ethanol) using Column CIP.

pH electrode storageFollow the instruction below to fill the pH flow cell with storage solution. The calibrationfunction is used to switch the pH valve. However, no calibration is performed.

ActionStep

Open the System Controlmodule and select System:Calibrate.1

Result: The Calibration dialog opens.

In theCalibrationdialog, selectpH from theMonitor to calibrate drop-downlist.

2

Press .3

Result: The pH valve switches to the calibration position.



ActionStep

Prepare at least 10 ml storage solution by mixing equal volumes of astandard buffer pH 4 and a 1 M Potassium Nitrate (KNO3) solution.

4

Fill a syringe with approximately 10 ml of the storage solution. Connect thesyringe to the pH valve port Cal, and inject the storage solution.

5

Press .6

Result: The pH valve switches back to the default position and theCalibrationdialog closes. No calibration is performed.

Log off or exit UNICORNFollow the instruction below to log off or exit UNICORN. This can be performed from anyof the UNICORN modules.


select File:Log off.log off UNICORN

Result: All open UNICORN modules close and the Log Ondialog opens.




select File:Exit UNICORN.exit UNICORN

Result: All open UNICORN modules close.

Note: If an editedmethod or result is open and not savedwhen you try to exitor log off UNICORN, you will see a warning. Click Yes to save,No to exitwithout saving, or Cancel to stay logged on.

Shut down the instrumentSwitch off the instrument by pressing the Power switch to the O position.



9 Evaluate and print the results

IntroductionThis chapter describes how to use the Evaluationmodule to evaluate and print theresults of a run. For further information, please refer to UNICORN 6 Evaluation Manual.


See pageSection

1169.1 View the results

1209.2 Peak integration

1259.3 Print the results


9 Evaluate and print the results

9.1 View the results

IntroductionA result holds a complete record of the run, including method, system settings,chromatogram, and run log. This section describes how to view a result and how tochange the view settings in the Evaluationmodule.

Open a resultFollow the instruction below to open a result.

ActionStep

Open the Evaluationmodule and click the Open Result Navigator icon.1

Result: The Result Navigator opens.

Select the Results tab.2

Select the result to open and click theOpen a Result button in the navigatortoolbar.

3

Result: The result is opened in a Chromatogram pane. Availablechromatograms and peak tables are displayed.


9 Evaluate and print the results9.1 View the results

Illustrationof theChromatogrampane

The Chromatogram pane displays the curves from the method run.

Customize ChromatogramTo change the chromatogram properties:


• Make appropriate selections under the tabs described in the table below.



CustomizationTab

Select curves to be displayed.Curves

Customize the appearance of the displayed curves.Curve style and color

Select base unit (time, CV or volume) and set X-axisscale.

X-axis

Select which y-axes to display and set Y-axis scale forthe different curves.

Y-axis

Select which parameters (variables, questions and/ornotes) to display in the header information at top of thechromatogram.

Header

• Click OK to save the changes and close the dialog.

Note: These operations determine which curves are displayed and theirappearance in theChromatogram pane and on printouts. The originalraw data curves can never be modified or removed from the result.

View details in ChromatogramFollow the instructions below to view selected parts of the chromatogram.


position the mouse pointer over the curve of interest.identify a curve

Result: The curve name is displayed.

click the curve of interest.select which curve theY-axis scale refers to Result: The Y-axis scale changes to the unit applicable for

the curve.

select the area of interest by moving the pointer with theleft mouse button held down.

zoom in on the curves

Result: The selected area is enlarged.

click the right mouse button and select Reset Zoom.zoom out again

Result: The original area is restored in one step.



View documentationTo view documentation of the run:

• Click the View Documentation icon to open the Documentation dialog.

• Select the tab of interest. Which tabs are displayed depends on the settings in theactive method.




9.2 Peak integration

IntroductionPeak integration is used to identify and measure curve characteristics, including peakareas, retention times and peak widths. This section describes how to perform peakintegration using the UNICORN software. For further information on peak integration,please refer to UNICORN 6 Evaluation Manual.

Perform peak integrationFollow the instruction below to integrate the curves in a result.

ActionStep

Open the Evaluationmodule and open a result from the Result Navigator.1

Click the Peak Integrate icon.2

Result: The Peak Integrate dialog opens.


9 Evaluate and print the results9.2 Peak integration

ActionStep

In the Peak Integrate dialog:3

• Select which curve to integrate (e.g., UV1_280 for proteins).

• Select a target peak table for storage of the result.

• Select Calculate baseline from the Baseline list.

• Click OK.

Result: The Peak Table is displayed beneath the active chromatogram, andthe start and end points of each peak are marked by vertical marks in thechromatogram.



Illustration of Chromatogramand Peak Table

The illustration below shows a Chromatogram pane including a Peak Table.



Customize Peak TableBy default, each peak in the Peak Table is displayed with its retention time, area andheight. Follow the instruction below to display other peak data.


• Under the Peak Table tab:

- Select which data to display in the peak tables from the Select peak tablecolumns list.

- Click OK to display the settings and close the dialog.



Study peak characteristicsThe peaks in the integrated chromatogramare automatically labeledwith their retentionvalues. The Peak Table is calculated using the same base unit (ml, CV ormin) as selectedin the chromatogram. Follow the instruction below to find the peak of interest.

ActionStep

Find the retention value above a peak in the Chromatogram.1

Search the Peak Table for the same retention value.2

On the same row, find the property of interest (e.g., Area).3

Save resultsFollow the instruction below to save your changes.


click the Save icon in the toolbar.save your changes tothe original result

• select File:Save As to open the Save Result As dialog.

• In the Save Result As dialog:

- select the location for the new result

- write a name for the new result

- click Save.

save the changed resultas a new result



9.3 Print the results

IntroductionThis section describes how to print a chromatogram and a standard format report.UNICORN uses the printers and printer settings that are installed on your computer.

Customize the viewBefore printing, make sure that the view is adjusted to show what is needed in thereport/printout. Customize the chromatogramand/or peak table and view details in thechromatogram according to the instructions above. Before printing, check that:

• correct curves are displayed

• correct data are displayed in the peak table

• a suitable scaling of the axes has been selected

• a suitable zoom factor has been selected


9 Evaluate and print the results9.3 Print the results

Print a chromatogram and apeak table

Follow the instruction below to print a chromatogram and, if peak integration has beenperformed, the related peak table.

ActionStep

Open the Evaluationmodule and open a result.1

Click the Print icon.2

Result: The Print Chromatograms dialog opens.

In the Print Chromatograms dialog:3

• Select Printer and Print Format.

• Click the Preview button.

Result: A previewof the chromatogramdisplays in theCustomize Reportwindow.



ActionStep

Are you satisfied with the layout?4


• If No, select File:Exit to return to the Print Chromatograms dialog andchoose another print format.

In the Customize Report window, click the Print icon.5


In the Print dialog, select Printer from the list and click OK.6

Result: Your chromatogram is printed.



Print a reportFollow the instruction below to print a standard format report.

ActionStep

Open the Evaluationmodule and open a result.1

Click the Report icon.2

Result: The Generate Report dialog opens.

In the Generate Report dialog:3

• Select any of the predefined formats from the list.

• Click the Preview button.

Result: A previewof the report displays in theCustomize Reportwindow.

Are you satisfied with the layout?4


• If No, select File:Exit to return to theGenerate Report dialog and chooseanother format.



ActionStep

In the Customize Report dialog, click the Print icon.5


In the Print dialog, select Printer from the list and click OK.6

Result: Your report is printed.



10 Maintenance

IntroductionThis chapter provides schedules for preventive maintenance that should be performedby the user of ÄKTAavant. Regular maintenance is essential for reliable function andresults. Refer to ÄKTAavant and UNICORN 6 User Manual for detailed instructions.

WARNINGAlways use appropriate personal protective equipment duringoperation and maintenance of ÄKTAavant system.


See pageSection

13110.1 Maintenance program


10 Maintenance

10.1 Maintenance program

IntroductionThis section describes the preventive maintenance to be performed on ÄKTAavant.

The maintenance is divided into:

• Daily maintenance

• Weekly maintenance

• Monthly maintenance

• Bi-annual maintenance

• Maintenance when required

Periodic maintenance programThe following periodic maintenance should be performed by the user of ÄKTAavant.

Maintenance actionInterval

Calibrate the pH monitorDaily

Calibrate pressure monitorsWeekly

Change pump rinsing solutionWeekly

Check the flow restrictorMonthly

Clean the UV flow cellBi-annual

Replace pH electrodeBi-annual


10 Maintenance10.1 Maintenance program

Maintenance when requiredThe followingmaintenance should beperformedby the user of ÄKTAavantwhen required.

Maintenance action

Clean the instrument externally

Perform System CIP

Perform Column CIP

Clean the Fraction collector

Replace tubing and connectors

Storage of pH electrode

Clean the pH electrode

Clean the conductivity flow cell

Calibrate the Conductivity monitor

Calibrate the UV monitor

Replace mixer

Replace online filter

Replace o-ring in mixer

Replace UV flow cell

Replace flow restrictor

Replace inlet filters

Clean the check valves

Replace check valves

Replace pump piston seals

Replace pump pistons

Replace pump rinsing system tubing

Replace valve modules


10 Maintenance10.1 Maintenance program

GE, imagination at work and GE monogram are trademarks of General ElectricCompany.

For local office contact information, visitwww.gelifesciences.com/contact ÄKTA, ÄKTAavant, Drop Design, and UNICORN are trademarks of GE Healthcare

companies.GE Healthcare Bio-Sciences ABBjörkgatan 30751 84 UppsalaSweden

All third party trademarks are the property of their respective owners.

© 2009 General Electric Company – All rights reserved.First published Sep. 2009

All goods and services are sold subject to the terms and conditions of sale ofthe company within GE Healthcare which supplies them. A copy of these termsand conditions is available on request. Contact your local GE Healthcarerepresentative for the most current information.www.gelifesciences.com/AKTAUNICORN: Any use of this software is subject to GE Healthcare Standard SoftwareEnd-User License Agreement for Life Sciences Software Products.

GE Healthcare Europe GmbHMunzinger Strasse 5, D-79111 Freiburg, Germany

GE Healthcare UK LimitedAmersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK

GE Healthcare Bio-Sciences Corp.800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA

GE Healthcare Bio-Sciences KKSanken Bldg.3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan

28-9440-69 AB 09/2009

imagination at work

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