KTA and UNICORN A short introduction

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

Current folder Current folder where the where the listed items listed items are saved are saved

Method files Method files

Result files Result files

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Creating New Users

Select User Setup Select User Setup from the from the Administration Administration Window Window

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Creating New Users

Select New Select New

Enter User Details Enter User Details

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

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Creating New Users

After the user has After the user has been created, Select been created, Select Edit Edit

Change the Group to Change the Group to the desired level of the desired level of access to the access to the instrument (see instrument (see below) * below) *

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

Select which folders Select which folders will be shared with will be shared with other users and which other users and which System the user is System the user is allowed to operate allowed to operate
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Handle files

Right-click on the file name!

Allows options such as copy, move, rename etc

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System control workspace

Run Data Run Data

Curves Curves

Flow scheme Flow scheme

Logbook Logbook

Manual instructions box Manual instructions box

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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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System Control, Manual

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

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kta Explorer 100 Air flowpath

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kta 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 Select the type of chromatography Selection 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 19 / GE / applicable) 02/12/2007

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

equilibration equilibration

Sample Sample application application

Wash 1 Wash 1

elution elution

Wash 2 Wash 2

reequilibration reequilibration

Methods are divided into logical parts Useful to import blocks from other methods
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Blocks
A group of instructions A method contains a main method and a set of blocks To use a block, it must Execute be : Block wash created called
Main Method Block equilibration

Main Method

Block equilibration Block wash Block sample application

Block equilibration Block wash Block sample application

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Text Method Mode

Method: desaltscout

Flexibility using Variables Flexibility using Variables Adapt value during Start Up Protocol

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Text Method Mode

Method: 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 KTA and UNICORN

US

ASK

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