ÄKTA™ and UNICORN™

A short introduction

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UNICORN Basics

UNICORN is divided into four main sections:

UNICORN Manager: Used to organise data (result files, user set up etc) and configure the system for different optional components

Method Editor: Create and edit methods to control the instrument automatically

System Control: View run data and control the instrument manually (in real time)

Evaluation: Present the chromatographic data post acquisition in flexible, user defined formats

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UNICORN Manager

Used to:• Create new users and define their level of access to the instrument. Once created, users will have their own folder where results and methods are automatically saved (highly recommended)

• Configure the software to recognise new hardware components

• Run Method Queues (methods run one after the other or to start at a certain time)

• Perform system diagnostics

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UNICORN Manager Layout

Method filesMethod files Result filesResult files

Current folder where the

listed items are saved

Current folder where the

listed items are saved

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Creating New Users

Select User Setup

from the Administration

Window

Select User Setup from the

Administration Window

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Creating New UsersSelect New…Select New…

Enter User DetailsEnter User Details

Create a new folder where the methods and results for that user will be saved

Create a new folder where the methods and results for that user will be saved

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Creating New UsersAfter the user has

been created, Select Edit…

After the user has been created, Select

Edit…

Change the Group to the desired level of

access to the instrument (see

below) *

Change the Group to the desired level of

access to the instrument (see

below) *

Select which folders will be shared with

other users and which System the user is allowed to operate

Select which folders will be shared with

other users and which System the user is allowed to operate

* An Administrator has full access to instrument including the creation of new users. To see the different levels of access select Access Groups

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Handle files

Right-click on the file name!

Right-click on the file name!

Allows options such as copy, move, rename etc

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System control workspace

CurvesCurves

Flow schemeFlow scheme

LogbookLogbook Manual instructions boxManual instructions box

Run DataRun Data

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Manual instructions

The system is receiving instructions from UNICORN™. These instructions are either :

• automatically sent (by a method)

• manually sent (by the user)

Menu 'Manual'

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Allows you to interact with the system manually, by giving appropriate instructions from a dialogue box, or interact with a running method.

Instructions are essentially the same as in Method Editor

System Control, Manual

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Äkta Explorer 100 Air flowpath

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Äkta Explorer 100 Air, main path

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Flow direction valve

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Injection valve, Load sample

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Injection valve, Inject sample

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Method Wizard

The Method Wizard is the easiest way to create chromatographic methods

The Wizard is interactive and flexible

Most of the parameters are defaulted to tried and tested values

The Wizard steps you through method creation in the same order as you would perform a run (technique selection, equilibration, sample injection, fraction collection, buffer gradient etc

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Select The Method Wizard

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Wizard Continued… Method and Column Selection Select the type of chromatography

Ion exchange, gel filtration etc

Select the column from the list (default

parameters such as flow rate and pressure

limits are set for that particular column)

Select the column position on the column

Valve (if applicable)

Flexible flow rates = flows that can be modified

at the beginning of each run

Flow Regulation = flows that will automatically

adjust downward if the column pressure

exceeds pre-set level

Buffer Prep = adding conc. buffer, salt, pH

modifier and water to four different buffer lines

allowing the system to mix the correct

proportion to each to attain a desired pH (if

applicable)

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Wizard Continued… Select Wavelength and Buffers

Select the three UV wavelengths to monitor

during the run (UV-900 modules only)

Select the pump inlets for buffer A and buffer B

(only applicable to AKTA units with buffer

selection or buffer prep valves)

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Wizard Continued… Equilibration

Select the starting concentration of buffer B

to equilibrate the column prior to injecting

the sample

Select the length of the equilibration (N.B. This

value is set as Column Volumes (CV) i.e if you

use a 1mL HiTrap column then it will

equilibrate for 5mL)

Instead of just using a Default value, you can

ask the system to ‘Watch Equilibration’. Select

the type of watch equilibration function from

the drop down menu (e.g. continue with the

method if the system reaches stable

conductivity)

A ‘Watch Function’ can be set on many different parameters – it

is simply defined as a conditional control that if a parameter is

met then the system proceeds to the next step

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Wizard Continued… Sample Injection

Select the injection technique – Manual

injection is available for all systems. If you

have a P-960 Sample Pump, then other

options such as direct loading of the column

become available using the drop down menu

(next page of this handout).

Select how much buffer will be used to empty

(flush) the loop onto the column. Good practice

is to enter a value here twice as much as the

loop volume attached to the injection valve.

For example if you have a 1mL loop, enter a

value of 2mL here.

Remember to keep the syringe in place in the

injection valve until after the system has

injected the sample on the column (avoids

sample loss or air getting in the system)

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Wizard Continued… Auto Sample Injection

If you have a Sample Pump (P-960), you can

select Sample Pump Direct Loading from this

drop down menu.

Select the volume of sample the sample pump

will inject on to the column (due to the dead

volume suggest values higher than 5mL).

Alternatively, if you have an Air Sensor on the

Sample Pump – tick the box ‘Inject All Sample’

– the system will continue to load until air is

detected

Tell the system which port your sample is

located (8-port valve designated line S1 to S8)

These two tick boxes tell the system to flush the sample line –

choose either to flush the lines to waste using (say) 20% ethanol

in sample line S8 or to flush the remainder of the sample onto

the column with 10mL of buffer A located in the S8 line

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Wizard Continued… Unbound Protein Fraction Collection

If you have a Frac-950, you can select the

rack from this drop down menu (e.g. the

yellow rack = 18 & 30mm tubes).

Select the Wash Out Unbound Sample with 2

CV – continues to run equilibration buffer

(buffer A usually) to remove the unbound

protein from the column before the gradient

begins.

Tell the system to collect this unbound

fraction, i.e. what volume is to be collected in

each fraction and the tube type. If you select

30mm tube it will collect this fraction in 50mL

Falcon tubes.

The system remembers where the last fraction was collected

from the previous run (if the same method is used). Tell the

system if you want to continue collecting fractions in the next

available tube or recycle back to the First Tube.

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Wizard Continued… Bound Protein Fraction Collection

Choose between Fixed Volume or Peak

Fractionation – Peak Fractionation only

collects when a peak is detected (user inputs

these parameters that define a peak on the

next step of the Wizard

Tell the system to collect this bound fraction,

i.e. what volume to be collected in each

fraction and the tube type. If you select 18mm

tube it will collect this fraction in 15mL Falcon

tubes in the centre of the yellow rack.

The system remembers where the last fraction was collected

from the previous run (if the same method is used). Tell the

system if you want to continue collecting fractions in the next

available tube or recycle back to the First Tube.

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Wizard Continued… Gradient Elution Profile

Select the Elution Profile. Choose between

Linear Gradient, Isocratic or Segmented

(Step) (user inputs these parameters that

define a Segmented gradient on the next

step of the Wizard

If you select Linear gradient, tell the system

the final concentration in terms of % Buffer B

and how many CVs it will take to reach this

concentration

Select to Clean After Elution with 100% B (5CV) to remove any

strongly bound contaminant from the column. Typically do not

select Reequilibration as this is performed at the beginning of

the next chromatography run

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Run Setup Mode

At the completion of the Method Wizard, the following page appears.

Notes and questions to give information to the users

Possibility to change all of the variables that were entered during the Method Wizard

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Blocks = Chromatographic steps

Methods are divided into logical parts

Useful to import blocks from other methods

equilibrationequilibration Sampleapplication

Sampleapplication

Wash 1Wash 1 elutionelution Wash 2Wash 2 reequilibrationreequilibration

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A group of instructions

Block equilibration

Main Method

Block equilibrationBlock wash

Block sample application

A method contains a main method and a set of blocks

Blocks

To use a block, it must be :

• created

• called

Main Method

Block equilibrationBlock wash

Block sample application

ExecuteBlock wash

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Text Method ModeMethod: desaltscout

Flexibility using VariablesFlexibility using Variables

Adapt value during

Start Up Protocol

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Text Method ModeMethod: desaltscout

Conditional Control using

Watch commands

IF a certain condition is met

THEN an action will follow

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Start a method

Start from Main Menu,

• Select Methods,

• Select the appropriate method using right mouse button,

• Select Run

• Follow the Start Up Protocol and fill in as appropriate

Start from System Control Menu

• Select File

• Select Run

• Select appropriate method

• Follow the Start Up Protocol and fill in as appropriate

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Open a Result in Evaluation

After the run, the result will be stored in Main Menu, Results

To open a Result from Main Menu

• Select the Result file with left mouse button and double click

To open a Result from Evaluation Menu

• Select File

• Select Open

• Select Result

• Select the appropriate Result file form the menu

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HELP

On using ÄKTA and UNICORN

ASK

US