Home

UNICORN™ 6.1 - GE Healthcare Life Sciences

1. Folder name System HCE DBILIAIUNICORNS Unicom Adam AnionE schangeblethod Demo System E Columnvashhethad Demo Spstem DesaltingM ethad Demo System Py DesaltwashandaleX i PA sari 3 To open the method queue e Click the Open button located in the toolbar of the Method Navigator pane Open or e double click the selected method queue Result The Method Queue dialog is opened with the details for the opened method queue Note If a method contained in the method queue has been altered since the last time it was saved an information dialog will be displayed Delete a method queue The table below describes how to delete a method queue from the database 236 UNICORN 6 1 Method Manual 28 9817 65 AB Step Action 7 Method queues 7 3 Edit a method queue 1 In the Method Editor Click the Open Method Navigator icon in the Toolbar or select File Open T New Method Ctrl N New Method Queue i Close or select View Method Navigator Toolbar Method Navigator X Gradient Phase Library Flow Scheme Restore to Default Result The Method Navigator is displayed Select the method queue to be deleted in the Folder name column Method Navigator Open irae Methods Method Qi E Folder name System HCE DBILIAUNICORN Unicom Adarn El AnionEschangeMethod Demo System Al Column ashk ethod Demo System al Desa
2. Select the phase to delete from the library e Click the Delete button at the bottom of the Phase Library pane or e Right click the phase and select Delete P A ea eo Supettoop 50 Insert K Export Result The phase is removed from the Phase Library 68 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 6 Scale or convert methods 3 6 Scale or convert methods Introduction UNICORN methods are always created specifically for a designated system and thus also for a specific system type e g AKTA avant 25 However it is often useful to convert a method that was originally created for a system of one type for use with another a system of another type e g AKTA avant 150 The converted method is created as a copy of the original method The original method remains saved When a method is converted it can also be scaled for use with a different column type than what it was originally created for Both processes are described in this section Tip If you wish to use a method for another system of the same AKTA avant system type that it was originally created for you only need to choose File Save As select the new system and save the method with another name To only change the selected column type you should edit the method change the column selection and save the edited method Prerequisites The following items should be considered to ensure that the method con
3. Parameters for Hold until Signal uv 1 v Test Greater than v Value 6000 0 6000 0 Var 0 0 mAU Timeout 0 00 9999 99 Var 5 002 cv C Infinite Note Click the Insert button Edit Variable Import Block Result The new instruction is inserted in the Text Instructions area Instructions that share the same breakpoint as the Hold until instruction but are placed after it in the method will be executed after the Hold until conditions have been met 342 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 3 Specific instructions 9 3 5 Messages and Set marks 9 3 5 Messages and Set marks When to use a message Messages are used to inform the operator of the progress of the run or to prompt the user for an action It is a good idea to issue messages at critical points in the method for example in combination with a Pause instruction to inform the operator that the inlet tube needs to be moved to another inlet Insert a Message instruction The Message instruction can be used to set up a message that will be displayed for the user during the execution of the method run The message can be for information in a screen only or it can require a signature before the user can control the system The messages are all added to the logbook text The table below describes how to add a Message instruction to the method Step Action 1 e Select
4. Ctrl 2 Ctri Result Mame amp Location Method Notes Zut Ctrl Copy Ctrl C Paste Ctri Mo Delete Del l Rename Fz or e Right click on the phase and select Delete from the context menu Result The phase is removed from the method UNICORN 6 1 Method Manual 28 9817 65 AB 49 3 Create and edit methods 3 3 Edit methods and phases 3 3 2 Edit the method outline Copy cut and paste phases ina Note It is only possible to copy cut and paste phases in the Method Outline pane not in the Phase Library The features copy cut and paste phases can be used to add delete and rearrange phases in the Method Outline The table below describes the copy cut and paste a phase select the phase and right click the phase and select Copy or use the shortcut Ctrl C or select Edit Copy select the phase and right click the phase and select Cut or use the shortcut Ctrl X or select Edit Cut Note The phase to be pasted will be pasted below the phase highlighted in the Method Outline Select the appropriate phase in the Method Outline Then right click the highlighted phase in the Method Outline and select Paste or use the shortcut Ctrl V or select Edit Paste method features copy a phase cut a phase paste a phase 50 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 4 Set general method options for the met
5. 1 In the Select Column Type area clear the Predefined types box Select Column Type Show by technique Ary ka Show by access label Predefined types Global types user created Personal types user created Result Only Global and Personal column types are displayed in the Column types list Select the column type s to be deleted in the Column types list To select several column types use the Ctrl or Shift keyboard keys In the Column Type Parameters tab click Delete Result The Confirm Column Type Delete dialog opens Confirm Column Type Delete A 4re you sure you want to permanently delete the selected Column Typetsi Click Yes to delete the column type Result The column type is permanently deleted from the database Export column types Note It is not possible to export predefined column types from the database The table below describes how to export Global and Personal column types from the database 254 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 2 Handling column types Step Action 1 In the Select Column Type area clear the Predefined types box Select Column Type Show by technique Ary w Show by access label Predefined types Global types user created Personal types user created Result Only Global and Personal column types are displayed in the Column types list Z Select the column type s to be exported in the Column types list To sele
6. SEHCI2DP3JD UNICORNAUNI FS Adam B Anne Bridget Charlie David E David T oo Show Global and Personal Procedures Francine J l mport as z Christine J F Gerald gt Close In the Import Evaluation Procedures dialog e Select the appropriate procedure to import in the Select procedure to import field e Itis possible to change the name of the procedure to be displayed in your method by changing the name in the Import as field e Click Import to import the procedure Note Only Global procedures and your own Personal procedures are shown in the list Note Itis also possible to import a procedure saved in another method by browsing to the appropriate folder and selecting the method containing the procedure The procedure will be listed in the Se lect procedure to import field and can be imported as described above Result The evaluation procedure is listed in the Evaluation Procedures dialog 58 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 4 Set general method options for the method Step Action 4 Evaluation Procedures Evaluation Procedure 1 Make sure the box in front of the evaluation procedure is checked to include it in the method Click Close Result The evaluation procedure is included in the method Note It is possible to edit an existing evaluation procedure by selecting it and clicking Edit The edits will
7. Note It is possible to add new responses to the experimental design in Evaluation These new responses will not be added to the method file as opposed to responses added in the Method Editor When all responses are defined select the Factors amp Design tab Design of Experiments Responses Factors amp Design Experiment Factors Name Abbreviation Range Method Variable Method Phase Click Add to define a factor Add Edit Delete Design selection Objective Screening v Design Advanced Total number of runs including center points UNICORN 6 1 Method Manual 28 9817 65 AB 131 5 Design of Experiments 5 2 Create an experimental design 5 2 1 Set up an experimental design Step Action 4 To add factors to the Design of Experiments click Add Result The Add Factor dialog opens For detailed information about how to define and add a factor see Sec tion 5 2 2 Add responses and factors to an experimental design on page 138 Add Factor Predefined Bed Height v User defined Abbreviation BeHe Unit Type Quantitative Quantitative multilevel O Qualitative Settings Low value High value Center point Method phase Method Settings Variable wt Don t connect the factor to a method variable 132 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 1 Set up an exp
8. Method Notes Unit selection Method Base Unit ey v Flow Rate Unit ml min Monitor settings Wavelengths 190 700 nm uv nm uv 2 254 nm guwa i nm Enable pH monitoring Enable air sensor alarm Inlet A Inlet B C Sample inlet Column Logbook Enable logging of C Column Performance Test Cy CiP Duration amp Variables 20 UNICORN 6 1 Method Manual 28 9817 65 AB 2 The UNICORN Method Editor 2 2 Methods in UNICORN 6 1 Method structure A method always starts with the Method Settings phase This phase contains general settings that affect the rest of the method e g Column type Flow rate and Method Base Unit If changing Column type UNICORN will automatically calculate correct set tings for volume flow rate and pressure limits Subsequent phases reflect steps included in the chromatography run The figure below shows a method with the different phases in the Method Outline in the Method Editor Method Settings Equilibration Sample Application Column Wash Elution Equilibration Working with methods It is recommended to create and edit methods using Phase Properties Phases can easily be dragged and dropped into the Method Outline from the Phase Library and the phases are easily rearranged Settings for each phase are set in the Phase Properties pane When working like this the text method is automatically built up in the Text Inst
9. Phase Library MDL versio in Column CIP Method Settings Column Performance Test Sample Application Column Preparation y Column Wash Column Wash v Elution a OP Peeesssssesesesssecccsssscses y Sample Application Column Wash System CIP y Equilibration System Preparation User Defined Delete Save Phase Method Overview UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 3 Edit methods and phases 3 3 2 Edit the method outline Step Action 2 e Click the Insert button located below the Phase Library Ingat or e double click the selected phase or e select Phases Insert Phase from Library Insert Phase From Library X Save Phase or e right click the phase in the Phase Library and select Insert UOUSUCESUSUCEGUSHCESESEEEOEUEE ES Equilibratian eert ELETT T n Sample Applicaii Result The phase is included in the method and highlighted in blue Continue with step 3 if adding the User Defined phase Column CIP Method Settings Soluma eer Vert ee Equilibration Column Preparation hd Column Wash Sample Application Elution v PPPS REE SESE LESTE TEESE ETE Column Wash Equilibration TTIPI Sette se seessrseseseses zat y Sample Application Elution System CIP y Column Wash System Preparation y User Defined Equilibration UNICORN 6 1 Method Manual 28 9817 65 AB 45 3 Create and edit methods 3
10. Choose which Export items to include by checking the appropriate boxes Choose whether to include All phases or only a specific Phase by selecting the appropriate Phase range option UNICORN 6 1 Method Manual 28 9817 65 AB 97 3 Create and edit methods 3 11 Import and export methods Step Action 5 To add further information to export click the Options gt gt button Result The Include options will be expanded Export Externally My Affinity Method Export tems Fhase range Test Instructions Al Variable List Phase Include Properties Gesign of experiment C Start protocol Signatures Method Duration BufferPro recipes Result name amp location Eval ET s EA a ha n t SACU E DeLay es ry ma pme Lalm 6 Select information to add to the text file by checking the appropriate options Note Information that is not included in the method will appear greyed out and cannot be selected 7 To save the text file with the selected information included click the Save As button Result The Export dialog opens Export Method to File Save in O Methods Folder My Recent Documents Filename My Affinity Method i Savesstype ASCI ase x 98 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 11 Import and export methods Step Action 8 Choose whether to save to an ASCII file or to an Excel file from the Save as type drop
11. Run Parameters Details Parameters Value Unit Parameters Unit Technique fi Anion Exchange Hardware diameter 1 6 cm Column volume 20 106 ml Bed height 10 0 Max pre column pressure 0 5 MPa Typical loading range 200 1400 mg Max delta column pressure 0 3 MPa Total liquid volume t 18 1 ml Default flowrate 3 0 ml min Void volume Vo ml Max flowrate 5 0 ml min Typical peak width at base 15 0 ml Default linear flowrate 89 52 cmh Average particle diameter 34 0 pm Max linear flowrate 149 21 cmh Molecular weight range Mr Min pH value short term 1 Ordering Information Max pH value short term 14 hice Value Name HiLoad 16 10 Q Sepharose HP Code number 17 1064 01 Medium name Q Sepharose High Performance Medium code number 17 1014 01 Hardware name XK 16 20 Hardware code number 18 8773 01 Min pH value long term 2 Max pH value long term 12 Function Select Column Type area Shows the available column types in the Column Handling dialog The list can be filtered to display column types for a specific technique and or access label Column Type Parameters tab Shows the parameters for the selected column type in the Column types list See Section 8 2 Handling column types on page 247 for more information UNICORN 6 1 Method Manual 28 9817 65 AB 243 8 Column Handling 8 1 Overview Function Column Logbook tab Shows available individual columns for the selected column type in th
12. Step Action 1 Open a new predefined Intelligent Packing method UNICORN 6 1 Method Manual 28 9817 65 AB 283 8 Column Handling 8 5 Intelligent Packing of AxiChrom columns 284 Step Action 2 In the Method Settings phase e Select the AxiChrom column type you created previously Note All the default settings for an Intelligent Packing method will be generated when the AxiChrom column is selected including target bed height and media The default column position will be selected It is recommended not to change any settings in the Method Settings phase of the method 3 In the Intelligent Packing phase e Select GE approved packing settings default or e Custom packing settings Tip The GE approved packing settings have been validated by GE Healthcare If you wish to use other settings for example other media or other bed height settings you must select Custom packing settings 4 If you selected to enter your own Custom packing settings you can edit the following settings e Select to Pack by Packing Factor and choose a packing factor value or Pack to the target bed height e Change the adapter velocity and e Select to use flow conditioning If you selected to use GE approved packing settings proceed with the step below Note It is not recommended to change the default position selections in the subsequent steps 5 If necessary select the Inlets for hydraulic chamber liquid and for the mobile phase 6 If necessa
13. e All types Prints information for all column types in the database e All shown types Prints information for all column types displayed in the Column types list e Selected types Prints information for the column type s selected in the Column types list 5 Select which type of information to include when printing the information e Check the Include the type s parameters box to include the information from the Run parameters Details and Ordering Information fields in the Column Type Parameters tab e Check the Include the associated columns box to include the Column ID and alias of the individual columns registered for the column type Check the Include column s parameters to include the parameters for each individual column registered for the column typeis 6 Click OK Result The selected information for the column type s is printed UNICORN 6 1 Method Manual 28 9817 65 AB 259 8 Column Handling 8 3 Handling individual columns 8 3 Handling individual columns Introduction Individual columns are handled on the Column Logbook tab in the Column Handling dialog The Column Logbook enables tracing of the run history for an individual column for example how many CIP runs have been performed using that column Individual columns are always connected to a particular column type Note The Column Logbook tab is only displayed if this option was selected when installing UNICORN Working with columns is primarily done i
14. ml min ml min cmh cmh Required information O Global Personal 248 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 2 Handling column types Step Action 2 If adding a column type for which the column hardware and medium are not made by GE Healthcare continue to step 4 3 e Select the GE Healthcare hardware type for the new column type in the drop down list To filter the drop down list to only show hardware types with certain diameters enter the diameter range in cm in the Min and Max fields for Show hardware types by diameter cm above e Select the GE Healthcare medium type for the new column type in the drop down list To filter the drop down list to only show medium types for a specific separation technique choose the appropriate technique in the Show medium types by technique drop down list above Result The following parameters are automatically filled in can be edited if appropriate Run Parameters Details Parameters Value Unit Parameters Value Unit Technique Affinity v Hardware diameter 1 0 cm Column volume ml Bed height cm Max pre column pressure 0 1 MPa Typical loading range mg Max delta column pressure MPa Total liquid volume Vt mi Default flowrate ml min Void volume Vo ml Max flowrate ml min Typical peak width at base ml Default linear flowrate cmh Average particle diameter 90 0 Max linear flowrate cmh Molecular weight range Min pH val
15. or e click the Save icon l LE Result The Save As dialog opens with the folder where the original method is saved open by default ie e Select the desired target folder e type anew method name in the Name field and e click the Save button Note The original method remains after the converted method is saved However the converted method will replace the original if you choose to save the converted method with the same name in the same folder as the original Note The flow rate and or pressure settings in the method will automatically be adjusted if the maximum flow rate and or pressure values for the target system is exceeded after the conversion The maximum settings for the target system will be used by default Note In case the original method contains instructions that are not supported by the new system this will be indicated in the method outline of the converted method as a red cross on the phase containing these instructions To be able to run the method on the new system you need to replace or re move the invalid instructions in the Text Instructions pane Invalid instructions are indicated with red square symbols in the text instructions You can also replace the phase with a predefined phase from the phase li brary The method after conversion The conversion will adjust the following settings to the appropriate values for the selected new system e Gradient delay e System wash v
16. 8 Column Handling 8 5 Intelligent Packing of AxiChrom columns 8 5 Intelligent Packing of AxiChrom columns Introduction UNICORN 6 1 features a solution for Intelligent Packing of AxiChrom columns The AxiChrom col umn family feature hands free packing using in ternal hydraulic axial compression Used with AKTA avant 150 systems Intelligent Packing of AxiChrom columns can be performed using either a predefined Intelligent Packing method or by creating a user defined method including an Intel ligent Packing phase The UNICORN 6 1 Method Manual provides an overview how to apply the Intelligent Packing method and how to create the individual AxiChrom column in UNICORN Column Handling More infor mation about the use of the AxiChrom column is available in the operating instructions for the columns UNICORN 6 1 Method Manual 28 9817 65 AB 2 9 8 Column Handling 8 5 Intelligent Packing of AxiChrom columns AxiChrom column types and individual AxiChrom columns In order to obtain correct default parameter values for the Intelligent Packing method phase a specific AxiChrom column type must be created based on the AxiChrom hardware and the selected media This is described below in Create an AxiChrom column type The default values for the created AxiChrom column type will be applied e g for the bed height when this column type is used in a method When the column is to be packed an individual AxiChrom
17. 90 3 Create and edit methods 3 9 Fraction collection Fractionation setup The following table describes the steps needed to set up fraction collection in the Phase Properties pane Step Action 1 Select the phase for which fractionation is required in the method outline and select the Phase Properties pane Method Sating Sample Ap picetion Te Ler hn saron few tat anim Meth Settings Fim nate LOWI mime P 8 Rubisco Elution ig 2 pct eamnple deen kaipi Md w L E a Equiibration OA Gavia dros cohen pop ipa andy eo i i ote ri FE keg wih Empty bop wath 1 00 ni Sangir vikaar nil A Uses the eres misti atin Method Semngi ire A AT 3 x irat ri an x O F es yere oath fe selected batten bution C iniri baigi iiiki a LY feel E000 6000 1 Cqulbration Fite Prachhnaie katara O i Cale Fiectronaton lype Fived volare haconston avai iF EE oe Firari dirila D dap rel plate w a C in waila eda att esisel Pesh frephernglen ce pirate Feed hasieran vuma 200 mi U0 220 Paak ribet valid si o a Note Text edited phases will show the fractionation options as vari ables in the Phase Variables list see Chapter 9 Text edit methods on page 286 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 9 Fraction collection Step Action 2 Using the Fractionate radio buttons select the fractionation type required for this phase e using outlet valve enables fraction collection using
18. A Eon the flow io ad mepes s E 00I Phate Platina Ourz dod B00 Phare Emabiialion Ouwa ma i n 2 Uva maruahy prepared butter wm o E kwi la 3 miad pa 3 E rahia ph monirig Bitak pari brain Taira Pm kfin im w Dihi A D lss Buttes hosi buie preparanon Apatin mr parar siam En Wa METHOD SETTINGS x oe belie Sea ee irin B Ted 1 oT Tanda ri pH ii 10 54 pomana z ped Care i 00 0109 Callan lataa Prd Loop Merag Eni kg of He Chora a i O iian Pime Tait i LGF It is possible to use the text editor in Text Instructions to create a phase from scratch and to edit methods Instructions are then created edited one by one This can be an option for fine tuning or optimization of a method If the text editor is used Phase Properties will subsequently only show a list of variables for the phase as shown in the following illustration This can always be restored by clicking on the Restore Phase Properties button Phases that have been edited in the text editor are noted with a blue letter T as shown in the illustration below 22 UNICORN 6 1 Method Manual 28 9817 65 AB 2 The UNICORN Method Editor 2 2 Methods in UNICORN 6 1 Cal Phase Properties Text Instructions T Elution T This phase has been test edited Phase Variables Block Variable Value Range ELUTION Inlet A AL y o Inlet B B1 v Flow rate ml min 1 500 0 000 25 000 Pressure control Pre column pressure v Start frac Elution Frac tube type Elution 96
19. Action In the New Method dialog e select a System e select a Predefined Method or select an Empty Method to be created from scratch e click OK Result The Method Outline pane shows the included phases for the chosen method and the default settings for the phase highlighted in the Phase Properties pane If an empty method was selected only the Method Settings phase is shown this phase is always included in all methods Phase Properties Test Instructions T Method Satire Cii bekatini Fana Mame b Larsin PENS Show by lechraye irem F thanga C mama E Coors Ga Bry k Mahad Han T Column yoma 0100 m Eemple Appike Prectune bral preu 200 MPa 00 00 TPP Freres ine debircotenn S000 Hie PODE 20 00 Method Basa Ev a E Use fines renc okma Wash Flees Fiat Lied fbi Caium poton Euer w x Horan irgi E bataia reska 130 700 nn Fleet rate TOO abn LON 2 000 ua 720 ra y Control the How bo avced cwerpnesnune Owe Colas Wah Ciwa mm CE Uia maily piigad butlers inaia fan T iiem 5 bel Enable oH moniizang Equititratice Coe Butero aema bullet peaporston Enable ai perio alur Pepa kak A in D C Sampe imes pi EIERN freconmended Core Dilemin iby neigen hot Padiri baller Dian lagik Eraihia kejgea of C Column Paionia Teal CL oF UNICORN 6 1 Method Manual 28 9817 65 AB 31 3 Create and edit methods 3 2 Create and open methods Open a method The table below describes
20. Import Column Type File name D Methods FoldersColumneH arylunts zip Select column type to import Blue Sepharose Ci0 40 DEAE for RbIC purification C Import as global 3 Make sure that the check boxes in front of the column types to be imported are checked If a column type should not be imported clear the corresponding check box 4 Check the Import as Global box if the column types should be global i e available for all users when imported Otherwise the column types will be imported as personal column types 5 Click OK Result The column types are imported into the database Note Ifa column type to be imported has the same name as an existing column type in the database you will be prompted to type a new name for that column type Type in a name and click OK UNICORN 6 1 Method Manual 28 9817 65 AB 257 8 Column Handling 8 2 Handling column types Import new column list Updated lists of predefined column types may be provided by GE Healthcare When a new list is imported it will replace all the predefined column types in the database with the updated column types Only predefined column types will be replaced User defined column types both personal and global will remain in the database Tip A column list is available in the Misc folder of the UNICORN 6 1 installation DVD The table below describes how to import a new column list into the database Step Action l In the Colu
21. Start Condition At queue start lad At ready command 1 2 3 CationExchangeMethod 4 Gi cn i E Add a Method and Start Condition to the Method list See Section 7 2 Create a method queue on page 231 Save the method queue Delete a method from a method queue The following table describes how to delete a method from the method queue for a system Step Action 1 UNICORN 6 1 Method Manual 28 9817 65 AB Open the Method Queue see Opening a method queue on page 235 Result The Method Queue dialog opens with the details for the chosen method queue In the Method Queue dialog select the method to be removed by clicking on its name in the Method list Method Queue lonExchangeMethodQueue Number of included systems System P2 9 01 v A Method Move Up Delete Start Condition 1 Colurmn WashProcedure At queue start 2 AnionExchangeMethod La Immediately after the previous method has ended 3 System P2 9 04 v Method Start Condition Ll At queue start Ja ready command pe Wait Oh 20min after the previous method has ended Gai m F ColumnwashProcedure2 1 2 3 CationExchangeMethod 4 5 239 7 Method queues 7 3 Edit a method queue Step Action 5 Delete the selected row by clicking on the Delete button Result The method will be deleted from the method queue Save the method queue Change order of methods ina method queue
22. Step Action 6 If outliers are detected try to identify why The table below gives a few ex amples of why outliers may be detected You may also look at the plots in the extended analysis to get more information about the experiment What to do Check the individual result and that correct response values have been entered If the run has failed consider per forming new experiments and re place the run Replace run results on page 167 for information about how to replace a run result The run may also be excluded from the experiment setup Results that are true outliers should be excluded Generate model on page 163 for information about how to exclude a run from the experiment Deviat ing ex peri ments Check that the correct response values have been entered Check the individual result Consider performing new experi ments to verify the deviation If the results are indeed valid the model may be inappropriate for the area See Section 5 4 2 Gener ate model on page 160 for how to check en tered response data Analyze and interpret the model basic analysis Before you can use the model and draw conclusions from it the model needs to be an alyzed to investigate if the model gives a good reflection of the experiment data Note The plots must be analyzed for each response A model can be good for one response but not for another In some cases a good model
23. Using this approach interaction effects between parameters might be missed that could lead to better optimization of a process In the DoE approach process parameters are allowed to vary simultaneously thus al lowing the effect of each parameter individually as well as the combined effect of pa rameters to be estimated Each parameter may have an optimum but when combining the parameters values may be found that together give a new optimum even better than the optima for the separate parameters The illustration below shows the different approaches in a graphical way Traditional approach DoE approach lt Optimum Optimum N ab 4 ab E o p o jae Parameter 1 Parameter 1 Parameter 2 Obtain reliable maps of the system Experiments are performed to assess the conditions for best processes or to obtain the product characteristics required In order to make the necessary decisions we need tools or ways to make this as intuitive and easy as possible In the evaluation of DoE results different plots are created from the model Decision making becomes more reliable when using tools that benefit from the created model This map of the process helps to decide on for example how to progress or whether the process is already optimized Is the process robust What experiments can be performed to verify the process DoE in UNICORN 118 In UNICORN DoE is used to systematically create an optimized s
24. 0 00 Inlet A Al itinlet A 0 00 Inlet B B1 iInlet B 0 00 System flow 1 000 Flow rate ml min Pre column pressure itPressure control 0 00 End_Block Mi 0 00 Phase Rubisco Elution Mi 0 00 Phase Equilibration amp Mi 0 00 Phase Sample Application Mi 0 00 Phase Column Wash Mi 0 00 Phase Elution 0 00 Phase Equilibration Instruction Box Breakpoint Instructions a F x Parameters for Outlet valve 0 00 F cy S Pumps and pressures a Denon Flow path i ni Var Injection valve out Column position Inlet A Inlet B pH valve Sample inlet Outlet valve Injection mark Monitors Fraction collection 3 Edit Variable Import Block 318 UNICORN 6 1 Method Manual 28 9817 65 AB Step Action 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables 2 e Locate the breakpoint or the required parameter in the Instruction box e Click the Var button 0 00 Column position By pass Column position Down flow 0 00 pH valve In line i tpH cell In line Flow restrictor 0 00 Alarm air sensors Enabled Air sensor alarm on inlet valve A Enabled Air sensor alarm on inlet valve B Disabled Air se 0 00 Inlet A A1 Inlet A 0 00 Inlet B B1 inlet B 0 00 System flow 1 000 Flow rate ml min Pre column pressure Pressure control F F ERE EEE gt F lt nstruction Box 0 00 End_Block 0 00 0 00 0
25. 1 ml inition will automatically be used for Volume pa rameter in the method block and thus used to calculate column volumes CV The Volume pa rameter may then not be edited manually The Column Type parameter can be defined as a variable This may be useful if it is desirable to change column type when starting the method run in the Variable List during the Start Protocol see Set up a Start Protocol on page 54 See Section 9 2 4 Method variables on page 315 for information about how to define variables 298 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 1 Base instruction Step Action 4 Click Change or Replace to save the settings for the selected Base instruction Result The parameters for the Base instruction are updated Note If the column type is changed the Column Value Update warning dialog opens displaying the changes that will be made in the method that will be made based on the column default values see diagram below If these changes are correct click OK oth erwise click Cancel Optimization0O1 Column Value Update The column type used by the method has been changed Update these instructions with the column default values Block Instruction Parameter Variable New Value Unit Alarm pre column Pre column pressure METHOD SETTINGS pressure High alarm limit 0 50 MPa Alarm delta column Delta column pressure METHO
26. 184 The Interaction plot shows if there is any interaction i e when the effect of one factor depends on another factor between two factors The illustration below shows an example of an interaction plot In this example there is an interaction between load mass LoMa and load conductivity LoCo The table below describes how to interpret different interactions plot in a schematic way UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 4 Analyze and evaluate the model extended analysis Description The two lines are parallel This plot shows an example of no interac tion between the two factors The two lines are not parallel This plot shows an example of inter action between the two factors The two lines are crossing This plot shows an example of strong interaction between the two factors UNICORN 6 1 Method Manual 28 9817 65 AB 185 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 4 Analyze and evaluate the model extended analysis Observed versus Predicted for each Response plot The Observed vs Predicted for each Response Y plot can be used to judge the quality of the model With a good model all the points will fall on the 45 line illustrated in the plot below Yield with Experiment Number labels Cbhserved Predicted W 11 B2 0 998 Be d9 0 996 DF 4 Q2 0 985 BSD 0 6455 18
27. 9 Text edit methods 9 3 Specific instructions 9 3 1 Gradients and eluent concentrations Insert a Gradient text instruction The table below describes how to insert a Gradient instruction Step Action 1 At a suitable Breakpoint in the method select the instruction line immedi ately before where you want to insert the gradient this decides when the gradient begins e Expand the Pumps and pressures item in the Instructions field of the Instruction Box e Select Gradient e Inthe Parameters for Gradient field select appropriate values for Target final eluent composition expressed in eluent B Length duration of the gradient Tip To form a step gradient set the Length parameter to zero Tip For many purposes it can be useful to define the length of the gradient as a variable When this is done breakpoints for instructions issued during or after the gradient in the same block are automatically shifted in proportion to the length of the gradient when the variable value is changed This is the same functionality as the Change button command in the Instruction Box Instruction Box Breakpoint Instructions Parameters for Gradient 0 00 cy I Pumps and pressures E Target 0 00 100 00 ystem flow i jas Var ats flow VAR 100 002 6 P h Length 0 00 100000 0 ump was System wash i 0 00 CV Wash flow BufferPro pH BufferPro setting Constant pressure flow Flow path E Moni
28. Delete 3 Type an appropriate name and click Return keyboard key Result The name of the phase is updated Equilibration CV UNICORN 6 1 Method Manual 28 9817 65 AB 47 3 Create and edit methods 3 3 Edit methods and phases 3 3 2 Edit the method outline Rearrange phases within a method The table below describes how to rearrange phases within a method Step Action 1 Select the phase to be moved in the Method Outline pane 2 e Drag and drop the phase to the requested position in the Method Outline pane Result The phase is moved to the requested position or e Right click the phase and select Move up or Move down Rename y Delete Save Phase Equilibration Move Down Cut Copy Paste Result The phase is moved one step up or down in the Method Outline Delete a phase from the Method Outline The table below describes how to delete a phase from the Method Outline Step Action 1 Select the phase to delete from the method in the Method Outline 48 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 3 Edit methods and phases 3 3 2 Edit the method outline Step Action 2 e Click the Delete button below the Method Outline pane Method Settings Equilibration Sample Application Column Wash Elution Equilibration 8CV Duration amp Varables or e Press the Delete key on the keyboard or e select Edit Delete
29. MS 94 544 0 g cate nee ene rene Recon enn ee ORS EO ern ere T 326 9 3 1 Gradients and eluent concentrations acsfedasesctocssseecsnsstiat cesctarislictinnsnsesntinddeiniecnnieencteeea teu 327 D ATT O EE S 330 ISE W TUON e E E A A T E ORR eT 555 934 Pause OF OIG a MEOD siririna iaei EEE EAE NE EEO 340 gs53 Messages ona eh sasies eer cere ee R 343 4 UNICORN 6 1 Method Manual 28 9817 65 AB Table of Contents 10 TROUDIESNOOUING sunicinicnconcanmcuntdudemouckanniamuminaaiunnn 346 10 1 Troubleshooting TST IO GS xcs saiagts se cease sersad actiestaleccyavessesseunsteatereeienai E 347 ilk OP ammo 10219 9 ll 4 fl 8 1 heen ene gase iener eee meine eA E ean nee me veers ERA ee 553 pe teas erespe teeta vete tee ouee ta pesaeasneeese co ntawasaesnea DEE E 358 UNICORN 6 1 Method Manual 28 9817 65 AB 3 1 Introducing the UNICORN Method Editor 1 Introducing the UNICORN Method Editor Introduction This chapter contains e A general introduction to creating methods using the UNICORN software e Information about the user documentation for UNICORN including an overview of related documents describing the use of the software Software declaration of conformity UNICORN 6 1 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 GEHC representative In this chapter This chapter contains these sections Section
30. Printing column type infor mation 258 Printing individual column information 275 Registering new individual columns 262 Scale a method for use with another column type 73 Unable to select for method scaling 351 View column history 274 Column types Create new column type 248 Deleting 253 Editing 251 Exporting 254 Importing 256 Predefined column types 248 Printing information 258 Column volume CV Base instruction 296 Column wash Predefined phase 88 Correlation matrix Design of Experiments 189 CV Column volume Base instruction 296 D Desalting Predefined method 83 Design of Experiments Add factors in experimental design 140 Add responses 138 164 Add runs 166 Analysis advanced 182 Analysis basic 174 ANOVA analysis of vari ance table 187 Change experimental de sign 145 Check data 170 Coefficient plot 180 Correlation matrix 189 Create report 200 359 Index 360 Delete responses from model 165 Edit model 190 Evaluation of results overview 158 Evaluation of single runs 160 Experimental design set up 129 Experimental designs 123 Fractional Factorial de Sign 122 Full Factorial design 122 Generate model 163 Interaction plot 184 Main effects plot 187 Model curvature 173 183 Models overview 125 Multiple scouting runs 150 Normal probability plot of residuals 173 179 Observed vs predicted plot 186 Open result 162 Optimiz
31. UNICORN 6 1 Method Manual 28 9817 65 AB 351 10 Troubleshooting 10 1 Troubleshooting methods Inappropriate inlet settings for CIP or preparation The table below describes how to ensure that the inlet settings are correct for a CIP or preparation phase Problem description Solution When a CIP or preparation phase CIP column Check which inlets are chosen in CIP system Prepare column or Prepare system Method Settings Choose the is started inlets that are not defined inthe phase same inlets as required for the are briefly used CIP or preparation phase Note This happens for a very short time and it will normally not cause any problems 352 UNICORN 6 1 Method Manual 28 9817 65 AB 10 Troubleshooting 10 2 System Error Reports 10 2 System Error Reports Introduction The Generate System Error Report Wizard is used to generate problem reports The report can provide useful background information for the support staff when trying to provide solutions to problems or suggestions for improved system performance Step 1 Create an error report This table below describes how to activate the Generate System Error Report Wizard and create a report Step Action 1 e Choose the Reports Create System Error Report menu command in the Administration module Result The Generate System Error Report Wizard opens Note If an error occurs during a method run you can also start the wizard by clicking the Report button
32. base Phosphate 0 1M NaCl pH 5 9 7 2 0 05 0 1 Titrated with conjugate acid Phosphate with HCI 0 1M NaCl pH 6 2 6 9 0 03 0 05 Titrated with strong acid Piperazine 0 1M NaCl low pH pH 5 5 6 4 0 03 0 05 Titrated with strong acid Piperazine 0 1M NaCl high pH pH 9 3 10 5 0 03 0 05 Titrated with strong base Succinic Acid 0 1M Nacl pH 3 4 5 6 0 03 0 05 Titrated with strong base Triethanolamine 0 1M Nacl Tris O 1M NaCl pH 7 4 8 4 pH 7 6 8 7 0 03 0 05 0 03 0 05 UNICORN 6 1 Method Manual 28 9817 65 AB Titrated with strong acid Titrated with strong acid 7 Method queues 7 Method queues Introduction This chapter describes how to create and edit method queues in UNICORN For informa tion on how to create and edit individual methods see Chapter3 Create and edit methods on page 25 In this chapter This chapter contains the following sections Section See page 7 1 Method queues overview 230 7 2 Create a method queue 231 7 3 Edit a method queue 235 UNICORN 6 1 Method Manual 28 9817 65 AB 229 7 Method queues 7 1 Method queues overview 7 1 Method queues overview Introduction A method queue in UNICORN is a linked set of methods to be run The method queue can contain methods to be run on up to three different systems Each system may have up to ten methods queued For example a method queue m
33. e 1 000 is the default value for the variable e ml min is the variable unit e Flow rate isthe variable name 316 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables Identifying variables in the Instruction Box Parameters that are defined as variables in the text method are also indicated in the Instruction Box for the selected instruction in the Text Instructions area When the instruction is shown in the Instructions field of the Instruction box the VAR button beside the parameter field is displayed in capital letters for variables that is VAR not Var Instruction Box i Breakpoint Instructions z Parameters for Column position 0 005 cy Pumps and pressures A ens Flow path Var Injection valve WARN By pass Column position Flow Direction Inlet 4 Inlet B Var Down flow Up flow pH valve Sample inlet Outlet valve Injection mark Monitors Fraction collection lt Edit Variable Import Block Variable name conventions Variables are defined with names that can be explicit descriptions of the variable function for example Sample volume and Gradient length Suitable choices of variable names can make the method easier to read and understand and also help the operator in setting variable values at the start of a method run When defining and or renaming vari
34. entire process on a system for example a purification run or a column performance test A method is comprised of one or several phases which are reusable sets of instruc tions Examples of phases are equilibration steps and elution steps The UNICORN Method Editor module is a comprehensive tool for creating or editing phases and methods UNICORN is delivered with templates for common procedures that can easily be edited for a specific process Using the Method Editor it is possible to e build a method from a library of phases e create custom phases e create method queues to run multiple methods on up to three separate systems e keep track of column types or individual columns using the Column Handling tool e design and optimize purification schemes using the Design of Experiments and Scouting tools e automatically mix and titrate buffers using the BufferPro tool etc UNICORN 6 1 Method Manual 28 9817 65 AB 9 1 Introducing the UNICORN Method Editor 1 2 About this manual 1 2 About this manual Introduction This section describes the purpose of the manual the general structure and conventions applied in the text and some prerequisites that should be fulfilled before you start to apply any of the procedures described in the following chapters The purpose of the UNICORN Method Manual The purpose of the UNICORN Method Manual is to provide a comprehensive guide to creating methods that can be run on an AKTA avant
35. ml min Pre column pressure Pressure control f System wash 15 Fill system Equilibration _1 ml Outlet valve Mi 0 00 Block Equilibrate_1 0 00 End_Block Breakpoint Instructions m EE gt Parameters for System wash 0 00 cy Pumps and pressures A Wash volume 10 999 System flow 7 mee Vat Sample flow 15 2 ml Clor ainen f Wash flow path to ump wasi ny System wash Outlet valve O Injection valve Wash flow BufferPro pH BufferPro setting Constant pressure flow Flow path Monitors v 2 Change the value for the appropriate variable s indicated by VAR 3 Click Change Result The settings are saved and the text instruction updated in the Text Instructions area UNICORN 6 1 Method Manual 28 9817 65 AB 325 9 Text edit methods 9 3 Specific instructions 9 3 Specific instructions Introduction This section describes some text instruction applications for example e Gradient instructions e Alarms e Conditional instructions e Messages set marks pause and hold instructions In this section This section contains these sub sections Section See page 9 3 1 Gradients and eluent concentrations S27 9 3 2 Alarm instructions 330 9 3 3 Watch instructions 333 9 3 4 Pause or hold a method 340 9 3 5 Messages and Set marks 343 326 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 3 Specific instructions 9 3 1 Gradients and eluent concentratio
36. pH 4 0 5 1 0 03 0 05 Titrated with strong acid Bicine 0 1M NaCl Bis Tris 0 1M NaCl pH 7 7 8 7 pH 6 0 7 1 0 03 0 05 0 03 0 05 Titrated with strong base Titrated with strong acid Bis Tris Propane 0 1M Nacl DH 6 6 7 7 0 03 0 05 Titrated with strong acid Carbonate 0 1M NaCl pH 9 2 10 5 0 05 0 1 Titrated with conjugate acid Carbonate with HCI pH 9 5 10 2 0 03 0 05 Titrated with strong acid 0 1M NaCl Citrate 0 1M NaCl pH 2 3 6 0 0 05 0 1 Titrated with conjugate acid Citrate with HCIO 1M gt pH 3 7 5 7 0 03 0 05 Titrated with strong acid Nacl Diethanolamine0 1M pH 8 6 9 7 0 03 0 05 Titrated with strong acid Nacl Ethanolamine 0 1M pH 9 2 10 2 0 03 0 05 Titrated with strong acid Nacl UNICORN 6 1 Method Manual 28 9817 65 AB 227 6 BufferPro 6 7 Predefined BufferPro recipes 228 Buffer system Formate 0 1M NaCl pH range at 252C pH 2 6 4 4 Concentration range M 0 05 0 1 Comment Titrated with conjugate acid Formate with HCI 0 1M NaCl pH 1 8 4 1 0 03 0 05 Titrated with strong acid HEPES 0 1M NaCl pH 6 9 8 1 0 03 0 05 Titrated with strong base MES 0 1M NaCl pH 5 6 7 0 0 03 0 05 Titrated with strong base Methylpiperazine 0 1M Nacl pH 4 6 5 3 0 03 0 05 Titrated with strong acid MOPS 0 1M NaCl pH 6 5 7 6 0 03 0 05 Titrated with strong
37. variable opens Insert Series For Flow rate x Enter variable ranges separated by commas For example 1 3 5 12 Range 0 000 25 000 O e Enter the appropriate range for example 1 3 5 7 Result The Scouting parameters table is updated Scouting parameters Method Settings METHOD SETTINGS Aun Included Flow rate ml min 1 1 000 2 2 000 3 3 000 4 5 000 5 6 000 E 7 000 110 UNICORN 6 1 Method Manual 28 9817 65 AB 4 Scouting 4 2 Setup and edit a Scouting scheme Step Action 8 Click OK in the Scouting dialog to save the scouting scheme Save the method Add delete or edit variables in the Scouting scheme The table below describes how to add delete or edit variables in the Scouting scheme Step Action 1 Open the Scouting scheme see block Set up a scouting scheme on page 106 Scouting Scouting parameters Method Settings METHOD SETTINGS Run Included Flow rate ml min 0 500 1 000 1 500 2 000 2 500 UNICORN 6 1 Method Manual 28 9817 65 AB 111 4 Scouting 4 2 Set up and edit a Scouting scheme Step Action 2 112 To add or delete variables in the Scouting scheme click in the Scouting dialog Result The Scouting Variables dialog opens Scouting Variables C Column C Column position C Column wash volume C Delta column pressure limit C Empty loop with C Equilibration volume C Equilibration volume_1 C Fill syst
38. 00 0 00 0 00 0 00 Phase Rubisco Elution Phase Equilibration Phase Sample Application Phase Column Wash Phase Elution Phase Eauilibration Breakpoint 10 00 Var Instructions TP d Parameters for Dutlet valve cy Pumps and pressures iti Flow path ee Injection valve Ws Out Waste Column position Inlet Inlet B pH valve Sample inlet Outlet valve Injection mark Monitors Fraction collection vs Result The New Variable dialog opens Mew Variable Variable name r isible in details only 3 e Typeaname for the variable see Variable name conventions on page 317 for information about how to name variables e Select the Visible in details only check box if you want to set the variable as a details variable Detail variables become visible in the Variable List if the Show details check box is selected This option can be used to simplify the workflow later Result The Var button changes to VAR to confirm the new variable Note UNICORN 6 1 Method Manual 28 9817 65 AB If a breakpoint or gradient length is defined as a variable changing the variable value in the Variable List tab when the method run is started will shift other instruction breakpoints ac cordingly This functionality is equivalent to using the Change button to alter a breakpoint or gradient length see Section 9 2 3 Working with text instructions on page 309 for how the
39. 28 9817 65 AB 191 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 5 Edit the model Step Action 4 Based on the previous analysis add the appropriate terms to the model e Add an interaction term by selecting the appropriate factors in the Fac tors table and clicking Add Interaction Tip Use the Ctrl or Shift keyboard key when to select multiple factors e Adda quadratic term to the model by selecting the appropriate factor in the Factors table and clicking Add Square Note Quadratic terms can be added if any of the plots in the analyses indicates that a quadratic term is missing in the Residuals vs Variables plot for example Note When you find curvature i e a quadratic term needs to be added in the model the design for the experiment in the DoE setup should be changed to an extended Full Fractional CCC or CCF design If the experimental setup is sufficiently stable the star point experiments alone can be added otherwise it is recommended to rerun all experiments Result The terms are added to the model If the model refinement gives higher R2 Adj and Q2 values the model refinement is justified If one model is fitted to several responses view the R2 Adj and Q2 values for all responses 5 To return to the original model settings click Reset 6 When you are satisfied with the editing click OK Result The Edit Model dialog is closed and the Analysis tab displayed showing the new plots f
40. 3 Edit methods and phases 3 3 2 Edit the method outline Step Action 3 When the User Defined phase has been added to the Method Outline the phase name is enabled for editing Type a name for the phase and press the Return keyboard key Note The User Defined phase is marked with the letter T meaning that it is text edited This phase contains only Base and End_Block instructions so any functional instructions must be added by hand To include instructions for the User Defined phase select the Text Instructions tab The Phase Properties tab will only show the variables used in this phase See Chapter 9 Text edit methods on page 286 for information about how to work with instructions in the text Instructions pane Rename phases Note It is only possible to rename phases in the Method Outline pane not in the Phase Library The table below describes how to rename a phase in the method Step Action l Select the phase to be renamed in the Method Outline pane 46 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 3 Edit methods and phases 3 3 2 Edit the method outline Step Action 2 e right click the phase and select Rename Equilibration K Delete Save Phase Move Up Move Down Cut Copy Paste or e press the F2 keyboard key or e select Edit Rename Chrl 2 Ctrl Result Mame amp Location Method Motes Zut Chrl Copy Ctrl C Paste Ctrl
41. 6 e When the objective is optimization Good design when Cond no lt 8 Questionable design when Cond no 8 12 Poor design when Cond no gt 12 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 4 Analyze and evaluate the model extended analysis Correlation matrix The correlation matrix gives a numerical presentation of the correlation between factors and responses and if the fit of the model is reasonable The linear correlation coefficients R between all the terms in the model and all the responses are displayed in the correlation matrix Process factors are log transformed scaled and centered and responses are log transformed The value of the correlation coefficient R represents the extent of the linear association between two terms The value of R ranges from 1 to 1 When R is near zero there is no linear relationship between the terms Correlation coefficients above the threshold between a term in the model and the responses are colored green The illustration below shows an example of the correlation matrix i o o o 0 0 72118 o i o o Oo 0 287094 o 0 1 o o 0 512175 o o o 1 0 0 353699 o o o o 1 0 0803863 0 72118 0 287094 0 512175 0 353699 0 0803863 1 UNICORN 6 1 Method Manual 28 9817 65 AB 189 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 5 Edit the model 5 4 5 Edit the model Introduction Editing of the
42. 9 7 Salt 4 0000 Nacl Achievable ranges with recipe Description Follow the safety instructions for each bulk chemical when preparing the BufferPro stock solutions 0 0300 0 0500 M HEPES 0 2000M 47 669 to prepare 1 litre Mw 238 3 NaOH 0 2000M Use ampule NaCl 4 000M 233 8g to prepare 1 litre Mw 58 44 Note Not recommended for AIEX Useful down to about UY 230nm 0 0000 1 0000 M 0 100 B 3 Click the Explore Proportions button 220 UNICORN 6 1 Method Manual 28 9817 65 AB 6 BufferPro 6 5 Calculate buffer composition using BufferPro Step Action 4 In the Explore Proportions dialog enter the pH Buffer concentrations the desired Gradient concentration and Temperature Explore Proportions Recipe HEPES 0 1M NaCl pH L 7 8 Buffer concentrations HEPES 0 0500 M Gradient concentration 25 0 wB Temperature EZ i Calculate Mixture concentrations HEPES NaOH Nacl Note The buffer concentrations may not exceed the limits of the recipe If this is the case the Calculate button will be grayed out 5 Click Calculate Note If the pH given is beyond the optimal buffering range of the buffer recipe a warning will be displayed 6 The actual concentrations of the components in the required buffer will be displayed Mixture concentrations HEPES o 0500 M NaH 0 0531 M Nac O 2500 M Note It is important that the molar amounts are as exact as possible when mixing
43. DoE report 202 Individual column informa tion 275 Method duration time and variables 60 Methods 79 Print screen 351 UNICORN 6 1 Method Manual 28 9817 65 AB Index Response surface plot DoE 194 Problem reports Generate system error re port 353 R Report System error report 353 Response surface plot Generating in DoE 194 Response values Optimize in DoE 197 Predicting in DoE 196 Result Name and location 52 Open DoE result 162 Reverse phase chromatogra phy RPC Predefined method 84 S Sample application Predefined phase 87 Scouting Add runs 114 Add variable 111 Change variable val ues 109 113 Delete runs 114 Delete variable 111 Edit variable 111 Overview 104 Usage 104 Variables in 111 Workflow 104 Scouting scheme Add runs 114 Delete runs 114 Editing 106 How to set up 106 View in DoE 155 Set mark Insert instruction 345 Usage 344 Start protocol Set up start protocol 54 Syntax errors Icons in text instruc tions 293 363 Index 364 In methods 348 In phases 348 In text instructions 348 System Components 348 Instrument configura tion 348 System CIP Maintenance method 86 Predefined phase 88 System preparation Maintenance method 86 Predefined phase 88 T Text editing Blocks 300 Help texts 309 Methods 286 Phases 300 Text instructions 288 Text instructions Add a new instruction 309 Alarm instructions 331 Base instr
44. Experiments 5 2 Create an experimental design 5 2 Create an experimental design Introduction This section describes how to set up a DoE in the Method Editor A Scouting scheme is generated as a result containing the method runs to be run in System Control In this section 128 This section contains the following sub sections Section See page 5 2 1 Set up an experimental design 129 5 2 2 Add responses and factors to an experimental design 138 5 2 3 Change design and design settings in a Design of Experiments setup 145 5 2 4 Divide the DoE runs into several scouting runs 149 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 1 Set up an experimental design 5 2 1 Setup an experimental design Introduction This section describes how to set up a Design of Experiments in the Method Editor Create a method Create a method for the process to be optimized The table below briefly describes how to create a method Step Action 1 Create a method for the process to be optimized See Chapter 3 Create and edit methods on page 25 for detailed information about how to create methods 2 Decide which run parameters that should be screened for or optimized in the experiment If the run parameters are not already defined as variables define the parameters as variables to be able to vary the values i the DoE setup and to connect them to the appropriate factors See
45. Other in the Instructions field of the Instructions box e Select Message in the instructions list 2 Type a message in the Message text box in the Parameters field Instruction Box Breakpoint Instructions OO Parameters for Message 0 00 cy End A Message Evaluate A S AVDE N T Var Hold Var J Check fraction collector _ 3 Loop Mode End Loo TE E E i e Yar Authorize M New chromatogram Sound Pause Ready Default sound 2 Set mark Snapshot Comment 3 3 Select one of the display options on the Mode menu e Screen thoat is only a text message is displayed e Noscreen that is the message will not be displayed but only inserted into the logbook e Authorize that is the message will require a signature from the user before the user can interact with the system again UNICORN 6 1 Method Manual 28 9817 65 AB 343 9 Text edit methods 9 3 Specific instructions 9 3 5 Messages and Set marks Step Action 4 e Select a sound on the Sound menu if desired e Click the Insert button Note If the Message instruction is inserted in a conditional block it will only be displayed if the conditions of the block for example a Watch is fulfilled When to use a Set mark Set mark instructions are useful text messages They can be used e to highlight certain stages in a method e to insert manual notes for example when a specific event occurs in a run only in System Control Set ma
46. Result The BufferPro Recipes dialog opens BufferPro Recipes Recipes Show All i v Stock Solutions Recipe name gt Inlet Stock solutions Concentration M Substance i Q1 Buffer substances 0 4000 Bicine Acetate with HCI 0 1M NaCl AIEX mix 0 1M NaCl Bicine 0 1M NaCl Bicine High Cone Bicine Low Conc Bis Tris 0 1M NaCl 0 7 Bis Tris Propane 0 1M NaCl 6 7 0 4000 NaOH Carbonate 0 1M NaCl Carbonate with HCI 0 1M NaCl CIEX mix 0 1M NaCl 0 7 4 0000 Nacl Achievable ranges with recipe Description 0 0600 0 1000 M 0 0000 1 0000 M 0 100 B m New J Leet Renee J Delete Emot tment _Pire _ _Eseloe Proportions 2 Click on the Import button Result The Import dialog opens 224 UNICORN 6 1 Method Manual 28 9817 65 AB 6 BufferPro 6 6 Export and import BufferPro recipes Stage Description 3 Browse to the zip file containing the BufferPro recipes on the computer disk and Open the file Result The Import BufferPro Recipes dialog opens Import BufferPro Recipes Select recipels to import Phosphate High 0 1M NaCl Global Phosphate Low O 1M MacliGlobal Phosphate High 0 1M Nal Global Phosphate Low 0 1M Nal Global Import as global 4 In the Import BufferPro Recipes dialog uncheck any recipe s that you do not wish to import Select whether the recipe s should be imported as global otherwise they will be imported as personal recipes 5 C
47. See Section 3 4 Set general method options for the method on page 51 for more information Method options intend Scouting ed to assist the user in optimizing runs in UNICORN Design Of Experiment DoE See Chapter 4 Scouting on page 103 for information See Chapter 5 Design of Experiments on page 116 for information BufferPro See Chapter 6 BufferPro on page 203 for information UNICORN 6 1 Method Manual 28 9817 65 AB 29 3 Create and edit methods 3 2 Create and open methods 3 2 Create and open methods Create a new method The table below describes how to create a new method Step Action 1 In the Method Editor e click the Create a new method icon in the Toolbar or e select File New Method New Method h Ctrl M Ea New Method Queue f 58 Open Ctrl Close Result The New Method dialog opens New Method System Create a new method by using the Predefined Method Anion Exchange Chromatography AlE Empty Method Method Description After equilibration and sample application negatively charged proteins are adsorbed to the column ligand After a wash to remove Unbound sample elution ie performed using a gradient of Increasing galt concentration of for example NaCl Finally the column i Washed and r equillbrated with start buffer 30 UNICORN 6 1 Method Manual 28 9817 65 AB Step 3 Create and edit methods 3 2 Create and open methods
48. System Setup dialog UNICORN 6 1 Method Manual 28 9817 65 AB 153 5 Design of Experiments 5 2 Create an experimental design 5 2 4 Divide the DoE runs into several scouting runs Step Action 12 In the Experiment tab check the boxes in front of the runs to be included in the second set of runs Responses Factors amp Design Experiment Experimental Plan Load Concentration Load Conductivity Load pH Included Sample Application S ample inlet Sample Application S ample inlet Sample Application S ample inlet oO 5 S2 2 52 6 52 oO 5 4 2 4 8 4 Fj 5 S 15 S7 6 S oO 5 3 15 S3 8 3 F 20 Not Enough Positions 1 2 Not Enough Positions 1 6 Not Enough Positions 1 oO 20 51 2 1 8 51 F 20 56 15 S6 6 S6 008 20 Buffer 15 Buffer 8 Buffer NO09 009 12 5 55 8 5 55 7 55 NO10 ws O 12 5 55 8 5 55 7 55 NO011 um o 12 5 55 8 5 55 7 55 Note In the example shown here one of the center points run 9 is also included 3 Click OK in the Design of experiments dialog Result Anew Scouting scheme is generated Click Yes and OK in any warning and messages dialog that appear 14 Save the method with a new name Result The two scouting runs are ready to be run in sequence Note In this example you must change samples in one of the sample inlets before starting the second scouting run It is not possible to just create a method queue start it and leave the s
49. The Print Preview dialog opens 3 Print Preview Print a te 4m Pagel of 12 gt gt i BA 75 v Help Close Page 1 of 12 Extended Report Experimert name Capto adhere User name Defaut Currert date amp time 6 5 2009 226 00 Ph Europe Sarndad Time Introduction and Background Factors and Responses Factors The following table contains the factors names abbreviation and settings Name Abbr Units Type Use Settings Transform Prec MLR Scale PLS Scale LoMa Quantitative Controlled 75 to None Free Orthogonal Unit Variance LoCo Quantitative Controlled 2to 15 None Free Orthogonal Unit Variance LopH Quantitative Controlled 6to 8 None Free Orthogonal Unit Variance Responses 2 Click the Print button Result The standard Print dialog opens 3 Select the appropriate printer and click the Print button Result The report is printed 202 UNICORN 6 1 Method Manual 28 9817 65 AB 6 BufferPro 6 BufferPro About this chapter This chapter describes how to create edit and use buffer recipes created using the BufferPro tool in UNICORN For information about how to prepare the system for using BufferPro see AKTA avant and UNICORN User Manual In this chapter This chapter contains the following sections Section See page 6 1 BufferPro Overview 204 6 2 Create a method using BufferPro 206 6 3 Create and edit BufferPro recipes 207 6 4 Print a BufferPro recipe 216 6 5 Calcul
50. The following table describes how to change the order of methods in an existing method queue Step Action 1 Open the method queue see Opening a method queue on page 235 Result The Method Queue dialog opens with the details for the chosen method queue In the Method Queue dialog select a method to be moved by clicking on its name in the Method list Method Queue lonExchangeMethodQueue Number of included systems 2 iv System P2 9 01 v Method Start Condition 1 ColumnwashProcedure At queue start 2 AnionExchangeMethod m Immediately after the previous method has ended 3 Move Up Move Down Lad Delete ad System P2 9 04 v Method Start Condition At queue start EJ At ready command Ld Wait 0h 20min after the previous method has ended LJ 1 Colurnn washProcedure2 ie DesaltingMethod oH 3 CationExchangeMethod To move the selected method up in the Method list click the Move Up button or To move the selected method down in the Method list click the Move Down button To change the order of further methods repeat steps 2 and 3 Save the method queue 240 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 Column Handling Introduction The Column Handling tool in UNICORN enables handling of column types and if selected during installation handling of individual columns using the Column Logbook The Column Handling tool can be opened from all available modules in
51. Troubleshooting 347 Unexpected results from converted methods 350 Variables 315 View variables 60 Method settings Predefined phase 87 Method variables Viewing 315 Model curvature Design of Experiments 173 179 183 N Notes Column notes 268 Method notes 55 P Pause Insert instruction 341 Text instruction 340 Phase properties Edit phase 37 Help 36 Phases CIP phases 352 Column CIP 88 Column performance test 88 Column preparation 88 Column wash 88 Deleting phases from li brary 67 Edit phases 37 Elution 88 UNICORN 6 1 Method Manual 28 9817 65 AB Equilibration 87 Export a phase 95 Help 36 Import a phase 99 Intelligent Packing 88 Method settings 87 Phase library 349 Phase properties 37 347 348 Predefined phases 24 87 Prepare phases 352 Sample application 87 Syntax errors in 348 System CIP 88 System preparation 88 Text editing 300 348 DH range BufferPro predefined buffers 227 Common buffers 205 Plots Coefficient plot DoE 180 Interaction plot DoE 184 Main effects plot DoE 187 Normal probability plot of residuals DoE 173 179 Observed vs predicted plot DoE 186 Replicate plot DoE 171 Residual vs run order plot DoE 184 Residual vs variable plot DoE 182 Response surface plot DoE 194 Summary of Fit DoE 176 Printing BufferPro recipe 216 217 Column performance re port 277 Column type informa tion 258 DoE methods 156
52. Type in a new name in the New name field and click Rename Check the Set visible in details only if the variable should be a detailed variable Uncheck the box to set it to a normal variable Click Delete to delete the variable Confirm that you want to delete the variable in the dialog that appears Click Close to close the dialog Edit a variable using the VAR button in the Instruction Box The table below describes how to edit a variable using the Instruction Box UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables Step Action 1 Select the instruction containing the variable to be edited in the Text Instruc tions area Result The parameters for the instruction are shown in the Instruction box 0 00 Phase Column Wash 5 0 0 00 Phase Equilibration 0 00 Base SameAsMain 0 00 Inlet A A1 Inlet A 0 00 Inlet B B1 Inlet B 0 00 Gradient 0 00 Percent B Equilibration _1 B 0 00 base 0 00 System flow 1 000 Flow rate ml min Pre column pressure Pressure control System wash 15 Fill system Equilibration _1 ml Outlet valve Mi 0 00 Block Equilibrate_1 0 00 End_Block Breakpoint Instructions Parameters for System wash 0 00 wv Pumps and pressures A Wash volume 10 999 System flow r Var Sample flow VAR 15 ml J of Gradient Wash flow path t
53. a color scale from blue to red For each color the response value is displayed The factors selected are displayed on the X axis and Y axis in the Contour Plot from Low to High as selected in the Factors at the plot axes area If you have more than two factors the other factors will have constant values The currently entered constant value s is displayed to the right of the contour plot This means that this value is kept constant while the factors on the X and Y axes are varied The red area indicates the area where the response is maximized using the factor settings within this area and the current constant value s 194 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 6 Use the model Step Action 2 It is possible to change the factors and their corresponding settings for the response surface plot as well as the constant values for the other factor s This is done per response if you have several responses In this way you can see what happens if constant values are changed and if other factors and or factor settings are set on the contour plot axes This will help you to decide if which complementary experiments need to be performed For example you may want to investigate which factor settings to use in new DoE setup to narrow down the area of interest The coefficient plot can be used to see which terms have the greatest positive or negative effect on the res
54. already defined in either the Method Settings phase or the predefined phases Note that some variables may be hidden or unused in the method New variables often do not need to be defined 106 UNICORN 6 1 Method Manual 28 9817 65 AB 4 Scouting 4 2 Setup and edit a Scouting scheme Step Action 2 In the Method Editor e Click the Scouting icon in the toolbar ir or e Select Tools Scouting Start Protocol Evaluation Procedures Design of Experiments Result The Scouting dialog opens with the Scouting Variables dialog dis played on top Scouting Scouting parameters Run Included Scouting Variables C Column C Column position C Column wash volume C Column wash volume_1 C Delta column pressure limit C Empty loop with C Equilibration volume C Equilibration volume_1 C Fill system E quilibration MI Fill sustarra IFE aniilikestion 1 tit i C Show details C Show unused variables Insert Run Series Remove Alun Note When editing a scouting scheme only the Scouting dialog is displayed UNICORN 6 1 Method Manual 28 9817 65 AB 107 4 Scouting 4 2 Set up and edit a Scouting scheme Step Action 3 e Inthe Scouting Variables dialog select the appropriate variable s to be varied by checking the appropriate box es Check the Show details box if you want to display variables defined as detailed variables in your method These are rare
55. and for Low alarm in the Param eters field Instruction Box Breakpoint Instructions re Parameters for Alarm system pressure 0 00S cy E Flow path A Mode Var Facta collection Disabled Spes G Ama High alarm 0 02 20 00 prae vaJ 20 00 MPa Alarm delta column pressure Low alarm 0 00 20 00 Prah ok id pressure acl s 000l MPa Alarm conductivity i i Alarm pH Sir air sebos v Note There are no high and low settings for Air sensors only enabled or disabled 4 Click the Enabled radio button 5 Click the Insert button Result The new Alarm instruction is inserted in the method UNICORN 6 1 Method Manual 28 9817 65 AB 331 9 Text edit methods 9 3 Specific instructions 9 3 2 Alarm instructions Available alarms The alarms available depend on the instrument configuration Alarms for the following monitor readings may be set 332 System pressure Sample pressure Delta column pressure Pre column pressure UV1 Conductivity DH Air sensors UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 3 Specific instructions 9 3 3 Watch instructions 9 3 3 Watch instructions Introduction Watch instructions allow the progress of a method run to be determined by events during the method run For example start collecting fractions when the first peak elutes The Instrument Configuration files include Watch instructions for each monitor defined in the system These instructi
56. cannot be obtained when several responses are included in the same model In this case try fitting an individual model for each response separately The table below describes how to perform a basic analysis of the model 174 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis Step Action 1 Select the Analysis tab if not already selected Result The Analysis tab opens showing four plots for each response the Replicate plot the Summary of Fit plot the Coefficient plot and the Normal probability plot of residuals To be able to see all plots use the vertical scroll bar Expeumerd Aniym Pinkin Djimon A Analysis Plot of Replicatiosas for Yid eth Espenment Number bets 10 i a0 pi l ry 2 i Massena Suisa a CarMate epen UNICORN 6 1 Method Manual 28 9817 65 AB 175 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis 176 Step Action 2 Scroll down to display the Summary of Fit plot If the experiment contains several responses the plot will contain the four bars shown below for each response summary of Fit R2 Ge Model Validity Reproducibility 0 9 0 6 ov 0 6 0 5 0 4 0 3 0 2 0 1 0 0 Yield W 11 Cond no 1 173 DF 5 Y miss 0 The bars in the plot describe diffe
57. coefficient plot for each response The coefficient plot can be used to see which factors that affect your response in which way they affect the response s and if there are any non significant terms in the model In the example below the following terms have been included in the model e the main effects that is the loading conditions for mass pH and conduc tivity LoMa LoCo and LopH e the two way interaction effects for LoMa LopH and LoCo LopH Note If an optimization design CCC or CCF has been used quadratic terms for the model will also be included in the coefficient plot scaled amp Centered Coefficients for Yield E E E E 5 s 5 5 5 8 s s N 11 B2 0 996 BR Adj 0 993 DF 5 Q2 0 984 BSD 0 8248 Conf lev 0 95 In the example above the confidence limits the black error bars shown on each green bar in the plot do not cross zero All of the terms are thus signif icant with the LoCo LopH two way interaction term being least significant Positive bars have a positive influence on the response in this example the Yield and negative bars a negative influence From the above plot it is evident that increasing the LoMa Load Mass and LoCo Load Conductivity values and decreasing the LopH Load pH value have a positive effect on the re sponse 180 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis S
58. column must be created This is described below in Create an individual AxiChrom column Registering this individual column for the column packing run will enable the results from the column performance tests to be registered in the column logbook for the column Two performance tests are part of the Intelligent Packing method one downflow and one upflow When the evalu ation of the tests are performed the actual packed bed height should be used This bed height will be registered in the column logbook The evaluation procedure is described in the UNICORN 6 1 Evaluation Manual To ensure that the actual packed bed height is applied when creating or running methods using the AxiChrom column type you must update the column type definition and replace the default value with the actual registered value of the individual column Until this is done the default bed height will be applied Tip If it is essential that the exact packed bed height is used you should create a column type for each individual AxiChrom column and update the bed height value after each time the column is repacked Create an AxiChrom column type The table below describes how to create the AxiChrom column type Note To ensure that the actual packed bed height is applied you must Step Action 1 Choose the Tools Column Handling menu command Result The Column Handling dialog opens 2 In the Column Type Parameters tab in the Column Handling dialog click
59. containing a high salt tion Chro concentration for example 2 M Ammo matogra nium Sulphate hydrophobic proteins Equilibration phy HIC are adsobed to the column ligand After y a wash to remove unbound sample Sample Application elution is performed using a gradient of decreasing salt concentration Final iii ly the column is washed and re equili brated with start buffer v Elution vy Column Wash v Equilibration Reversed After equilibration and sample applica Phase Chro tion hydrophobic proteins adsorb to matogra the column ligand After a wash to re phy RPC move unbound sample elution is per Equilibration formed by generating a gradient of a non polar organic solvent such as Sample Application Acetonitrile Finally the column is m washed and re equilibrated BO v Elution v Column Wash v Equilibration WARNING Fraction collector Do not fractionate flammable liquids When running RPC methods or other procedures using organic solvents collect fractions through the Outlet valve 84 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 8 Predefined methods and phases Predefined maintenance methods A number of predefined methods for preparation and cleaning are available These maintenance methods are used to prepare the system clean the system and to fill the system with storage solution The table below describes the available predefine
60. displayed Design of Experiments Responses Factors amp Design Experiment Experimental Plan inckided Load pH Load Conductivity jami Sample Application S ample inlet Sample Application S ample inlet Edit System Setup Click OK to generate a scouting scheme The Run column shows the run order for the optimized Scouting scheme that is generated from the DoE when clicking OK Note If excluding any of the runs in the Experimental Plan the results may not be reliable for use in the DoE evaluation 134 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 1 Set up an experimental design Step Action 7 In some cases it may be necessary to divide the DoE runs into two or more scouting runs for example if there are too few sample inlet valves Some of the runs can be excluded the first time and run during further rounds If limitations in the hardware exist this will be indicated in the Experimental Plan on the Experiment tab by the text Not Enough Positions For informa tion about how to proceed when for example the available sample inlet positions are not sufficient see Section 5 2 4 Divide the DoE runs into several scouting runs on page 149 Design of Experiments Responses Factors amp Design Experiment Experimental Plan ineluces Load pH E Load Conductivity Load Concentration Sample Application S ample inlet Sample Appli
61. experimental design Step Action 5 Select to which phase the factor is connected in the Method phase drop down list Method phase Sample Application k For example if adding the predefined factor Load pH the pH at loading is controlled in the method phase Sample application 6 Select to which Variable the factor is connected in the Variable drop down list C Don t connect the factor to a method variable For example if adding the predefined factor Load pH the sample pH at loading is controlled by the Sample inlet valve position Note Variables connected to factors will be included in the Scouting scheme that is generated when completing the DoE setup 7 If the factor is not connected to anything that can be controlled by UNICORN e g if the experiment is performed in a cold room or in room temperature check the box Don t connect the factor to a method variable 8 Click OK to add the factor to the Design of Experiments Result The factor will be listed on the Factors amp Design tab Design of Experiments Responses Factors amp Design Experiment Factors Abbreviation Unit Method Variable Method Phase Ztos Sample inlet Sample Application Design selection Objective Screening 4 Design Total number of runs including center points 9 To add more factors repeat this procedure 144 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimen
62. fix e ne ere ER SHE Bf fess Poe Add delete responses Insert missing run results 2 Analyze and evaluate the model Check raw data Analysis and evaluation of model Basic analysis Extended analysis Replicate plot Summary of Fit plot Interaction plot Residuals vs variable plot iL AO EEN Raw data OK l A ad Normal probability plot 7 Main effects plot Observed vs predicted plot Normal probability plot aac S ANOVA A table i Residuals vs run order plot Raw data not OK e g outlier found CO ot ess Tm f Exclude replace outlier R Q i w E Hd Correlation matrix i i 1 a r i i i ammm bama m odel not OK Edit model p gt 0 05 RQ Response surface plot Coefficient plot Use Prediction list only optimization Use Optimizer only optimization 3 Use the model UNICORN 6 1 Method Manual 28 9817 65 AB 159 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 2 Generate model 5 4 2 Generate model Introduction This section describes how to open single DoE runs for evaluation how to open DoE results and how to generate a model Evaluate the results of the single DoE runs Before opening the DoE result in the Evaluation module it is recommended to evaluate the single runs included in the Scouting run The table below describes how to open and evaluate single runs in the Evaluation module Step Action 1 In the Evaluation modu
63. for editing Elutian Equilibration Type a name for the phase and press the Return keyboard key Note The User Defined phase is marked with the letter T meaning that it is text edited This phase contains only Base and End_Block instructions so any functional instructions must be added by hand To include instructions for the User Defined phase select the Text Instructions tab The Phase Properties tab will only show the variables used in this phase See Chapter 9 Text edit methods on page 286 for information about how to work with instructions in the text Instructions pane UNICORN 6 1 Method Manual 28 9817 65 AB 43 3 Create and edit methods 3 3 Edit methods and phases 3 3 2 Edit the method outline Add a phase to the method outline using a button or menu command The table below describes how to add a phase to the method outline using a button or menu command Step Action 44 1 Select the appropriate phase e g Equilibration in the Phase Library Select the appropriate phase e g the Method Settings phase in the Method Outline to determine where to place the new phase Note When adding a phase to the Method Outline using a button or menu command the new phase is always inserted below the currently selected phase in the Method Outline Result The selected phase in the Phase Library is indicated by a blue dotted frame and the selected phase in the Method Outline is highlighted in blue
64. instruction begins 2 To insert a Hold instruction select Other Hold in the Instructions field of the Instruction Box Breakpoint Instructions a00 s r Block Parameters for Hold re Continue Var End_Block End Evaluate eS Loop End Loop Message New chromatogram Pause Read v 3 To insert a Pause instruction select Other Pause and enter the Time for the method to be paused in the Time field To pause the method for infinite time check the Infinite box Breakpoint Instructions Block Continue Var End_Block nd 0 00 cy Evaluate Hold Loop End Loop Message New SESE Ready v Var FT Parameters for Pause Time 0 00 399 90 0 00 min C Infinite UNICORN 6 1 Method Manual 28 9817 65 AB Edit Variable Import Block 9 Text edit methods 9 3 Specific instructions 9 3 4 Pause or hold a method Step Action 4 To insert a Hold until instruction e select Watch Hold until in the Instructions field of the Instruction Box e select the appropriate parameters for the Hold until instruction in the Parameters for Hold until area See Section 9 3 3 Watch instructions on page 333 for descriptions of the available settings Instruction Box Breakpoint Instructions i 0 007 cy Pumps and pressures T Flow path Var Monitors Fraction collection Alarms Watch parameters Watch atch Watch off E Other
65. into blocks of information with separating lines The blocks are identified by a label extending into the margin such as the label Document Structure above This makes it easier for you to quickly scan a page to find the exact topic you are looking for Typographical conventions Menu commands field names and other text items from the software are quoted exactly as they appear on the screen in a bold italic typeface Example Method Navigator Search paths are shown in a bold italic typeface with a separating colon between each level Example Edit mport mport Phase i e the menu option Import Phase in the sub menu Import from the Edit menu Controls on the instrument computer or keyboard keys are shown with a bold regular typeface Example Press the Delete key Text that the user must either type exactly as shown in the manual or that UNICORN displays as a response not a regular part of the graphic user interface is represented by a monotype typeface within quotation marks Example Connection change Prerequisites The following prerequisites must be fulfilled before you can use this manual the way it is intended e You need to have a general understanding of how your PC and Windows work In most cases universal computer functions will not be explained e UNICORN must be installed and configured correctly on your computer e Your user profile and access rights must be set up and you must be a
66. mix 0 1M NaCl Bicine 0 1M NaCl Bicine Low Conc Bis Tris 0 1M NaCl Bis Tris Propane 0 1M NaCl Carbonate 0 1M NaCl Carbonate with HCI 0 1M NaCl CIEX mix 0 1M NaCl Acetate with Hcl OAM Nach 4 0751 PO Achievable ranges with recipe pH Bicine 0 0600 0 0000 o 100 Stock Solutions Inlet Stock solutions Concentration M Substance Q1 Buffer substances 0 4000 Bicine Q2 Acid or Base 0 4000 NaOH Q3 water Q4 salt 4 0000 Nacl Description m re Let Cherone Dette JIC Emot imeat J _Pint _ J Esploro Pronortione Note Several recipes may be exported to the same zip file To select a continuous range click on the first recipe then Shift click the last To add single recipes to a selection Ctrl click them 3 Click Export Result The Export dialog opens 4 Choose a location on the computer disk and a filename for the zip file 5 Save the file UNICORN 6 1 Method Manual 28 9817 65 AB 223 6 BufferPro 6 6 Export and import BufferPro recipes Importing BufferPro recipes The following table illustrates the steps required to import one or several recipes Stage Description 1 In the Method Editor select Tools BufferPro Recipes Start Protocol Evaluation Procedures Scouting Design of Experiments Column Handling 9 Ctrl b BufFerPro Recipes ae Administration ES System Control 4a Evaluation Options Change Passwords
67. model may be necessary after analysis of the model if the current model does not give a good fit In the analysis you may for example e find insignificant terms that need to be removed e find that the model may have curvature and that a quadratic term needs to be added The refined model can be analysed to see if it better fits the data This section describes how to edit the model Edit the model The table below describes how to edit a model Step Action 1 In the Analysis tab click Edit Model Result The Edit Model dialog opens Edit Model Factors Model terme Load hl as LoM r Constant Load Conductivity Loha 0 00002 Load pH EE LoCo 0 00060 LopH 0 00006 LoMa LoCo 0 66102 Add Square ONNA EORR ee LoCo LopH 0 05096 Use Ctrl or Ctrl Shift and left mouse button to select multiple factors when adding an interaction Response and model coefficients Select a response Yield R2 Adi gz 190 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 5 Edit the model Step Action 2 Note the R2 Adj and Q2 values for the response s before starting to edit the model Select different responses in the Select a response drop down list When editing the model the R2 Adj and Q2 values are updated Higher values indicate a better model See also Analyze and interpret the model basic analysis on page 174 for a descr
68. model validity gt 0 25 indicates a good model A model validity lt 0 25 indicates a significant lack of fit that is the model error is significantly larger than the pure error reproducibility Reproducibility A reproducibility lt 0 5 indicates that there is a large pure error and poor control of the experimental setup high noise level If the Summary of Fit plot does not look good there may be several reasons for this The table below lists a few UNICORN 6 1 Method Manual 28 9817 65 AB 177 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis 178 Action Summary of Fit plot value Low Q and model validity Possible cause Non significant two way interactions may be present What to do Look at the coefficient plot see step 3 and the Interac tion plot to see if there are in teraction effects Curvature in the mod el Is there a need of adding quadratic terms to the model Look at the Residual vs vari able plot see Residuals versus variables plot on page 182 and the ANOVA table see ANOVA table on page 187 to see if these also indicate cur vature in the model If you suspect curvature try adding a quadratic term to the mod el Model with moderate R 0 6 and Q 0 4 Important factors may be missing Are there uncontrolled factors that may affect the experiment If needed perform mor
69. number of times Total no of runs This field lists the total number of runs to be performed based on the number of center points and replicates e The Settings button is only active if a CCC design using star points is selected To change the default star point distance in relation to the de sign box click Settings Result The Star Distance dialog opens Star Distance Enter the star distance for the CCF design Star distance 0 5 1 414 a Change the Star distance as appropriate and click OK To return to the default value click Default UNICORN 6 1 Method Manual 28 9817 65 AB 147 5 Design of Experiments 5 2 Create an experimental design 5 2 3 Change design and design settings in a Design of Experiments setup Step Action 5 In the Change Design dialog click OK Result Changes in the Change Design dialog are saved and the settings in the Design setup area in the Design of Experiments dialog are updated Note If additional variables have been defined in the scouting scheme for a previously saved DoE method these will be lost and need to be redefined 148 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 4 Divide the DoE runs into several scouting runs 5 2 4 Divide the DoE runs into several scouting runs Introduction If hardware limitations exist for example too few sample inlet valve positions are available for the number of runs t
70. oO O06 Cap shen method noes SMAI eo oO ite Capto adhere method ingest OHEA TIHA Oo i mi Capo adhere methoding esl MOHE TIIA 2 oH Caph kiari thong as UHE TIH O wa Capii bhar wahodd del 2OEHG 12 VIH O a A hnnga Dame Rapora 2 To insert the missing runs click the Insert Result button Result The Insert Result dialog opens 166 Insert Result Folder name Capto adhere method 1 tar Capato adhere met ad Capto adhere meth xd Capto adhere meth xq Capto adhere meth Ti Capto adhere meth fx Capto adhere meth al Capto adhere meth al Capto adhere meth fx Capto adhere meth ad Capto adhere meth Capto adhere method 2 Project 7 fa Project 2 Project 3 EA Project 4 Project 5 Project 6 alt i Last modified 2009 05 12 20 00 2009 05 14 12 51 2009 05 12 19 34 2009 05 12 19 36 2009 05 12 19 38 2009 05 12 19 40 2009 05 12 19 42 2009 05 12 19 44 2009 05 12 19 46 2009 05 12 19 48 2009 05 12 19 51 2009 05 12 20 00 2009 06 22 13 47 2009 06 22 13 47 2009 06 22 13 47 2009 06 22 13 48 2009 06 22 13 48 2009 06 22 13 48 2 See ee i Pa Re ESE EOFT er BOP eo E Cancel UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 2 Generate model Step Action 3 Browse and select the run s that should be inserted Tip The run order number i
71. of increasing salt concentration of e g NaCl Finally the column is washed and re equilibrated with start buffer Sample Application v Column Wash v Elution v Column Wash y Equilibration UNICORN 6 1 Method Manual 28 9817 65 AB Predefined purification method Chromatofo cusing CF 3 Create and edit methods 3 8 Predefined methods and phases Principle After equilibration and sample applica tion elution is performed using a pH gradient The proteins separate and elute according to their isoelectric points Finally the column is re equili brated Included phases Method Settings Equilibration v Sample Application v Elution v Equilibration Desalting After equilibration and sample applica tion the proteins are eluted isocratical ly This technique is commonly used for buffer exchange Method Settings Equilibration hd Sample Application v Elution Gel filtra tion GF UNICORN 6 1 Method Manual 28 9817 65 AB After equilibration and sample applica tion proteins separate and elute ac cording to their size largest first Method Settings Equilibration Sample Application v Elution 83 3 Create and edit methods 3 8 Predefined methods and phases Predefined Principle Included phases purification method Hydropho After equilibration and sample applica bic Interac tion use a buffer
72. only change the procedure that is included in the method See the UNICORN 6 1 Evaluation Manual for information about how to edit an evaluation proce dure UNICORN 6 1 Method Manual 28 9817 65 AB 59 3 Create and edit methods 3 4 Set general method options for the method View and print the method duration time and variables The table below describes how to view and print an estimation of the method duration time and the variables in the method Step Action 1 In the Method Editor e click the Duration amp Variables button below the Method Outline pane or e select View Duration amp Variables Toolbar Method Navigator Gradient we Phase Library Flow Scheme Restore to Default Refresh H Duration amp variables h Result The Method Duration amp Variables dialog opens displaying the Method Duration tab 60 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 4 Set general method options for the method Step Action 2 Method Duration amp Variables Method Duration Variable List 0 00 Base CV 0 100 ml Any Column HM 0 00 Phase Method Settings Base SameAsMain Alarm pre column pressure Enabled 20 00 Pre column pressure limit MPa 0 00 MPa Alarm delta column pressure Enabled 20 00 Delta column pressure limit MPa 0 00 MPa Wavelength 280 U V1 nm Off HUY2 nm Off HUV3 nm Noise reduction UY 0 2 HUY averaging time sec Injectio
73. optional In the Column Handling dialog View column performance report In the Start Protocol 4 Scan column barcode i or Select column manually The report lists Column Logbook data If the appropriate column type is not available in the database If the appropriate column is not available in the database Create new column type Register new column import column import column type T teed Lees s te ee der fetenere ws In the Column Handling dialog In the Column Handling dialog or Start Protocol Create a new column type Register new column or a Click b Scan column barcode or type in ID manually Import the column type c Enter column information and notes or Selected performance graphs Run history and path to result files for all runs performed using the column In the Column Handling dialog Import the column 246 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 2 Handling column types 8 2 Handling column types Introduction When you create a new method and select a column type in the Method Settings phase the volume flow rate and pressure limits are automatically set Most of the work regard ing handling of column types is performed in the Method Editor The column type to be used ina method is selected when creating the method as shown in the illustration below C Phase Properties Text Instructions iT Method Settings Column select
74. peak setting in the Watch parameters helps the software to detect valleys peaks and peak maxima and to filter noise in the chromatogram The Delta peak value should be set e large enough so that signal noise does not activate the conditions and e small enough so that the condition is activated close to the valley or peak As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height If you set a too high value you can prevent a new peak from being detected after a local minimum Use of the Delta peak setting The Delta peak setting in the Watch parameters e sets the threshold for signal increase after a local minimum that will be interpreted as a valley for the Less than or valley condition A valley and a new peak are detected when the signal increases to 102 of the local minimum plus the Delta peak value Note A valley is detected only after a Peak max has been detected Example If there is a local minimum at 50 mAU and a Delta peak of 10 mAU a valley will be detected at 1 02 x 50 10 61 mAU e sets the threshold for signal decrease after a local maximum that will activate the Peak max condition Peak max is detected when the signal falls to the specified fraction of the most recent peak maximum minus the Delta peak value The schematic figure below illustrates the Delta peak setting where Peak max is detected when the signal falls by Delta peak from a local maximu
75. performed when the conditions have been met Watch off Cancels the watch on the specified signal Breakpoint Instructions ia Parameters for Watch 0 00 cy Pumps and pressures Signal Flow path Var Monitors UV v Fraction collection Tes Alarms Watch parameters Greater than Ni w i Value 6000 0 6000 0 ache 0 0 mAU z Watch off Action a Other Var LS uilbrate v 3 Select a signal for the watch from the Signal drop down menu See Monitor signals to watch on page 335 for available signals that can be selected 4 For watch types Hold until or Watch select the appropriate Parameters for Watch e Test See Test options in the Parameters field on page 335 for a description of the different Test options e Value Slope Minutes Factor depending on the selected test e select an appropriate Action See Actions when a Watch condition is met on page 336 for a description of the different Watch Action options 334 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 3 Specific instructions 9 3 3 Watch instructions Step Action 5 Click the Insert button Result The new Watch instruction is inserted in the Text Instructions area Note A Watch off instruction can be added to the method at a breakpoint where the watch no longer is needed Note Watch parameters may be set as variables so that the method easily can be adjusted for different run conditions Monitor signals
76. previous method has ended 4 5 ka 7 Repeat steps 4 to 6 to add further methods to the Method list for each re quired system UNICORN 6 1 Method Manual 28 9817 65 AB 233 7 Method queues 7 2 Create a method queue Step Action 8 Click Save or Save As to save the completed method queue Note An error dialog will be displayed if any of the methods could be incompatible with the system on which they are queued 234 UNICORN 6 1 Method Manual 28 9817 65 AB 7 Method queues 7 3 Edit a method queue 7 3 Edita method queue Introduction This section describes how to open delete and edit existing method queues Methods can be inserted and deleted from a method queue and their order in the queue can be changed Opening a method queue The table below describes how to open an existing method queue in the database Step Action 1 In the Method Editor e Click the Open Method Navigator icon in the Toolbar or e select File Open New Method Ctrl N EL New Method Queue or e select View Method Navigator Toolbar Method Navigator X Gradient Phase Library Flow Scheme Restore to Default Result The Method Navigator is displayed UNICORN 6 1 Method Manual 28 9817 65 AB 7 Method queues 7 3 Edit a method queue Step Action 2 Select the method queue to be opened in the Folder name column Method Navigator Open eet amp Methods Method Gu
77. section describes the plots in the extended report and gives information about how to evaluate the plots Open and view plots for extended analysis To be able to view the plots for extended analysis create an extended report See Create a report on page 200 for information about how to create an extended report Residuals versus variables plot The Residuals Plot vs Variable shows the residuals i e the minimized error between the measured and theoretical data according to the model for one factor and one re sponse The residuals should be randomly distributed with no pattern When a curved pattern can be seen in the plot this may indicate that a quadratic term is missing in the model In this case try to add a quadratic term to the model and see if the model is improved See Section 5 4 5 Edit the model on page 190 for information about how to add a quadratic term to the model The illustration below shows an example of a plot indicating that a quadratic term is missing in the model 182 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 4 Analyze and evaluate the model extended analysis Load pH vs Residuals for Aggregate Content with Experiment Number labels Deleted Studentized Residuals 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 76 79 60 541 Load pH The illustration below shows the plot for the same experiment when a quadratic term h
78. select Tools BufferPro Recipes Evaluation Procedures Scouting tel Design of Experiments Column Handling Ctrl L TA System Control vg Evaluation Options Change Passwords 212 UNICORN 6 1 Method Manual 28 9817 65 AB Step Action 6 BufferPro 6 3 Create and edit BufferPro recipes 6 3 2 Rename a BufferPro recipe 2 In the BufferPro Recipes dialog select the recipe to be renamed BufferPro Recipes Recipes Show All Recipe name Acetate 0 1M Nat AIEX mix 0 1M NaCl Bicine 0 1M NaCl Bicine High Cone Bicine Low Conc Bis Tris 0 1M NaCl Bis Tris Propane 0 1M NaCl Carbonate 0 1M NaCl Carbonate with HCI 0 1M NaCl CIEX mix 0 1M NaCl Achievable ranges with recipe pH Bicine 0 0600 0 0000 o 100 Stock Solutions Inlet Stock solutions Concentration M Substance Q1 Buffer substances 0 4000 Bicine Q2 Acid or Base 0 4000 NaOH Q3 water Q4 salt 4 0000 Nacl Description m re Let Chenna Dette JIC Emot C imeot J _Pint _ J Esploro Proportions Note The available recipes may be filtered by type All Predefined Global or Personal by using the Show drop down list 3 Click Rename and enter the new name AIEX mix 1M NaCl PD Bicine 0 1M MaCl PO E High Con P 117 120 P Bis Tris O 1h NaCl Po UNICORN 6 1 Method Manual 28 9817 65 AB 213 6 BufferPro 6 3 Create and edit BufferPro recipes 6 3 3 Delete a BufferPro
79. system to be used with the target system as described in the applicable instruction above i e with or without scaling of the column size 78 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 7 Print a method 3 7 Printa method Introduction This section describes how to print a method s text instructions and variables UNICORN uses the printers and printer settings that are installed on your computer Print a method The table below describes how to print an opened method Step Action 1 e Click the Print icon in the Toolbar or e select File Print New Method Ctrl M New Method Queue Close New Folder Save Ctrl 5 Save Os Scale or Convert Method Sign Method Ctri P Result The Print dialog opens UNICORN 6 1 Method Manual 28 9817 65 AB 79 3 Create and edit methods 3 7 Printa method Step Action 2 In the Print dialog Print REX Hilrap Chelating Printer Printer outside office Print items Phase range Test instructions G All phases Vartable list Phase e select Printer e select which Print items to be printed e select to print all phases in the method or a specific phase in the Phase range field 3 By default information about the overall method settings as well as any Signatures and specific columns used in the method are printed To exclude or add information click Options gt gt and check uncheck the appropr
80. table below describes the different parts in the method Description The main block contains the complete method It contains the Base instruction mandatory and the appropriate phases with instructions to be executed in a method Blocks at the highest level in the method represents the major steps in the process flow and are called phases Each phase can contain sub blocks that is blocks at a lower level Note If the method has not been text edited proper ties for the phase can be set in the Phase Properties pane New phases can only be added to the Method Outline using the Phase Library It is however possible to copy and paste an existing phase in the Text Instructions pane Each block starts with a Base instruction continues with the appropriate instructions and always ends with an End_Block instruction Sub block A sub block is a block at a lower level than a phase that may contain conditional instructions or other instructions for specific events within a phase Each sub block starts with a Base instruction continues with the appropriate instructions and always ends with an End_Block instruction UNICORN 6 1 Method Manual 28 9817 65 AB Description of icons and text formats in the text method 9 Text edit methods 9 1 Overview 9 1 2 The Text Instructions pane The table below describes the icons and text formats that may appear in the the Text Instructions pane Iicon text format
81. the Method Outline Method blocks Block length 300 Instructions in each block are executed in the order they are written The instructions within a block are executed until the block is finished or the End_Block instruction Is executed Any settings made in a block are valid throughout the method until the settings are changed However if a conditional instruction e g a Watch instruction controlling the start of a sub block is included in a phase the instructions in the sub block are executed when the condition for that Watch is met e g when a particular monitor signal meets a given condition The length of a block is determined by the breakpoint of the last instruction in the block Even if all breakpoints are set to 0 the instructions might take some time volume because they are executed sequentially The illustration below shows an example of a method where Equilibration has a breakpoint set to 5 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 2 Working with phases and blocks 0 00 Phase Equilibration 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 D Hi Base SameAsM ain Inlet A AT nlet A Inlet B B1 Inlet E Gradient 0 00 Percent B Equilibration 28 0 00 base System flow 1 000 Flow rate ml min Pre column pressurej Pressure control System wash 25 HFill system E quilibration ml Outlet va
82. the buffers It has been found that four decimal places in molar concentration gives reproducible results 7 The buffer composition can be printed by pressing the Print button UNICORN 6 1 Method Manual 28 9817 65 AB 221 6 BufferPro 6 6 Export and import BufferPro recipes 6 6 Export and import BufferPro recipes Introduction BufferPro recipes are stored internally in the UNICORN database It is possible to export these recipes to a zip file on the local computer so that the recipe can be imported again later into the same database installation or imported into another This section describes how to export and import BufferPro recipes Exporting BufferPro recipes The following table illustrates the steps required to export one or several recipes Note Predefined recipes can not be exported since these recipes will always be found in a UNICORN installation Stage Description 1 In the Method Editor select Tools BufferPro Recipes Evaluation Procedures Deal Scouting Design of Experiments H Column Handling Ctrl L Ho Administration System Control a Evaluation Options Change Passwords 222 UNICORN 6 1 Method Manual 28 9817 65 AB Stage Description 6 BufferPro 6 6 Export and import BufferPro recipes 2 Choose the recipe to be exported from the list in the BufferPro Recipes dialog BufferPro Recipes Recipes Show All Recipe name Acetate 0 1M Nat AIEX
83. the outlet valve The Fractionation settings will change to reflect this choice and the outlet valve position can be selected as the Fractionation destination Fractionate Fractionation settings 5 using outlet valve Fractionation type Fixed volume fractionation v using fraction collector Fractionation destination Es v pede in waste do not collect Peak fractionation destination Fixed fractionation volume 2 00 mil 0 01 20000 00 Peak fractionation volume ml 0 01 20000 00 e using fraction collector enables fraction collection in the fraction collec tor The Fractionation settings will change to reflect this choice and the desired Fractionation destination can be chosen from the drop down list Fractionate Fractionation settings using outlet valve Fractionation type Fixed volume fractionation v Settings using fraction collector Fractionation destination 96 deep well plate v ino in waste do not collect Peak fractionation destination Fixed fractionation volume 3 ml Bs n Peak fractionation volume E 50 ml tubes C only for isocratic e 50 ml tubes full rack e in waste do not collect will send the eluent to the waste 3 Edit the Fractionation settings as appropriate For detailed information on these settings see the online help for the phase refer to Getting help when editing Phase Properties on page 36 UNICORN 6 1 Method Manual 28 9817 65 AB 91 3 Create and edit methods
84. to watch The monitor signals that can be watched differ depending on the Instrument Configu ration but may include the following pH Cond UV 1 2 and 3 Pressure System Sample Pre column and Delta column Flow System and sample Air sensor System pump A and B sample pump The buffer concentration may also be set as a watch parameter Test options in the Parameters field The table below describes the Test options that are available for the Watch instruction in the Parameters for Watch field Explanation Greater than The signal exceeds a certain value Less than The signal falls below a specified value Slope greater than The rate of change of the signal exceeds a specified value expressed in monitor units minute for example mAU min Slope less than The rate of change of the signal falls below a specified value expressed in for example mAU min UNICORN 6 1 Method Manual 28 9817 65 AB 335 9 Text edit methods 9 3 Specific instructions 9 3 3 Watch instructions Less thanor valley Explanation The signal falls below a specified value or a valley is detected A valley is detected only after a Peak max has been detected and the valley is defined by a local minimum followed by an increase to 102 of the local minimum value plus the Delta peak value see The Delta peak setting on page 338 Peak max The signal falls to a specified fraction of the most recent peak maximum minu
85. when preparing 226 DH ranges 227 Predefined 205 C Cation exchange Predefined method 82 Chromatofocusing Predefined method 83 CIP Column maintenance method 85 Column predefined phase 88 System maintenance method 86 System predefined phase 88 Column CIP Maintenance method 85 Predefined phase 88 Column Handling Barcodes 261 Column types 247 Individual columns 260 Overview 242 UNICORN 6 1 Method Manual 28 9817 65 AB Registering new individual columns 262 Workflow 246 Column list Importing predefined col umn types 258 Column performance test Maintenance method 85 Predefined phase 88 Column preparation Maintenance method 85 Predefined phase 88 Columns Adding or editing notes 268 Barcodes 261 Column Handling Overview 242 Column Handling Work flow 246 Column performance test 276 Column types 247 Create an AxiChrom column type 280 Create new column type 248 Deleting column types 253 Deleting individual columns 270 Editing column type 251 Editing individual columns 268 Exporting column types 254 Exporting individual columns 271 Find an individual col umn 266 Handling individual columns 260 Importing column types 256 Importing individual columns 271 Importing new column lists 258 Notification limits 269 Performance report 277 Predefined column types 248 UNICORN 6 1 Method Manual 28 9817 65 AB Index Preparing an Intelligent Packing method 283
86. with the main tasks are Individual columns Register new columns scan or manually type in the barcode add notes optional Tip New columns can be registered using the Column Handling dialog or before the run is started Select columns to be used in the run view column history optional view performance report optional scan column barcode Tip Individual columns to be used for a specific run can only be selected via the Start Protocol Before selecting a previously used column it is possible to view the run history and a perfor mance report if available in the Col umn Handling dialog Edit columns add edit notes set notification limits delete unused columns Print column information Generate a performance report Export and import columns from UNICORN UNICORN 6 1 Method Manual 28 9817 65 AB 245 8 Column Handling 8 1 Overview Illustration of Column Handling workflow The illustration below shows a possible workflow when working with column types and individual columns 1 Create method 2 Select column for the run and start the run 3 Generate report optional Select column type in the In the Column Handling dialog In the Evaluation module Method Settings phase gS p View column history optional If a column performance test HETP has been run evaluate the results in the Evaluation module View column performance Performance Report report if available
87. 0 10 10 1 9414 10 10 40 Experiment It is possible to see the combination factor settings that will give a certain response The number of iterations for optimization is indicated in the Iter column Lower or more negative Log D values the logarithm of the distance to the target indicate better results UNICORN 6 1 Method Manual 28 9817 65 AB 199 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 7 Create and print reports 5 4 7 Create and print reports Introduction This section describes how to create basic and extended reports and how to print the reports Create a report The table below describes how to create a report Step Action In the Analysis tab click Report Result The Report dialog opens displaying the Basic report by default It displays the Replicate Summary of Fit Normal probability and Coefficient plots Basic Report Experiment name Capto adhere User name Default Current date amp time 6 5 2009 2 25 59 PMV Europe Standard Time Replicate Plot Plot of Replications for Yield with Experiment Number labels Replicate Index Basic Report Extended Report 200 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 7 Create and print reports Step Action 2 To display the extended report select the Extended Report radio button Basic Report Extended Rep
88. 0 HR HiPrep 26 10 Desalting Ri HiPrep 26 10 Sepharose 6 FF HiPrep 26 60 Sephacryl S 100 HR HiPrep 26 60 Sephacryl S 200 HR HiPrep 26 60 Sephacryl S 300 HR HiPrep 26 60 Sephacryl S 400 HR gt Lill 266 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 3 Handling individual columns 8 3 3 Find an individual column Step Action 3 To select several column types use the Ctrl and Shift keyboard keys Result The individual columns registered for the selected column type s are displayed in the Columns list The Column Logbook area to the right shows parameters and information for the selected column Column Type Parameters Column Logbook Select Column IDs Code lot exp ID FindbyID Find by alias Columns Column Logbook Column Data Value 17 5091 01 10021091 2015 05 0509 Project 4 Mono Q 1 Mono G 10 100 GL Medium Batch ID 17 0719 01 Medium expiration date 2015 05 31 Date of first use 2010 05 09 Number of runs 7 Max of delta pressure MPa 0 32 Number of runs since last CIP 2 Number of runs since last Column performance test 7 Measured bed height Resolution 0 00 Asymmetry 0 00 Plate height cm 0 00 Plates per meter N m 0 0 Kay 0 00 Retention factor 0 00 Performance validation result Not accessible Calculated based on the last Column Performance test Delete Column History Performance Report Notification Limits Tip To show
89. 0 Pham iutan s E 0 00 Phare Epin T Equittratc Fem arate tow Binh Le a E Othe Peek v a Me EQUILIBRATION Das esk i Evakiaie Hold Lisi ed Log Weri Hs hacer er i at Deis Dee Save Peace Cei b Vaate Evaa e Biek e mi T a Phm Blot oo pea pis hard The table below describes the different areas in the Text Instructions pane UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 1 Overview 9 1 2 The Text Instructions pane Description Text Instructions area Shows the method as a list of individual text instruc tions The instructions are grouped into blocks denoted by blue square symbols in the figure below to obtain a logical overview of the method Instruction Box Shows the available instructions It can be displayed using the Auto Hide function see Auto Hide optional panes on page 18 for more information Use the Instruction Box to e insert change replace and delete blocks and instructions in the method e delete phases e specify breakpoints parameters and variables Note It is not possible to add phases using the Instruction Box For information about how to add phases see Section 3 3 2 Edit the method outline on page 43 Structure of the text method A method in the Text Instructions area consists of a Main block that contains the Base instruction mandatory and the appropriate phases and blocks to be used in the method Blocks c
90. 037 Gel filtration 28 9288 13 20090415 0000 00 0032 Desalting Rb 28 9288 13 20080704 0000 00 1043 Desalting general use 28 9288 13 20090376 0000 00 0017 Anion excha 3 Make sure that the check boxes in front of the individual columns to be im ported are checked If a column should not be imported clear the correspond ing check box 4 Click OK Result The columns are imported into the database Note If an individual column to be imported has the same barcode or alias name as an existing column in the database a dialog will be displayed saying that the column already exists in the database and that it will not be imported UNICORN 6 1 Method Manual 28 9817 65 AB 213 8 Column Handling 8 3 Handling individual columns 8 3 6 Print and view individual column information 8 3 6 Print and view individual column information Introduction This section describes how to view the run history for an individual column and how to print column information View individual column history It is possible to view the run history for an individual column to see how many runs that have been performed using the column The path to the result files for each run is also displayed If the run was a column performance test or CIP run this is shown as a remark The table below describes how to view the Column History for a column Step Action 1 Select the individual column for which to view Column History in the Col
91. 1 07 1 System Francine 187 2009 1 24 17 Default ma a we a mo r lt System SystemC 4 Browse to the required folder in the database and type in a new Name if necessary 5 Select a System for the method from the drop down menu 6 Click the Import button to import the method Result The imported method will be opened in the Method Editor Note If the imported method contains instructions that are not sup ported by the selected system the phases containing these in structions will be marked with an error symbol 102 UNICORN 6 1 Method Manual 28 9817 65 AB 4 Scouting 4 Scouting Introduction Scouting is used to repeat a series of method runs automatically using different settings or with predetermined changes in the values for one or more Variables A Scouting scheme is defined as part of the method This chapter gives an overview of scouting and the scouting workflow and describes how to set up and edit a Scouting scheme Scouting is ideal for relatively simple variable combinations When designing experiments to analyse several variables at the same time it is advantageous to use the Design of Experiments DoE tool This tool applies statistical methods for generating scouting runs that provide the most information with as few runs as possible thus economising on time and sample amounts For details on DoE see Chapter 5 Design of Experiments on page 116 In this chapter This chapter co
92. 1 User Manual for information 104 UNICORN 6 1 Method Manual 28 9817 65 AB 4 Scouting 4 1 Overview Note The Start protocol will only be displayed before the first run in the Scouting experiment e Evaluate the results of the scouting run This is performed in the Evaluation module All results from Scouting runs performed at any one time are stored in the same folder See UNICORN 6 1 Evaluation Manual for information UNICORN 6 1 Method Manual 28 9817 65 AB 105 4 Scouting 4 2 Set up and edit a Scouting scheme 42 Setup and edit a Scouting scheme Introduction Any parameter can be scouted provided that it can be defined as a variable in the method This section describes how to set up and edit a Scouting scheme Set up a scouting scheme The table below describes how to set up a Scouting scheme where the flow rate is varied In this example the flow rate is varied between 0 5 and 3 ml min Note The Start protocol will only be displayed before the first run in the Scouting experiment Step Action 1 Create a method and decide appropriate run parameters to be varied in the experiment The run parameters to be varied should be defined as Variables in your method See Chapter 3 Create and edit methods on page 25 for information about how to create methods See Section 9 2 4 Method variables on page 315 for information about how to define new variables Tip Many variables that can be used for scouting are
93. 17 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis Step Action 4 Look at the normal probability plot of residuals The residuals or minimized errors between the measured data and the theoretical data calculated ac cording to the model should normally be distributed as shown in the diagram below Yield with Experiment Number labels M Probability 14 4 12 10 08 06 O4 O2 00 032 0 4 0 6 0 6 1 0 12 14 Standardized Residuals W 11 Be 0 5958 Be Adg 0 956 LDF 4 Q2 0 559 BSED 0_ 6455 In anormal distribution of residuals for a good model the experiments should be distributed close to a straight line and also should lie within a Standard ized Residuals range of 4 to 4 SD standard deviations Single experiments that deviate from this may be outliers A non linear distribution of experiments may also indicate the presence of insignificant missing terms for example curvature of the model See Analyze and interpret the model basic analysis on page 174 and Section 5 4 4 Analyze and evaluate the model extended analysis on page 182 5 If the raw data is OK continue with the basic analysis of the model described in Analyze and interpret the model basic analysis on page 174 UNICORN 6 1 Method Manual 28 9817 65 AB 173 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis
94. 2 5 5 8 5 5 7 55 8 Clear the Included boxes in front of all runs UNICORN 6 1 Method Manual 28 9817 65 AB 151 5 Design of Experiments 5 2 Create an experimental design 5 2 4 Divide the DoE runs into several scouting runs Step Action 9 Click the Edit System Setup button Result The Edit System Setup dialog opens Sample inlet Position Load Concentration Load Conductivity Load pH 152 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 4 Divide the DoE runs into several scouting runs Step Action 10 Change the position for the inlet that did not have any position before to a valid position The inlet that previously had the position must also be changed Example Change the position for in this example the sample inlet position indicated by Not Enough Positions 1 to Buffer position 2 rere 3 Sample inlet Position Load Concentration Load Conductivity Load pH 51 w 20 2 8 2 5 2 6 3 v5 1S 8 S4 v5 z 8 5 12 5 8 5 f S6 wv 20 15 6 57 v5 15 6 Buffer v 20 2 6 v 20 15 8 Then change the sample inlet position originally set to Buffer to Not Enough Positions 1 Position ie Concentration Load Conductryity Load pH 51 wv 20 2 a 52 5 2 E 53 5 15 4 54 5 2 5 55 125 8 5 T 5B wv 20 15 E S7 wi 15 E Jot 20 2 E Buffer 20 15 a The two inlet positions have been changed 11 Click OK in the Edit
95. 3 10 Sign methods electronically 3 10 Sign methods electronically Introduction Methods can be signed electronically to enhance data file security Once a method has been signed it is not possible to edit the method Tip To edit a signed method create a new method using the settings in the signed method by selecting File Save As and save the method with a new name Sign a method electronically in the Method Editor The table below describes how to sign a method electronically in the Method Editor Step Action l Select File Sign Method New Method Ekri New Method Queue Close New Folder Save Ctrl 5 Save AS Scale or Convert Method Result The Sign Method dialog opens 92 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 10 Sign methods electronically Step Action 2 Sign Method Method 1 Sign method View signatures Signing user David T Log on password Full name Dave Tomkins Job title Senior Protein Specialist Signature description laa for ruin Signature password e The Signing user field shows the user currently logged on If you are to sign the method but are not the one logged on to UNICORN select your user name in the Signing user drop down list Your Full name and Job title are displayed e Type your Log on password to UNICORN e Type a Signature description if appropriate e Type your Signature password
96. 4 below UNICORN 6 1 Method Manual 28 9817 65 AB 63 3 Create and edit methods 3 5 Save methods and phases Step Action 2 Browse for an appropriate folder or create a new one eet G Methods Folder name Ea Bridget H Charlie E Christine RuBisCo H DavidE EG David T Fa DefaultH ome Ea Francine Ga Gerald H Greta Ga Helen Hame Rubisco MonoG Tris 1 Last modified Created by 5 18 2009 1 225 Default 571872009 1 231 Default 1572009 1 25 0 Default 5 18 2009 1 24 5 Default 5 18 2009 1 23 3 Default 5 18 2009 1 23 24 Default 25 2008 1 00 12 Syste 5 18 2009 1 24 71 Default 5 18 2009 1 239 5 Default 571872009 1 29 5 Default 5 18 2009 1 24 2 Default System SystemC mM Save Cancel 3 e Select the folder in which to save the results e Entera method Name e Select for which System to save the method 64 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 5 Save methods and phases Step Action 4 Click Save Result The method is saved in the database Note An error message will appear if you are trying to save the method for e asystem using another instrument configuration and or another component configuration than the method originally was created for and e the settings in the method depends on the component con figuration e g if an extra inlet A valve is used in the method this setting cannot be used in a sys
97. 53 2 Buffer 2 S1 15 56 15 Not Enough Positions 1 Load pH Sample Application S ample inlet 6 S2 8 S4 6 S7 8 3 6 Buffer 8 S1 6 SB 8 Not Enough Positions 1 Edit System Setup Click OK to generate a scouting scheme If limitations in the hardware exist this will be indicated in the Experimental Plan by the text Not Enough Positions for the run s in the Design of exper iments dialog These runs are also excluded from the Experimental Plan Clear the Included box in front of the experiments to be excluded in the first set of runs In the example below Run 009 and Run 010 are excluded from the first set of runs Responses Factors amp Design Experiment Experimental Plan Load Concentration Included Sample Application S ample inlet 5 2 5 54 5 S 5 3 20 Buffer 20 51 20 56 KS KSP SPSS IES 08 d 20 Not Enough Positions 1 NO09 oo9 O 12 5 55 N010 005 12 5 55 N011 011 12 5 55 Load Conductivity Sample Application S ample inlet 2 2 2 54 15 57 15 3 2 Buffer 2 51 15 56 15 Not Enough Positions 1 8 5 S5 8 5 55 8 5 55 Load pH Sample Application S ample inlet 6 52 8 S4 6 57 8 53 6 Buffer 8 51 6 56 8 Not Enough Positions 1 7 55 7 55 7 55 Include at least one center point runs 5 9 and 11 in the above example in each scouting run to
98. 6 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 4 Analyze and evaluate the model extended analysis Main effects plot The main effects plot displays the predicted response values when a factor varies from its low to its high level all other factors in the design being set on their averages Main Effect for Load pH resp Yield g2 90 55 T a 66 54 my 50 59 6061 62 63 64 65 66 67 66 69 FO 74 72 73 F4 75 76 77 FB 79 80 61 Load pH W 11 BO 5958 Be Adg 0 956 D F 4 Q2 0 559 BSD 0_ 6455 Conf lew 0 95 ANOVA table The ANOVA analysis of variance table gives a numerical presentation of the variance analysis The illustration below shows an example of the ANOVA table y MassOut MassIn DF ss MS variance F p SD Total 10 79468 2 7946 82 Constant 1 780570 5 780570 5 Total Corrected 9 897 072 99 7413 9 98706 Regression 6 893 347 148 891 103 271 0 001 12 2021 Residual 3 4 32527 1 44176 1 20073 Lack of Fit 1 O 0786042 0 0786042 0 0370193 0 865 0 280364 Model Error Pure Error 2 4 24666 2 12333 1 45717 Replicate Error N 10 Q2 0 988 Cond no 3 635 DF 3 R2 0 995 Y miss 0 R2 Adj 0 986 RSD 1 201 UNICORN 6 1 Method Manual 28 9817 65 AB 187 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 4 Analyze and evaluate the model extended analysis 188 When looking at the ANOVA table the p values for regression Lack of Fit mode
99. 65 AB 7 Method queues 7 2 Create a method queue 7 2 Create a method queue Creating a method queue The following table describes how to create a method queue Step Action 1 In the Method Editor e click the New Method Queue icon in the Toolbar A a or e Select File New Method Queue view Phases Tools J New Method Ctrl M Result The Method Queue dialog opens 2 In the Method Queue dialog choose the Number of included systems from the drop down list Method Queue Number of included systems no A Start Condition v Result A separate method queue block will be added to the dialog for each additional system if required UNICORN 6 1 Method Manual 28 9817 65 AB 231 7 Method queues 7 2 Create a method queue Step Action 5 Choose a system for each method queue block from the System drop down list System ne N l System F2 9 01 Method mn Result The Select Method dialog opens In the Select Method dialog browse to the required method and click OK Result The method is added to the method queue Note For reasons of system compatability the individual methods should be saved for the system on which they are queued UNICORN 6 1 Method Manual 28 9817 65 AB 7 Method queues 7 2 Create a method queue Step Action 6 Select a Start Condition for the method from the drop down list Note The first Method for the first System will a
100. 65 AB 303 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 2 Working with phases and blocks Step Action E e Click Yes to rename the block after insertion A new block is created The variables in the block will get new names so the variable values can be changed without affecting the original block Or e Click No to just insert the copied block with the same name The block and variables names in the block are copied If changing variable values in the pasted block the values will be changed in the original block as well Result The block is inserted in position Note The pasted block is inserted with the same breakpoint value as the block or instruction selected for point of insertion When a Phase is copied and pasted the Rename dialog is not opened Import blocks The table below describes how to import blocks from other methods Step Action 1 Click the Import Block button Result The Import Block dialog opens 2 Locate and select the method you wish to import the block from in the Method folder structure Import Block Method Selectblock Folder name System Last modified Cre Stop frac Sample Appl Start frac Column Wash SEHCSJ2DP3JDSUNICORNAUNI ras E Adam 5 18 2009 1 23 0 Stop frac Column Wash Anne 5 18 2009 1 22 4 Sect weak EJI DAOCS NickelColumn SystemC 6 4 2009 2 04 58 Stop frac Elution Bridget 5 18 2009 1 22 5 Eel Ch
101. 8 9817 65 AB 9 Text edit methods 9 1 Overview 9 1 1 Working with text instructions Phase Properties Text Instructions iT Column Wash iT This phase has been text edited Method Settings Equilibration v Sample Application y Column Wash T p Phase Variables Block Variable Value Range COLUMN WASH Inlet B B1 v Elution COLUMN WASH Percent B Column Wash B 0 00 0 00 100 00 COLUMN WASH Flow rate ml min 1 000 0 000 25 000 v Pressure control Pre column pressure Y Equilibration Start frac Column Wash Outlet frac start position Column Wash Dwl 0 M Outlet frac volume Column wash ml 20000 00 0 01 20000 00 y Wash Column wash volume Cv 20 00 0 00 999999 00 User Defined iF C Show details Edit Variable Considerations when text editing a method Before starting to text edit a method consider the following e Editing instructions in the Text Instructions pane is only recommended for advanced users e f the text instructions for a method are edited manually the phase properties will no longer show all optional settings but only the Phase Variables To restore the phase properties you have to undo the edited text instructions by clicking the Restore Phase Properties button which is displayed in the Phase Properties tab after text instructions have been edited e Several phases may be labelled as text edited when editing a single phase in the Text Instructions pane This is the cas
102. Blue square beside text Blue square with a red cross Description A block containing instructions that can be run A block containing one or more instructions that are not possible to run due to instrument configuration incompat ibility syntax errors Bold text Instructions that can be run Red dot Instructions that are not possible to run All such instruc tions must be deleted or changed before a method can be run See Section 9 2 3 Working with text instructions on page 309 The errors in the instructions may be of the following types e Instructions that apply to a different instrument con figuration can occur if a method is written for one system and saved for another e Instructions for deselected components in the System Setup e References to blocks that are not defined in the method e g a Watch instruction but no instructions to be executed when the Watch is activated Normal text Instructions that will not be run Instructions with a red dot are formatted as normal text instead of bold text Unused instructions are also formatted as normal text Instead of deleting instructions they can be moved to unused Instructions below the text method UNICORN 6 1 Method Manual 28 9817 65 AB 293 9 Text edit methods 9 1 Overview 9 1 2 The Text Instructions pane Iicon text format Text with a red loop symbol 2 Description When a block is called from within itself this wi
103. Capto Viral 5 ml HiTrap DEAE FF 1 ml HiTrap DEAE FF 5 ml Hil rap O FF 1 ral Hil rap GFF 5 mil UNICORN 6 1 Method Manual 28 9817 65 AB By default the same column type that was selected in the original method is shown in the Column Type field ha 3 Create and edit methods 3 6 Scale or convert methods Step Action 6 e Select the column type from the list of available column types and e click the OK button Result The Select Column Type dialog closes and the selected column is shown in the Column Type field of the Scale or Convert Method dialog Scale or Convert Method Method AIE ARTA avant 25 Convert method to systern Convert and Scale up down method Target System AKTA avant 150 iw Column Type Scale 16 20 Capto DEA Select Column Keep linear flow on new column 7 If desired select the Keep linear flow on new column option Note This option is applicable only if linear flow was selected in the original method If linear flow is not selected the default flow settings for the selected column type will be used UNICORN 6 1 Method Manual 28 9817 65 AB 75 3 Create and edit methods 3 6 Scale or convert methods Step Action 8 Click the OK button Result The method is scaled as an untitled copy UNTITLED converted The Method Notes dialog opens showing basic information about the con version Method Notes Method DefaultH
104. Change button affects instructions within gradients 519 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables Step Action 4 Click Change Result The variable is saved and displayed in the Text Instructions area Edit variables Editing a variable includes renaming and deleting the variable and choosing whether the variable should be a detailed variable or not For information about how to edit the variable values see Edit variable values on page 324 Edit a variable using the Edit variable button The table below describes how to edit a variable using the Edit Variable button 320 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables Step Action 1 e Inthe Instruction Box click Edit Variable Breakpoint Instructions a Parameters for Column position 0 002 cy Pumps and pressures Position Flow path Var Injection valve UAR by par Column position Flow Direction Inlet Inlet B Down flow Up flow pH valve Sample inlet Outlet valve Injection mark Monitors Fraction collection L868 lt Edit Variable Import Block e Alternatively if the phase containing the variable has been text edited select the Phase Properties tab to display the phase variables select the variable and click Edi
105. D SETTINGS ptessure High alarm aa k 0 30 MPa METHOD SETTINGS Noise reduction UY Averaging time UY averaging time 5 0 sec METHOD SETTINGS System flow Flow rate Flow rate 8 000 ml min EQUILIBRATION System flow Flow rate Flow rate 8 000 ml min Inject System flow Flow rate Flow rate 8 000 ml min COLUMN WASH System flow Flow rate Flow rate 8 000 rolmin ELUTION System flow Flow rate Flow rate 8 000 ml min COLUMN WASH_1 System flow Flow rate Flow rate i 8 000 ml min EQUILIBRATION _1 System flow Flow rate Flow rate 8 000 ml min UNICORN 6 1 Method Manual 28 9817 65 AB 299 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 2 Working with phases and blocks 9 2 2 Working with phases and blocks Introduction This section describes how to add delete and edit phases and blocks in the text method It also describes how to import blocks from other methods Phases vs blocks Because phases are blocks at the highest level in the text method the same editing op erations can be performed In this section the name block will be used both for phase blocks and other blocks unless otherwise stated Exception It is not possible to add a phase using the Instruction Box A new phase must be added from the Phase Library The User Defined phase is intended for this purpose but any phase may be text edited See Section 3 3 2 Edit the method outline on page 43 for information about how to add phases to
106. Experiments 5 4 3 Analyze and evaluate the model basic analysis Step Action 2 For each response look at the replicate plot This plot displays the variation in the response for replicated experiments and the variation among the replicates in relation to the variation across the entire design reproducibil ity Plot of Replications for Yield with Experiment Number labels eld Replicate Index Each arrow in the plot represents an experiment In a good replicate plot as shown in the example above the replicate runs should show as small a variation as possible experiments 9 10 and 11 There should normally be some variation across the dataset of non replicate experiments However a single experiment should not deviate dramatically from the rest Note When a robustness test has been performed variations in the data should instead be as small as possible UNICORN 6 1 Method Manual 28 9817 65 AB 171 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis Step Action 5 172 The replicate plots can also be used to identify outliers If for example a single experiment deviates a lot from the rest of the experiments see plot to the left below or if a replicate deviates a lot from the rest of the replicates see plot to the right below this run could be an outlier hi UNICORN 6 1 Method Manual 28 98
107. GE Healthcare Life Sciences UNICORN 6 1 Method Manual Table of Contents Table of Contents 1 Introducing the UNICORN Method Editor cccsssssssssssesscessssesseessseeeeees 7 11 jJABOUE Me UNICORN Method EALO rsson R E 8 Lo Poou aN aE E E EE ERE N E 10 Ls UNICORN 6 1 user documentation ssssssssssssssssssssssssssssrssssssssssrsrriisressnnssnsstneetrerirnrnnsnsnnssnsrnnn 12 The UNICORN Method Editor cccssccsscssccsrcesscssscssessccsscesscsssesesesscesseesees 13 L TT TOG E OT aos E E T E EEE EEOAE AAE 14 22 Methods m UNICORN bI ssccassarecsstexstocecnspssrresvencttseoetonaiunsensnncarvoysentivenaatcenitaasientiameneaianienninnt 20 Create and edit MEtNOdS odicncceteiyveadciscunienetiaettceeaienauntmanemieene 25 3 1 Working with methods Overview sssssssssssssssssssssssssssssssssssssssssrssseesreernsererrnrerierrinrrnrrnnennnrnnsns 26 32 Qredte ond open Menos sesers E A 30 33 Edit FNS TAOS ana SOS sssri oii cee uses ee vad ches EEEE E ETO Nina 34 soa PNA NOT e a E E entry Pe near 35 3 3 2 Edit the method outline ssssssssssssissssssssssssssssrsisssssssssssssnssssserrrssesssnnnsnsstnerrirnrrnnnnnnnnnntnnntnrrrnnnnnnnnns 43 3 4 Set general Method options for the Method oo ccecccccsseessessseesssesssessseessecssecsseesseesseessseesneeees 51 a9 Save MEetMNods and PNOSOS seniii RANN 63 36 Scdeorconyart mengas sirikan oaaae ARAR aapka Omaa ANAE oaRRa 69 A Pan a A nto meeaie se umn a unme
108. ICORN There are several types of designs available in UNICORN The table below describes three design variants illustrated by the design box Different designs are used based on the objective and the experiment setup The center point experiments are always included in all designs UNICORN 6 1 Method Manual 28 9817 65 AB 121 5 Design of Experiments 5 1 Introduction to Design of Experiments Design type Description Fractional Factorial In the Fractional Factorial design some of the corner point experiments are excluded as illustrated to the left i e the white circles This design will not give as much information as when using all corner point exper iments but by excluding the corner points as shown in the illustration the information loss is minimized Information about which factors are important main effects and some information about factor interaction effects are obtained This design type is good to use when you need to obtain information about the parameter settings and reduce the number of factors in your experiment before optimization Fractional designs are suggested when performing e Screening because the information provided using this design is often enough to find the factors affecting the process e Robustness Testing because then optimal factor settings have al ready been found and only minor changes in the factor settings are studied Less experiments are needed compared to using Full Factorial and O
109. Mew Result The New Column Type dialog opens UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 5 Intelligent Packing of AxiChrom columns Step Action 3 e Select the appropriate AxiChrom column hardware in the GE Healthcare hardware type drop down list e Select the GE Healthcare medium type for the new AxiChrom column type in the drop down list Tip Only some of the available media are approved by GE Healthcare for use in the Intelligent Packing of AxiChrom columns Click the GE approved media button in the Intelligent Packing phase to view a list of the approved media and bed heights Other media can also be selected but the packing procedure will then be performed with custom packing settings with a set of general default settings Result Based on the selections some of the column type parameters are automatically filled in 4 Enter the remaining parameter values for the new column type in the Run Parameters Details and Ordering Information tabs for example e target bed height e max flow rate e max delta column pressure based on chosen media Fields marked with must be filled in Values in the gray fields are calculated and automatically filled in based on entered values for the corresponding parameters 5 Select whether the the new column type should be Global available for all users or Personal only available for the current user 6 Click Save As to save the column type Resul
110. NICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 3 Handling individual columns 8 3 6 Print and view individual column information Print information about individual columns The table below describes how to print information for individual columns Step Action l Select the appropriate column s for which to print information in the Columns list in the Column Logbook tab See Section 8 3 3 Find an individual column on page 266 for information about how to find and select a column To select several columns in the Columns list use the Ctrl and Shift keys 2 In the Column Logbook tab click Cei Result The Print dialog opens Printer TOSHIBA 2 5 Print items Selected columns All shown columns All columns 3 Select Printer 4 Select for which individual column s to print information e Selected columns Prints information for the column s selected in the Columns list e All shown columns Prints information for all columns displayed in the Columns list e All columns Prints information for all columns in the database Click OK Result The Column ID alias and parameters for the columns are printed UNICORN 6 1 Method Manual 28 9817 65 AB 215 8 Column Handling 8 4 Column performance 8 4 Column performance Introduction Column performance can be tested by measuring the height equivalent to a theoretical plate HETP and asymmetry factor As values Tests should be
111. Section 9 2 4 Method variables on page 315 for information about how to define new variables Note In the DoE setup factors are connected to the variables in the method 2 Save the method UNICORN 6 1 Method Manual 28 9817 65 AB 129 5 Design of Experiments 5 2 Create an experimental design 5 2 1 Set up an experimental design Set up a new Design of Experiments 130 The table below describes how to set up a new Design of Experiments in the Method Editor Step Action 1 In the Method Editor e click the Design of Experiments icon in the Toolbar or e select Tools Design of Experiments Start Protocol Evaluation Procedures Scouting Design of Experiments P Column Handling ctrl L Result The Design of Experiments dialog opens Design of Experiments Responses Factors amp Design Experiment Responses Name Abbreviation Unit Click Add to define a response Delete UNICORN 6 1 Method Manual 28 9817 65 AB Step 5 Design of Experiments 5 2 Create an experimental design 5 2 1 Set up an experimental design Action To add responses to the Design of Experiments click Add Result The Add Response dialog opens For detailed information about how to define and add a response see Sec tion 5 2 2 Add responses and factors to an experimental design on page 138 Add Response fx Predefined Actvty S O User defined Abbreviation Act Unit
112. See page 1 1 About the UNICORN Method Editor 8 1 2 About this manual 10 1 3 UNICORN 6 1 user documentation 12 UNICORN 6 1 Method Manual 28 9817 65 AB 7 1 Introducing the UNICORN Method Editor 1 1 About the UNICORN Method Editor 1 1 About the UNICORN Method Editor Introduction This section is a brief introduction to creating methods in UNICORN and a description of the scope of this manual What is UNICORN UNICORN is a complete software package for e control and supervision of chromatography systems e evaluation and analysis of the results from separation runs This manual describes UNICORN 6 1 which is designed for AKTA avant chromatography systems This software version is not compatible with other AKTA systems Workflow The workflow in UNICORN can be divided into four distinct stages The flow chart below shows the work flow stages 1 Create a method 2 Run the method 3 Evaluate the results 4 Compile a report This manual describes step 1 of this workflow Tip Step 2 how to perform method runs is described in the KTA avant and UNICORN 6 1 User Manual Step 3 evaluate the results and step 4 compile a report are described in the UNICORN 6 1 Evaluation Manual 8 UNICORN 6 1 Method Manual 28 9817 65 AB 1 Introducing the UNICORN Method Editor 1 1 About the UNICORN Method Editor Create a method A method in UNICORN is a user defined set of instructions that can be used to run an
113. Select the row s in the Scouting parameters table and click Result The selected runs are removed from the Scouting scheme or click Result All runs are removed from the Scouting scheme No scouting will be performed when starting the run e To exclude a run from being used in the Scouting experiment but keep it in the Scouting scheme Clear the Included box in front of the appropriate run 3 Click OK in the Scouting dialog to save the changes to the scouting scheme Save the method UNICORN 6 1 Method Manual 28 9817 65 AB 115 5 Design of Experiments 5 Design of Experiments About this chapter This chapter describes how to set up an experimental design plan using the Design of Experiments tool in the Method Editor and how to evaluate the results of the runs in the Evaluation module It also gives a brief overview of Design of Experiments and de scribes some basic terms and concepts used in the Design of Experiments tool in UNI CORN In this chapter This chapter contains the following sections Section See page 5 1 Introduction to Design of Experiments 117 5 2 Create an experimental design 128 5 3 Run a scouting created with DoE 155 5 4 Evaluation of Design of Experiments 157 116 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 1 Introduction to Design of Experiments 5 1 Introduction to Design of Experiments Introduction This section gives a brief introduction to the basic
114. Show drop down list 3 Click Delete A dialog will appear asking you to confirm the deletion UNICORN 6 1 Method Manual 28 9817 65 AB 215 6 BufferPro 6 4 Print a BufferPro recipe 6 4 Print a BufferPro recipe Introduction This section describes how to print a BufferPro recipe from UNICORN A recipe can be printed from the Phase Properties tab in Method Editor or from the BufferPro Recipes dialog It is also possible to include the BufferPro recipes when printing the whole method See Section 3 7 Print a method on page 79 Printing from Method Editor The following table describes how to print a recipe from the Phase Properties tab in 216 Method Editor Step Action 1 In the Phase Properties tab in the Method Editor click the BufferPro Prop erties button Use Butter Pro automatic buffer preparation Recipe CIE mis 0 1M Nall pH 2 7 PO w BufferPro Proja i pH 2 0 7 0 recommended Conc Defined by recipe for multicomponent buffers In the BufferPro Properties dialog click the Print button Result The Print dialog opens Choose a printer from the drop down list in the Print dialog and click OK UNICORN 6 1 Method Manual 28 9817 65 AB 6 BufferPro 6 4 Print a BufferPro recipe Printing from BufferPro Recipes dialog The following table describes how to print a recipe from the BufferPro Recipes dialog Step Action 1 In the Method Editor select Tools Buf
115. Tag label UNICORN 6 1 Method Manual 28 9817 65 AB 261 8 Column Handling 8 3 Handling individual columns 8 3 2 Register a new individual column 8 3 2 Register a new individual column Introduction In order to take advantage of the column handling features of UNICORN each individual column needs to be registered in the software Note It is essential that the individual column is registered before a column perfor mance test is performed Otherwise the results will not be entered in the Column Logbook It is not possible to enter the performance test results af terwards Register an individual column The following table describes how to register an individual column in the Column Logbook Step Action 1 Select the Column Logbook tab and then click New Comin lype Pasaman Column Logbook Select Colne Case jal N oi Zh by rar abal Find by iC Tind by aise fo Peona typan user created Colmens Column hps i Abys HFiep SF FF 16 00 A Hep SPSL IRAD 3 Hce 16 2 Cano ULALE Irasanadi Wiadin Baiah 1 Hipcete Ie 20 Came HHC erie M Hiiiraan Rup FF Hadum sapasin dse Hi is FP Hiensen Capto adhera Date ot ird um Higes Lopo DEAE Hiierman iis ureter of rune Wim of cies pretties Fa Hidereen Mac FF Number of runs sirce beet CP l Hune of runes arce bai Caium palace leat Hiipean Hab Tele SuPle id Laleulsted bani en ihe bi Coann Fabeae tel sed i 1 F ae bhie Tran LD Hew dre Poa Expat Cim damhs Fedarn
116. The optimization designs will give enough information to create the quadratic part of the model 126 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 1 Introduction to Design of Experiments DoE workflow in UNICORN The main steps when performing a Design of Experiments in UNICORN are Step Action 1 Create a method for your process to be screened optimized or tested for robustness This includes defining the appropriate variables if not already defined that should be connected to the factors in DoE See Chapter 3 Create and edit methods on page 25 for information about how to create a method 2 Set up an experimental design This is performed in the Method Editor in the Design of Experiments tool See Section 5 2 Create an experimental design on page 128 for more informa tion 3 Perform the runs in the Scouting scheme generated from DoE See AKTA avant and UNICORN 6 1 User Manual for information about starting scouting runs Note The Scouting scheme generated from DoE does not normally need to be edited If for some reason this is absolutely necessary care must be taken so that the results can be used during evalu lation of the DoE results 4 Perform statistical evaluation of a DoE scouting This is performed in the Evaluation module See Section 5 4 Evaluation of Design of Experiments on page 157 for more information UNICORN 6 1 Method Manual 28 9817 65 AB 127 5 Design of
117. UNICORN This chapter gives an overview of the Column Handling and Column Logbook tools In this chapter This chapter contains the following sections Section See page 8 1 Overview 242 8 2 Handling column types 247 8 3 Handling individual columns 260 8 4 Column performance 276 8 5 Intelligent Packing of AxiChrom columns 279 UNICORN 6 1 Method Manual 28 9817 65 AB 241 8 Column Handling 8 1 Overview 8 1 Overview Introduction This section gives an overview of the Column Handling tool and suggests a workflow when working with column types and individual columns Definitions Description Column type A type of column consisting of a particular hardware and media Column An individual column of a column type or Individual col umn Example A laboratory has two Mono Q HR 16 10 columns used in different projects Both columns are of column type Mono Q HR 16 10 However each individual column may be treated and logged separately using Column Logbook in UNICORN assuming this option has been enabled during installation Note When creating methods and performing method runs the bed height for the column type will be used If you wish to use the actual bed height of a custom packed column e g an AxiChrom column you must create a specific column type that is equal to the individual column using the packed bed height Open the Column Handling dialog To open the Column Handling dialog e sel
118. UNICORN 6 1 Method Manual 28 9817 65 AB 125 5 Design of Experiments 5 1 Introduction to Design of Experiments Graphical illustration Description Constant N A by is the unknown constant term It is the response bo of Y when the main effects are 0 Linear main effects Undistorted plane The main effects are described by the linear terms bX bX b3X In the graphical illustration this part of the model can be viewed as an undistorted plane It will give an overall idea of where the optimum for your process is but not details on how the sampling plane is twisted or which curvature the plane has This part of the model usually gives sufficient infor mation when the objective is screening or robust ness testing The fractional factorial designs will give enough input to create the linear part of the model Two way interaction Twisted plane The two way interaction terms describe how the bioX1X gt by3 1X3 Fe effect of one factor depends on the level of a sec bo3XoX ee Pe ond factor In the graphical illustration this part of the model can be viewed as twisted plane This part is added to the model when the objective is screening The fractional and full factorial de signs will give input to the two way interaction part of the model Quadratic Curved plane The curvature of the sampling plane is described b X24 b x bx aes Aa terms E This part is added to the model when the objective is optimization
119. Upper Repeat this procedure until you are satisfied Optimize response values and factor settings It is possible to optimize the response values using the optimizer When using the opti mizer criteria for the response values and factor settings are entered e g Yield gt 90 and factor settings are calculated In this way the experimental region can be moved to an optimum The table below describes how to use the optimizer Step Action 1 Select the Optimization tab in the Design of Experiments box Result The Optimization Criteria and Result tables are displayed Expenment Ar Prediction Optintinstion Optimization Catena Faime Fach Eak we a eta pa E Conshard 350 r Tise amp a Fina wt 15 Ancol _ Calas Optimal Seninge Cem Losfigi Led Conduct Tieki Arep F gemo Losi Man UNICORN 6 1 Method Manual 28 9817 65 AB Enpe Becpenc riora Wagh bii Tera Max Megs W 1 a k Himes 7 P EES E Fiy P Arpa 197 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 6 Use the model 198 Step Action 2 In the Responses area select the Criteria for the response Responses Response Criteria Weight Min Target Max CO T hd aximize Target Exclude The following choices are available e Minimize The response value should be minimized Enter Target value and Max value for the response e Maximize The response value should be m
120. a BufferPro method In this section This section contains the following sections Section See page 6 3 1 Create and edit a BufferPro recipe 208 6 3 2 Rename a BufferPro recipe 212 6 3 3 Delete a BufferPro recipe 214 UNICORN 6 1 Method Manual 28 9817 65 AB 207 6 BufferPro 6 3 Create and edit BufferPro recipes 6 3 1 Create and edit a BufferPro recipe 6 3 1 Create and edit a BufferPro recipe General considerations The concentration of the buffer stock will affect the pH range and the settable concen tration range in the method The pH range will in general increase with increasing buffer concentration and decrease when lowered The titrant and buffer agent concentrations should be the same since there may other wise not be sufficient titrant to reliably obtain the entire pH range For recipes titrated with strong acid base the concentration range that can be achieved is 15 25 of the buffer stock concentration For conjugate acid base titrants the corresponding range is 25 50 of the buffer stock concentration The following table describes how to create a new recipe and how to edit existing recipes Create edit a recipe 208 The following table describes how to create or edit a BufferPro recipe Step Action 1 In the Method Editor select Tools BufferPro Recipes Start Protocol Evaluation Procedures we Scouting Design of Experiments Column Handling Ctrl L ah ae Adm
121. able below describes how to edit variable values in the Phase Properties tab for a text edited phase Step Action 1 Select the Phase Properties tab to display the Phase Variables list Phase Properties Text Instructions t Method Senings 7 Equilibratom T This phata has bean tant ed ad Phase Variabiet Block Vatieble Valve flange FO BRATION Inder Al v C Show detai Ect Variable lt gt l Restore Phare Properes 2 Change the variable value for the appropriate variable in the Value field by choosing a new value in the drop down list or typing in the field Tip To show detailed variables check the Show details box Result The variable value is updated 3 Repeat this procedure for the appropriate variables Edit variable values in the Instruction Box The table below describes how to edit variable values in the Instruction Box UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables Step Action 1 Select the instruction containing the variable value to be edited in the Text Instructions area Result The parameters for the instruction are shown in the Instruction box H 0 0 00 Phase Column Wash 5 0 0 00 Phase Equilibration 0 00 Base SameAsMain 0 00 Inlet A Al inlet A 0 00 Inlet B B1 i Inlet B 0 00 Gradient 0 00 Percent B Equilibration _1 B 0 00 base 0 00 System flow 1 000 Flow rate
122. ables consider the following e The names can be up to 50 characters long and the following characters can be used Letters a z Digits 0 9 The underscore character _ The Space character e The case of letters is retained but not significant The names Flow Rate and FLOW RATE are treated as identical For information about defining and renaming variables see Define new variables on page 318 and Edit variables on page 320 UNICORN 6 1 Method Manual 28 9817 65 AB 317 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables Define new variables Only one variable that affects block length breakpoint or gradient length may be defined within each block However any number of parameters may be defined as variables within a block The table below describes how to define a new variable Step Action 1 Select the instruction where you want to define the variable in the Text In structions area Result The parameters for the instruction are shown in the Instruction box 0 00 Wavelength 280 U 1 nm Off HUY2 nm OfFHUY3 nm 0 00 Noise reduction UY 0 2 UY averaging time sec 0 00 Injection valve Manual load 0 00 Column position By pass Column position Down flow 0 00 pH valve In line pH cell In line Flow restrictor 0 00 Alarm air sensors Enabled Ai sensor alarm on inlet valve Enabled Air sensor alarm on inlet valve B Disabled Air se
123. about how to find and select a column 2 In the Column Logbook tab click Result The Notes dialog for the selected column opens Notes 28 9288 13 20090415 0000 00 0037 Desalting RbIC Pr fx l 3 SG Column meant for use for desalting after ammonium sulphate precipitation step Storage 102 ethanol Cold room 24 IF you want to borrow me PLEASE contact Gus in room Gb4 3 3 Add edit notes by typing in the dialog and click OK Result The notes for the column are updated 268 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 3 Handling individual columns 8 3 4 Edit individual columns Set notification limits for an individual column Notification limits can be set for individual columns Once the limit is reached the user receives a message stating what action should be taken before the column is used Ex amples of such limits are the Medium expiration date that the column has been used a given number of times since the last CIP or column performance test were performed or that the column performance parameters are poor so the medium should be repacked Note Warnings related to the number of times a column has been used for example since the last CIP was performed are only issued when the start protocol is performed In a method queue this may not always be shown for every run Each run will however be noted in the column history The table below describes how to set notification limits fo
124. added to the method file as opposed to responses added in the Method Editor 138 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 2 Add responses and factors to an experimental design Step Action 3 e To add a predefined response Select the response to be added in the Predefined drop down list e To add a user defined response Select User defined and type in your own response Result Abbreviation is automatically filled in e f applicable enter unit for the response Add Response Predefined Elution Conductivity User defined Abbreviation Unit C 4 Click OK Result The selected response is added to the Responses list in the Design of Experiments dialog Design of Experiments Responses Factors amp Design Experiment Responses Abbreviation Unit Add UNICORN 6 1 Method Manual 28 9817 65 AB 139 5 Design of Experiments 5 2 Create an experimental design 5 2 2 Add responses and factors to an experimental design Add factors The table below describes how to add factors to a Design of Experiments Step Action 1 Select the Factors amp Design tab Design of Experiments Responses Factors amp Design Experiment Factors Name Method Variable Method Phase Click Add to define a factor Edit Delete Design selection Objective Screening Total number of r
125. all registered individual columns select all the available column types by checking the boxes for Predefined Global and Personal types then select all the Column Types in the list 4 e Ifyou have a short list of individual columns registered for the column type just select the appropriate column in the Columns list To select several columns use the Ctrl and Shift keyboard keys e lf you have many individual columns registered find and select the ap propriate column as described below position the cursor in the first position of the Code field scan the column barcode or UniTag and click Find or type in the barcode in the Find by ID field and click Find or type in the alias in the Find by alias field and click Find Result The individual column is selected in the Columns list UNICORN 6 1 Method Manual 28 9817 65 AB 267 8 Column Handling 8 3 Handling individual columns 8 3 4 Edit individual columns 8 3 4 Edit individual columns Introduction This section describes the ways in which individual columns may be edited This includes adding and editing notes setting notification limits and deleting individual columns Add edit notes for a column The table below describes how to add edit notes for an individual column Step Action 1 Select the individual column for which to add edit notes in the Columns list in the Column Logbook tab See Section 8 3 3 Find an individual column on page 266 for information
126. also have these options e Click the Preview button to open the report in Notepad e Click the Print button to print the report without any preview 5 e Click the Finish button Result The report is generated and saved and the wizard dialog closes UNICORN 6 1 Method Manual 28 9817 65 AB 357 Index Index 358 A Add runs DoE 166 Scouting 114 Affinity chromatography Predefined method 81 Alarms Available monitors 332 General description 330 Insert alarm in method 331 Anion exchange Predefined method 82 ANOVA analysis of variance table Design of Experiments 187 Auto Hide Optional Method Editor panes 18 B Barcodes Column identification 261 Base instruction CV Column volume 296 Edit settings 297 Text instructions 296 Time 296 Volume 296 Blocks Adding 302 Copy paste 303 Deleting 308 Import from other meth ods 304 Moving 306 Rename 307 Text editing 300 Breakpoints In methods 350 BufferPro Considerations when preparing buffers 226 Create method using 206 Create recipe 208 Delete recipe 214 Edit recipe 208 Explore proportions 219 Export recipe 222 Import recipe 224 Overview 204 pH ranges 227 Predefined buffers anion exchange 205 Predefined buffers cation exchange 205 Predefined recipes 227 Print recipe 216 217 Rename recipe 212 Workflow 204 Buffers Anion exchange 205 Calculate composition of 219 Cation exchange 205 Considerations
127. aluation Manual and adjust the actual bed height according to the results if necessary UNICORN 6 1 Method Manual 28 9817 65 AB 285 9 Text edit methods 9 Text edit methods Introduction Normally methods are created and edited using the Method Outline Phase library and Phase Properties panes in the Method Editor see Chapter 3 Create and edit methods on page 25 for information about how to create and edit methods using the Phase Properties pane However in some cases you may want to edit a method or phase using the Text Instructions This can be an option for fine tuning or optimization of a method This chapter gives an overview of the Text Instructions pane and describes how to use the Text Instructions pane to create and edit methods It also describes some text in struction applications and how to access information about the text instructions In this chapter This chapter contains these sections Section See page 9 1 Overview 287 9 2 Working with methods in the Text Instructions pane 295 9 3 Specific instructions 326 286 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 1 Overview 91 Overview Introduction This section gives an overview of working with text instructions and a description of the Text Instruction pane In this section This section contains the following sub sections Section See page 9 1 1 Working with text instructions 288 9 1 2 The Text Instructions pan
128. ame for the block in the Block field e Click the Insert button Result The block is inserted after the block that was selected in step 1 302 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 2 Working with phases and blocks Copy and paste blocks The table below describes how to copy and paste a block Step Action 1 Select the block you want to copy e click the Copy icon in the Toolbar or e right click the block and choose Copy or e select Edit Copy Ctrl C 2 Select the instruction line just above the point where you want the block to be pasted e click the Paste icon in the Toolbar Import Block Method _ Select block Folder name System Last modified SEHCSJ2DP3 D UNICORNAUNI Adam 5 18 2009 1 23 0 Stop frac Column Wash E Ane 5 18 2009 1 22 4 Eb a a ek EJ DAOCS NickelColumn SystemC 6 4 2009 2 04 58 Stop frac Elution Bridget 5 18 2009 1 22 5 pees ate E Charlie 5 18 2009 1 23 1 Call from Christine 5 18 2009 1 23 0 METHOD SETTINGS David E 5 18 2009 1 23 3 Call at David T 5 18 2009 1 23 2 0 00 CV DefaultHome 9 25 2008 1 07 71 Block name TZ CR ate ata a ma s 1 Start frac Column Wash or e right click the instruction line and choose Paste or e select Edit Paste Ctrl V Result A Rename dialog opens UNICORN 6 1 Method Manual 28 9817
129. ample injection Run Included Sample inlet 1 1 52 53 S4 51 55 sa 3 Click Cancel or OK to close the Scouting dialog Print method including DoE Before starting the run it is useful to print the method information to see for example which sample positions are used for the different runs See Section 3 7 Print a method on page 79 for information about how to print the method 156 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 Evaluation of Design of Experiments Introduction This section describes how to perform statistical evaluation of a DoE scouting In this section This section contains the following sub sections Section See page 5 4 1 Workflow 158 5 4 2 Generate model 160 5 4 3 Analyze and evaluate the model basic analysis 170 5 4 4 Analyze and evaluate the model extended analysis 182 5 4 5 Edit the model 190 5 4 6 Use the model 193 5 4 7 Create and print reports 200 UNICORN 6 1 Method Manual 28 9817 65 AB 157 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 1 Workflow 5 4 1 Workflow Introduction This section describes the workflow when evaluating a Design of Experiments scouting Workflow The main steps when performing statistical evaluation of an experimental design are 1 Generate model This includes evaluating single DoE runs opening the DoE result and entering re sponse data Th
130. ance Fecet Haini Voter ea E T ol as Result The first New Column dialog opens New Column Add a new column by entering a Column ID either manually or with a barcode scanner Code lot exp ID coum O Jf ll __ o Add column by manually entering UniT ag lot and ID hil ag has tired values for Code and exp UniT ag has fixed values for Cod d 262 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 3 Handling individual columns 8 3 2 Register a new individual column Step Action 2 Register the column using the 2D barcode scanner as follows e Make sure that the mouse pointer is placed in the first position of the Code field e Pointthe 2D barcode scanner towards the data matrix tag on the column label or the UniTag label e Press and hold the trigger mb T e When the 2D barcode scanner beeps the column ID is registered and the second New Column dialog opens 3 If no 2D barcode scanner is available enter the column ID manually e fthecolumn hasa column label enter the column ID shown in the Code field e Ifthe column has a UniTag label check the box Add column by manu ally entering UniTag lot and ID and manually enter the number for the lot and ID fields e Click Continue to open the second New Column dialog Note The lot field should contain eight digits and the ID field should contain four digits If the lot or ID numbers of the column contains fewer than e
131. and Empty methods Method Description Predefined Predefined methods include a number of relevant phases appropriate for the purification or maintenance to be performed You may use the predefined methods as they are or with adjusted settings as needed See Section Section 3 8 Predefined methods and phases on page 81 for descriptions of the Predefined methods supplied with the software Note The Predefined methods are included in the instrument configuration files for each specific instrument Empty methods include the mandatory phase Method Settings Other phases are then added by the user and settings adjusted as needed Predefined phases UNICORN provides a number of Predefined Phases e g Equilibration Column CIP and User Defined that can be used when building editing methods in the Method Editor A predefined phase contains all necessary instructions to be run except Method Settings which is mandatory in all methods and User Defined that are special phases See Section 3 8 Predefined methods and phases on page 81 for descriptions of the pre defined phases supplied with the software See also Chapter 3 Create and edit methods on page 25 24 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 Create and edit methods About this chapter This chapter describes how to create edit and handle chromatography and maintenance methods in UNICORN 6 1 using the Phase Properties pane It a
132. and click OK Result The method has been signed UNICORN 6 1 Method Manual 28 9817 65 AB 95 3 Create and edit methods 3 11 Import and export methods 3 11 Import and export methods Introduction UNICORN methods are stored internally in the UNICORN database It is however possible to export entire methods or individual phases to a zip file on the local computer so that they can be imported again later into the same database installation or imported into another database installation Alternatively methods or phases can be exported as plain text files or Excel files which may be useful for documentation purposes 94 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 11 Import and export methods Export a phase or method to UNICORN The following table outlines the steps needed to export a method or a phase for later import into UNICORN Step Action 1 In the Method Navigator select the method to be exported Method aA Open rear A Methods Method Q E Folder name System A SEHCA2DPSI0SUNICORNSUNI l Adam Anne My Affinity Method System Bridget ao PA Chsrlis or In the Phase Library pane select the phase to be exported B Method Editor My Affinity File Edit Views Phases Tac MEL version A X i Rubisco Elution z a a od Note Only Personal or Global phases may be exported Note Only single methods or phases may be exporte
133. ane e Repeat until all runs are included using the correct variable values Note The scouting scheme can also be edited just prior to starting the method run in the Start Protocol Here variable values can be changed and individual runs included or excluded 5 To insert a series of runs click in the appropriate variable column in the Scouting parameters table and click Series This button is activated for variables with continuous values such as flow rates or pressure limits Result The Insert Series dialog for the selected variable opens Insert Series For Flow rate x Enter start value how much the value shall be Increased for each run and how many rung to add Range 0 000 25 000 Start value Shep by Number of runs 0 000 Set as integer values UNICORN 6 1 Method Manual 28 9817 65 AB 109 4 Scouting 4 2 Set up and edit a Scouting scheme Step Action 6 In the Insert Series dialog e Enter Start value Step by and Number of runs In this example 0 5 0 5 and 6 Result The Scouting parameters table is updated Scouting parameters Method Settings HETHOD SETTINGS Aun Included Flow rate ml min 1 0 500 2 1 000 3 1 500 4 2 000 5 2 500 E 3 000 7 Alternatively to enter either consecutive or non consecutive integer values e Check the Set as integer values box in the Insert Series dialog Result The following alternative Insert Series dialog for the selected
134. ange Design Linear Linear Linear Linear Second Interaction Interaction Design setup of the factor levels Main effects and all interactions are clear of each other not confounded Default No ot Center Points 3 runs for Full Factorial 2 levels is 4 i Replicates 0 x Total no of runs 3 To change to another design select the appropriate design in the Design table Result The Description field shows a short description of the selected design For a description of which designs are supported by UNICORN and when they may be proposed see Designs supported by UNICORN on page 123 146 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 3 Change design and design settings in a Design of Experiments setup Step Action 4 The Design setup area shows the settings for the selected design e Change the settings for No of Center Points and Replicates as appropri ate The table below describes the different settings Setting Description No of Center The No of Center Points means that the center Points point experiment will be run the selected number of times It is recommended to use at least three center points to be able to estimate the pure error that is the variation in the measurements Replicates Replicates means that the whole experiments series corner and center points will be replicat ed the selected
135. anual 28 9817 65 AB 3 Create and edit methods 3 11 Import and export methods Import a method into UNICORN Methods that have previously been exported as zip files can be imported back into UNICORN Plain text files or Excel files cannot be imported since there is no guarantee that they contain all the information UNICORN needs to recreate the method The follow ing table outlines the steps needed to import a method Step Action 1 Select File Import Import Method Result The Import dialog opens Look in e Methods Folder ia Affinity Method zip My Recent Documents B Desktop I My Documents My Computer File name My Network Files of type Zip Files zip 2 Browse to the required zip file in the Import dialog UNICORN 6 1 Method Manual 28 9817 65 AB 101 3 Create and edit methods 3 11 Import and export methods Step Action 3 Open the file by selecting it and clicking the Open button or by double clicking on the file name Result The Import Method dialog opens Import Method Veer Methods z z Folder name System Last modified Created by SEHCV2DPSIDSUNICORNSUNI H Adam 5 18 2009 1 23 0 Default FA Anne 187 2009 1 224 Default Bridget 187 2009 1 2 5 Default Charlie 5187 2009 1 231 Default Christine BY 182009 1 23 00 Default David E 571872009 1 23 35 Default David T 571872009 1 23 2 Default DefaultHome 25 2008
136. arlie 5 18 2009 1 23 1 Salomi Christine 5 18 2009 1 23 0 METHOD SETTINGS E DavidE 5 18 2009 1 23 3 David T 5 18 2009 1 23 2 DefaultHome 9 25 2008 1 07 1 o rw mnnm sa me Block name E tart frac Column Wash Result All available blocks are listed in the Select block field 3 Select a block to import from the method in the Select block list 304 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 2 Working with phases and blocks Step Action 4 e Select the block where the imported block will be inserted in the Call from drop down list e Type the breakpoint that the imported block will be called at in the Call at text box e f necessary type a new name for the block in the Block name text box optional 5 e Click the Import button e Confirm if you also want to import sub blocks if any E A The block selected for import contains one or more sub blocks Do you want to import the sub blockis too Result The block is imported into the method you are editing Unless you have specified a breakpoint that is earlier the block will be inserted at the end of the block that it is called from Note If you are importing a block with the same name as a block that already is included in the method you are editing a warning will open and you will be asked to confirm that you wish to replace the original block with th
137. as been added to the model Now the residuals are randomly distributed with no pattern Load pH vs Residuals for Aggregate Content with Experiment Number labels a oo 5 i E a T 3 i ii F a D Oo 59 6061 62 63 64 65 66 6 7 66 69 70 71 72 Ta F 4 75 76 77 FB 79 00 51 Load pH Note When you find curvature in the model the design for the experiment in the DoE setup should be changed to design allowing quadratic terms to be added to the model Full factorial 3 levels CCC CCF Box Behnken Rechtschaffner or Doehlert If the experimental setup is sufficiently stable the star point ex periments alone can be run otherwise it is recommended to rerun all exper iments UNICORN 6 1 Method Manual 28 9817 65 AB 183 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 4 Analyze and evaluate the model extended analysis Residual versus run order plot The Residuals Plot vs Run order shows the residuals for the run order and one response The residuals should be randomly distributed with no pattern A pattern in the plot indi cates a change in residuals over time This could for example be the result when ran domization errors exist in the experiment The illustration below shows a plot where the residuals are randomly distributed with no pattern Yield with Experiment Number labels Standardized Residuals Run Order W 11 B2 0 996 BR Adj 0 993 DF 5 Q2 0 984 BSD 0 8248 Interaction plot
138. ase lint CV e E Cvate E Use flow seetictoe E Claes Column Wash Flow Pinto Lindt win E Deteuthome Epia Cokin penton Bp pasa Mi Mister anllinagyt Bex 7 EHra Dampa Appears Eki Wavelength 190 700 i E lawns ee Flew pale 100 olan O00 25 000 iP i 200 9 Been een ce F Contrat the flow to iid overnnectune gow a B Eroina Equilibratean Oa iji See Sytem Progen ate E i Ba Line manually prepared buller u BD Lda TERETI Wea a e wa m arr pet mentored EI birt lotta Une BufterPrp automatic buffer preparation Enable ae tensor alarm a E Mavis Pedant Pham Pee Acetate DIt Pi TECLA Phy fist inda A s A D iea Propers irb E bi a 3 i peery Sample riat t EI Mathes iam a LA i pH Aki 78 5 4 it o a p diee a i Mos i Bare Gr ian Phase Block oi oo Method Settings Mar aH 10 2 7 Lit mG to s 4 0 20 10 a y EJ 14 1 Fai F a Description Method Navigator optional pane Shows all the user folders methods and method queues that are available in the database Phase Library optional pane Contains all available phases Method Outline Shows the phases included in the opened method 4 Phase Properties tab Select to display the Phase Properties Phase Properties shows the settings for the highlighted phase in the Method Outline 16 UNICORN 6 1 Method Manual 28 9817 65 AB 2 The UNICORN Method Editor 2 1 The Method Editor Descr
139. ases 3 3 Edit methods and phases About this section This section describes how to edit the phase properties for a phase and how to edit the method outline of a method that is determine which phases that should be included in the method and determine the order of the phases in the method In this section This section contains the following sub sections Section See page 3 3 1 Edit phase properties 35 3 3 2 Edit the method outline 43 34 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 3 Edit methods and phases 3 3 1 Edit phase properties 3 3 1 Edit phase properties Introduction When editing Phase Properties for a phase the changes affect either e the whole method when editing the Method Settings phase or e only the phase that is being edited when editing phases other than the Method Settings phase UNICORN 6 1 Method Manual 28 9817 65 AB 55 3 Create and edit methods 3 3 Edit methods and phases 3 3 1 Edit phase properties Getting help when editing Phase Properties 36 The table below describes how to get help information for the properties in a phase Step Action 1 Select a phase in the method to be edited for example Equilibration Method Settings Ste leiiiisle licen v Sample Application v Column Wash v Elution v Equilibration Result The properties for the selected phase are displayed in the Phase Properties pane Phase Properti
140. at name in the method Import Block The method already contains a block with the name Start Frac Column Wash Do you want to replace the existing black UNICORN 6 1 Method Manual 28 9817 65 AB 305 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 2 Working with phases and blocks Move blocks Blocks can be moved by drag and drop within the method You can also use Cut and Paste as described below Step Action l Select the block you want to move e click the Cut icon in the Toolbar 5 or e right click the block and choose Cut or e select Edit Cut Select the instruction line just above the point where you want the block to be moved e click the Paste icon in the Toolbar wi or e right click the instruction line and choose Paste or e select Edit Paste Result The block is now removed from its original breakpoint and pasted at the new breakpoint Note The pasted block is inserted with the same breakpoint value as the block or instruction selected for point of insertion 306 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 2 Working with phases and blocks Rename blocks The table below describes how to rename a block Step Action 1 Right click the block in the text pane and select Rename uk Copy Delete Print What s This Result The block name is highlighted i
141. ate buffer composition using BufferPro 219 6 6 Export and import BufferPro recipes eee 6 7 Predefined BufferPro recipes 226 UNICORN 6 1 Method Manual 28 9817 65 AB 203 6 BufferPro 6 1 BufferPro Overview 6 1 BufferPro Overview Introduction This section gives an introduction to the BufferPro tool in UNICORN and includes a brief overview of the BufferPro recipes that are predefined What is BufferPro The BufferPro tool allows automatic mixing of buffers during a run Four stock solutions are generally used in a recipe the buffering agent a titrant a salt stock solution and water BufferPro facilitates Scouting or Design of Experiments runs using pH as a variable BufferPro is optimized for use with anion or cation exchange chromatography but can also be used with gel filtration where the salt concentration may also be used as a variable during Scouting or Design of Experiements Commonly used buffer systems have predefined recipes in UNICORN from which new recipes can easily be created New or edited recipes may be stored as personal or global recipes UNICORN uses a robust algorithm to calculate pH ranges for optimal buffering taking into account the buffer type concentration temperature and ionic strength Once an optimal buffer has been found it is possible using BufferPro to calculate the buffer composition for the production of bulk scale buffer solutions if required For details on Scouting Cha
142. ate the model extended analysis on page 182 for information about how to evaluate these plots This section describes how to use the model Generate response surface plot and edit settings The response surface plot graphically displays the experimental region It is helpful when you want to get an overview of how different factor settings affect the response find the interesting experimental area get help in deciding where to start a new investigation get help in deciding where to make verifying experiments understand the impact of large interactions Note The underlying model must be good and have a high Q value See Sec tion 5 4 3 Analyze and evaluate the model basic analysis on page 170 UNICORN 6 1 Method Manual 28 9817 65 AB 193 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 6 Use the model The table below describes how to generate a response surface plot and how to evaluate the plot Step Action i In the Analysis tab click _ Response Surface Piot Result The Response Surface Plot dialog opens Response Surface Plot Contour Plot Load Conductivity 6 5 150 200 Load Mass Response Yield x Constant factors Factors at the plot axes eens Low High Factor Settings x axis Load Mass w 75 300 Reset default Load Conductivity i CENTER Y axis Load pH vl 6l 8 Reset default The Contour Plot shows a map of the model The plot has
143. ated with the changes 6 Add new scouting runs to the scouting scheme as required 7 Click OK in the Scouting dialog to save the scouting scheme Save the method UNICORN 6 1 Method Manual 28 9817 65 AB 113 4 Scouting 4 2 Set up and edit a Scouting scheme Add delete runs in the Scouting scheme The table below describes how to add runs and series of runs to the Scouting scheme and how to delete runs Step Action 1 Open the Scouting scheme see Set up a scouting scheme on page 106 Scouting Scouting parameters Method Settings METHOD SETTINGS Run Included Flow rate ml min 0 500 1 000 1 500 2 000 2 500 114 UNICORN 6 1 Method Manual 28 9817 65 AB 4 Scouting 4 2 Setup and edit a Scouting scheme Step Action e To inserta run after an existing run Select the appropriate row in the Scouting parameters table and click Insert Run Result A new row is added below the selected run to the Scouting pa rameters table The variable value from the selected row is copied to the new run Edit the variable value as appropriate e To insert a new series of runs Click in the appropriate variable column in the Scouting parameters table and click EE Set up a series in the Insert series dialog and click OK see Set up a scouting scheme on page 106 Result The new set of runs are inserted in the Scouting scheme with the values provided e To delete runs from the Scouting scheme
144. ation designs 123 Optimize response val ues 197 Outliers 170 Overview 117 Predict response values 196 Print method 156 Print report 202 Replace run results 167 Replicate plot 171 Report creating 200 Report printing 202 Residual vs run order plot 184 Residual vs variable plot 182 Response surface plot 194 Summary of Fit plot 176 View scouting scheme 155 Workflow 127 Documentation AKTA avant 12 Downscale Scale a method for a small er column type 73 E Edit BufferPro recipe 208 Column individual 268 Column type 251 DoE model 190 Method notes 55 Method outline 43 Method phases 37 Method queue 235 Scouting scheme 106 Text instructions 311 Variables 320 Electronic signature Signing a method 92 Elution Predefined phase 88 End_Block Text instruction 300 Equilibration Predefined phase 87 Evaluation DoE results overview 158 Single DoE runs 160 Evaluation procedures Include after run 57 Experimental design Add factors in DoE 140 Add responses in DoE 138 Change design in DoE 145 Design types in UNI CORN 123 Setup in DoE 129 Exporting BufferPro recipes 222 Column types 254 Individual columns 271 Methods 95 Phases 95 F Fractional Factorial design Description 122 Fraction collection Overview 89 Setup 90 Full Factorial design Description 122 UNICORN 6 1 Method Manual 28 9817 65 AB G Gel filtration Predefined method 83 Gradients Gradient breakpoi
145. ave the edited column type Result The Save As dialog opens Save As sess Column type name e Edit the Column type name and click Save Result The column type is saved in the database and displayed in the Column types list 6 If editing parameters for a Global or Personal column type the column type can be saved with a new name see step 5 above or the changes can be applied to the current column type name e Click Save Result The changes for the column type are saved Note When editing parameters for Global column types It is recom mended to save the edited column type with a new name Other users may otherwise not be aware that the parameters have been changed for that column type Note Methods that use the edited column type should be updated Delete column types Note It is not possible to delete predefined column types from the database If a column type has any registered columns it can not be deleted unless the individual columns are first deleted See Section 8 3 Handling individual columns on page 260 for information about how to delete individual columns If an individual column of a certain type has been used it will not be possible to delete either the individual column or the column type The table below describes how to delete Global and Personal column types from the database UNICORN 6 1 Method Manual 28 9817 65 AB 253 8 Column Handling 8 2 Handling column types Step Action
146. aximized Enter Target value and Min value for the response e Target The response value should be optimized to reach the Target value Enter Min Target and Max values for the response e Exclude The response should not be included in the optimization if you have several responses Result The entered values are displayed Responses Response Criteria Weight Min Target Max Yield Maximize E 90 l UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 6 Use the model Step Action 3 In the Factors area select Role and settings for each factor Factors Factor Role Value wae ae Free w 100 300 Constant 15 le 75 e lf the role Free is selected the factor settings to be calculated for the response can have values within the entered Low Value and High value range Enter the Low Value and High value as appropriate to get an idea of the new region of interest use the response surface plot e Ifthe role Constant is selected the factor setting is constant Enter the factor value in the Value field 4 In the Result area click the Calculate Optimal Settings button Result The results are displayed in the Experiment table Resuk Calculate Optimal Settings HH Factor Ml Response Load Mass Load Conductivity LoadpH Yield Iter Log D 1 9455 1
147. bes how to generate a system error report describing performance problems In this chapter This chapter contains these sections Section See page 10 1 Troubleshooting methods 347 10 2 System Error Reports 553 346 UNICORN 6 1 Method Manual 28 9817 65 AB 10 Troubleshooting 10 1 Troubleshooting methods 10 1 Troubleshooting methods Introduction This section describes how to solve the following method problems The Phase Properties tab only shows a variables table You cannot find settings that you need in the Phase Properties There are red instructions in a method Breakpoints are not calculated correctly Volumes are smaller or larger than expected after a method is converted A column cannot be selected for scaling The Print Screen command does not send a copy of the screen to the printer Undefined inlets are used briefly for CIP or preparation phases The Phase Properties tab only shows a Phase Variables table The table below describes how to restore the options and settings to the Phase Properties tab Problem description The Phase Properties tab shows only a variables table and not the regular options and settings for the selected phase The phase is marked with the letter T in the method outline UNICORN 6 1 Method Manual 28 9817 65 AB Solution The phase has been edited in the Text Instruc tions tab Click the Restore Phase Properties button to return to the default settings and
148. ble to log on to UNICORN and access a database e You need to understand the general concepts of liquid chromatography Terminology and functionalities will be explained only when they differ from normal practice UNICORN 6 1 Method Manual 28 9817 65 AB 11 1 Introducing the UNICORN Method Editor 1 3 UNICORN 6 1 user documentation 1 3 UNICORN 6 1 user documentation Introduction This section describes the user documentation that is delivered with an AKTA avant system User documentation The user documentation listed in the table below is available from the Help menu in UNICORN or on the AKTA avant and UNICORN User Documentation CD Document Main contents KTA avant and UNICORN 6 1 Site preparation stand alone installation and test Installation Guide procedure Getting Started with KTA System overview and instructions to perform a basic avant and UNICORN 6 1 run KTA avant and UNICORN 6 1 Instructions for safe handling of the system Descrip User Manual tions of components Information about how to run and maintain the system UNICORN Help Dialog descriptions for UNICORN from the Help menu UNICORN 6 1 Method Manual Overview and detailed descriptions of the method creation features in UNICORN Instructions on how to use the software Workflow descriptions for common operations UNICORN 6 1 Evaluation Man Overview and detailed descriptions of the evaluation ual features in UNICORN I
149. by UNICORN Design type Description L designs L designs are a type of Fractional Factorial design Different variants are available in UNICORN The table below gives a short description of the designs L design Description Fractional design at three levels for up to four factors You can estimate square terms but not all interactions Fractional design with one factor at two levels and with up to 7 factors at three levels Fractional design at three levels for up to 13 factors You can estimate square terms but not all interactions Fractional design at three levels for up to 13 factors You can estimate square terms but not all interactions L designs are useful when performing Screening or Robustness Testing UNICORN 6 1 Method Manual 28 9817 65 AB 123 5 Design of Experiments 5 1 Introduction to Design of Experiments Design type Description Plackett Burman Plackett Burman is a type of Fractional Factorial design with a lower resolution This means that it is not possible to estimate any two factor interactions using this design Plackett Burman designs are useful when performing Screening or Robustness Testing Rechtschaffner Rechtschaffner is a saturated fraction of the 2 and 3 factorial designs that supports all the first order interactions and quadratic terms Rechtschaffner is useful when performing Optimization and you have at least three factors in your experimental plan Full Fac
150. cation S ample inlet Sample Application S ample inlet 8 56 2 SB 5 S6 6 57 15 7 5 S7 8 Not Enough Positions 1 15 Not Enough Positions 1 5 Not Enough Positions 1 6 S2 2 2 20 2 8 54 2 S4 20 54 6 Buffer 15 Buffer 20 Buffer 8 53 15 53 20 53 7 55 8 5 55 12 5 55 7 5 8 5 55 12 5 55 7 55 8 5 55 12 5 S5 y O Edit System Setup Click OK to generate a scouting scheme UNICORN 6 1 Method Manual 28 9817 65 AB 135 5 Design of Experiments 5 2 Create an experimental design 5 2 1 Set up an experimental design Step Action 8 To view the system setup click Edit System Setup Result The Edit System Setup dialog opens Edit System Setup Sample inlet Position Load pH Load Conductivity E In this example the variable connected to both factors is the Sample inlet valve The Load pH and Load Conductivity values are set for each sample inlet To change the position for a certain combination of Load pH and Load Conductivity e Select the appropriate position in the corresponding Position drop down list Note It is not possible to change to a position already used Result The changes are saved and you return to the Design of Experiments dialog 136 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 1 Set up an experimental design Step Action 9 e Inthe Design of Experiments dial
151. ct several column types use the Ctrl or Shift keyboard keys 5 In the Column Type Parameters tab click Erot Result The Export Column Type dialog opens Export Column Type Save in 9 Columns G ColumnsInColdroomSep09 zip My Recent Documents Gp Fiera B MyNetwok Saveastyps Zip Fies f zip B 4 Select in which folder to save the information and type a name for the zip file to be exported Result The column type information is exported This information can be imported into another database UNICORN 6 1 Method Manual 28 9817 65 AB 255 8 Column Handling 8 2 Handling column types Import column types The table below describes how to import column types into the database Step Action 1 In the Column Type Parameters tab in the Column Handling dialog click Result The Import dialog opens Look in My Recent Documents amp D rat g D Ja na sT My Network 256 O Columns Apl ColumnsInColdroomSep09 zip ki Harry Jun09 zip File name Harun0s zip Files of type Zip Filet zip Cancel UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 2 Handling column types Step Action 2 Locate the zip file with the column type information to be imported and click Open Result The Import Column Type dialog opens displaying the names of the column types included in the zip file
152. d Note If the flow is changed the slope of the gradient will also change End_Block The gradient formation will continue uninterrupted unless a new Gradient instruction is issued For example this means that a block can be called conditionally during gradient formation without interrupting the gradient UNICORN 6 1 Method Manual 28 9817 65 AB 329 9 Text edit methods 9 3 Specific instructions 9 3 2 Alarm instructions 9 3 2 Alarm instructions Introduction This section is a description of how alarms work in UNICORN and of the Alarms text in structions It also describes the differences between Alarms and Warnings Alarms and Warnings The Alarms parameter settings define the high and low Alarm limits for process monitor signals You can define these limits either in the system settings or as part of a method Settings in the method will override the system settings The limits that will generate a Warning from the system are defined in the instrument configuration files and you cannot edit these settings Conditions can also be applied to process monitor signals such that a block of instructions will execute when a particular condition is satisfied for example when the absorbance of the eluent exceeds a certain limit This is done using Watch instructions which are described in Section 9 3 3 Watch instructions on page 333 The table below describes the general difference between Alarms and Warnings If the si
153. d it is not possible to write multiple methods or phases to a single zip file UNICORN 6 1 Method Manual 28 9817 65 AB 95 3 Create and edit methods 3 11 Import and export methods Step Action 2 Choose File Export to UNICORN Export Method to UNICORN Result The Export dialog opens Export to Another UNICORN Database B Methods Folder E My Recent Documents W File name My Affinity Method zip My Network Save as type Zip Files zip fi Note In the case of phases it is also possible to right click on the phase name and select Export from the context menu 3 Choose a file name and location and click the Save button to save the zip file 96 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 11 Import and export methods Export a method to a plain text file The following table outlines the steps needed to export a method as a plain text file or to Excel Step Action 1 4 In the Method Navigator select the method to be exported Open reer A Methods Method E Folder name System A SEHCI 2DP3ID UNICORNAUNI HI Adam Hi Anne ELMy Affinity Method SystemC HI Bridget B Fa Fhada Choose File Export Export Method Externally Result The Export Externally dialog opens Export Externally My Affinity Method Export items Phase range Text Instructions G All Variable List Phase
154. d Outline and or edit the Phase Properties for the appropriate phases 3 Save the method UNICORN 6 1 Method Manual 28 9817 65 AB 27 28 3 Create and edit methods 3 1 Working with methods Overview Illustration of workflow when creating or editing anew method The illustration below shows the workflow in the Method Editor when creating or editing a method 1 Create method 2 Edit method 3 Save method Create new method Build edit Method Outline Save method Add delete rearrange phases n and or Edit Phase Properties Dasa biis kiia Oelaa Delad Delma Delos Delma Chrintine _ Centre Centre Orire Y Save phase optional UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 1 Working with methods Overview Overall method options In addition to creating editing and saving the method in the Method Editor a number of more general method options are available These are settings for the method and are saved with the method Overall method settings can be divided into two groups The table below shows the dif ferent groups Method option Description General method options setting result name and the location of the results setting up start protocols adding editing notes to the method choosing to include evaluation procedures to be per formed after the run viewing and printing an estimate of the method dura tion time and the variables in the method
155. d be used either to equilibrate the column before purification or to r equilibrate the onurnn after the punitication Select this to recet autozera the UV montor before and after the equilibration This setting is selected by default Shows the equilibration flow rate De select the opten Use the same flow rate as in Method Settings to t t the flow rate Se v i View and edit phases using Phase Properties The following table describes how to edit a method phase in the Phase Properties tab Step Action 1 Make sure the Phase Properties tab is selected UNICORN 6 1 Method Manual 28 9817 65 AB 37 3 Create and edit methods 3 3 Edit methods and phases 3 3 1 Edit phase properties Step Action 2 e Select the Method Settings phase if you want to edit basic settings af fecting the whole method e g Column type Flow rate and Method Base Unit Continue with steps 3 4 Note You can also edit the Result name amp Location the Start Protocol and Method Notes from the Method Settings phase These are overall method options that also can be set using the corresponding Toolbar options and not described in this section See Section 3 4 Set general method options for the method on page 51 for information on how to edit these settings or e Select any other phase to edit the properties for that specific phase Continue with step 5 38 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit method
156. d click the Remove button When attaching the UNICORN Log a separate dialog will open Choose the time period for the attached log Attach UNICORN Log To 6 3 2008 356 UNICORN 6 1 Method Manual 28 9817 65 AB 10 Troubleshooting 10 2 System Error Reports Step Action 3 To include more information in the report select the appropriate check boxes in the System information field Computer and Operating System information A summary of the computer and operating system information for example type of processor processor speed RAM hard disk capacity and printer Integrity check When UNICORN is installed a checksum calculation is performed on the stationary files dll and exe for the system An integrity check means that a new checksum calculation is performed for the same files in their folders This new calculated value is compared to the checksum value obtained during installation The results of the comparison are presented in the report and any deviations are included e Click the Next button Result The Generate System Error Report dialog is displayed 4 Go to step 3 below Step 3 Generate and save the report The table below describes how to generate and save the report Step Action 1 By default the report is saved as a zip file in the UNICORN folder on your local computer If you want to save the report in another location click the Browse button and select a destination folder 2 You
157. d edit methods 3 4 Set general method options for the method Include Evaluation Procedures after the run The table below describes how to include an evaluation procedure in the method The evaluation procedure will be performed automatically after the run has finished The evaluation procedures must have been defined in the Evaluation module see the UNI CORN 6 1 Evaluation Manual Step Action 1 In the Method Editor e click the Evaluation Procedures icon ce or e select Tools Evaluation Procedures Tools Ew Start Protocol Evaluation Procedures be Scouting TE Design of Experiments Result The Evaluation Procedures dialog opens Evaluation Procedures Selected evaluation procedures will run at the end of the method e fthere are no evaluation procedures listed in the Evaluation Procedures dialog click the Import button to import an evaluation procedure Result The Import Evaluation Procedure dialog opens Continue with step 3 e Ifan evaluation procedure that should be used in the run has been saved in the method earlier it is shown in the Evaluation Procedures dialog Continue with step 4 UNICORN 6 1 Method Manual 28 9817 65 AB 57 3 Create and edit methods 3 4 Set general method options for the method Step Action 3 Import Evaluation Procedure hA Methods E Select procedure to import Folder name System Evaluation Procedure 1
158. d maintenance methods Prede fined mainte nance method Column CIP Principle The column is filled with a cleaning solution Select inlet positions Enter the solution identity volume flow rate and incubation time By adding steps several cleaning so lutions can be used Suggestions for clean ing steps are available for a number of col umn types Included phases Method Settings Column CIP Column Perfor mance Test After equilibration of the column sample is injected via a capillary loop and eluted iso cratically A non adsorbing sample like acetone or salt should be used After the run calculate column performance in the Evaluation module The efficiency of the column is determined in terms of height equivalent to a theoretical plate HETP and the peak asymmetry factor A The result is logged in the column logbook Method Settings Equilibration v Column Performance Test Column Prepara tion The column is filled with buffer solution Select inlet positions Enter the solution identity volume flow rate and incubation time By adding steps several preparation solutions can be used Method Settings Column Preparation UNICORN 6 1 Method Manual 28 9817 65 AB 85 3 Create and edit methods 3 8 Predefined methods and phases Prede Principle Included phases fined mainte nance method Intelli Packs AxiChrom columns with a predeter gent mined col
159. deep well plate v Frac volume Elution ml 2 00 0 00 2 20 Last tube filled action Elution Pause v Linear gradient Gradient target Elution B 100 0 0 0 100 0 Gradient length Elution Cv 15 00 0 00 100000 0 C Show details v lt _ Restore Phase Properties The phase User Defined is an empty phase designed for text editing methods Such phases will only be displayed as a variable list in Phase Properties and may be saved in the personal or global phase library for reuse in other methods See Chapter 9 Text edit methods on page 286 for information about text editing methods Note Do not mix text edited and non text edited phases unless you clearly under stand the implications for the entire method of the instructions in the text edited phases UNICORN 6 1 Method Manual 28 9817 65 AB 23 2 The UNICORN Method Editor 2 2 Methods in UNICORN 6 1 Predefined and Empty methods In UNICORN a number of Predefined methods for different separation techniques and maintenance applications e g preparation and cleaning of the system and columns are supplied When creating new methods it is possible to use one of the Predefined methods or create a user defined method starting with an Empty method The phase Method Settings is mandatory in all methods See Chapter 3 Create and edit methods on page 25 for information about how to create new methods The table below gives a general description of Predefined
160. details box to view variables classified as detailed The letter D will be shown to the left of the detailed variables e Check the Show unused variables box to view unused variables in the method The letter U will be shown to the left of unused variables 6 To print the information in the Method Duration amp Variables dialog click the Print button Result The Print dialog opens 7 Select a Printer from the drop down list and click OK Result The information is printed UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 5 Save methods and phases 3 5 Save methods and phases Introduction Methods and phases are saved in the UNICORN database Individual edited phases may be saved to the Phase Library for later use in other methods on systems having the same instrument configuration and component config uration Note You cannot save an edited method phase to replace a predefined method or phase If you want to save an edited variant of a predefined method or phase with specific settings you must save it under another name A prede fined method or phase can not be overwritten Save a method The table below describes how to save a method in UNICORN Step Action 1 e Click the Save the Method icon or e select File Save or File Save As Result e lf the method has been named and saved previously the changes are saved immediately If not e The Save As dialog opens Proceed with steps 2
161. dling column types 252 Action In the Column Type Parameters tab click Edt Result The Edit Column Type dialog opens Edit Column type GSTrap FF 1 ml If the column hardware and medium are made by GE Healthcare select the name of the hardware and medium to have most of the parameters filled in automatically Show hardware types by diameter cr Show medium types by technique Min Lg Mas ooo Any ha GE Healthcare hardware type GE Healthcare medium type Glutathione Sepharose 4 Fast Flow Run Parameters Details Ordering Information Parameters Value Technique Affinity Column volume Mas pre column pressure Max delta column pressure Default flowrate Max flowrate Default linear flowrate Max linear flowrate Min pH value short tern Max pH value short term Min pH value long term Man pH value long tern Aequired information O Global Personal Edit the column type parameters as appropriate on the Run Parameters Details and Ordering Information tabs Select whether the edited column type should be Global available for all users or Personal only available for the current user UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 2 Handling column types Step Action 5 If editing parameters for a predefined column type the column type must be saved with a new name e Click Save As to s
162. down menu 9 Choose a file name and location and click the Save button to save the zip file Import a phase into UNICORN Phases that have previously been exported as zip files can be imported back into UNI CORN Plain text files or Excel files cannot be imported since there is no guarantee that they contain all the information UNICORN needs to recreate the phase The following table outlines the steps needed to import a phase Step Action 1 Select File Import Import Phase Result The Import dialog opens Look in Methods Folder G JMy Affinity Method zip H Rubisco Elution zip My Recent Documents My Documents File name Rubisco Elution zip My Network Files of type Zip Files zip J 2 Browse to the required zip file in the Import dialog UNICORN 6 1 Method Manual 28 9817 65 AB 99 3 Create and edit methods 3 11 Import and export methods Step Action 3 Open the file by selecting it and clicking the Open button or by double clicking on the file name Result The Import Phase Library dialog opens Import Phase Library Phase Mame Rubisco Elution Global Personal 4 Type a new Phase Name if required and select whether the phase should be imported as Global or Personal Global phases are available to all users Personal phases only to the currently logged on user 5 Click the OK button to import the phase 100 UNICORN 6 1 Method M
163. e for a process parameter in the method it must be de fined as a variable Qualitative factors are character ized by being discrete discontinu ous for example column type wecignyneand binercubeanee Low and high values are entered for the quantitative factors The factors will be varied within this range Response DoE design When evaluating the DoE runs the measured response values for each experiment are entered in UNICORN The output parameter s from a process For example capacity yield and purity The design is the setup of experiments with different combinations of factor settings resulting in a minimum number of experiments to be run to obtain as much information as possible UNICORN suggest a suitable design to be used in the experiment based on the e number of factors e type of factors quantitative or qualitative e experimental objective screening optimization or robustness testing There are different objectives and design types included in the DoE tool in UNICORN See the following blocks for more information about design objectives and design types UNICORN 6 1 Method Manual 28 9817 65 AB 119 5 Design of Experiments 5 1 Introduction to Design of Experiments Design objectives The table below describes the different design objectives Design objective Used when you want to Screening Determine which factors are important in your process and the appropriate ranges for these
164. e Column types list The parameters for the selected column in the Columns list are shown in the Column Logbook area to the right See Section 8 3 Handling individual columns on page 260 for more information Coki Type Pacameter Cmn Logbook Select Come IDs Code ki fody O Fred by abas Cokan HMiLead 16 10 P Sepharose HP l Load 16 50 Superdex 30 pg NLoed 1T Superge 290 pg 1707190 VLead 16 00 Saperdes 75 pp 6Lcad 2070 Pharyi Sepharose IP msn iLcad 21 70 0 Sepharose HP Load 210 SF Sepharose HP mowy y aw Calouisted based on the isst Commn Peiamance best New Due Poe Egoa iagoa Cekme Matory Padamacefapat Notteatontews Note HPrep Och FF 16710 Myer Pheri FE hh na IENA Main Column Handling tasks 244 There are many possible workflows when working with column types and individual columns in UNICORN The table below lists the main tasks that are performed in the Column Handling tool or the Select columns dialog in the Start Protocol when starting the run in System Control When working with the main tasks are Column types e Create new column types e Import export column types Used to transfer column type data between differ ent databases e Edit column types Edit parameters and delete column types e Print information about column types UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 1 Overview When working
165. e 290 UNICORN 6 1 Method Manual 28 9817 65 AB 287 9 Text edit methods 9 1 Overview 9 1 1 Working with text instructions 9 1 1 Working with text instructions Introduction Phases are normally edited in the Phase Properties pane If you have selected a phase in the Method Editor and the Text Instructions tab is selected the corresponding phase block is selected in the Text Instructions Changes made in the Phase Properties pane are automatically updated on the Text Instructions pane However if text editing the method in the Text Instructions pane the settings in the Phase Properties pane will be replaced by a list of phase variables Text editing a method Adding editing or deleting any blocks or instructions in a phase in the Text Instructions area means text editing of the method When a method has been text edited one or several of the phases displayed in the Method Editor window are affected depending on the type of editing performed When text editing a method the settings in the Phase Properties pane will be replaced with a list of phase variables that may be changed The letter T next to the phase name in the Method Editor window indicates that the phase has been text edited The illustration below shows the Phase Properties pane when a method has been text edited and the indication T on the phase that has been text edited The Phase Properties pane shows a list of phase variables 288 UNICORN 6 1 Method Manual 2
166. e EA 242 e2 Handing column Ypes sanmensa iE POE DT oa RUNNER NTT os RD ORT 247 83 Handing WIN UC OMANI Soe es sect cegeseceny O 260 831 TACIVIGUG COMI dent COO srasni EEE 261 832 Registeranew individual COlUTIN sseisesiiiaorstiiiesiiie aii ii 262 GSS FIO GR TACIVIGUG COUT sisii E A 266 COG GI WICIVIGUG COMING retre ine ELONE ET NA 268 8 53 9 Exportana import individual COUMNS srecen TE 27 1 8 3 6 Print and view individual column information ssssesssessessssssssssssssssssssssssssssssrssreesn 274 A Column TET OM IANO Sa crscsticacs Sects cepeseoe vac earecucveeci EEAO 276 85 Intelligent Packing of AXiChrom COMMIS ssctszisoscessziccscssssrescessniecticndacesscinnbideneiactine 279 9 Text edit Method ccsscsssssccsscssscsssessccssccsscsesesecsssecsecsssesssesscssccsesseesseesses 286 S OC N E S ciate eee 287 91 1 Working With text INStrUCtIONS ssissississiscisisisircerercteriietiniieiiiiiiiiiiiaidinini 288 Ce TET gis T PINE e E 290 9 2 Working with methods in the Text Instructions pane sssesrrssssssrssssssssssssssssssssssssss 295 Jea BEIT UCO a E E E 296 9 2 2 WORKING with phases and DIOCKS sssssssssssesssssssrsssrssrrsiirrrrirrierrirrrrersrssssssrssssssssssssssssssssssss 300 92 53 Working WIth text INSTrUCUONS siisisssioiieniieiitocarn Teen eas i brii 309 9 2 4 Method variables aesivcticsvicccovertisness events decosnspsneoes as hsosioenepedenensiteanbs Soe tavesneseesesovsianelsdeveteeesaeiveesinn 315
167. e System Control toolbar iP Note If the pause is set to Infinite the method must be resumed manually by clicking the Continue icon Hold instruction 340 The Hold instruction suspends the execution of the method but continues to pump eluent at the current flow rate and concentration settings For example this instruction is useful for giving the operator time to load a sample loop Resume the method The method may be resumed if you click the Continue icon on the System Control toolbar le Note With the Hold instruction the method must always be resumed manually by clicking the Continue icon UNICORN 6 1 Method Manual 28 9817 65 AB Hold until instruction 9 Text edit methods 9 3 Specific instructions 9 3 4 Pause or hold a method The Hold until instruction is a special kind of Watch instruction The method is put on hold until a specific condition is met Signal Test or Value or the Timeout is reached Thereafter the remaining instructions in the method are executed See Section 9 3 3 Watch instructions on page 333 for a description of Watch instructions Insert a Pause Hold or Hold until instruction The table below describes how to insert a Pause Hold or Hold until instruction Step Action 1 At a suitable Breakpoint in the method select the instruction line immedi ately before where you want to insert the Pause Hold or Hold until instruc tion this decides when the
168. e ex periments The model is good for one response but not the other It might be difficult to fit the same model to all responses Consider dividing the experi ment in two or more to be able to fit one model re sponse UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis Step Action 3 Look at the normal probability plot of residuals If the model describes the experimental data well the experiments should be distributed close to a straight line and lie within a Standardized Residuals range of 4 to 4 SD standard deviations See Check the raw data on page 170 If the centre points points 9 10 and 11 in the illustration below are not lin early distributed this may indicate curvature in the model rather than true outliers A low Q model validity and significant lack of fit may also indicate curvature Aggregate Content with Experiment Number labels M Probability 1 0 1 Standardized Residuals W 11 B2 0 768 Be d9 0 5356 DF 5 Q2 0 003 BSD 0_ 9788 If you Suspect curvature try adding a quadratic term to the model See Section 5 4 5 Edit the model on page 190 for more information UNICORN 6 1 Method Manual 28 9817 65 AB 179 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis Step Action 4 Look at the
169. e in the text method 4 To view the variables in the method click the Variable List tab Result The Variable List is displayed UNICORN 6 1 Method Manual 28 9817 65 AB 61 3 Create and edit methods 3 4 Set general method options for the method Step Action 5 Method Duration amp Variables Method Duration Variable List Phase Block Variable Value Method Settings METHOD SETTINGS Inlet 4 Al Inlet B B1 Flow rate ml min 10 000 Pressure control Pre column pressure Rubisco Elution ELUTION_1 Percent B Elution _1 B 0 0 Rubisco Elution Start frac Elution 1 Frac tube type Elution _1 96 deep well plate Frac volume Elution _1 ml 2 00 Frac start position Elution _1 Next tube Last tube filled action Elution _1 Pause Rubisco Elution Linear gradient_1 Gradient target Elution _1 B 100 0 Gradient length Elution _1 Cv 20 00 Rubisco Elution Gradient delay_1 Gradient delay volume_1 ml 3 00 Equilibration EQUILIBRATION Percent B Equilibration B 0 0 Fill system E quilibration ml 15 Equilibration Equilibrate Equilibration volume CV 5 00 lt M Show details V Show unused variables The Variable List shows the variables in the method It is also possible to see in which phases the variables are included and the different values Variables with an ellipsis after their name are used in multiple phases or blocks It is not possible to change any values in this dialog e Check the Show
170. e individual column for which to generate a Performance Report in the Columns list in the Column Logbook tab See Section 8 3 3 Find an individual column on page 266 for information about how to find and select a column UNICORN 6 1 Method Manual 28 9817 65 AB 217 8 Column Handling 8 4 Column performance Step Action i In the Column Logbook tab click Performance Report Result The Performance Report dialog for the selected column opens Performance Report 28 9269 78 70090119 2009 01 0001 1111110 Printer Microsoft Office Document Image Writer Properties Performance graphs Preview page 1 3 fh 3 Max of delta pressure MPa Resolution Asyrnmetry v Plate height cm Plates per meter N m Retention factor 3 Select Printer 4 Check the appropriate boxes in the Performance graphs area to include the corresponding graphs in the report Note The parameters and the corresponding values from the Column Logbook are always included on the first page in the report to gether with the latest performance test results All runs are listed in the Run History at the end of the report including Column Performance Test and CIP runs which are labelled 5 A preview of the report is shown on the right side of the dialog Use the buttons above the report to scroll the preview 6 Click Print to print the information 7 Click Close to close the dialog 278 UNICORN 6 1 Method Manual 28 9817 65 AB
171. e software will then generate a model 2 Analyze and edit the model This includes checking that the raw data is OK and performing a basic analysis of the model The model may need refinement by removing insignificant terms which should be done with care Extended analysis can be performed for additional infor mation 3 Use the model This includes generating a response surface plot as well as using the predictor and the optimizer e Generate response surface plot to get a map of the experimental area and infor mation about how to proceed with new experiments e Use the predictor to predict response values based on entered factor settings optimization experiments only e Use the optimizer to optimize responses based on entered criteria for factors and responses optimization experiments only for example maximizing response 1 and minimizing response 2 while keeping factor 1 constant and allowing the other factors to vary within a defined range Basic and extended reports can also be created for the experiment 158 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 1 Workflow Illustration of workflow The illustration below shows a possible workflow for evaluating a Design of Experiments scouting 1 Generate model A Evaluate single runs B Open DoE result C Check experiment setup D Software generates model included in DoE and enter response values V
172. e the File Save As menu command to save the converted method or e click the Save icon Result The Save As dialog opens with the folder where the original method is saved open by default e Select the desired target folder e type anew method name in the Name field and e click the Save button Note Note Note 72 The original method remains after the converted method is saved However the converted method will replace the original if you choose to save the converted method with the same name in the same folder as the original The flow rate and or pressure settings in the method will automatically be adjusted if the maximum flow rate and or pressure values for the target system is exceeded after the conversion The maximum settings for the target system will be used by default In case the original method contains instructions that are not supported by the new system this will be indicated in the method outline of the converted method as a red cross on the phase containing these instructions To be able to run the method on the new system you need to replace or re move the invalid instructions in the Text Instructions pane Invalid instructions are indicated with red square symbols in the text instructions You can also replace the phase with a predefined phase from the phase li brary UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 6 Scale or convert methods Convert and scale a me
173. e the same concentration This is set as default for Acid or Base concentra tion 6 Choose a Salt from the drop down list and edit its Concentration if required Note The salt concentration of the stock solution should be four times larger than the desired maximum salt concentration for the gradient T Enter a description of the buffer Description Low concentration Bicine boffer recipe Bicine 0 1000M 16 32g Bicine to prepare 1 litre Mw 163 2 NaGH 0 1000M Use ampule Maci 4 000M 233 89 to prepare 1 litre Mw 58 44 Mote Mot recommended for AIE Note Although the description is optional it is highly recommended to add the recipe details for future reference 8 Select to save the edited recipe as Global or Personal and click Save as or Save K Personal Note Recipes can be changed from Personal to Global and vice versa by editing the recipe changing the type then clicking on Save Result The Save As dialog opens 9 Enter a name and click Save E icine Low Cone Save Cancel UNICORN 6 1 Method Manual 28 9817 65 AB 211 6 BufferPro 6 3 Create and edit BufferPro recipes 6 3 2 Rename a BufferPro recipe 6 3 2 Rename a BufferPro recipe Introduction The following table describes the steps for renaming a BufferPro recipe Note Predefined recipes shown as PD in the Type column can not be renamed Rename a recipe Step Action 1 In the Method Editor
174. e when editing for example the phase Method Settings because several parameters are used in other phases e Donot mix text edited and non text edited phases unless you clearly understand the consequences UNICORN 6 1 Method Manual 28 9817 65 AB 289 9 Text edit methods 9 1 Overview 9 1 2 The Text Instructions pane 9 1 2 The Text Instructions pane Introduction This section gives an overview of the Text Instructions pane in the Method Editor and the structure of a text method Illustration of the Text Instructions pane 290 The Text Instructions pane consists of two areas the Text Instructions area and the Instruction Box The illustration below shows the Method Editor window with the Text Instructions tab selected The phase Equilibration is selected in the Text Instructions area and the cor responding phase is highlighted in blue in the Method Outline and the Gradient panes E Hesihaid Lditsr UNTIL E Menj 2 R is Cv 0 100 fel jing ji E O00 Pham Method Sating Eo GOO iasa Samah am TD Dre AC Atja A irls M jiri jisi 0 7 0 Dioden 01 Percent B Egan 58 0 00 nat O00 Spetem ire j OE ew tale een Pee cohen perce eee cana Sasphe Appain O00 Syatiee wash 258A epiter Equi ston lel Outlet vabre O00 Watch Uf pacsmetenc DOD mAh 100 fend TOD Hold ue UF Stable tonai 007 eed ia Dean B00 Ato smn Le TOD Erd Birk T gt o s 000 Phase Coben Wash Elution E O0
175. ect Tools Column Handling in any of the UNICORN modules or e click the Column Handling icon in the Toolbar where available 242 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 1 Overview Illustration of the Column Handling dialog The illustration below shows the Column Handling dialog displaying the Column Type Parameters tab Column Handling Select Column Type Show by technique Any Show by access label Predefined types Global types user created Personal types user created Column types AxiChrom 50 300 20um SS Capto Q 20 cr AxiChrom 50_20 Capto DEAE Transamina AxiChrom 50_20cm_CaptoMMC_insulin GSTPrep FF 16 10 GSTrap 4B 1 ml GSTrap 4B 5 ml GSTrap FF 1 ml GSTrap FF 5 ml GSTrap HP 1 ml GSTrap HP 5 ml HiLoad 16 60 Superdex 30 pg HiLoad 16 60 Superdex 200 pg HiLoad 16 60 Superdex 75 pg HiLoad 26 10 Phenyl Sepharose HP HiLoad 26 10 Q Sepharose HP HiLoad 26 10 SP Sepharose HP HiLoad 26 60 Superdex 30 pg HiLoad 26 60 Superdex 75 pg HiLoad 26 60 Superdex 200 pg HiPrep 16 10 ANX FF high sub HiPrep 16 60 Sephacryl S 100 HR HiPrep 16 60 Sephacryl S 200 HR HiPrep 16 60 Sephacryl 300 HR HiPrep 16 60 Sephacryl 400 HR HiPren 16761 Senhaenl S RON HR lt 2x HiScreen Capto DEAE 20 cm Transami A AxiChrom 50 300 10um SS MabSelect 20 olumn Type Parameters Column Logbook Name HiLoad16 10Q Sepharose HP Predefined
176. ed instruction but does not change the breakpoint of any other instruction Replace can change the relative order of instructions in the method 312 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 3 Working with text instructions Effects of the Change button and the Replace button on gradient length The Length parameter in the Gradient instruction affects the length of a gradient The change will have different results depending on which button is used The table below describes this Command Function If this button is used to change the length of a gradient the breakpoints for any instructions issued during the progress of the gradient will be adjusted proportionately so that they are always placed at the same relative posi tion within the gradient Instructions issued after the end of the gradient will be shifted by the amount of the change Since the gradient works over time any instruc tion that you want to insert after a gradient should be placed after the combined breakpoint and gradient length Note Moving the End_block instruction in a gradient block with the Change button does not affect the length of the gradient Replace If this button is used to change the length of a gradient other instructions are not affected Move instructions A selected instruction may be dragged and dropped in a new location to change the order of in
177. eed man ual adjustments after the conversion are updated and Review all text edited phases to locate system parameters that must be edited For more information see Section 3 6 Scale or convert methods on page 69 UNICORN 6 1 Method Manual 28 9817 65 AB 10 Troubleshooting 10 1 Troubleshooting methods A column cannot be selected when converting and scaling a method The table below describes how to solve problems when a column cannot be selected for conversion and scaling of a method Problem description Solution When converting the method The reason for this may be that either the option including scaling of the col Scale was not selected or that the Any column was umn the field for column selected in the original method If Scale was selected scaling is inactive either e select a column in the original method and re peat the conversion or convert the method to the new system first and select a column in the converted method after wards Print screen does not send a copy of the screen to the printer The table below describes how to solve a printing problem Problem description Solution The Print Screen command only If you want to print the view on the screen makes a copy of the screen tothe press the Print Screen key and paste the im clipboard and not to the default age from the clipboard into an appropriate printer program such as Microsoft Paint and then print out the image
178. em Equilibration C Fill system Equilibration _1 al Elin rata Show details Show unused variables e Toadda variable to the Scouting scheme check the appropriate box in front of the variable e To delete a variable from the Scouting scheme clear the box in front of the variable If you can not find the appropriate variable e Check the Show details box to display variables defined as detailed variables in your method e Check the Show unused variables box to display variables currently not used in the method To define a new variable see Section 9 2 4 Method variables on page 315 for information Click OK Result The Scouting parameters table is updated with the changes UNICORN 6 1 Method Manual 28 9817 65 AB 4 Scouting 4 2 Setup and edit a Scouting scheme Step Action 4 To edit a variable value for a run e Select the appropriate row and the variable value cell in the Scouting parameters table e Type anew value for the variable Result The variable value is updated Note Changing variable values in the scouting scheme does not change the values in the Variable List in the Duration and Variables dialog or in the text instructions The actual variable values used for each run in the scouting scheme are saved in the result file To change the default values the variable values must be edited in the Phase Properties pane 5 Click OK Result The Scouting parameters table is upd
179. ent 0 1M NaCl The ionic strength of the mixed solution affects the ap parent pKa of the buffering agent For pH outside the recommended range the buffering capacity is unreliable and should be avoided UNICORN will display a warning in case either the required concentrations or pH will not provide adequate buffering If in doubt check the pH of the eluent after running an experiment using a reliable lab pH meter Certain buffer substances are not recommended for anion exchange and others not for cation exchange For example phosphate buffers are not suitable for anion ex change Buffer suitability is noted in the predefined recipes in UNICORN 226 UNICORN 6 1 Method Manual 28 9817 65 AB 6 BufferPro 6 7 Predefined BufferPro recipes pH and concentration ranges for predefined recipes The following table gives the optimal pH and concentration ranges for buffer recipes that are predefined in BufferPro at 25 C Buffer system pHrangeat Concentration Comment AIEX mix 0 1M NaCl 25 C pH 5 8 8 9 range M Fixed at 25 of the concentra tion of the stock solution Broad range buffer sys tem for Anion exchange chromatography CIEX mix 0 1M NaCl pH 2 0 7 0 Fixed at 25 of the concentra tion of the stock solution Broad range buffer sys tem for Cation ex change chromatogra phy Acetate 0 1M NaCl pH 3 8 5 4 0 05 0 1 Titrated with conjugate acid Acetate with HCI 0 1M NaCl
180. er of experi ments that can be afforded The process can be iterated and the initial screening results from one DoE can logically be used as input for the next DoE and so on 120 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 1 Introduction to Design of Experiments Design types A design can be graphically illustrated by a box The design box in the following examples illustrates designs where three different factors X4 X gt and X3 are varied simultaneously Each corner point is the experiment for a specific combination of the settings of the three factors e g low value for X4 and X and high value for X3 The center point is the experiment where the different factors have the closest distance to all other factor set tings that is the mean value The corner points are used to assess factor interaction effects The center points are used to estimate the pure error and detect curvature See Model on page 125 for detailed information about the terms interaction curvature and pure error Illustration of the design box The illustration below to the left shows a design box with corner and center points for the different factors X4 X and Xz The illustration below to the right shows the factor values for one of the corner points The arrow directions along the box edges denote the parameter change from low to high X Low X High X High Corner point Center point Different designs in UN
181. ered e Enter notes for the column by clicking the Notes button and enter notes in the Notes dialog that opens Result The entered information is saved and the registered column is dis played in the Column Handling dialog 264 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 3 Handling individual columns 8 3 2 Register a new individual column Step Action Wir T ary LD od ii E Ce ee a Beet Cig Cer Hoto Fodomanse Reson Hoileainte Haee l m UNICORN 6 1 Method Manual 28 9817 65 AB 265 8 Column Handling 8 3 Handling individual columns 8 3 3 Find an individual column 8 3 3 Find an individual column Introduction Many features of the Column Handling tool require an individual column to be selected This section describes how to find a column Find and select an individual column The table below describes how to find select a registered column in the Column Logbook Step Action 1 Select the Column Logbook tab 2 Filter the list of column types by choosing the required technique in the pull down menu Show by technique then select the column type to which the individual column belongs Select Column T ype Show by technique Any v Show by access label Predefined types Global types user created Personal types user created Column types HiPrep 16 60 Sephacryl S 300 HR HiPrep 16 60 Sephacryl S 400 HR HiPrep 16 60 Sephacryl S 50
182. erimental design Step Action 5 When all responses and factors have been defined select the objective for the Design of Experiments e Inthe Design selection area in the Factors amp Design tab select the ap propriate objective from the Objective drop down list Design selection Objective Screening v Design Full factorial 2 levels 1st choice yl Screening RobustnessT esting Result Depending on the selected objective UNICORN suggests a suitable design to obtain sufficient resolution with as few experiments as possible in the Design list The total number of runs are displayed for the suggest ed design Design selection Objective Screening w Design Full factorial 2 levels 1st choice v Total number of runs including center points 7 e Itis possible to select the 2nd choice design in the Design drop down list if appropriate The 2nd choice design usually either requires a higher number of runs to be performed or the resolution of the design is lower For information about how to view details for the selected design and or to change to another design than the 1st or 2nd choice designs see Sec tion 5 2 3 Change design and design settings in a Design of Experiments setup on page 145 UNICORN 6 1 Method Manual 28 9817 65 AB 133 5 Design of Experiments 5 2 Create an experimental design 5 2 1 Set up an experimental design Step Action 6 Click the Experiment tab Result The Experimental Plan is
183. es Text Instructions T Equilibration Reset UY monitor recommended if the equilibration occurs before the purification Use the same flow rate as in Method Settings Flow rate 1 000 ml min 0 000 25 000 Equilibrate until the total volume is Cv the following condition is met Lonductivity greater than Conductivity greater than Accepted pH fluctuation Accepted UY fluctuation Accepted conductivity fluctuation Signal stable for Maximum equilibration volume Use the same inlets as in Method Settings Inlet 4 Al v Inlet B B1 v 0 00 B 0 00 100 00 Fill the system with the selected buffer 000 mS cm 0 00 1000 00 3 10 0 00 14 00 010 mau 0 00 6000 00 1 10 mS cm 0 00 300 00 1 00 min 0 02 1000 00 10 00 CY oa R i UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 3 Edit methods and phases 3 3 1 Edit phase properties Step Action 2 Click anywhere in the Phase Properties area to make it the active area in the software 5 e Press the F1 keyboard key or e Select Help Contextual Help Result The Online help for the selected phase is displayed roi y i UNICORN Phase Help x BB UNICORN and AKTAsvare Online Help and Dr 1 ow o Method phase Equilibration With the exception for CIP or preparation methods most standard chromatography methods include one of more Equilibration phases The equilibration phase coul
184. et of experiments to be run Depending on the objective and the number of parameters a suitable design is suggested An experimental plan is presented and a Scouting scheme is generated as a result from DoE containing the method runs to be performed When the runs have been performed the results can be analyzed in the Evaluation module A model is cre ated and a number of plots are generated to aid evaluation of the results The model can be used to predict responses for new parameter settings and to optimize the param eter settings for a desired combination of responses e g optimize the response combi nation minimize the level of impurities and maximize the yield UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 1 Introduction to Design of Experiments Factors and responses Factor The table below lists the definitions of the DoE terms factors and responses and how to use them in UNICORN Definition The different parameters that may af fect the process to be run In UNICORN The factors are connected to a variable in the method For example the factor pH may be connected to the variable BufferPro pH In pre defined methods most useful parameters are already defined as variables Note Factors may be either quantitative or qualitative e Quantitative factors are character ized by being found on a continuous scale for example pH flow rate and conductivity To be able to vary a valu
185. factors Optimization Find the optimal factor settings for your process that is factor settings that give the desired responses Robustness Testing Determine the process robustness by making minor ad justments of the factor settings and see if the responses are within the set specification limits If the responses do not vary significantly due to the factor changes the pro cess is considered to be robust Example of how to use DoE for different objectives To obtain maximum amount of protein after purification of a sample using a minimum number of runs use DoE to find e important parameters e g pH conductivity and the appropriate parameter ranges affecting this process e the optimal parameter settings and any dependencies interaction between the parameters affecting the response of the product or process e g yield or impurity level When the parameters affecting the process as well as their settings have been determined it is appropriate to test if the process is robust that is not affected by minor changes in the parameter settings Neither the parameter settings selected or their interactions should affect the process if the process Is to be considered robust A specific DoE setup is required for each step i e screening for parameters or parameter settings for optimization of parameter settings and for robustness testing Each setup is a balance between the amount of information obtainable and the numb
186. faultHome Folder name for Design of Experiments or Scouting Result name Name Variable Method name Date C Add unique identifier In the Result Name amp Location dialog e Set Result location by clicking the Browse button and select a folder in which to save the results By default the results will be saved in your home folder e Select Result name Name The result name can be typed in manually Variable The result name will be generated from the chosen variable see Section 9 2 4 Method variables on page 315 Method name default The result name will be generated from the name of the method Date The result name will be generated from the date of the run e Check the Add unique identifier box if you want to include a unique identifier number to the file name The number will be generated by UNICORN based on the run time of the method e Click OK to confirm and close the dialog UNICORN 6 1 Method Manual 28 9817 65 AB 53 3 Create and edit methods 3 4 Set general method options for the method Set up a Start Protocol The table below describes how to set up a Start Protocol to be displayed before the run starts Step Action 1 In the Method Editor e click the Start Protocol icon Scouting Evaluation Procedur s Design of Experiments e click the Method Settings phase and click the Start Protocol button in the Phase Properties tab Start Prot
187. ferPro Recipes Start Protocol Evaluation Procedures Scouting Design of Experiments f Column Handling Ctrl L BufFerPro Recipes Administration System Control g Evaluation Options Change Passwords Z BufferPro Recipes Recipes Show All Stock Solutions Recipe name S Inlet Stock solutions Concentration M Substance Acetate DIM NaCl S804 PD Q1 Buffer substances 0 4000 Bicine Acetate with HCI 0 1M NaCl AIEX mix 0 1M NaCl Bicine 0 1M NaCl Bicine High Cone Bicine Low Conc Bis Tris 0 1M NaCl Bis Tris Propane 0 1M NaCl 6 Q2 Acid or Base 0 4000 NaOH Carbonate 0 1M NaCl g l 3 Carbonate with HCIO IMNaCl 95 10 Q3 Water CIEX mix O 1M NaCl 2 0 7 Q4 Salt 4 0000 Nacl Achievable ranges with recipe Description pH Bicine 0 0600 0 1000 M 0 0000 1 0000 M 0 100 B 3 Click the Print button Result The Print dialog opens UNICORN 6 1 Method Manual 28 9817 65 AB 217 6 BufferPro 6 4 Print a BufferPro recipe Step Action 4 Choose a printer from the drop down list in the Print dialog and click OK 218 UNICORN 6 1 Method Manual 28 9817 65 AB 6 BufferPro 6 5 Calculate buffer composition using BufferPro 6 5 Calculate buffer composition using BufferPro Introduction This section describes how to calculate an exact buffer composition for a buffer previ ously optimized using BufferPro This is desirable when scaling up a puri
188. fication procedure in order to prepare bulk scale buffer solutions for example ion exchange A and B buffers Calculating buffer composition The following table describes the steps needed to calculate the buffer composition of a BufferPro recipe at a particular pH buffer and gradient concentration and temperature In the examples shown in the table a pH optimization scouting run has been performed The buffer at which optimal separation was obtained was 50 mM HEPES pH 7 8 at 25 C and the required peak eluted at 25 of the gradient Step Action 1 In the Method Editor select Tools BufferPro Recipes o Start Protocol Evaluation Procedures Scouting tel Design of Experiments 2 Administration System Control 4a Evaluation Options Change Passwords UNICORN 6 1 Method Manual 28 9817 65 AB 219 6 BufferPro 6 5 Calculate buffer composition using BufferPro Step Action 2 Choose the appropriate recipe from the list in the BufferPro Recipes dialog BufferPro Recipes Recipes Show All Stock Solutions Recipe name Stock solutions Concentration M Substance Citrate 0 1M NaCl 3 6 Buffer substances 0 2000 HEPES Citrate with HCI 0 1M NaCl 7 5 Diethanolamine 0 1M NaCl 6 9 z Ethanolamine 0 1M NaCl Formate 0 1M NaCl Formate with HCI 0 1M NaCl HEPES 0 1M Natl MES 0 1M NaCl Acid or Base 0 2000 NaOH Methylpiperazine 0 1M NaCl 6 5 MOPS 0 1M NaCl 5 7 Water Phosphate 0 1M NaCl
189. gator is hidden and only the Method Navigator tab is displayed 2 Method Settings 10Je IAeN poyga Equilibration e To display the Method Navigator again move the mouse pointer over the Method Navigator tab e To turn off the Auto Hide function click the horizontal pin symbol in the top righthand corner of the Method Navigator pane Result The Method Navigator pane is displayed statically Getting help on the Toolbar and panes in the Method Editor The table below describes how to find detailed information about the Toolbar and the different panes in the Method Editor by opening the Online Help Step Action 1 To display detailed information about the Toolbar and different panes in the Method Editor interface select Help Help For Method Editor Help For Method Editor N Contextual Help Fi Instruction Set About UNICORN Result The online help opens displaying the Method Editor help start page 2 To display help for a specific pane click in the pane and press the F1 key board key Result The online help page describing that pane is opened UNICORN 6 1 Method Manual 28 9817 65 AB 19 2 The UNICORN Method Editor 2 2 Methods in UNICORN 6 1 2 0 Methods in UNICORN 6 1 About methods The program instructions for a chromatography run are defined in a Method The instruc tions are specific for each instrument configuration and component set up and follow certain syntactical and
190. gbook tab click Erot Result The Export Column Type dialog opens 3 Select in which folder to save the information and type a name for the zip file to be exported Result The individual column information is exported The column information can be imported into another database Import individual columns Individual columns that have been exported and saved locally can be imported into another database The table below describes how to import individual column information to a database UNICORN 6 1 Method Manual 28 9817 65 AB 271 8 Column Handling 8 3 Handling individual columns 8 3 5 Export and import individual columns Step Action 1 In the Column Logbook tab click _Import _ Result The Import dialog opens Import Look in CO Column Handling O 5 3 E fal CcolindExp colTypeExp My Recent i ColumnHandlingLotta Documents 0 ColtypeExp zip Q dColurnindividuals zip File name My Network Files of type Zip Files zip w of ane 272 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 3 Handling individual columns 8 3 5 Export and import individual columns Step Action 2 Locate the zip file with the column type information to be imported and click Open Result The Import Column dialog opens displaying the barcodes and aliases of the individual columns included in the zip file Import Column Select column to import 28 9288 13 20080603 0000 00 0
191. gnal exceeds then Alarm limits an alarm sounds an alarm message is displayed the process is paused i e the method execution is suspended and all pumps are stopped e the alarm is noted in the Run log The situation must be acknowledged and corrected before the process can be continued Warning limits e awarning message is displayed e the process continues e the warning is noted in the Run log Note The Alarms are not active unless the mode is set to Enabled 330 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 3 Specific instructions 9 3 2 Alarm instructions Alarms in a network Alarms and warning messages are displayed on all stations with a connection to the concerned system This is regardless of the activity that is currently performed in UNICORN and regardless of the identity and access rights of the current user Alarms and warnings can however only be acknowledged from the station that is con nected in control mode Insert an Alarm text instruction The table below describes how to insert an alarm instruction into the method Step Action 1 Select the instruction line immediately before where you want to insert the Alarm at a suitable Breakpoint in the method This will decide when the alarm conditions begin e Select Alarms in the Instructions field of the Instruction Box e Select the desired alarm from the list 3 Select appropriate values for High alarm
192. gura tion 348 UNICORN 6 1 Method Manual 28 9817 65 AB Problem description Syntax errors appear because the method was connected to the wrong system That is the instrument config uration of the system Is incompatible with the method 10 Troubleshooting 10 1 Troubleshooting methods Solution Edit the method so it can be run on the currently chosen system Red instruc tions must be removed Save the method for a system that has all components installed Note The red instructions must be replaced Reselect the required component under System Properties in the Administra tion module if the component is actual ly present on the system Reopen the method and replace the red instructions with the corresponding instruction for the added component Syntax errors appear because the system s instrument configuration has been updated with a new instrument configuration that differs in the instruc tion set Syntax errors appear because the method was converted for use with a system with a component set up differ from the component set up of the system for which the method was originlly created Select the red instruction and either delete it or replace it with a corresponding instruc tion if available from the Instruction box Repeat this for all red instructions before saving the method Select the red instruction and either delete it or replace it with a corresponding instruc tion if available fr
193. have control of experimental variations UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 4 Divide the DoE runs into several scouting runs Step Action 4 Click OK Result The following warning dialog opens Design of Experiments You have excluded one or several experiments Note that all experiments must be performed to achieve a reliable model 4re you sure you want to exclude experiments 5 Click Yes in the warning dialog Result The following message is displayed Method Editor A Design of Expenments has been created for this method Changes to the method or scouting scheme may affect the design C Do not show this message again 6 Click OK and save the method i To define the second set of runs open the Design of experiments dialog again and select the Experiment tab Result The Experimental Plan is displayed Responses Factors amp Design Experiment Experimental Plan eed Load Concentration Load Conductivity Load pH Sample Application S ample inlet Sample Application S ample inlet Sample Application S ample inlet 5 2 2 82 6 52 5 54 2 54 8 54 5 S7 15 S7 6 S7 5 53 15 3 8 53 20 Buffer 2 Buffer 6 Buffer 20 1 2 51 8 51 20 SE 15 S6 6 S6 20 Not Enough Positions 1 15 Not Enough Positions 1 8 Not Enough Positions 1 NOOS wos O 12 5 5 8 5 5 7 5 NO10 005 12 5 55 8 5 55 7 55 Non 011 1
194. he correct run Replace Result C It is not possible to replace the result For the current experiment The run number and Factor settings must match in order to replace a result Note The run to be inserted must have the appropriate factor settings UNICORN 6 1 Method Manual 28 9817 65 AB 169 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis 5 4 3 Analyze and evaluate the model basic analysis Introduction This section describes how to perform basic analysis of the model and how to evaluate the model Check the raw data Before starting to analyze the model the raw data must be checked to ensure that the correct conclusions can be drawn in the analysis and evaluation of the model The table below describes how to perform some initial checks that the raw data is OK Step Action 1 Select the Analysis tab if not already selected Result The Analysis tab opens showing 4 plots for each response the Replicate plot the Summary of fit plot the Coefficient plot and the Normal probability plot of residuals To be able to see all plots use the vertical scroll bar Feed eben Pirkei Cpedaten 5 Analysis Plot of Replcatiosa for Yid eth Espenmernt Humber beta l a 4 a T J E Mlesgonse Surasa Mt E d kioci Napar 170 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of
195. he first and mandatory phase in any method Defines common parameters used in the subsequent phases The Method Settings phase defines e Column type Pressure limits Flow rate e Option to control the flow to avoid overpressure Note Default values for pressure limits and flow rate are given for the selected column type Column position Flow restrictor use Buffer preparation Manual or BufferPro automatic buffer preparation Unit selection for Method base and Flow rate Monitor settings UV monitor pH monitor Air sensor alarm settings Settings for Column Logbook Start Protocol e Result name and location Note Some of these options may not be required by certain methods Equilibration Equilibrates the column before purification or re equilibrates the column after purification Sample Applica Applies sample to the column Defines the sample application tion technique the sample volume and the handling of flowthrough UNICORN 6 1 Method Manual 28 9817 65 AB 87 3 Create and edit methods 3 8 Predefined methods and phases 88 Phase Name Column Wash Elution Description Washes out unbound sample after sample application or removes strongly bound proteins after elution Elutes the sample from the column Defines parameters for the elution and fractionation settings Column Prepara tion Prepares the column before use by removing the storage solution and equilibrat
196. he right side of the Parameters field additional param eters are available Instruction Box Breakpoint Instructions 3 Parameters for System flow 0 00 cy E Pumps and pressures f Flow rate 0 000 25 000 Sample flow 0 500 ml min Gradient Pressure control Pump wash System wash Of Wash flow BufferPro pH BufferPro setting Constant pressure flow Flow path Monitors y Edit Variable Import Block Click the Insert button Result The instruction will be inserted in the block e atthe position of the breakpoint of the new instruction if there are no other instructions at that breakpoint e immediately after the currently highlighted instruction if the highlight is at the same breakpoint as the new instruction e as the last instruction at the breakpoint if there are several instructions at the same breakpoint and none of these is highlighted Note Once a method has been edited in text editing mode the phases affected by the edited instruction are indicated with the letter T and the Phase Properties pane changes to show a variable list as shown below To restore the Phase Properties again you can click the Restore Phase Properties button to return the method to the state before the text edit Any changes that were made in the Text Instructions pane will be removed New phases from the Phase Library may be inserted in the method after text editing and the settings for these new phases ca
197. hen you want to find out how detailed factor settings influence the response s in an optimization experiment Factor settings are entered and response values are calculated when using the Prediction list The table below describes how to use the Prediction list Step Action 1 196 Select the Prediction tab in the Design of Experiments box Result The Prediction list opens Design of Experiments Capto adhere Read Only Experiment Analysis Prediction Optimization Prediction Load Mass Load Conductivity Load pH Yield Lower Upper Enter the appropriate settings for the different factors in their respective fields Click the Predict button Result The response value is calculated and displayed in the Yield field to gether with the Lower and Upper confidence limits The larger the confidence interval the more uncertain the calculation is Prediction Load Mass Load Conductivity Load pH Yield Lower Upper 15 6 1 E Delete row UNICORN 6 1 Method Manual 28 9817 65 AB Step Action 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 6 Use the model 4 To enter other factor settings enter the settings in the empty row below and click Predict In this way it is possible to compare different response values for different factor settings Prediction Load Mass Load Conductivity 250 15 300 15 Load pH 6 1 6 Yield Lower
198. hide instructions in blocks The table below describes how to view or hide blocks and instructions in the Text Instruc tions pane If you want to expand all blocks inthe double click Main method UNICORN 6 1 Method Manual 28 9817 65 AB 301 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 2 Working with phases and blocks If you want to view the instructions in a block click the symbol or double click the block name hide the instructions in a block click the symbol or double click the block name Add phases Phases can be added to a text method by e adding any phase to the method from the Phase library The phase User Defined is designed for use in creating text methods from scratch and consists only of Base and End_block instructions See Section 3 3 2 Edit the method outline on page 43 for information about how to add a phase to the Method Outline or e by copying pasting an existing phase in the text method and then edit it See Copy and paste blocks on page 303 for information about how to copy and paste blocks Note It is not possible to add a new phase using the Instruction Box Add blocks in a phase The table below describes how to add blocks in a phase Step Action 1 Select the instruction or block after which you want to insert the new block 2 Select Other Block in the Instruction Box 3 e Enter a n
199. hierarchical rules Instructions are combined into blocks Individual instructions and minor blocks are combined into the major method blocks called Phases Each phase reflects a step in the chromatography run for example equilibration or sample application A number of settings are available for each type of phase By building methods in this way methods are easily created and edited See Chapter 3 Create and edit methods on page 25 for information about creating and editing methods in the Method Editor The illustration below shows the phases in a method in the Method Outline and the corresponding settings for the highlighted phase in the Phase Properties pane Method Settings Equilibration vy Sample Application v Column Wash v Elution Equilibration 2 Phase Properties Method Settings Column selection Show by technique Affinity v Column type Any v Column volume 0 100 ml Pressure limit pre column 20 00 MPa 0 02 20 00 Pressure limit delta column 20 00 MPa 0 02 20 00 Use flow restrictor Column position By pass lt Flow rate 1 000 ml min 0 000 25 000 Control the flow to avoid overpressure Use manually prepared buffers Inlet B BI v Inlet a vi Use BufferPro automatic buffer preparation Recipe pH 1 6 3 8 5 4 recommended Cone 0050 M 0 050 0 100 Text Instructions iT Result Name amp Location Start Protocol
200. hod 3 4 Set general method options for the method Introduction This section describes how to set and view options for an entire method The following are covered in this section Defining the name and location for the results How to set up a Start Protocol that will be displayed before each method run Adding or changing method notes How to include evaulation procedures which can be executed during the run Viewing the method duration time and volume Viewing the variables used in the method UNICORN 6 1 Method Manual 28 9817 65 AB 51 3 Create and edit methods 3 4 Set general method options for the method Define Result Name amp Location The table below describes how to define the name of the result file created after the run and how to specify the folder in which to save the result file 52 Step Action 1 In the Method Editor click the Result Name amp Location icon Ag or select Edit Result Name amp Location Ctrl 2 li Result Name amp Location X Method Motes Eon click the Method Settings phase and click the Result Name amp Location button in the Phase Properties tab Result Name amp Location Result The Result Name amp Location dialog opens UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 4 Set general method options for the method Step Action 7 Result Name amp Location C No Result Result location De
201. hod 63 Saving a phase 65 Tools available in 14 Viewing help topics 19 Method notes Add to method 55 Edit 55 Method outline Description 21 Duration time and vol ume 60 361 Index 362 Edit method outline 43 Method queues 229 Change method order 240 Create 231 Delete method from 239 Deleting 236 Editing 235 Insert method into 238 Mulitple systems 231 Opening 235 Methods Add notes 55 Affinity chromatography 81 Anion exchange 82 Breakpoints in 350 BufferPro using 206 Cation exchange 82 Chromatofocusing 83 CIP mehods 352 Convert a method to anoth er system type 69 Convert and scale a method for another system type and column type 73 Create new method 30 Definition 20 Desalting 83 Duration time and vol ume 60 Edit method outline 43 Edit methods 37 Edit notes 55 Electronic signature 92 Empty methods 24 Evaluation procedures in clude 57 Export a method 95 Export as text 97 Gel filtration 83 Hydrophobic interaction chromatography HIC 84 Import a method 101 Intelligent Packing 86 Maintentance methods predefined 85 Method outline 43 Open a method 32 Predefined methods 24 81 Prepare methods 352 Print duration time and vol ume 60 Printing 79 Print variables 60 Result name and loca tion 52 Reverse phase chromatog raphy RPC 84 Saving 63 Saving a phase 65 Start protocol setup 54 Structure of 21 Syntax errors in 348 Text editing 286
202. how to open an existing method in the database Step Action 1 In the Method Editor e Click the Open Method Navigator icon in the Toolbar ze or e select File Open EI Mew Method Ctrl M New Method Queue Close or e select View Method Navigator Toolbar Method Navigator h Gradient Phase Library Flow Scheme Restore to Default Result The Method Navigator is displayed 2 Select the method to be opened in the Folder name column Folder name system fi UNICORN Database x Eric Methods E Meta Spend El Method Systema Fy Results 32 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 2 Create and open methods Step Action 3 To open the method e Click the Open button located in the toolbar of the Method Navigator pane Open or e double click the selected method or e Right click on the method name and select Open from the context menu Result The method is opened and displayed in the Method Outline pane with included phases You can continue to edit the phases of the method using Phase Properties See Section 3 3 Edit methods and phases on page 34 for more information about how to edit a method Method Settings Equilibration hd Sample Application v Column Wash v Elution v Column Wash v Equilibration UNICORN 6 1 Method Manual 28 9817 65 AB 55 3 Create and edit methods 3 3 Edit methods and ph
203. iate boxes Print REX Hilrap Chelating Print items Phase range Text instructions G All phases Variable list Phase a Include Properties Design of Experiments Start protocol Scouting Method duration BufferPro recipe Result name and location Evaluation procedures Note Only options that are used in a method can be printed Options that are not used are greyed out in the Print dialog 4 Click OK Result The method is printed 80 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 8 Predefined methods and phases 3 8 Predefined methods and phases Introduction A predefined method contains a set of phases each phase reflecting a specific stage of a chromatography or maintenance run You can select additional phases from the phase libraries and add these to an existing method or remove an undesired phase The predefined purification methods have default values with suitable running conditions for the chosen column type such as flow and pressure limits Other settings e g sample application technique sample volume elution profile and fractionation are set on the Phase Properties pane in the appropriate phases This section describes the predefined methods and phases Predefined purification methods The Method Editor has predefined methods for different separation techniques The methods include a number of relevant phases The table below describes the available predefined pur
204. ification methods and which phases that are included Predefined Principle Included phases purification method Affinity After equilibration and sample applica Chromatog tion the protein of interest is adsorbed raphy AC to the column ligand After a wash to remove unbound sample elution is Equilibration performed either by using a buffer v containing a competitor to displace the Sample Application protein of interest or by changing the pH or ionic strength Finally the column Canai is re equilibrated with start buffer a Elution hd Equilibration UNICORN 6 1 Method Manual 28 9817 65 AB 81 3 Create and edit methods 3 8 Predefined methods and phases Predefined purification method Principle Included phases Anion Ex After equilibration and sample applica change tion negatively charged proteins are Chromatog adsorbed to the column ligand After a raphy AIEX wash to remove unbound sample Equilibration elution is performed using a gradient m of increasing salt concentration of e g Sample Application NaCl Finally the column is washed m and re equilibrated with start buffer E v Elution Column Wash hd Equilibration Cation Ex After equilibration and sample applica change tion positively charged proteins are Chromatog adsorbed to the column ligand After a raphy CIEX wash to remove unbound sample Equilibration elution is performed using a gradient
205. ight be useful on a single system when a wash procedure is programmed in a separate method This method can then be linked to a series of dif ferent process methods ensuring the same wash procedure is used in each process On multiple systems the product of a separation on the first system might be the starting material for a separation on the next allowing fully automatic multi step processing Note When a method queue is started an option is available to run the start pro tocol for the method queue only once Notification limit warnings related to the number of times a column has been used for example since the last CIP was performed are only issued when the start protocol is performed See Set notification limits for an individual column on page 269 Ina method queue this may therefore not always be shown exactly when the notification limit is reached Each run will however be noted in the column history which should be checked before critical runs in a method queue See View individual column history on page 274 Main steps when creating a method queue The main steps when creating a method queue are Step Action 1 Create methods for the required system s See Chapter 3 Create and edit methods on page 25 2 Create open a method queue e Create a new method queue or e Open an existing method queue that can be edited and saved with a new name 3 Save the method queue 230 UNICORN 6 1 Method Manual 28 9817
206. ight or four digits respectively insert leading zeros before the number Note If the column has no GE Healthcare label and you have run out of UniTag labels check the box Add column by manually enter ing UniTag lot and ID then enter an arbitrary lot and ID This procedure is possible but not recommended UNICORN 6 1 Method Manual 28 9817 65 AB 263 8 Column Handling 8 3 Handling individual columns 8 3 2 Register a new individual column Step Action 4 In the second New Column dialog New Column Add a new column by entering a Column ID either manually or with a barcode scanner Code Exp ID Column ID 28 9281 Lega 578 sne a eee Alias optional Capto G for Ribosome project Technique Anion Fachanae Column type Set medium expiration date Friday April 30 2010 e Enter an Alias optional Tip Alias can be used for easy identification of an individual col umn e Select Technique and Column type Note For prepacked GE Healthcare columns with a matrix barcode these are filled in automatically e Check the Use medium batch ID and type in the batch number of the medium e Check the Set medium expiration date and select expiration date for the medium to get a notification in UNICORN when this date is reached Note The expiration date cannot be set or changed after a column has been regist
207. in the error message dialog e Click the Next button 1 You can also create an error report from System Control by choosing System Create System Error Report Asystem must be connected The report will be created for the connected system and step 2 in the instruction above will be omitted UNICORN 6 1 Method Manual 28 9817 65 AB 353 10 Troubleshooting 10 2 System Error Reports Step Action 2 The Select Systems dialog is displayed showing all accessible systems Tip In this and all subsequent dialogs you can always click the Back button to return to previous dialogs and change the entries e Select the system that the error is connected to e Click the Next button Note If the problem is general related to the UNICORN software and cannot be connected to a specific system you do not have to select a system in this step Ensure that the empty space above the first system is selected as illustrated below and click Next to proceed However it is an advantage in subsequent trou bleshooting if at least one system can be referenced Select Systems Select system to make a report on SystemC hs 3 The Error Description dialog opens Add the following information e A short description of the problem e The circumstances under which the problem occurs e The consequences of the problem Click the Next button 354 UNICORN 6 1 Method Manual 28 9817 65 AB 10 Troubleshooting 10 2 System Error Reports Step Actio
208. ing the column By adding steps several prepara tion solutions can be used sequentially Column CIP Cleans the column after purification runs by rinsing the column with a cleaning solution to remove unspecifically bound proteins By adding steps several cleaning solutions can be used sequen tially System Prepara tion Prepares the system before a run by removing storage solution and filling the system and inlets with buffer solution One prepa ration solution is used per phase System CIP Cleans the system after purification runs by rinsing the system with a cleaning solution One cleaning solution is used per phase Column perfor mance test Tests the efficiency of a packed column in terms of height equivalent to a theoretical plate HETP and the peak asymmetry factor AJ Intelligent Pack ing A flow of hydraulic liquid pushes the adapter down The user ini tiates the start of compression at the exact point when the adapter reaches the consolidated bed surface The adapter compresses the bed according to the packing factor or target bed height as selected Only available for AKTA avant 150 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 9 Fraction collection 3 9 Fraction collection Introduction For many purification schemes it is convenient to collect fractions of the eluent Several of the predefined phases and methods include options for fraction collectio
209. inistration ES System Control 4a Evaluation Options Change Passwords UNICORN 6 1 Method Manual 28 9817 65 AB 6 BufferPro 6 3 Create and edit BufferPro recipes 6 3 1 Create and edit a BufferPro recipe Step Action 2 To create a new recipe click New in the BufferPro Recipe dialog To edit an existing recipe select the recipe to be edited from the list and click Edit Note The available recipes may be filtered by type All Predefined Global or Personal by using the Show drop down list BufferPro Recipes Recipes Show All i v Stock Solutions Recipe name Inlet Stock solutions Concentration M Substance i Q1 Buffer substances 0 2000 Bicine Bis Tris 0 1M NaCl Bis Tris Propane 0 1M NaCl Carbonate 0 1M NaCl Carbonate with HCI 0 1 M NaCl La gt p Q2 Acid or Base 0 2000 NaOH CIEX mix 0 1M NaCl 2 0 7 3 Citrate 0 1M NaCl foe Q3 water Citrate with HCI 0 1M NaCl Erg Q4 salt 4 0000 Nacl Achievable ranges with recipe Description Follow the safety instructions for each bulk chemical when preparing the BufferPro stock pH Ot TR solutions piane 0 0500 M Bicine 0 2000M 32 64g Bicine to prepare 1 litre Mw 163 2 NaOH 0 2000M Use ampule Nacl 4 000M 233 8g to prepare 1 litre Mw 58 44 Note Not recommended For AIEX 1 0000 M 100 B UNICORN 6 1 Method Manual 28 9817 65 AB 209 6 BufferPro 6 3 Create and edit BufferPro recipes 6 3 1 Create and edi
210. io athe 111 SLATS 7 St PM Capio adhere M SAAS TEH Mi Lapo ahea 006 SATUS aoe Pa Caps odes 00S Soh ois 7B Pl Capte adhere OHO SENTO 754 9P Capac sha 0 Sue Pan Pe Caio shira UH SS T EPM Canis adhe 063 STE Pd 2ah8 2 ia Oe oee Ti c BES Delete responses The table below describes how to delete a response from the experiment Step Action 1 2 In the Design of Experiments tab in the Evaluation module click the Delete Response button Result The Delete Responses dialog opens Delete Responses Select the responses you want to delete Responses Delete Cancel Check the box in front of the response to be deleted and click Delete Result The response is deleted from the experiment UNICORN 6 1 Method Manual 28 9817 65 AB 165 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 2 Generate model Insert new runs Runs can be inserted if there are runs missing in the experiment This can be the case if the DoE run has been divided into two scouting runs In that case there will be two DoE results The table below describes how to add a missing run result to the experiment Step Action 1 If runs are missing in the experiment the rows for the missing runs are blank Capesiment D isi F Menon Aur tor apiy adhere mhed ey ext m a Dapy adhere irah ey cel SOOT 15 2005 CI fh apy siere method ng ei SUT See a ra Capio shere rated eet SORT T9 ee ee
211. ion Result Name amp Location t tHiLoad 16 10 0 Sepharose Column type HiLoad 16 10 Q Sepharose HP y vj batted Noles Column volume 20 106 mi Pressure limit pre column 0 50 MPa 0 02 20 00 Unit selection Pressure limit delta column 0 30 MPa 0 02 20 00 Method Base Unit Cy v Use flow restrictor Flow Rate Unit ml min w Column position By pass v antaas Wavelengths 190 700 nm Flow rate 2 500 ml min 0 000 25 000 Uy 290 nm Control the flow to avoid overpressure m uy2 nm U 3 214 nm Use manually prepared buffers Inlet At InletB B wa Enable pH monitoring Column types are either globally available to all users or only personally available A number of column types are predefined in UNICORN see below for more information about predefined column types Note When creating methods and performing method runs the bed height for the column type will be used The measured bed height of a custom packed indi vidual column e g an AxiChrom column may differ from the bed height of the parental column type If you wish to use the correct bed height for method creation you can edit the column type and enter the measured bed height of the individual column This section describes how to add edit and delete column types It also describes how to import and export column types and how to print information about selected column types UNICORN 6 1 Method Manual 28 9817 65 AB 247 8 C
212. iption Text Instructions tab Select to display the Text Instructions Text Instruc tions shows the method in a text format The illustration below shows the Text Instructions pane b b OOD Pha Start thee Sapi Appi sE omo pho kya Toolbar optional pane Shows the toolbar icons Gradient optional pane Shows the programmed gradient and break points for included phases and blocks Flow Scheme optional pane Illustrates the flow path of the instrument graphically Note For detailed information on the Toolbar and the different panes in the Method Editor see Getting Help on the Toolbar and panes in the Method Editor below Display optional panes The optional panes in the Method Editor are displayed by selecting them in the View menu To restore the appearance of the Method Editor to display the default panes select Restore to Default in the View menu Then the Toolbar Gradient and Phase Li brary are displayed The appearance of the optional panes can also be controlled using the Auto Hide function see below for more information Note Settings made by a user are automatically remembered by the software next time the same user opens the Method Editor The illustration below shows the View menu with the default panes selected UNICORN 6 1 Method Manual 28 9817 65 AB 17 2 The UNICORN Method Editor 2 1 The Method Editor Toolbar Phase Library Flow Scheme Method Navigator Gradien
213. iption of the values 3 Non significant terms may have been found in the analysis of the model for example in the coefficient plot To remove a non significant term select the term in the Model terms table and click Remove If the P value gt 0 05 the term is not significant Note Always remove non significant terms from the model one by one starting with the least significant interaction or quadratic term When the first term has been removed the significance of the other terms changes The P value can be used to determine which term to be removed next Note If you fit a model to two or more responses a model term that is not significant for one response may be significant for another response Then the term should not be removed Before removing a term always check that the term is not significant for any of the other responses by selecting the response in the Select a response drop down list and checking the P value for the term you want to remove Note If a main term is not significant but one of its interaction terms is significant the main term should not be removed Note If a main term is removed its interaction terms are also removed Result The term is removed from the model and the R2 Adj and Q2 values are updated If the model refinement gives a higher Q2 value the model re finement is justified If one model is fitted to several responses view the R2 Adj and Q2 values for all responses UNICORN 6 1 Method Manual
214. is h i biin bed echted Cohn peker Fecull Hare amp Location Shere by ledrepa Amon Fae Png sa ae Emuuilibraticn iT Sha Prodan Method Hoti k Hiuk Yarab Yaban Farmy Sampi Applikati A sri aurir Ary or He TROD 3E 1 TINGS Pre culuinn perce lent HF ad Fal n DUGG S00 i mnes beat MPa mo oun S Leama pheri Hypes Ea Columa Waah an aaea ee nar e ee j a METHOD SETTINGS ried A Al ka pi k a iudi 25 tate Elution conte Fre cohen pierna y Coumi Wi aah y Equilibration C Stew delais EITE Enable automate bula pepaston Hulan Ciam geak Harg iris kap of C Cohn Peshormancce Tesi CoP Core Deine by aciga fu mulicomponent buie e Dursion amp vanasbles l Faita Phasa Propeta Identifying variables in the Text Instructions area Parameters that are defined as variables in the text method are indicated in the Text Instructions area The parameter is given as the default value in parentheses followed by the variable name The illustration below shows an example of this 0 00 Phase Equilibration 0 00 Base SameAsHMain 0 00 Pump A inlet Al inlet A 0 00 Pump B inlet B1 Inlet B 0 00 Gradient 0 00 Percent B for equilibration ZB 0 00 base 0 00 System wash 25 45 ystemash olume ml Outlet valve 0 00 Watch UY parameters 0 00 mAU 0 10 mA 0 00 Hold until UY 1 Stable signal 0 10 min Infinite base 0 00 Auto zero UY 0 00 Block Equilibrate For example in 1 000 Flow rate ml min
215. l error and condition number give important information about the model The table below describes these values in more detail Regres sion p value Description The regression p value is a measure of the significance of the regression model Interpretation p lt 0 05 indicates a significant regres sion model Lack of Fit p val ue Cond no con dition number The lack of fit p value is a measure comparing the model error with the replicate error This value is used in the calcu lation of Model Validity in the Summary of Fit plot The condition number can be used to investigate if the de Sign is appropriate to use espe cially if any of the default de Signs suggested in the Method Editor have been altered Depending on the design differ ent condition numbers are ex pected for the model to be good p gt 0 05 indicates a good model If p lt 0 05 this indicates that the model does not describe the relation between Y and X and that a quadratic term may be missing See Section 5 4 5 Edit the model on page 190 for information about how to add a quadratic term to the model A low p value may also be due to other reasons for example terms missing or that there is no correlation between X and Y that can be modelled e When the objective is screening and robustness testing Good design when Cond no lt 3 Questionable design when Cond no 3 6 Poor design when Cond no gt
216. le click the Open Result Navigator icon in the Toolbar Result The Result Navigator is displayed 160 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 2 Generate model Step Action 2 Browse for the result and double click the result name single runs are indi cated by the chromatogram icon Result Navigator Open wa Results DoE Results E Folder name Created by Last modified D DoE results KP Default FYB 2009 4 40 47 tl DoE ID DoE _1 Default of1 2 2009 1 43 2 IA IDoE 1D DoE_1 001 Default 1 13 2009 3 21 4 lel DoE ID DoE_1 002 Default 171472009 7 31 00 lel DoE ID DoE_1 003 Default 1271872008 5 12 lel DoE ID DoE_1 004 Default 12 18 2008 5 18 lel DoE ID DoE_1 005 Default 12 18 2008 5 26 lel DoE ID DoE_1 006 Default 12 18 2008 5 31 lel DoE ID DoE_1 007 Default 1221872008 537 lel DoE ID DoE_1 008 Default 12 18 2008 5 42 lal DoE ID DoE_1 009 Default 12 18 2008 5 47 lel DoE ID DoE_1 010 Default 1271872008 5 52 lel DoE ID DoE_1 011 Default 1271872008 5 57 Result The result of the run is opened and displayed in the Evaluation module 3 Inspect the results visually and check that the runs have been performed as expected 4 Evaluate the results for the run as appropriate See UNICORN 6 1 Evaluation Manual for information about how to perform evaluation 5 Save any changes Tip It is possible
217. lick OK to import the recipe s UNICORN 6 1 Method Manual 28 9817 65 AB 225 6 BufferPro 6 7 Predefined BufferPro recipes 6 7 Predefined BufferPro recipes Introduction This section describes in detail the predefined buffer systems in BufferPro that are deliv ered with UNICORN General points The following points should be taken into consideration The pKa of certain buffer substances can vary significantly with temperature This means that the working pH range for optimal buffering will vary with temperature It is possible to estimate the appropriate pH ranges using the Explore Proportions tool see Section 6 5 Calculate buffer composition using BufferPro on page 219 The two broad range buffer systems AIEX mix 0 1M NaCl and CIEX mix 0 1M NaCl may only be used at fixed concentration since these are multi component buffers The working concentration for buffers that are mixed using conjugate acid base pairs is 25 50 of the stock solution concentration For buffers mixed using strong acid or base solutions the working concentration is 15 25 of the stock solution concentration Although it may be possible to mix solutions outside this range UNI CORN will show a warning since the pH of the resulting buffer may not be reliable If in doubt check the pH of the eluent after running an experiment using a reliable lab pH meter The pH range given is based on the narrowest range for effective buffering for the entire gradi
218. list use the Ctrl and Shift keys 2 In the Column Logbook tab click Delete Result The Confirm Column Delete dialog opens Confirm Column Delete A 4re you sure you want to permanently delete the selected Columns 3 Click Yes in the Confirm Column Delete dialog Result The individual column is deleted Note Individual columns that have been used cannot be deleted 270 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 3 Handling individual columns 8 3 5 Export and import individual columns 8 3 5 Export and import individual columns Introduction The information for individual columns is stored in the UNICORN database This informa tion may be exported to a zip file in order to move the information to another UNICORN installation This section describes how to export individual columns from UNICORN and how to import previously exported columns Export individual columns Individual columns can be exported from the database to a zip file The columns can then be imported to another database if appropriate The table below describes how to export individual columns from the database Step Action 1 Select the individual column s to be exported in the Columns list in the Column Logbook tab See Section 8 3 3 Find an individual column on page 266 for information about how to find and select a column To select several columns in the Columns list use the Ctrl and Shift keys In the Column Lo
219. ll generate a potentially infinite loop It is not possible to run such a method 294 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 Working with methods in the Text Instructions pane Introduction This section describes how to create or edit methods using specific text instructions The general structure of the text method syntax is described including the major hierarchy of the text method parts phases and blocks In this section This section contains these sub sections Section See page 9 2 1 Base instruction 296 9 2 2 Working with phases and blocks 300 9 2 3 Working with text instructions 309 9 2 4 Method variables 315 UNICORN 6 1 Method Manual 28 9817 65 AB 295 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 1 Base instruction 9 2 1 Base instruction Introduction Every method block must start with a Base instruction defining the base for calculating breakpoints see also Structure of the text method on page 291 Different blocks can use different bases This section describes how to choose and edit settings for the Base instruction What base should I use Depending on the experiment different bases should be used Use the base that most closely suits the purpose of the block Column volume CV is recommended as the base for most steps in a run In some situations however it ma
220. lso describes overall method options how to sign methods electronically how to print methods how to convert and scale methods from one AKTA avant system type to another and how to import export methods Descriptions of the predefined methods and phases supplied with the software are also included Note It is recommended to work with phases using the Phase Properties pane This chapter does not cover how to edit methods using the Text Instructions pane For information about text editing methods see Chapter 9 Text edit methods on page 286 In this chapter This chapter contains the following sections Section See page 3 1 Working with methods Overview 26 3 2 Create and open methods 30 3 3 Edit methods and phases 34 3 4 Set general method options for the method 51 3 5 Save methods and phases 63 3 6 Scale or convert methods 69 3 7 Print a method 79 3 8 Predefined methods and phases 81 3 9 Fraction collection 89 3 10 Sign methods electronically 92 3 11 Import and export methods 94 UNICORN 6 1 Method Manual 28 9817 65 AB 25 3 Create and edit methods 3 1 Working with methods Overview 3 1 Working with methods Overview Introduction In UNICORN 6 1 methods are built up using phases where each phase corresponds to a step in a chromatography run with a number of properties associated to that phase By building methods in this way methods are easily created and edited See Section 2 2 Methods in UNICORN 6 1 on page 20 fo
221. lt in some loss of information UNICORN 6 1 Method Manual 28 9817 65 AB 163 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 2 Generate model Step Action 4 Click the Analysis tab Result A model is fitted to the entered data For information about how to analyze the model see Section 5 4 3 Analyze and evaluate the model basic analysis on page 170 Add responses Note Responses added in the Evaluation module will not be included in the original method The table below describes how to add a new response to the experiment Step Action il In the Design of Experiments tab in the Evaluation module click the Add Response button Result The Add Response dialog opens Add Response Predenet S O User defined Abbreviation Act Unit 2 Select the response to be added in the Predefined drop down list or define your own response by selecting User defined and type in your own response Note Abbreviation is automatically filled in 3 Enter the Unit for the response if appropriate 164 UNICORN 6 1 Method Manual 28 9817 65 AB Step 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 2 Generate model Action Click OK Result The response is added to the DoE experiment Epaien Anian Frecketion Opterataben I pmnan E si D Meccan Aer Dale pated Caps ates 17 SETS 7 SP apio she C5 Say Paaa Pa Canis aches UE STATS fue Pe Tap
222. ltinghethod Demo System 1 DesaltwashdndAlEX iS FE Awe UNICORN 6 1 Method Manual 28 9817 65 AB 237 7 Method queues 7 3 Edit a method queue Step Action 3 To delete the method queue e select Edit Delete or e press the Delete key Or e right click the selected method queue and select Delete from the context menu Result A dialog will appear asking to confirm the delete operation Insert a method into a method queue The following table describes how to insert a method into the Method list for a system Step Action 1 Open the method queue see Opening a method queue on page 235 Result The Method Queue dialog opens with the details for the chosen method queue 2 In the Method Queue dialog select the position in the list at which a method will be inserted by clicking on the Method column Method Queue lonExchangeMethodQueue System P2 9 01 v Method Start Condition 1 ColumnwashProcedure At queue start 2 AnionExchangeMethod Lad Immediately after the previous method has ended Hd Inston ka Delte _ la System P2 9 04 v Method Start Condition 1 ColumnwashProcedure2 At queue start 3 4 5 lad a C Coen CO 238 UNICORN 6 1 Method Manual 28 9817 65 AB Step 7 Method queues 7 3 Edit a method queue Action 3 4 Insert a new row by clicking on the Insert Row button Result An empty row will be inserted System P2 9 04 v Method ColumnwashProcedure2
223. lve Watch UY parameters 0 00 mAU 1 00 mAU Hold until U 1 Stable signal 0 03 min Infinite base Auto zero UY Block Equilibrate 0 00 Base SameAsMain 5 00 E quilibration volume End_ Block 0 00 0 00 Auto zero UY End_ Block In the example above the value 5 00 will be 5 column volumes CV if the Base in the Main block is set to CV 5 minutes if Base is set to Time or 5 ml if Base is set to Volume To extend the length of a block without performing any other operation set the breakpoint of the End_block instruction appropriately for example as in the illustration below 0 00 Phase Equilibration 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 H 0 00 0 00 0 50 End_ Block 0 00 Phase Saana igebcalion Base SameAsMain Inlet A Al Inlet A Inlet B B1 Inlet B Gradient 0 0 Percent B Equilibration B 0 00 base System flow 0 000 Flow rate ml min Pre column pressure Pressure control System wash 25 Fill system Equilibration ml Outlet valve Watch UY parameters 0 00 mAU 1 00 mAU Hold until UY 1 Stable signal 0 03 min Infinite base Auto zero UY Block Equilibrate Auto zero UY In this example the block will end after 0 5 ml since Base is set to Volume An estimation of the time for running the method can be obtained in the Method Duration and Variables window See View and print the method duration time and variables on page 60 View
224. lways have its Start Condition set to At queue start Available Start Conditions are e At queue start The method will begin at the start of the method queue Only available for the first method for each system e Immediately after the previous method has ended The method will start when the previous has ended on the queue for that system e Wait The method will start after a specified Wait time has elapsed since the previous method in the queue for the system has ended A separate di alog will open where the Wait time can be specified in Hours and Min utes The delay time will be shown in the Method Queue dialog once entered e At ready command The method will start when a Ready instruction in a method on another system has been executed Using this start condition it is possible to connect methods running on different systems A separate dialog will open where the System and Method can be chosen An arrow to the left of the method queues will show the connected methods as shown in the diagram Method Queue lonExchangeMethodQueue Number of included systems 2 v System P2 9 01 v Method Start Condition 1 ColumnwashProcedure At queue start 2 AnionExchangeMethod kaa Immediately after the previous method has ended Insert Row s Eo System P2 9 04 v Method Start Condition 1 ColumnwashProcedure2 la At queue start 2 DesaltingMethod ow At ready command 3 CationExchangeMethod Lad Wait Oh 30min after the
225. ly prepared buffers Inlet Inlet E Use BufferPro automatic buffer preparation Recipe Bicine O 1M MaCl pH 7 r 8 7 PD ButterFro Properties pH 80 7 8 7 recommended Cone 0 050 MM 0 030 0 050 Note It is not necessary to have the Enable pH monitoring option checked The output from the pH monitor is not used by the BufferPro algorithm 2 Select Recipe and enter pH and buffer concentration Conc within the specified range Note For broad pH range multi component buffers the concentration is fixed For further information see Section 6 7 Predefined BufferPro recipes on page 226 Note To obtain an even gradient the gradient should run for at least 10 minutes and the Flow rate should be not lower than 1 ml min for AKTA avant 25 and 2 ml min for AKTA avant 150 3 Save the method 206 UNICORN 6 1 Method Manual 28 9817 65 AB 6 BufferPro 6 3 Create and edit BufferPro recipes 6 3 Create and edit BufferPro recipes Introduction This section describes how to create edit rename and delete BufferPro recipes Prede fined recipes may not be overwritten renamed or deleted Edited recipes including edited predefined recipes can be saved as global or personal recipes Global recipes are available for all users personal recipes only for the current user Note The predefined recipes can be used in the majority of cases There is often no need to create a new recipe before creating
226. ly used as scouting variables Check the Show unused variables box if you want to display variables currently not used in the method Result The Scouting dialog is updated with the selected variable s and their default value s Scouting Scouting parameters Method Settings METHOD SETTINGS Run Included Flow rate ml min 1 1 000 It is possible to insert runs one by one see step 4 or insert series of runs see step 5 108 UNICORN 6 1 Method Manual 28 9817 65 AB 4 Scouting 4 2 Setup and edit a Scouting scheme Step Action 4 To insert runs one by one e Inthe Scouting dialog select a row in the Scouting parameters table and click _Insert Run Result A new row is added below the selected run The variable value from the selected row is copied to the new run All chosen variables are displayed in a separate column Scouting parameters Method Settings METHOD SETTINGS Run Included Flow rate ml min 1 1 000 S 1 000 e Inthis example click in the Flow rate ml min column for the appropriate run and edit the flow rate value Note Changing variable values in the scouting scheme does not change the values in the Variable List in the Duration and Variables dialog or in the text instructions The actual variable values used for each run in the scouting scheme are saved in the result file To change the default values the variable values must be edited in the Phase Properties p
227. m if the Peak max Factor is set to 1 in Watch Watch Parameters for Watch UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 3 Specific instructions 9 3 3 Watch instructions Delta peak Peak max detected vc re ee Valley detected Delta peak 2 Watch test parameter for air sensors Two Watch conditions are available for air sensors The table below describes the con ditions and their explanations Explanation j Air detected Note To use the Watch parameters for an air sensor the corresponding Alarm air sensors setting must be disabled UNICORN 6 1 Method Manual 28 9817 65 AB 539 9 Text edit methods 9 3 Specific instructions 9 3 4 Pause or hold a method 9 3 4 Pause or hold a method Introduction A method can be programmed to be delayed at critical points There are three instructions for this purpose Pause Hold and Hold until These instructions are described below Pause instruction The Pause instruction suspends execution of the method and stops the pumps so that the system comes to a standstill The valves remain in the position they were in before the pause The pause may be defined as Infinite or for a specified number of minutes Resume the method It is possible to define the pause time for the method in the Pause instruction The method will continue when the set time has elapsed The method may also be resumed if you click the Continue icon on th
228. mn Type Parameters tab in the Column Handling dialog click m Result The Import dialog opens Locate the zip file with the column list to be imported and click Open Result The Import confirmation dialog opens explaining what will will happen when the zip file is imported Import p The selected file contains predefined column settings Woa J Importing this file will update all existing predefined column hardware and media settings Any global or personal column settings will remain unaltered Do you wish to continue x Click Yes Result The new list of predefined column types Is imported into the database Print information about column types The table below describes how to print information about column types Step Action 1 Select the column type s for which to print information in the Column types list To select several column types use the Ctrl or Shift keyboard keys UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 2 Handling column types Step Action 2 In the Column Type Parameters tab click Print Result The Print dialog opens Printer TOSHIBA e STUDIO451 cSeriesPCL5c l Print items All types Include the type s parameters All shown types Include advanced parameters when available Selected types Include associated columns Include the columns parameters 2 Select Printer 4 Select for which column types to print information
229. n 4 The Error Reproducibility dialog opens e Specify whether the problem is reproducible or not Select one of these alternatives e Yes Provide a short description in the text box of how the problem can be reproduced No Unknown e Click the Next button Result The File Attachment dialog opens Go to step 2 below Step 2 Attach example files You can attach results methods and or UNICORN log files to the problem report The table below describes how to attach a file Step Action 1 The File Attachments dialog box is displayed File Attachments Files Result Method System Log UNICORN Log System information Computer and Operating System information C Integrity check UNICORN 6 1 Method Manual 28 9817 65 AB 355 10 Troubleshooting 10 2 System Error Reports Step Action 2 e Depending on the character of the file to be attached select the appro priate tab Result Method System Log or UNICORN Log e Attach a file Click the Add button Selecta file in the dialog and click the Attach button Result The selected file is added to the tab in the File Attachments dialog File Attachments Files Result Method System Log UNICORN Log Capto adhere 007 Capto adhere 008 Capto adhere 009 System information Computer and Operating System information Integrity check Tip To remove a file from the tab select the checkbox an
230. n Functions like signing methods electronically and importing exporting methods are also included Tools in the Method Editor The table below describes the different tools included in the Method Editor Description Design of experiment DoE is used to find out in a systematic way which run DoE parameters affect a process to be run and how to find optimal values for these parameters to obtain the best possible result using a minimum number of runs When creating a method and setting up an experimental design using DoE an optimized Scouting scheme will automatically be created See Chapter 5 Design of Experiments on page 116 for more information Scouting Scouting is used to repeat a series of Method runs auto matically where the user can change the values of prede termined variables before starting the method A Scouting scheme is defined as part of the method See Chapter 4 Scouting on page 103 for more information 14 UNICORN 6 1 Method Manual 28 9817 65 AB 2 The UNICORN Method Editor 2 1 The Method Editor Description BufferPro BufferPro allows a buffer of defined pH and with defined salt concentrations to be prepared from four stock solu tions one Buffer stock solution one Titrant Water and a Salt stock solution pH and salt concentration can be used as variable scouting parameters included in a Scouting scheme or in a Design of Experiments DoE BufferPro is optimized for cation and anion e
231. n phases in your method changing settings in one of them will not affect the other To be able to see that they are different it is recommended to rename one of them See Section 3 3 2 Edit the method outline on page 43 for information about how to rename a phase e Repeat steps 5 6 until the appropriate phases have been edited Result The method is updated with the new settings The edited settings remain in place while subsequent phases are edited If the method Is closed and not saved the settings will revert back to the earlier values v Save the method 42 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 3 Edit methods and phases 3 3 2 Edit the method outline 3 3 2 Edit the method outline Introduction The Method Outline shows the phases that are included in the method and the order of the phases in the method Phases can be added rearranged renamed and deleted from the Method Outline Add a phase to the method outline using drag and drop The table below describes how to add a phase to the method outline using drag and drop Step Action 1 Select the appropriate phase in the Phase Library pane and drag and drop the phase to the requested position in the Method Outline pane Result The phase is included in the method at the requested position If the User Defined phase was added continue with step 2 2 When the User Defined phase has been added to the Method Outline the phase name is enabled
232. n a box 2 Type in a new name Note If the block you renamed is called in a Block or Watch instruction the block name in these instructions will be changed automati cally UNICORN 6 1 Method Manual 28 9817 65 AB 307 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 2 Working with phases and blocks Delete blocks The table below describes how to delete a block Step Action 1 e Right click a block and choose Delete or e Selecta block and click Delete in the Instruction box or e Selecta block and press the Delete key on the keyboard Result A dialog will appear asking if the block should be deleted permanently or moved to unused blocks Text Instructions The block can either be permanently deleted from the method or be moved to the Unused section in the method Move to Unused Note If deleting a phase the phase will be deleted right away 2 e Click Delete to delete the block permanently e Click Move to lt Unused gt to delete the block from the method and place it after the method 308 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 3 Working with text instructions 9 2 3 Working with text instructions Introduction Instead of editing the method in the Phase Properties pane instructions may be edited one at a time in the Text Instructions pane The instructions in a block are alway
233. n be edited in the Phase Properties pane or Text Instructions pane UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 3 Working with text instructions Phase Properties Text lrir T Method Sanings iT Equilibration IT T hii phata hai Boen banediad Mock Vasieble Value Mange EQUIUSAATION lisi A Al ge Semple Application abe Bt w EULA TION Percend D Equilrstond 58 Mitai 200 iaol 7 EQUUSFRATIJN Figa rites ib Trn 1 00 o 25 0 Peeteute ganiga Pre piini pisii we Column Wash COULRATION Fil apitar Eguitration imi 15 EE E qiia ate Eguia vod Cy 50 Q00 Serer F Elution T Colima Waal T Equilibration T Li She detali Edit V ariabi CY Feiere Phata Propertier Change or replace an instruction The table below describes how to edit instructions in the Text Instructions area Step Action 1 Select an instruction in the text method Result The current Breakpoint and parameters for the selected instruction is displayed in the Instruction Box Z Edit or select parameter values in the Instruction Box Instruction Box Instructions z Parameters for Fractionation Fraction collection Base Fractionation Peak fractionation Time Volume Stop fractionation Tube type Stop peak fractionation Last tube filled LYAR 96 deep well plate Feed tube Fraction size 0 00 2 20 Fractionation in outlet valve ja St
234. n in the Phase Properties pane This section describes briefly the various options available for fractionation in predefined methods and phases and how to set up fraction collection when editing a method More detailed information for individual settings can be found using the online help for the phase see Getting help when editing Phase Properties on page 36 Fractionation overview Fractionation is available in the Phase Properties pane in the predefined phases Sample Application Column Wash and Elution These three phases are included in many of the predefined methods in UNICORN This option will also be available in personal or global phases derived from these See Section 3 3 1 Edit phase properties on page 35 for details on how to edit methods and phases For each phase fractions can either be collected using the outlet valve or the fraction collector If there is no risk of sample loss the eluate may be sent to the waste and not collected When fractionating to the outlet valve a specific outlet valve position is selected When collecting fractions in the fraction collector a tube or plate type is chosen and the fractions will be collected in the first available tube or plate of that type WARNING Fraction collector Do not fractionate flammable liquids When running RPC methods or other procedures using organic solvents collect fractions through the Outlet valve UNICORN 6 1 Method Manual 28 9817 65 AB 89
235. n the Method Editor and System Control de pending on the task to be performed In this section This section covers the following Section See page 8 3 1 Individual column identification 261 8 3 2 Register a new individual column 262 8 3 3 Find an individual column 266 8 3 4 Edit individual columns 268 8 3 5 Export and import individual columns 271 8 3 6 Print and view individual column information 274 260 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 3 Handling individual columns 8 3 1 Individual column identification 8 3 1 Individual column identification Matrix barcode Most pre packed GE Healthcare columns are marked with a matrix barcode on the col umn label This barcode can be scanned using the 2D barcode scanner to register new individual columns or to find columns in the database Columns can also be labeled with UniTag labels A UniTag label is a unique identifier for individual columns that are not pre labelled with a matrix barcode such as HiTrap columns manually packed columns or columns from other sources A number of UniTag labels are supplied with the system and they can also be purchased separately The diagram below shows an example of a column label and a UniTag label with their matrix barcodes Label No 28 9366 32 AC HiPrep 26 60 Sephacryl S 400 HR Code 28 9356 05 _ Jni R lot 20090421 Unitag ae est exp 2010 10 lot 20090220 ID 0005 ID 0001 Column label Uni
236. n valve Manual load Outlet valve Out Waste Column position By pass Column position Down flow pH valve In lineJHpH cell In line Flow restrictor Alarm air sensors Enabledj Air sensor alarm on inlet valve Enabled Ai sensor alarm on Inlet A A1 Inlet A Inlet B B1 Inlet B System flow 1 500 Flow rate ml min Pre column pressure Pressure control E End_Block M 0 00 Phase Rubisco Elution 0 00 Base SameAsMain 0 00 Inlet A Alltinlet A lt Base Time min Volume ml Run 2 3 Total Yolume approximately 28 50 ml Please note The actual time and volume may differ from calculated values when wash watch or Total Time fappendniatek 20 58 min programmed hold instructions are included in the Run Yolume approximately 9 50 ml method Run Time approximately 6 33 min The Method Duration tab shows an estimation of the accumulated method time and volume for the current method below the text method If the method includes a Scouting series an estimation of the accumulated method time and volume for the total series of runs is displayed below the text method Note Click the arrow buttons to display the different scouting runs The accumulated time volume is an approximation and does not take into account time or volume for Watch blocks Wash commands or programmed Hold 5 e Select Time as Base to show the time in minutes in the text method e Select Volume as Base to show the volum
237. nek Preckcbon Optnetaton F opman Fais Rire Pun Gate crested ios Awu E w HHHH 120 E mg H 1751 0F zl E ASA 1217 a8 fl wr TURE 120 i mo HHH 12516 E mi FEMA 175R 25 Fl 003 SE Pde Fl w5 H TAE T E poz DOH Tita al fi FEMA IF 44 1 zl Cei eer Nen Chepe iaire 2 Click the Replace Result button Result The Replace Result dialog opens Replace Result Folder name Last modified 2 E Usel 2009 05 20 10 12 1 E Use2 2009 05 20 10 18 1 E Use3 2009 05 20 10 22 0 E Usen 2009 05 20 10 31 0 2 E Uses 2009 05 20 10 32 4 E Labi 2009 05 20 12 44 4 E Laz 2009 05 20 13 00 3 E Las 2009 05 20 13 49 1 E Labs 2009 05 20 13 52 4 Babs 2009 05 20 13 55 2 a Lab5 Manual 2009 06 08 16 14 3 amp Column Handling Work Station 2009 06 09 12 35 2 Ga Back up 2009 06 09 13 24 17 2 amp DoE Workstation 2009 06 09 18 45 0 ay Capto adhere 001 2009 06 04 12 40 3 mi New run Capto adhere 002 2009 06 04 12 42 2 mi Capto adhere 002 2009 06 04 12 42 2 rel Capto adhere 003 2009 06 04 12 44 0 3 Browse and select the run that should replace the selected run 4 Click OK Result The new run is listed in the Experiment table UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 2 Generate model Step Action 5 If the run does not match the run to be replaced an error message will be displayed Repeat from step 2 to insert t
238. ns 9 3 1 Gradients and eluent concentrations Introduction Gradient instructions allow definition of an A and B buffer mix The starting point for the Gradient is always the current eluent composition The instruction can be read as follows form a Gradient to reach Target after Length Linear gradients and step gra dients can be created using Gradient instructions Gradient instructions are given in the Text Instructions editor of the Method Editor This type of instruction defines gradients and immediate changes in eluent concentration Linear gradients A gradient can be defined as a linear gradient The eluent composition changes linearly over time Example of instruction 10 00 Gradient 50 B 20 base The example instruction above forms a gradient to 50 B Target starting at breakpoint 10 with duration 20 method base units Length The example instruction will finish at breakpoint 30 If the current eluent concentration is greater than 50 the gradient will be negative Step gradients A gradient can be defined in several steps A step gradient is an immediate change in eluent composition To form a step gradient set the Length parameter to 0 in the Gra dient instruction Example of instruction 10 00 Gradient 50 B O base The example instruction above forms a step from the current eluent composition to 50 B at breakpoint 10 The method continues with 50 B UNICORN 6 1 Method Manual 28 9817 65 AB 327
239. nstruction Watch parameters instructions are used to define accepted limits and fluctuations for a signal in a Watch instruction Watch parameters instructions should therefore be in serted just before the Watch instruction on which the limits are required Step Action 1 Select the instruction line immediately before the Watch instruction to which the parameters will apply 2 e Expand Watch parameters in the Instructions field of the Instruction Box e Select the desired watch parameters from the list e Select appropriate values for the Accepted fluctuation and Delta peak for the Watch UV parameters and Watch cond parameters instructions in the Parameters field For information about the Delta peak setting and how to use it see The Delta peak setting on page 338 Breakpoint Instructions FT Parameters for Watch UV parameters 0 00 cy eee sit pressures Delta peak 0 00 6000 00 E Flow pa aaa Var Monitors L 0 00 mau ee Accepted fluctuation 0 00 6000 00 Alarms a Watch parameters 0 10 z mAU Watch flow parameters Watch pH parameters Watch pressure parameters amp Watch 3 Click the Insert button Result The new Watch parameters instruction Is inserted in the method in the text area UNICORN 6 1 Method Manual 28 9817 65 AB 337 9 Text edit methods 9 3 Specific instructions 9 3 3 Watch instructions The Delta peak setting 538 The Delta
240. nstructions on how to use the software Workflow descriptions for common operations UNICORN 6 1 Administration Network setup and complete software installation and Technical Manual Administration of UNICORN and the UNICORN database 12 UNICORN 6 1 Method Manual 28 9817 65 AB 2 The UNICORN Method Editor 2 The UNICORN Method Editor About this chapter This chapter gives an introduction to the Method Editor in UNICORN 6 1 It gives a brief description of the Method Editor interface and describes the concept of methods in UNICORN 6 1 For information about how to create open and edit methods as well as signing methods and importing exporting methods see Chapter 3 Create and edit methods on page 25 In this chapter This chapter contains the following sections Section See page 2 1 The Method Editor 14 2 2 Methods in UNICORN 6 1 20 UNICORN 6 1 Method Manual 28 9817 65 AB 13 2 The UNICORN Method Editor 2 1 The Method Editor 2 1 The Method Editor Introduction The Method Editor provides complete facilities for e creating and editing methods e copying saving and deleting methods e converting methods for use with different types of KTA avant systems e g from an KTA avant 25 system to an KTA avant 150 system The Method Editor also provides a number of tools to assist the user in optimizing runs and a tool for handling column types and individual columns see below for more infor matio
241. ntains the following sections Section See page 4 1 Overview 104 4 2 Set up and edit a Scouting scheme 106 UNICORN 6 1 Method Manual 28 9817 65 AB 103 4 Scouting 4 1 Overview 41 Overview Introduction Scouting can be used to generate a series of method runs where one or more Variable parameters are varied in the same method The resulting Scouting scheme is defined and saved in the method This section gives an overview of scouting and the scouting workflow When to use scouting Some typical situations where scouting is useful are for instance when the objective is to e screen for the best column e find the optimal pH e test column capacity sample volume e find the optimal flow rate for binding and elution e optimize gradient length and slope e optimize step gradients Scouting workflow overview To perform a scouting experiment the following steps must be performed e Create a method and decide appropriate run parameters i e variables to be varied in the experiment See Chapter 3 Create and edit methods on page 25 for information about how to create methods e Setup a scouting scheme This includes selecting variables inserting runs series of runs with different variable settings To define new variables for a method see Section 9 2 4 Method variables on page 315 for information e Start and monitor the scouting run This is performed in System Control See AKTA avant and UNICORN 6
242. nts 329 Length as variable 328 Linear 327 Step 327 Text instructions 329 H Help Text instructions 309 Viewing 19 Hold Insert instruction 341 Text instruction 340 Hold until Insert instruction 341 Text instruction 341 Hydrophobic interaction chro matography HIC Predefined method 84 Importing Blocks from other meth ods 304 BufferPro recipe 224 Column types 256 Individual columns 271 Methods 101 Phases 99 Predefined column types 258 Individual columns Adding or editing notes 268 Column Logbook 260 Column performance test 276 Deleting 270 Editing 268 Exporting 271 Finding 266 Importing 271 Notification limits 269 Performance report 277 Printing information 275 View column history 274 UNICORN 6 1 Method Manual 28 9817 65 AB Index Instrument Components 348 Instrument configura tion 348 Intelligent Packing Predefined method 86 Predefined phase 88 lon exchange Predefined method 82 M Maintenance Generate system error re port 353 Maintenance methods Column CIP 85 Column performance test 85 Column preparation 85 System CIP 86 System preparation 86 Message Text instruction 343 Method blocks Adding 302 Block length 300 Copying 303 Deleting 308 Hide instructions 301 Import from other meth ods 304 Moving 306 Rename 307 View instructions 301 Method Editor General interface descrip tion 15 Optional panes 17 Overview 26 Saving a met
243. o Pump wash System wash Outlet valve O Injection valve Wash flow BufferPro pH BufferPro setting Constant pressure flow Flow path Monitors v Edit Variable Import Block 2 Click the VAR button for the appropriate variable Result The Edit Variable dialog opens Edit Variable Variable name isible in details only 3 Do one or several of the following as appropriate e Type in a new name in the Variable name field e Check the Visible in details only if the variable should be a detailed variable Uncheck the box to set it to a normal variable e Click Clear to delete the variable 4 Click OK 5 To save the changes click Change in the Instruction Box Result The text instruction is updated UNICORN 6 1 Method Manual 28 9817 65 AB 323 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables Edit variable values To edit default variable values you can either e edit the value in the Phase Properties tab if the text method has been edited or e edit the instruction in the Instruction box of the Text Instructions Changes made in the Phase Properties tab are automatically updated on the Text In structions tab and vice versa Edit variable values in the phase variables list If the phase containing the variable value to be edited has been text edited it is possible to edit the variable value on the Phase Properties tab The t
244. o be performed the DoE runs can be divided into several scouting runs This section describes how to divide a DoE run into several smaller runs Divide DoE runs into several scouting runs directly in the Scouting scheme It is possible to include exclude runs directly in the generated Scouting scheme and edit for example the sample inlet positions However for complex experimental plans it is recommended to create multiple DoE methods each using the same design but with different sub sets of scouting runs see below As long as the designs are identical the results can then be merged for analysis UNICORN 6 1 Method Manual 28 9817 65 AB 149 5 Design of Experiments 5 2 Create an experimental design 5 2 4 Divide the DoE runs into several scouting runs Divide DoE runs into several scouting runs in the DoE setup The table below describes how to identify hardware limitations in a DoE run Step Action 1 150 In the Design of experiments dialog select the Experiment tab Result The Experimental Plan is displayed Design of Experiments en Responses Factors amp Design Experiment Experimental Plan itied Load Concentration Sample Application S ample inlet 5 S2 5 S4 5 S 5 S3 20 Buffer 20 51 20 SB 20 Not Enough Positions 1 12 5 55 12 5 55 12 5 S5 SUES SPEAKS ESTES d Load Conductivity Sample Application S ample inlet 2 52 2 54 15 S7 15
245. ocol Result The Start Protocol dialog opens In the Start Protocol dialog e Select items to display at method start When selecting a method item a description is shown to the right Result Name and Location is selected by default e Click OK to confirm and close the dialog 54 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 4 Set general method options for the method Add edit Method Notes The table below describes how to add edit notes to a method Step Action 1 In the Method Editor e click the Method Notes icon or e select Edit Method Notes Ctrl 2 or e click the Method Settings phase and click the Method Notes button in the Phase Properties tab Result The Method Notes dialog opens UNICORN 6 1 Method Manual 28 9817 65 AB 55 3 Create and edit methods 3 4 Set general method options for the method Step Action 2 Method Notes In the Method Notes dialog e Enter edit notes about the method If notes already have been entered it is possible to search for specific words using the Find button e Click OK to confirm and close the dialog Note Information will automatically be added to the Method Notes if the method has been converted for use with another AKTA avant system type than what it was originally created for or scaled for another column type than what was originally selected 56 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create an
246. og click OK Result The following dialog opens Method Editor A Design of Expenments has been created for this method Changes to the method or scouting scheme may affect the design Do not show this message again Result A Scouting scheme is generated with the runs to be performed The method is displayed in the Method Editor Note If you change the Scouting scheme the DoE experimental plan is changed and the results may not be reliable for use in the DoE evaluation 10 Save the method including DoE UNICORN 6 1 Method Manual 28 9817 65 AB 137 5 Design of Experiments 5 2 Create an experimental design 5 2 2 Add responses and factors to an experimental design 5 2 2 Add responses and factors to an experimental design Introduction This section describes how to add responses and factors to the Design of Experiments setup in the Method Editor Add responses The table below describes how to add responses to an experimental design Step Action 1 Select the Responses tab Design of Experiments Responses Factors amp Design Experiment Responses Name Abbreviation Unit Click Add to define a response Delete 2 To add a response click Add Result The Add Response dialog opens Add Response Predefined Active I O User defined Abbreviation Unit Note It is possible to add new responses to the experimental design in Evaluation These new responses will not be
247. olume i e Fill system value UNICORN 6 1 Method Manual 28 9817 65 AB 77 3 Create and edit methods 3 6 Scale or convert methods e Volume for finalization of sample application only applicable when Inject sample directly onto column and then Inject all sample using air sensor is selected The following settings may remain to be adjusted manually after the conversion e Sample volume e Fractionation volumes e User defined volumes in System CIP and System preparation e System related settings in text edited phases However column related settings will be scaled also in text edited phases provided they have been defined as variables Converting a method for use in a different database The table below describes the necessary steps to be performed if you wish to convert a method for use with another AKTA avant system type in another database than where the original method was created Tip This procedure must be followed in order to convert methods from one stand alone system to another stand alone system Step Action 1 Set up a new system in the target database Use the same instrument con figuration as the system for which the method was originally created Tip This system is created for the conversion only and can be set up inactivated 2 Export the method from the original database 3 Import the method into the target database for use with the new inactivated system 4 Convert the method from the inactivated
248. olumn Handling 8 2 Handling column types Predefined column types A number of GE Healthcare column types are predefined in UNICORN For each column type as many individual columns as needed can be registered Parameters for the pre defined column types can be edited by saving the column type with a new name and as a Personal or Global column type The complete list of predefined column types can be found in the Column Handling dialog Create a new column type The table below describes how to add a new column type with the Column Handling tool Step Action 1 In the Column Type Parameters tab in the Column Handling dialog click New Result The New Column Type dialog opens New Column Type IF the column hardware and medium are made by GE Healthcare select the name of the hardware and medium to have most of the parameters filled in automatically Show hardware types by diameter cm Min GE Healthcare hardware type Any Parameters Technique Column volume Mas pre column pressure Default flowrate Max flowrate Default linear flowrate Max linear flowrate Min pH value short tern Max pH value short term Min pH value long term Mas pH value long term Mas delta column pressure Max 0 00 E Show medium types by technique Any v GE Healthcare medium type Ary Run Parameters Details Ordering Information ml MPa MPa
249. om the Instruction box Repeat this for all red instructions before saving the method A phase is marked as incorrect with a red cross This may appear if e the instrument configuration has been changed components have been removed or e the method was converted from a system with a different compo nent set up UNICORN 6 1 Method Manual 28 9817 65 AB Replace the phase with a compatible phase from the Phase Library This phase will au tomatically be adapted to the current instru ment configuration and component set tings 349 10 Troubleshooting 10 1 Troubleshooting methods Breakpoints are not correctly calculated The table below describes how to solve problems with calculation of breakpoints in the method for example in the Method Duration and Variables dialog Problem description Method breakpoints are not calcu lated All values are shown as zero Solution If the method block uses volume or column volume base the breakpoints are calculated from the pump flow rate Check that the flow rate is not zero A converted method generates unexpected results The table below describes how to solve problems when a converted method generates unexpected results Problem description Solution When running the method volumes are generally smaller or larger than expect ed 350 Ensure that the method uses Column Volume CV as base unit Verify that all parameter settings that n
250. ome AlEX_AKTA avant 25 was sucessfully converted from AKTA avant 25 MOL461 6 1 0 0 to ARTA avant 150 MOHO64 6 1 0 0 Date of conversionecalng 6 16 2010 Method Settings Column volume changed from 0 962 ml to 41 479 ml Pressure limit pre column changed from 0 5 MPa to 2 MPa Pressure limit delta column changed from 0 3 MPa to 2 MPa Innon text edited phases system related parameters such as wash and delay volumes are automatically adjusted for the new system tupe Motel This information was valid at the time of scaling converting the method Modifications made afterward are not logged here For more details see user documentation CK Note The information shown in the Method Notes will not include de tails about scaled system related parameters e g wash or delay volumes or notes concerning method instructions that may have become invalid as a result of conversion between systems with different components and instrument configurations You must verify that there are no phases with invalid instructions i e phases marked with a red cross in the new method before it can be used See note below this instruction 3 e Type any additional notes you wish to add in the text field and e click the OK button to close the Method Notes dialog 76 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 6 Scale or convert methods Step Action 10 e Choose the File Save As menu command
251. ons are used to monitor method runs and instruct the system to call a specified block or an instruction when a particular signal meets a given condition As long as the condition is not met the block is not activated Note Watch instructions available for the instrument configuration are listed in the Instruction box When is a Watch active The breakpoint when the Watch instruction is issued determines when the watch begins not when the block is activated A watch is active from the point at which it is issued until e the Watch condition is met e anew watch is set for the same monitor e a Watch off instruction is issued for the monitor or e the method ends Insert a Watch text instruction The table below describes how to insert a watch instruction in the text method Setting up additional Watch parameters is described afterwards see Insert a Watch parameters instruction on page 337 Step Action 1 At a suitable Breakpoint in the method select the instruction line immedi ately before where you want to insert the watch this decides when the watch begins UNICORN 6 1 Method Manual 28 9817 65 AB 333 9 Text edit methods 9 3 Specific instructions 9 3 3 Watch instructions Step Action 2 e expand Watch in the Instructions field e select the desired Watch type Hold until Subsequent instructions in the block will execute when the conditions have been met Watch A specified action will be
252. ontaining valid instructions are denoted by blue square symbols for a description of other icons that may appear see Description of icons and text formats in the text method on page 293 Structuring the method into blocks enables reuse of instructions in the method It also makes it possible to perform a sequence of instructions using watches see Section 9 3 3 Watch instructions on page 333 for more information about watch instructions The illustration below shows an example of a method in the Text Instructions area UNICORN 6 1 Method Manual 28 9817 65 AB 291 9 Text edit methods 9 1 Overview 9 1 2 The Text Instructions pane 292 E Main 0 00 Base C 0 100 ml Anyj Column H E 0 00 Phase Method Settings SM 0 00 Phase Equilibration 0 00 Base SameAsMain 0 00 Inlet A A1 HInlet A 0 00 Inlet E B1 Inlet B 0 00 Gradient 0 00 Percent B Equilibration 2B 0 00 base 0 00 System flow 1 000 Flow rate ml min Pre column pressure Pressure control 0 00 System wash 25 HFill system Equilibration ml Outlet valve 0 00 Watch UY parameters 0 00 mAU 1 00 mAU 0 00 Hold until UY 1 Stable signal 0 03 min Infinite base 0 00 Auto zero UY 0 00 Block Equilibrate 0 00 Base SameAsMain 5 00 E quilibration volume End_Block 0 00 Auto zero UY 0 00 End_Block E 0 00 Phase Column Wash E 0 00 Phaze Elution E 0 00 Phase Equilibration 2 E 0 00 Phase Sample Application The
253. op frac in outlet valve ji 2 00 mil Peak frac in outlet valve Stop peak frac in outlet valve Peak fractionation parameters J Next tube i 1 Next tube ESM Skip two tubes Start position Edit Variable Import Block UNICORN 6 1 Method Manual 28 9817 65 AB 311 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 3 Working with text instructions Step Action 3 To add the edited or a new instruction to the method click one of the follow ing buttons e Insert e Change e Replace Note The Insert button adds the edited instruction immediately below the instruction that was selected in the method The Change and Replace buttons are equivalent unless changes are made to the breakpoint or gradient length Both buttons re place the highlighted instruction with the newly edited instruction The differences are explained below Effects of the Change button and the Replace button on breakpoints The table below describes the difference in function between the Change and Replace buttons when changing breakpoints Function This button shifts all subsequent instructions in the block according to the change in the breakpoint Change does not affect the relative order of instructions in the method You cannot change the breakpoint of an instruction to earlier than the nearest previous breakpoint in a block Replace This button moves the select
254. or the edited model 7 Perform an analysis of the edited model to see if the new model is OK See Analyze and interpret the model basic analysis on page 174 for information about how to analyze the model 192 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 6 Use the model 5 46 Use the model Introduction When you have found a good model use the model to draw conclusions and to decide if more experiments are needed and what experiments to perform The following plots and tools can be used in the evaluation Response surface plot Generate a response surface plot to get a graphical representation of the experimen tal region From this the most interesting area can be used to plan new experiments verifying experiments and to better understand the impact of large interactions be tween factors Prediction Use the predictor to predict response values for entered factor settings Optimization Use the optimizer to enter response and factor settings criteria and obtain suitable factor setting combinations for the set response criteria Note Information about significant terms and how they influence the response values have already been found in the analysis of the model by looking at the coefficient plot interaction plot main effects plot and correlation matrix See Analyze and interpret the model basic analysis on page 174 and Sec tion 5 4 4 Analyze and evalu
255. ort Result The Extended Report opens in the Report dialog This report includes all available plots as well as the experiment setup objective and design used in the experiment Extended Report Experiment name Capto adhere User name Default Current date amp time 6 3 2009 11 06 29 AM W Europe Standard Time Introduction and Background Factors and Responses Factors The following table contains the factors names abbreviation and settings Name Abbr Units Type Use Settings Transform Prec MLR Scale PLS Scale Dead Mass Loma Quantitative Controlled 75 to 300 None Free Orthogonal Unit Variance ead conductivity Loco Quantitative Controlled 2 to 15 None Free Orthogonal Unit Variance LoadpH LopH Quantitative Controlled 6 to 8 None Free Orthogonal Unit Variance Responses The following table contains the responses names abbreviation and settings Name Abbr Units Transform MLR Scale PLS Scale Type Yield viel None None Unit Variance Regular Objective The objective determines the choice of the model and desian The following objectives are available Basic Report Extended Report 3 To view the information in the report use the vertical scrollbar UNICORN 6 1 Method Manual 28 9817 65 AB 201 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 7 Create and print reports Print a report The table below describes how to print a report Step Action 1 In the Report dialog click the Print button Result
256. ow by technique Ary w Show by access label Predefined types Global types user created Personal types user created Column types GSTrap 46 5 ml gt G5Trap FF 5 mil G5Trap HP 1 ml G5Trap HP 5 ml HiLoad 16 10 Phenyl Sepharose HP HiLoad 16 10 Q Sepharose HP HiLoad 16 10 SP Sepharose HP HiLoad 16 60 Superdex 30 pg HiLoad 16 60 Superdex 200 pg Hiload 16 60 Superdes 75 pg Result The parameters for the selected column type are displayed in the Column Type Parameters tab to the right Name GSTrap FF 1 ml Predefined Run Parameters Details Parameters Value Parameters Value Unit Technique Affinity Hardware diameter 0 7 cm Column volume 0 962 ml Bed height 2 5 cm Max pre column pressure 0 5 MPa Typical loading range 1 10 mg Max delta column pressure 0 3 MPa Total liquid volume Vt 0 86 ml Default flowrate 1 0 ml min Void volume vo ml Max flowrate 4 0 ml min Typical peak width at base 1 0 ml Default linear flowrate 155 91 cm h Average particle diameter 90 0 pm Mas linear flowrate 623 63 cm h Molecular weight range Mr Min pH value short term 3 Ordering Information Max pH value short term 13 Pamaen Value Min pH value long term 3 TR GSTrap FF 1 mi Man piiivalue long term Me Code number 17 5130 01 Medium name Glutathione Sepharose 4 Fast Flow Medium code number 17 5132 01 Hardware name HiTrap 1 ml Hardware code number UNICORN 6 1 Method Manual 28 9817 65 AB 251 8 Column Handling 8 2 Han
257. ponse This information can be tested by changing the contour plot settings and updating the plot 3 To change the Contour Plot settings e Select the response for the contour plot in the Response drop down list e Select factors for the X axis and Y axis and their corresponding ranges To return to the default values click the Reset default button e Select or enter values for the Constant factors by choosing in the Set tings drop down list as shown in the diagram below If selecting CUSTOM click in the Value field and enter a value Response Yield v Constant factors Factors at the plot axes z rect Low High Factor Value Settings x asis Load Mass v 75 300 Reset default Load Conductivity 85 CENTER J Y axis Load pH v 6 8 Reset default HIGH stow M 4 Click the Update Plot button Result The Contour Plot is updated 5 When you have obtained the appropriate information to help you in the decision on how to proceed it is possible to print the Contour Plot e Click the Print button Result The Print Preview dialog opens e Click Print Result The standard Print dialog opens e Select the appropriate printer and click Print UNICORN 6 1 Method Manual 28 9817 65 AB 195 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 6 Use the model Predict response values It is possible to predict response values based on entered factor settings using the model This is useful w
258. pter 4 Scouting on page 103 Design of Experiments Chapter 5 Design of Experiments on page 116 Workflow e frequired create a new BufferPro recipe Tip Generally the predefined recipes will be sufficient e Create a method including BufferPro e Save the method 204 UNICORN 6 1 Method Manual 28 9817 65 AB 6 BufferPro 6 1 BufferPro Overview pH ranges for predefined buffers The following diagrams show the optimal pH ranges for buffers commonly used in anion and cation exchange chromatography Recipes for these buffers are predefined in UNI CORN Anion exchange chromatography pH 4 5 6 7 8 9 10 AIEX mix Se Methuylpiperazine Piperazine m Bis Tris c Bis Tris Propane aae Triethanolamine G Tris paaa Diethanolamine ee Ethanolamine es Piperazine D Cation exchange chromatography pH 2 3 4 5 6 7 8 9 10 CIEX mix A Formate E Citrate r Succinic Acid LS Acetate MES _ Phosphate MOPS HEPES Bicine UNICORN 6 1 Method Manual 28 9817 65 AB 205 6 BufferPro 6 2 Create a method using BufferPro 6 2 Create a method using BufferPro Introduction This section describes the how to use BufferPro recipes in a method For details on how to edit methods see Chapter 3 Create and edit methods on page 25 Creating a BufferPro method Step Action 1 In the Method Settings phase of a method select the Use BufferPro auto matic buffer preparation option Use manual
259. ptimization designs Full Factorial The Full Factorial design uses all corner point experiments This design is often suggested when performing Screening Information about which factors are important main effects and more information about factor interaction effects are obtained 122 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 1 Introduction to Design of Experiments Design type Description Optimization designs For optimization studies and especially if curvatures are detected the Full Factorial design can be extended with additional experiments out side the box called star point experiments The design box illustrates the experimental space the low and high values of the different factors and experiments outside the box that is star points enhancing the detection capability for curvatures The default star point distance CCC design see below can be edited in UNICORN This design results in a higher number of experiments but more infor mation can be obtained It may be suggested when performing Opti mization but often not as the first choice because a higher number of experiments must be performed Information about which factors are important main effects informa tion about factor interaction effects and curvature are obtained See also Model on page 125 for information Designs supported by UNICORN The table below briefly describes the design types that are supported
260. r an individual column Step Action 1 Select the individual column for which to set Notification Limits in the Columns list in the Column Logbook tab See Section 8 3 3 Find an individual column on page 266 for information about how to find and select a column 2 In the Column Logbook tab click Notification Limits Result The Notification Limits dialog for the selected column opens Notification Limits 17 0686 01 20090119 2009 01 0001 Medium expiration date C Number of runs since last CIP C Number of runs since last Column Performance test C Plate Height cml Plates per meter M m Resolution Retention factor Asymmetry C Kav 3 Check the appropriate boxes and enter notification values When the values are reached or a value is outside the defined range a warning will be displayed that action should be taken 4 Click OK Result The settings are saved and the dialog is closed UNICORN 6 1 Method Manual 28 9817 65 AB 269 8 Column Handling 8 3 Handling individual columns 8 3 4 Edit individual columns Delete individual columns The table below describes how to delete an individual column from the database Step Action 1 Select the individual column to be deleted in the Columns list in the Column Logbook tab See Section 8 3 3 Find an individual column on page 266 for in formation about how to find and select a column To select several columns in the Columns
261. r more information about method structure definitions and concepts of methods in UNICORN 6 1 There are two different ways of creating and editing methods in UNICORN 6 1 e Creating and editing methods using phases and the phase properties in the Phase Properties pane the recommended workflow described in this chapter or e Creating methods from scratch by text editing methods creating and editing text instructions one by one Main steps when creating a new method The main steps when creating a method are 1 Create open a method e Create a Predefined method including a set of phases that may be edited or e Create a new method from scratch Empty method containing only the Method Settings phase or e Open an existing method that can be edited and saved with a new name or overwritten 2 Build edit the Method Outline and or edit the Phase Properties for the appropriate phases e Predefined methods use as they are or edit the Method Outline and or Phase Properties e Empty methods add phases to the method i e build the Method Outline and edit Phase Properties for the phases as appropriate e Opened methods edit the Method Outline and or Phase Properties 3 Save the method 26 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 1 Working with methods Overview Main steps when editing a method The main steps when editing a method are 1 Open the method to be edited 2 Edit the Metho
262. re store the Phase Properties options and settings Note that if the text edited settings also involve subsequent phases and the general Method Settings all these phases are changed as well and you must restore them all individually 347 10 Troubleshooting 10 1 Troubleshooting methods Options are not available in the phase properties The table below describes what to do if the standard settings available in the Phase Properties for a phase are not suitable for your specific application needs Problem description Solution Options that you need are not avail Add a User Defined phase to the method able for selection or editing in the and edit the properties in the Text Instruc Phase Properties tions tab or Text edit the phase where the option is required There are red instructions in a method Red instructions instructions with a red dot in a method are syntax errors and may have several causes A phase containing syntax errors is marked in the method outline with a red cross The table below describes some solutions to syntax error problems Problem description Solution The method instructions do not corre Check your system components under spond to the components you have System Properties in the Administration chosen for your system module and that the correct instrument configuration is selected Syntax errors are not corrected by Close and reopen the method changing the component confi
263. recipe 6 3 3 Delete a BufferPro recipe Introduction The following table describes the steps needed to delete a BufferPro recipe Note Predefined recipes shown as PD in the Type column can not be deleted Delete a recipe Step Action 1 In the Method Editor select Tools BufferPro Recipes Evaluation Procedures Scouting tel Design of Experiments Column Handling Ctrl L TA System Control vg Evaluation Options Change Passwords 214 UNICORN 6 1 Method Manual 28 9817 65 AB Step Action 6 BufferPro 6 3 Create and edit BufferPro recipes 6 3 3 Delete a BufferPro recipe 2 In the BufferPro Recipes dialog select the recipe to be deleted BufferPro Recipes Recipes Show All Recipe name Acetate with HCI 0 1M NaCl AIEX mix 0 1M NaCl Bicine 0 1M NaCl Bicine High Cone Bicine Low Conc Bis Tris 0 1M NaCl Bis Tris Propane 0 1M NaCl Carbonate 0 1M NaCl Carbonate with HCI 0 1M NaCl CIEX mix 0 1M NaCl Achievable ranges with recipe pH Bicine 0 0600 0 0000 o 100 7 Stock Solutions Inlet Stock solutions Concentration M Substance Q1 Buffer substances 0 4000 Bicine Q2 Acid or Base 0 4000 NaOH Q3 water Q4 salt 4 0000 Nacl Description m re Let Cherone Dette JIC Emot C imeot J _ Print J Explore Proportions Note The available recipes may be filtered by type All Predefined Global or Personal by using the
264. rent statistical calculations for each re sponse measuring how good the model is It is the contribution of all values that together indicate if the model is good A good model has high values for all parameters as seen in the plot above The table below gives a descrip tion of the parameters UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 3 Analyze and evaluate the model basic analysis Step Action Coefficient value Description for R describes how well the model fits the current data It can vary between 0 and 1 where 1 equals a perfect model and 0 corresponds to no model at all A high R value is necessary for a good model but not sufficient on its own A value of 0 75 indicates a rough but stable and useful model Note R2 Adj is the fraction of variations in the response data that is explained by the model adjusted for degrees of freedom R does not take into account degrees of freedom Q describes how well the model will predict new data It can vary between and 1 The higher Q value the better indicator of how well the model will predict new data 0230 5 is good and 0230 9 is excellent Q is a better indicator of the usefulness of the model than R Note R should not exceed Q by more than 0 2 0 3 for a good model Model Validity Model validity is only available if replicated exper iments have been performed A
265. ribed in Section 8 3 2 Register a new individual column on page 262 and e click Continue Result The second New Column dialog opens UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 5 Intelligent Packing of AxiChrom columns Step Action 3 In the second New Column dialog Enter an Alias optional Tip Alias can be used for easy identification of an individual col umn Select Technique and the AxiChrom Column type you created before Check the Use medium batch ID and type in the batch number of the medium in the column Check the Set medium expiration date and select expiration date for the medium to get a notification in UNICORN when this date is reached Note The expiration date cannot be set or changed after a column has been registered 4 Enter notes for the column by clicking the Notes button and typing infor mation in the Notes dialog that opens 5 Click OK Result The entered information is saved and the registered column is dis played in the Column Handling dialog Note Since the packing has not been performed at this point the bed height for the column will be a target bed height based on the selected AxiChrom col umn type This value is adjusted after the column performance tests are evaluated and the Column Logbook is updated Prepare an Intelligent Packing method Use the predefined method Intelligent Packing to prepare a method for packing the AxiChrom column
266. riment at three different temperatures 4 C 10 C and 25 C Qualitative Qualitative factors are discrete discontinuous process parameters or categorical data e g column type and type of salt used 142 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 2 Add responses and factors to an experimental design Step Action 4 Enter settings for the selected factor e Quantitative factors Enter a Low value and a High value for the factor The center point is automatically calculated Settings Center point e Quantitative multilevel factors Enter the discrete values for the factor in the different rows Toadd more rows click the Add button Acenter point is automatically selected To select another center point choose the appropriate one in the Center drop down list Settings 1 4 2 6 3 18 4 10 5 Add e Qualitative factors Select or type in the parameters in the different rows To add more rows click the Add button Acenter point is automatically selected To select another center point choose the appropriate one in the Center drop down list Settings 1 HiLoad 1610 Q Sepharose w 2 HiFrep O FF 1610 w 3 Hi creen Capto O w 4 HiScreen G FF w 5 i w Add Center HiPrep Q FF 1610 w UNICORN 6 1 Method Manual 28 9817 65 AB 143 5 Design of Experiments 5 2 Create an experimental design 5 2 2 Add responses and factors to an
267. rks differ from Messages in that they are inserted into the chromatogram at set points as well as into the logbook during a method run Example of a Set mark The illustration below shows an example where Set marks are used to highlight the start and end of fractionation in a method 0 00 Set mark Fractionation starts E 0 00 Block StartFlowthroughFractionation l E 0 00 Block Inject 0 00 Injection valve Manual load l E 0 00 Block StopFlowthroughFractionation 0 00 Base SameAsMain 0 00 Stop frac in outlet valve i 0 00 End_Block 0 00 End_Block 344 UNICORN 6 1 Method Manual 28 9817 65 AB Insert a Set mark 9 Text edit methods 9 3 Specific instructions 9 3 5 Messages and Set marks Set marks are inserted from the Instructions box The table below describes how to do this Step Action 1 Select Other Set mark in the Instructions field Instruction Box Breakpoint Instructions O oo 2 ee End FT h e e Var ga Fractionation stops Loop End Loop Message New chromatogram Pause Read Snapshot p Comment v 2 Type the message in the Mark text field 3 Click the Insert button Result A new line with the Set mark is added to the text method UNICORN 6 1 Method Manual 28 9817 65 AB 345 10 Troubleshooting 10 Troubleshooting Introduction This chapter describes different problems which may arise when creating methods in UNICORN and how to solve the problems It also descri
268. ruc tions pane and settings for blocks and instructions are updated accordingly The illustration below shows the phase properties settings and the text instructions for the Method Settings phase UNICORN 6 1 Method Manual 28 9817 65 AB 21 2 The UNICORN Method Editor 2 2 Methods in UNICORN 6 1 Phos Properties Test Instructor Phas Properti Tast pautna cas a a paii A koth Dein E Haa es 00 Base CV 0100 fel Ang BC m emi Emer Fest fame amp Lacan 1 00 Bar Sammtaklem Thy ahai Airy DO Ala pre cohen permas nabis 70 Mire cobaan pernas beet MPa O00 Gara DDD Alem doka tohen porman Enabied 20 00H cha tohar pornn baa MPa 0 HFa oimn bpa Ary DDD Whavederegiiy MOAL Grom CUE fem CTA rm DOD Hee miwiti UY 0 BUY meore Geer oei Eakin raima Drin mi 0 00 Inection valve Manual jani Parnas brat peecciumn mid Hy oe Soy DOO Datel walker Duw aia lira ieir LM Cohen peice Ep palo paiia Doen iss Peecnee imi deb rcoimn m Papi oy babs Pease iri ov 3 D00 p i wahis lire ial cal a iaa i lee a FT Ute gee ari 5 LOD Alem am rormnmn Erato for pea n n miel yan AL Ense Be ri alee on onde yale H pashe z Flow Fiano Un niian MOD indst Ac AT Pde A DOG inia E 0 iiede 8 imnpnisn Hpprmi baal PEEP y 000 Spite ioe i DOJ lee aie mlin Pe cohen pore Eine oara i 1 00 Emi Miri Wing 0 T a BO fha Uqukination r B00 Pham Sample Appia Tiss uate en G PL ai Wi 0 m iE Phare Colmes Wash
269. run directly after packing or obtaining a new column regularly during the lifetime of the column and when sepa ration performance is seen to deteriorate By regularly monitoring the performance of an individual column UNICORN can generate appropriate warnings when a cleaning procedure needs to be applied or even when the column lifetime is approaching its end For a description on how to set such notification limits see Set notification limits for an individual column on page 269 This section describes the workflow to run a Column Performance Test and how to generate a performance report for a specific column Column performance test 2 6 The following table describes the workflow for generating and analyzing a Column Per formance Test result Step Action 1 Create a Column Performance Test method or a method containing a Column performance test phase For details how to create methods see Chapter 3 Create and edit methods on page 25 Note The option Enable logging of Column Performance Test should be automatically selected in the Phase Properties for the Method Settings phase when this method is created This can be dese lected if logging of the performance test is not desired but it should normally be kept selected Column Logbook Enable logging of Column Performance Test C CIP UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 4 Column performance Step Action 2 Run the method For de
270. rv 193 547 Crea and pni 1010 gc erai E EE ER 200 UNICORN 6 1 Method Manual 28 9817 65 AB a Table of Contents O Jill gg we mennecee reer teenteteataee mr tartan eptrenereeser emer Ser later erste A rr erere eet te 203 Gl BuferPro OVS OW siiis es easlesccapiessnepetirstelncanaereasenatiideintedseeerenmudieeiuiieaes 204 6 2 Create a method using BufferPro isacscctecistnccsttneccstinzescesseesSaassecnsesassbeserescrbusbesentaentnaeetecrieiee 206 6 3 Create and edit BufferPro recipes we eceecsesssesssessseesseesssesssecssecssscssscssucsssesssessuecssecssecssecsseceseesseeen 207 6 3 1 Create and edit a BufferPro reCipe ais cars ahaha ttt cskcehe dees ctbeccosseteccestevacerantnt ite SSoieasateetes 208 6 3 2 Rename a BufferPro recipe eases caps ecceccttststbarstocesscsediuattussestencoansseticeeevtsilneanstitecenis 212 63 5 Delete g BunerPro recipe oeresarteniniir iranan E EE 214 64 Punca IT recipe eiaa E NE e 216 6 5 Calculate buffer composition using BufferPro ssesrirrsrrsrsssssssssssssssssssssssssssrssss 219 6 6 Export and import BufferPro recipes ssssssssssssssssssssssrsssrssiriiiisrrirrrerisrrrsresrsnssssssnssnnsnnnnssnnssnans 222 67 Preoefined BUTTERPFO TeCIDES spessore resrreeEn ian EEEE EEEE 226 T Meinod QGUCUES oosa ETT EEEE 229 7 1 Method queues OVerviEW sses iE aaaea oseaan Nias 230 Ll Crge a 0 9111 10 100 U 6 sien EAE 231 To Fota menodGuUCUE seii 255 6 COT FMI seese Enn E 241 odl 2 0 2 LV ae centr
271. ry select the column position for the hydraulic chamber only column position A is used Tip Click the Column Connection button to view information about the connections including an illustration UNICORN 6 1 Method Manual 28 9817 65 AB Step 8 Column Handling 8 5 Intelligent Packing of AxiChrom columns Action 10 14 Enter the slurry start concentration to generate a slurry recipe which is shown in a summary in the Start Notes at the start of the method run You can view this recipe by clicking the Slurry Recipe button Note The accuracy of the slurry preparation will affect the packed bed height Note This function is not available when Custom packing settings is selected Verify the settings in the Equilibration phase Verify the settings in the downflow Column Performance Test phase Verify the settings in the upflow Column Performance Test phase Save the method Run the method and evaluate the packing Once the Intelligent Packing method is ready you can proceed to perform the actual packing of the individual column you have registered Refer to the AxiChrom operating instruction for instructions how to prepare the column connect it to the AKTA avant 150 system and perform the packing run The Intelligent Packing method includes two Column Performance Test phases evalu ating both the column upflow and downflow performance Evaluate the results from these test as described in the UNICORN 6 1 Ev
272. s 3 3 Edit methods and phases 3 3 1 Edit phase properties Step Action 3 To edit the properties for the Method Settings phase click Method Settings in the Method Outline Method Settings technique Anion Exchange ssa w up HiLoad 16 10 Q Sepharose HP E Enable ai sensor lam inet Fy ample inlet Column Logbook UNICORN 6 1 Method Manual 28 9817 65 AB 39 3 Create and edit methods 3 3 Edit methods and phases 3 3 1 Edit phase properties Step Action 4 Edit the settings for the Method Settings phase in the Phase Properties pane as appropriate If changing Column type UNICORN will automatically calculate correct settings for volume flow rate and pressure limits Note Settings in this phase will affect the whole method Note Allowed parameter ranges are shown in parenthesis beside the text boxes Result The method is updated with the new settings 40 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 3 Edit methods and phases 3 3 1 Edit phase properties Step Action 5 Select a phase in the method to be edited for example Equilibration Result The properties for the selected phase are displayed in the Phase Properties pane UNICORN 6 1 Method Manual 28 9817 65 AB 41 3 Create and edit methods 3 3 Edit methods and phases 3 3 1 Edit phase properties Step Action 6 e Edit the settings as appropriate Note If there are for example two predefined Equilibratio
273. s 3 6 Scale or convert methods Step Action 5 Click the OK button Result The method is converted as an untitled copy UNTITLED converted The Method Notes dialog opens showing basic information about the con version Method Notes MOL461 6 1 0 0 to ARTA avant 150 MOHOB4 6 1 0 0 Date of conversionscaling 6 16 2010 In non text edited phases system related parameters such as wash and delay volumes are automatically adjusted for the new system type Motel This information was valid at the time of scaling converting the method Modifications made afterwards are not logged here For more details see user documentation Note The information shown in the Method Notes will not include de tails about scaled system related parameters e g wash or delay volumes or notes concerning method instructions that may have become invalid as a result of conversion between systems with different components and instrument configurations You must verify that there are no phases with invalid instructions i e phases marked with a red cross in the new method before it can be used See note below this instruction 6 e Type any additional notes you wish to add in the text field and e click the OK button to close the Method Notes dialog UNICORN 6 1 Method Manual 28 9817 65 AB 71 3 Create and edit methods 3 6 Scale or convert methods Step Action e Choos
274. s on page 116 for more information e change parameter values in the Start Protocol immediately before the start of a method run without using the Method Editor allowing one method to be used for runs under a variety of conditions Each parameter defined as a variable is assigned a default value which is used if no changes are made to variable values at the start of a run For information about how to change variable values in the Start Protocol see KTA avant and UNICORN 6 1 User Manual Viewing method variables All variables in a method are listed on the Variable List tab in the Method and Duration dialog grouped according to the phase and block in which they appear For information about how to view the variables in a method see View and print the method duration time and variables on page 60 for more information If the method has been text edited the phase variables for the selected phase will be displayed in the Phase Properties pane If the Method Settings phase has been edited some additional parameters e g enabling of BufferPro and Column Logbook settings will also be displayed It is possible to edit variable names values and the other settings displayed in the Phase Properties pane UNICORN 6 1 Method Manual 28 9817 65 AB 315 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables Phaze Properbes Text tnetrochong IT Mathcad Saminga Method Gating T i T hii pha
275. s exe cuted sequentially This section describes the general principles for how to edit instruc tions Help texts for the instructions It is possible to display help texts for the instructions that can be inserted in the Instruc tion Box The table below describe how to display the help text for an instruction Step Action 1 In the Instruction Box select the appropriate instruction for which to display help text 2 Press F1 on the keyboard Result A dialog with help text for the selected instruction will be displayed Insert a new instruction The table below describes how to insert a new text instruction in the Text Instructions area Step Action 1 Select a block and display the instructions within the block 2 Select the instruction in the block after which you want to add the new in struction UNICORN 6 1 Method Manual 28 9817 65 AB 309 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 3 Working with text instructions 310 Step Action 3 Open the Instruction Box if it is hidden Do the following e Set the appropriate breakpoint in the Breakpoint box e Choose the instruction type and the instruction in the Instructions field For basic help on each instruction select the instruction and press F1 e Type values for instruction parameters in the Parameters text boxes The allowed range is shown in brackets beside the text box If a scroll bar appears at t
276. s found at the end of the result name This number is the same as in the Run column This makes it easier to locate the runs to be inserted 4 Click OK Result The runs are inserted in the experiment 5 If a run does not match any of the missing runs in the experiment an error message will be displayed Repeat from step 2 to insert the correct run Insert Result x It is not possible to insert the result For the current experiment The run number and Factor settings must match in order to insert a result Note The run to be inserted must have the appropriate factor settings Replace run results Runs can be replaced with a new run if for example the run has failed Note If a run has failed there is always a risk that experimental conditions that cannot be controlled may have affected the result e g temperature in the lab different batches of buffer preparation etc Therefore it is not always a good idea to replace a failed run with a new one Re running a centre point experiment will help in Keeping track of uncontrolled variations The table below describes how to replace a run result UNICORN 6 1 Method Manual 28 9817 65 AB 167 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 2 Generate model 168 Step Action 1 In the Experiment tab of the Design of Experiments box select the result to be replaced in the Result column in the Experiment table _ A
277. s the Delta peak value Stable signal The signal is stable within the accepted fluctuation given by the relevant Watch parameters instruction see Insert a Watch parameters instruction on page 337 for the period specified by the minutes parameter Air sensor test parameter explained below Note In order to set a valid slope value use the Differentiate function in the Eval uation module to measure the slope of the test chromatogram Actions when a Watch condition is met The selection in the Action drop down list will determine what happens when the condi tion of a Watch instruction is met The table below describes the possible actions Block name Calls the named block Note All available method blocks are listed in alpha betical order in the drop down list before the general actions which are listed below Continue Continues the method if paused or held End_block Ends the current block and return to the point from which the block was called Hold Holds the method the flow continues See Hold instruction on page 540 End_method Ends the method Next_breakpoint Indicates that the run may execute the next breakpoint 536 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 3 Specific instructions 9 3 3 Watch instructions Instruction Effect Pauses the method the flow is stopped See Pause instruc tion on page 340 Insert a Watch parameters i
278. selected using the same parameter settings as the Main block this is displayed in the Instruction Box Parameters for Base Base e Select the appropriate Base from the Base drop down list Volume the unit depends on which Instrument Configuration used Time minutes CV column volume the corresponding volume in for example ml can be defined numerically or taken from the Column Type list SameAsMain does not apply for the main block The block will inherit the base defined in the main block Result The settings in the Parameters for Base area are updated UNICORN 6 1 Method Manual 28 9817 65 AB 297 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 1 Base instruction Step Action 2 Select the appropriate Column Type in the drop down list The table below gives a short descriptions of the available options Column parameter Description Any column can be used in the block If the Column Type is set to Any and the Base is set to CV enter the column volume in the Volume field ColumnSameAs The same column as in the main block will be Main used When the Base is set to Volume but the flow still goes through the column the Column Type can be set to SameAsMain to provide information on for example pressure limits for the column Named column The named column type will be used in the block type e g HiTrapQ The volume specified in the selected column def HP
279. ses Step Action 4 In the For system field the system that was selected when the current method was set up will be displayed by default To save the phase for another system choose the appropriate system from the For system drop down list Note Only systems using the same instrument configuration and component configuration as the system that was selected when the current method was set up will be displayed in the For system field 5 e Select if the phase shall be Global available for all users or Personal for your own use only Note It is not possible to replace a predefined phase by saving an ex isting phase Result The phase is saved and is available in the Global Phases or Personal Phases panel of the Phase Library Phase Library MDL versio A X Sample Application Superooap 50 Delete a phase from the Phase Library It is possible to delete personal and global phases from the Phase Library Predefined phases cannot be deleted The table below describes how to delete a personal or global phase from the phase library UNICORN 6 1 Method Manual 28 9817 65 AB 67 3 Create and edit methods 3 5 Save methods and phases Step Action 1 Select the appropriate phase library Personal Phases or Global Phases at the bottom of the Phase Library pane Result The phases in that phase library are displayed Phase Library MDL versio A X Sample Application Supenooap 50
280. simplexes Doehlert designs are useful when performing Optimization 124 UNICORN 6 1 Method Manual 28 9817 65 AB Model 5 Design of Experiments 5 1 Introduction to Design of Experiments A model is created in the Evaluation module based on the response values measured or entered for each experiment in the DoE setup The model is a mathematical fit to all data Multiple Linear Regression MLR and can be expressed as Yh f X4 Xo soss Xal where Y is response and X is factor The model can be explained in a graphical way as shown in the illustration below In this case the model is a linear relationship The residual error between the measured data and theoretical data according to the model is minimized Response Y Measured data Model in this case a Pa linear relationship Y b b X Residual Factor X Model details A more detailed description of the model is provided by the following formula as shown in the illustration below The example is valid for three factors X4 X and X respectively As seen in the illustration above the model can be divided into four parts the Constant Term the Linear Terms main effects the Two way interaction terms and the Quadratic terms The b values are determined by the selected model The Y values are the response values that are entered in UNICORN The table below gives a brief description on how to interpret the different terms in the model
281. structions The symbol shown in the illustration below will be displayed if the instruction cannot be dropped in a specific location 0 00 Block Equilibrate O Dimi uriti E ss 5 00 H E quilibration volume End_Block UNICORN 6 1 Method Manual 28 9817 65 AB 313 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 3 Working with text instructions Delete instructions The table below describes how to delete method instructions in the Text Instructions pane Step Action 1 Select the instruction in the Text Instructions pane 2 e Right click the instruction and choose Delete or e Click the Delete button in the Instruction box or e Press the Delete key on your keyboard End_Block instruction If you delete the End_Block instruction the block will end at the last instruction in the block If a gradient is currently being formed the gradient will continue into the next block 314 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables 9 2 4 Method variables Introduction Variables are used when you want to vary parameter values in a method Variables must be defined when you want to e perform scouting and Design of Experiments DoE where different parameters are varied to find for example optimal settings for a process See Chapter 4 Scouting on page 103 and Chapter 5 Design of Experiment
282. system It covers the features and tools included in the Method Editor module of the UNICORN software with practical in structions The manual covers the following e how to create methods and phases e how to use BufferPro e howto design and optimize experiments using Design of Experiments and Scouting e how to use method queues e how to handle column types and individual columns e how to convert and scale methods created for KTA avant 25 systems to be used with AKTA avant 150 systems or conversely For advanced users an overview of how to edit methods at the level of individual instruc tions is also given Note The Method Manual does not describe the functions of every command in all panes and dialogs of the user interface Refer to the online help for infor mation about commands that are not described in this manual The online help in the Method Editor module is accessed either by clicking Help buttons in software dialogs by pressing the F1 key or selecting Help Help for Method Editor 10 UNICORN 6 1 Method Manual 28 9817 65 AB 1 Introducing the UNICORN Method Editor 1 2 About this manual Document structure Each chapter starts with a brief overview that presents the contents and the headings for the sections that the chapter contains Most sections begin with an introduction that Summarizes the content Some sections are divided into sub sections each with an overview of the contents A section is divided
283. t Restore to Default Auto Hide optional panes 18 The optional panes may either be displayed statically in the position where they open or the Auto Hide function can be selected to automatically hide display the pane when moving the mouse pointer over the pane The table below describes how to turn on the Auto Hide function and how to hide display in this example the Method Navigator pane Step Action 1 If not already displayed open the Method Navigator in the Method Editor by selecting View Method Navigator Result The Method Navigator pane is displayed Method Navigator Open amp Methods Method Qe gt Folder name System Last A j uMIcORN Database H Anita Wa Anna 12 1 To turn on the Auto Hide function click the vertical pin symbol in the top righthand corner Method Navigator Open i Methods Method Qu Folder name System Last UNICORN Database Anita 1 714 Anna 12 1 Result The pin symbol is rotated to horizontal position and a tab named Method Navigator is displayed to the left Method MNAE T Open re Methods Method Qi ae EE EE E SS 2 Folder name System Last mar IE UNICORN Database gu gl Anita Ww sal GajAnna 12 1 20 UNICORN 6 1 Method Manual 28 9817 65 AB 2 The UNICORN Method Editor 2 1 The Method Editor Step Action 3 Click outside the Method Navigator Result The Method Navi
284. t The Save As dialog opens Save As Column type name UNICORN 6 1 Method Manual 28 9817 65 AB 281 8 Column Handling 8 5 Intelligent Packing of AxiChrom columns Step Action 7 Type in a Column type name and click Save Tip To simplify identification it is recommended to choose a name for the column type comprised of hardware name media name and bed height However the Method Editor will recognize the column from the selected hardware irrespective of the name Result The AxiChrom column type is saved in the database and displayed in the Column types list Create an individual AxiChrom column 282 Once a column type consisting of the AxiChrom hardware and selected media is created you can proceed to register an individual column Note Step It is essential to create an individual column before the column packing since it is impossible to afterwards manually register the results from the column performance tests in the Column Logbook Action 1 Select the Column Logbook tab and then click New Result The first New Column dialog opens New Column Add a new column by entering a Column ID either manually or with a barcode scanner Code lot Exp l D camo O O O _cex_ c Add column by manually entering UniT ag lot and ID UniT ag has fited values for Code and exp Continue Cancel e Register the column either by scanning a UniTag or manually as de sc
285. t 79 3 8 Predefined methods and phases ou eecescsessssssssessseesseesssesssecssecsscssscssecesscssecssussssecssecssecsseceseceseesses 81 39 Fraction Colectoare EE 89 340 Signa metnods SIO CUOAICGNY sesira er AA oE 92 311 Importanad export Metos emissaris iseiti enn n 94 E N aa E E 103 4 1 DE E E O EA A 104 42 Set poand editascouting Schene sesrsssansarsrsroiseiiedn aei 106 Design of Experiments ccsccssscesscessscsssesscessesssccsssesssesseesseessssesssessseesseeseees 116 5 1 Introduction to Design of EXperiMents cceccesssesssessssessssessecssecssecssecsseesssesssecssecssecssecssecsseceseesses 117 Da Ceedeceon SRS ASIC SS UT ssn A 128 52d Sa uUparexpenmenai design srci da E E i 129 5 2 2 Add responses and factors to an experimental design irs 138 5 2 5 Change design and design settings in a Design of Experiments setup 145 5 2 4 Divide the DoE runs into several scouting TUS s 149 53 Runascoutingcredted with DOE seisine et 155 54 Eval dtionof Design of Experiments xiccecccscincescccesessssnsstecsucstedactsteesnsnoncststindwoiancseutabsnectatindn 157 SAL WOKOW ripiru iii Ea EA ESAE AEE Ai E EEEE EAEE EE 158 De GCI 0G tasers E 160 5 4 3 Analyze and evaluate the model basic analysis u 170 5 4 4 Analyze and evaluate the model extended analysis ceccccssesssesssessssecsseesseessecssessseesseeeses 182 io ho Eoee mode sueri ee A E A TT Torr 190 SAD Uee Model ariran e a E AA E TONT Ce
286. t Variable Vatiable Value Nange v EQUILIBRATION inlet Al Sample Appication ae d 81 v EQUIUDAATION Percent D Equitestion 48 ow 10 00 00 00 EQUILIBRATION Flow rate riirn 1 000 0 000 25 000 Pressure contol Pre column pretoute wv Column Wash CQUILIDRATION Fil aystarn E qu abon Iri 15 10 999 Equitxate Equitation voksee CV 50 0 00 33335353 0 v Elution v Column Wash v Equilibration C Show detas Ect Variatie lt gt Result The Edit Variable dialog opens displaying all variables if opened from the Text Instructions pane or the phase variables if opened from the Phase Properties tab UNICORN 6 1 Method Manual 28 9817 65 AB 321 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 4 Method variables 322 Action Edit Variable Ei sensor alarm on inlet valve A Air sensor alarm on inlet valve B Air senor alarm on sample inlet valve Column Column postion Column wash volume Column wash yolume_1 Delta column pressure limit Empty loop with Equilbration volume Equilibration volurrie_1 Fill system E quilibration Fill yster E quilibration _1 Flow rate Flow restrictor Frac start position E lution Frac tube type Elution Frac volume Elution New name Air sensor alarm on inlet valve 4 Set visible in details only Select the variable to be edited if not already selected Do one or several of the following as appropriate
287. t a BufferPro recipe Step Action 5 Select a Buffer substance from the drop down list Edit BufferPro Recipe Bicine 0 1M NaCl Achievable ranges with recipe Stock Solutions Inlet Stock solutions Concentration M Substance ph x Q1 Buffer substances 0 2000 Bicine 0 0500 M Q2 Acid or Base 0 2000 ane Q3 water Q4 Salt 4 0000 Nacl 0 0000 1 0000 M 0 100 B Description O Goba Personal Note Up to five buffer substances may be included in the recipe for the Q1 inlet If more than one substance is used the concentra tion of the final buffer in BufferPro will be fixed and is then de pendent on the concentration of the stock solutions Note To choose a conjugate acid base pair as the buffer select the base form as Buffer substance apart from phosphate where the acidic or basic form may be chosen The conjugate acid or base will appear as an option in the Acid or Base drop down list 4 Select the concentration and edit the value Stock Solutions Inlet Stock solutions Q1 Buffer substances 02 acid or Base Q3 Water Q4 Salt 210 Concentration M Substance Bicine 0 1000 NaCH 4 0000 Nacl UNICORN 6 1 Method Manual 28 9817 65 AB 6 BufferPro 6 3 Create and edit BufferPro recipes 6 3 1 Create and edit a BufferPro recipe Step Action 5 Choose a titrant Acid or Base from the drop down list and if required edit its Concentration Note The titrant and the stock solution should generally hav
288. tails on running a method see AKTA avant and UNICORN 6 1 User Manual Suitable samples that can be used to monitor the column performance are for example 1 acetone measuring the absorbance at 280 nm or 2 0M NaCl and eluting with 0 5 M NaCl Note A sample volume between 0 5 and 3 of the column volume and a flow rate between 15 and 30 cm h is recommended The calculated number of plates and the asymmetry factor will in part depend on the selected flow rate To ensure that test re sults are comparable always use the same flow rate for the tests 3 Evaluate the Column Performance Test see UNICORN 6 1 Evaluation Manual 1 The individual column must be selected when the method run is started in order to register the results from the column performance test in the column logbook The result cannot be logged for the individual column at a later time The individual AxiChrom column must also be selected when performing an Intelligent Packing method run since this method includes column performance tests which should be logged Create a performance report A column performance report can be created before using an individual column to ensure that it is in good condition for use The performance report contains the following infor mation e Run and performance parameters e Notes e Performance graphs optional e Run history The table below describes how to generate a column performance report Step Action 1 Select th
289. tal design 5 2 3 Change design and design settings in a Design of Experiments setup 5 2 3 Change design and design settings in a Design of Experi ments setup Change design in a Design of Experiments setup The design suggested by UNICORN can be changed to another design in the setup of the Design of Experiments The settings for a selected design can also be edited The table below describes how to change the default design and design settings i e the number of center points and replicates in a Design of Experiments setup Step Action 1 In the Factors amp Design tab of the Design of Experiments dialog the sug gested design is displayed in Design drop down list Design selection Objective Screening b Design Full factorial 2 levels 1st choice Total number of runs including center points 7 UNICORN 6 1 Method Manual 28 9817 65 AB 145 5 Design of Experiments 5 2 Create an experimental design 5 2 3 Change design and design settings in a Design of Experiments setup Step Action 2 Click the Advanced button to e change to another design than the 1st or 2nd choice design available in the Design drop down list continue with step 3 and or e edit the settings for the currently selected design in the Design table continue with step 4 Result The Change Design dialog opens displaying the designs that may be used for the current experimental setup and selected objective Ch
290. tem lacking the extra inlet A valve It will still be possible to save the method but the phases in the method will be marked with an error symbol In order to be able to subsequently run the method either the method must be text edited or the component configuration of the system changed in the Administration module Save a phase The table below describes how to save a phase to the Phase Library Step Action 1 Select the phase to be saved in the method outline Note A Method Settings phase can not be saved as a separate phase with a new name If editing properties for the Method Settings phase the changes will be saved with the method UNICORN 6 1 Method Manual 28 9817 65 AB 65 3 Create and edit methods 3 5 Save methods and phases Step Action 2 click the Save Phase button below the Method Outline pane or select Phases Save Phase Phases Insert Phase From Library Save Phase be or right click the phase and select Save Phase Rename Delete Move Up Move Down uk Copy Result The Save Phase to Phase Library dialog opens Save Phase to Phase Library 66 Phase name For system s ystems ei C Global Personal Type a Phase name or Choose a phase from the Phase name drop down list This phase will be replaced by the phase with the new settings UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit methods 3 5 Save methods and pha
291. tep Action 5 Non significant terms can be identified by the confidence limits for a coeffi cient the black error barrs crossing zero The diagram below shows the extreme example where no terms are significant 200 200 Lola Lota LopH LoMa loca LoMa LopH Lot o LopH Insignificant terms should be removed from the model one at a time before reanalysing the model 6 If the model does not look good or non significant terms are present edit the model or continue with the extended analysis before editing the model See Section 5 4 4 Analyze and evaluate the model extended analysis on page 182 and Section 5 4 5 Edit the model on page 190 for more information 7 If the model looks good and all terms are significant continue with Sec tion 5 4 6 Use the model on page 193 UNICORN 6 1 Method Manual 28 9817 65 AB 181 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 4 Analyze and evaluate the model extended analysis 5 4 4 Analyze and evaluate the model extended analysis Introduction If you want to perform further analysis of the model in order to decide how to proceed an extended report can be generated The following plots and tables are displayed in the extended report in addition to the basic analysis e Residuals versus variables plot e Residual versus run order plot e Interaction plot e Observed versus Predicted e Main effects plot e ANOVA table e Correlation matrix This
292. terms and concepts used in Design of Experiments DoE What is Design of Experiments Design of Experiments DoE is a way to systematically vary several parameters simul taneously to obtain as much information about a process with as few experiments as possible Why use DoE Maximize the amount of information using a minimum number of runs When trying to find optimal conditions for a process to obtain the best results it is usu ally not possible to perform all experiments needed due to time or cost using a random experiment approach The number of runs to be performed needs to be minimized at the same time as the information from the runs are maximized DoE facilitates this by using a systematic approach for experimental set up and statistical modelling for the results For example it could be enough to obtain 80 information about a process This level of information can be reached using a significantly lower number of experiments using DoE than using a random experiment approach as illustrated in the figure below DoE structured approach Information 8 X 8 16 52 64 128 Number of experiments UNICORN 6 1 Method Manual 28 9817 65 AB 117 5 Design of Experiments 5 1 Introduction to Design of Experiments Estimate parameter interactions In the simplest traditional approach to optimization experiments one parameter is varied while all others are fixed In this way optimal values may be found for each parameter
293. thod up or down The table below describes how to convert a method to be used with another system type and at the same time scale the method to be used with another column size Step Action 1 Open the method you want to convert and scale in the Method Editor 2 Choose the menu command File Scale or Convert Method Result The Scale or Convert Method dialog opens 3 Select the option Convert and Scale up down method 4 Choose the system to which the method should be converted in the Target System list UNICORN 6 1 Method Manual 28 9817 65 AB 73 3 Create and edit methods 3 6 Scale or convert methods 74 Step Action Click the Select Column button to select a new column type Result The Select Column Type dialog opens Select Column Type Show by technique Anion Exchange Show by access label Predefined types Global types user created Personal types user created AxiChrom 507300 20um 55 Capto O 20 cm HiLoad 16 10 O Sepharose HP HiLoad 26 10 O Sepharose HP HiPrep 16 10 4N FF high sub HiPrep DEAE FF 16 10 HiPrep O FF 16 10 HiPrep OQ L 16710 HiScale 16720 Capto DEAE HiScreen Capto adhere HiScreen Capto DEAE HiScreen Capto Q Hiscreen O FF HiScreen Q HP HiTrap AN FF high subj 1 ml Hil rap AN FF high sub 5 mil Hil rap Capto adhere 1 ml Hil rap Capto adhere 5 ml Hil rap Capto DEAE 7 ml Hil rap Capto DEAE 5 ml Hil rap Capto Q 1 ml HiT rap Capto Q 5 ml Hil rap
294. to generate a model for the DoE result Step Action 1 Responses defined in the method appear in the result in the Design of Ex periments box To add or delete responses to the experiment use the Add Response and Delete Response buttons See Add responses on page 164 and Delete re sponses on page 165 for more information 2 To enter response data e Click ina response cell for the appropriate response and experiment and type in the data the Yield column in the example shown here Result The data is entered in the cell Cgemert dean fredchon Optmenahon Replace Pend wt Rena AdsRepowe Delete Hewporces Tip Response data can be obtained from external measurements e g biological activity peak data from UNICORN e g HETP tests or resolu tion e Repeat this procedure for all experiments and responses 2 Select the runs to be included in the calculations for generating the model by checking the Included box for the appropriate runs usually all runs To insert a new run or to replace a failed run with a new run use the Replace Result and Insert Result buttons See Replace run results on page 167 and Insert new runs on page 166 for more information about inserting and replacing runs Tip Instead of replacing a failed run with a new run the run can be excluded from the model calculations by clearing the Included box in front of the appropriate run This will however often resu
295. to have a Scouting run result open at the same time even if a DoE result is open 6 Repeat this procedures for all the runs included in the DoE result UNICORN 6 1 Method Manual 28 9817 65 AB 161 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 2 Generate model Open the DoE result 162 The table below describes how to open a DoE result Step Action 1 In the Evaluation module click the Open Result Navigator icon in the Toolbar Result The Result Navigator is displayed 2 Browse for the DoE result and double click the result name DoE results are indicated by the design box icon Results Recent Runs Find TA o 5 G Results DoE Results E Folder name C Katti eS DoE results 1 Capto adhere al Capto adhere 001 xd Capto adhere O02 fo Capto adhere O03 a Capto adhere O04 al Capto adhere 005 x Capto adhere 006 xl Capto adhere 007 mi Capto adhere 008 xd Capto adhere 009 i Capto adhere 010 fa Capto adhere 011 Result The Design of Experiments box opens displaying the DoE scouting BRSSERS F Bes S12 2008 7 SPM DIAA 7 SS Pe STE Fa FP RAO fo Pe 12200 7 a SP Su 22008 7 43 02 Po Epira imar iia S122 T7 1FFH Se edi Pd Si Fh Pe Erpen r 73604 Po popopp00 poog 3 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 4 Evaluation of Design of Experiments 5 4 2 Generate model Generate model The table below describes how
296. torial 2 Full Factorial 2 levels is an orthogonal balanced design with all combinations of levels the factor levels Main effects and all interactions are clear of each other not confounded Full Factorial 2 levels designs are useful when performing Screening or Robustness Testing Full Factorial 3 Full Factorial 3 levels is a full factorial design with every factor varied at three levels levels You can estimate the full quadratic model Full Factorial 3 levels designs are useful when performing Screening Optimization or Robustness Testing They are however not the primary choice for Screening or Robustness Testing The Central Composite Circumscribed CCC design is composed of a full or fractional factorial design and star points CCC designs are useful when performing Optimization The Central Composite Face CCF design is composed of a full or fractional fac torial design and star points placed on the faces of the sides CCF designs are useful when performing Optimization Box Behnken Box Behnken is a three level Response Surface Modelling RSM design All design points except the center points are located at the center of the edges of the hy percube and are also on the surface of a sphere You can estimate the full quadratic model Box Behnken is useful when performing Optimization and you have at least three factors in your experimental plan Doehlert Doehlert is a RSM design constructed from regular
297. tors v Edit Variable Import Block 328 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 3 Specific instructions 9 3 1 Gradients and eluent concentrations Step Action 3 Edit the Breakpoint for the gradient if appropriate Note The breakpoint for a Gradient instruction defines the time or volume according to method base for the start of the gradient A gradient with a non zero duration occupies time and volume in the method and breakpoints for other instructions may be set to occur before the gradient is completed The instruction is simply carried out at the requested breakpoint while the gradient is forming 4 Click the Insert button Result The new Gradient instruction is inserted in the method in the Text Instructions area Instruction after a gradient Any instruction that you want to insert after a gradient should be placed after the com bined breakpoint and gradient length since gradients function over time Instructions that affect gradients The table below describes the instructions that affect the gradient Instruction Effect Gradient Anew gradient will start at the requested breakpoint The remaining duration of any previous gradient is ignored Flow The eluent flow rate will change at the requested break point If the current base is volume or column volume the duration of the gradient will be changed If the method base is time the volume of the gradient will be change
298. uction 296 Change 312 Change and Replace differ ences 312 Delete instructions 314 Delta peak settings 338 Edit an instruction 311 End_Block 300 Gradient breakpoints 329 Gradient instructions 329 Help 309 Hold instruction 340 Hold until 341 Icons and text formats 293 Linear gradient 327 Message 343 Message instructions 343 Move an instruction 313 Pause instruction 340 Replace 312 Set mark 344 Step gradient 327 Syntax errors in 348 Text editing a method 288 Text instructions pane 290 Text method symbols 293 Watch instructions 333 Watch parameters 337 Watch parameter test op tions 335 Time Base instruction 296 Tools In Method Editor 14 Troubleshooting Methods 347 U Upscale Scale a method for a larger column type 73 V Variables Breakpoints or gradient lengths 319 Defining 318 Deleting 320 Edit values 324 Identification in text instruc tions 316 317 Method variables 315 Renaming 320 Scouting use in 111 Variable names 317 Viewing 315 Volume Base instruction 296 W Warnings Compared to Alarms 330 Watch instructions Actions 336 Air sensors 339 Delta peak settings 338 Description 333 Insert instruction 333 Parameter test options 335 Signals available 335 Watch parameters Delta peak settings 338 Watch parameters instructions Insert text instruction 337 UNICORN 6 1 Method Manual 28 9817 65 AB For local office contact information
299. ue short term 3 Max pH value short term 13 Ordering Information Min pH value long term 4 Parameters Value Max pH value long term 12 Name Code number Medium name Blue Sepharose 6 Fast Flow Ese Medium code number 17 0948 01 Required information Hardware name C 10 40 Hardware code number 19 5003 01 4 Enter the remaining parameter values for the new column type in the Run Parameters Details and Ordering Information tabs Fields marked with must be filled in Values in the gray fields are calculated and automatically filled in based on entered values for the corresponding parameters 5 Select whether the the new column type should be Global available for all users or Personal only available for the current user UNICORN 6 1 Method Manual 28 9817 65 AB 249 8 Column Handling 8 2 Handling column types Step Action 6 Click Save As to save the column type Result The Save As dialog opens Save As Column type name ii Type in a Column type name and click Save Result The column type is saved in the database and displayed in the Column types list 250 UNICORN 6 1 Method Manual 28 9817 65 AB 8 Column Handling 8 2 Handling column types Edit parameters for a column type The table below describes how to edit parameters for a column type Step Action 1 Select the appropriate column type for which to edit parameters in the Col umn types list Select Column Type Sh
300. umn type by a flow of hydraulic Method Settings Packing liquid that pushes the adaptor down The user initiates the start of compression at Intelligent Packing the exact point when the adapter reaches v the consolidated bed surface The adapter Column Performance Test compresses the bed according to the v packing factor or target bed height as se elua Doctors Test lected Two Column Performance Test up flow downflow phases are automatically performed after the AxiChrom column has been packed Only available for AKTA avant 150 The system is filled with cleaning solution Select for example inlets outlets and col umn positions to be cleaned Three System CIP phases are included in the method to System CIP facilitate the use of three different cleaning solution Additional System CIP phases can siio be added from the Phase Library if desired v System CIP System Thesystem is filled with preparation solu Prepara tion Select for example inlets outlets and tion column positions to be prepared Two Sys tem Preparation phases are included in the System Preparation method Additional System Preparation phases can be added from the Phase Li System Preparation brary if desired 86 UNICORN 6 1 Method Manual 28 9817 65 AB Predefined phases 3 Create and edit methods 3 8 Predefined methods and phases The table below describes the predefined phases Phase Name Method Settings Description T
301. umns list in the Column Logbook tab See Section 8 3 3 Find an individual column on page 266 for information about how to find and select a column In the Column Logbook tab click Column Histor Result The Column History dialog for the selected column opens Column History 17 5167 01 30324242 2010 10 0001 Project 4 Mono Q 1 Run history 4 17 2009 2 32 PM 02 00 AIEX purification standard method 001 DefaultHome Project 4 AlEX purification standard method 001 4 17 2009 2 39 PM 02 00 AIEX purification scouting col pos 001 DefaultHome Project 4 Test 3 different columns AIEX purification scouting col pos 001 4 17 2009 2 40 PM 02 00 AIEX purification scouting col pos 005 DefaultHome Project 4 Test 3 different columns AIEX purification scouting col pos 005 4 17 2009 2 40 PM 02 00 AIEX purification scouting col pos 006 DefaultHome Project 4 Test 3 different columns AIEX purification scouting col pos 006 4 17 2009 2 47 PM 02 00 Column CIP Mono Q 1 001 001 DefaultHome Project 4 Column CIP Mono Q 1 001 001 CIP 4 17 2009 2 52 PM 02 00 Column Performance Test AIEX 001 DefaultHome Project 4 Column Performance Test AIEX 001 Column Perfo 4 17 2009 2 54 PM 02 00 AIEX purification standard method 002 DefaultHome Project 4 AIEX purification standard method 002 The runs performed using the column are listed The date result name and location and any remarks for the run are displayed Click Close to close the dialog 274 U
302. uns including center points Advanced 140 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 2 Create an experimental design 5 2 2 Add responses and factors to an experimental design Step Action 2 To define factors click Add Result The Add Factor dialog opens Add Factor Bed Height eo s lt L BeHe UNICORN 6 1 Method Manual 28 9817 65 AB 141 5 Design of Experiments 5 2 Create an experimental design 5 2 2 Add responses and factors to an experimental design Step Action 3 To add a predefined factor e Select the factor to be added in the Predefined drop down list Result Abbreviation and the correct Type radio button Is selected e If applicable type in the Unit for the factor To add a user defined factor e Select User defined and type in your own factor Result Abbreviation is automatically filled in e lf applicable type in the Unit for the factor e Select what kind of factor it is by selecting the appropriate Type radio button The table below describes the different types of factors Type of factor Description Quantitative Quantitative factors are process parameters that can be measured and have values on a continuous scale e g flow rate and pH values Quantitative multi To specify more than two levels for a factor level select the Quantitative multilevel type For ex ample if your are performing an expe
303. version and scaling is successful e The original method should use column volume CV as base e All parameters that will require scaling should be defined as variables e f linear flow is to be maintained in the scaled method it must have been applied in the original method as well e Scaling of the column type is not possible if the Any column type was selected in the original method e Text edited phases will not be automatically updated during the conversion Convert a method to another system type The table below describes how to convert a method to be used with another system type Step Action 1 Open the method you want to convert in the Method Editor UNICORN 6 1 Method Manual 28 9817 65 AB 69 3 Create and edit methods 3 6 Scale or convert methods Step Action 2 Choose the menu command File Scale or Convert Method Result The Scale or Convert Method dialog opens Scale or Convert Method Method AEs ARTA avant 25 Convert method to system Convert and Scale up down method Target System Column Type 3 Select the option Convert method to system 4 Choose the system to which the method should be converted in the Target System list The list will show all available active systems Deactivated systems are not shown Tip You can convert methods that originally were created for systems that now are deactivated 70 UNICORN 6 1 Method Manual 28 9817 65 AB 3 Create and edit method
304. visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden www gelifesciences com unicorn imagination at work GE imagination at work and GE monogram are trademarks of General Electric Company AKTA AxiChrom Drop Design HiTrap Mono Q and UNICORN are trademarks of GE Healthcare companies All third party trademarks are the property of their respective owners 2009 2011 General Electric Company All rights reserved First published Sep 2009 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare repre sentative for the most current information UNICORN Any use of this software is subject to GE Healthcare Standard Software End User License Agreement for Life Sciences Software Products GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham 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 Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 28 9817 65 AB 04 2011
305. xchange chromatogra phy but can also be used when running other chromato graphic techniques See Chapter 6 BufferPro on page 203 for more information Column Handling Column Handling enables handling of column types and individual columns See Chapter 8 Column Handling on page 241 for more in formation Illustration of the Method Editor The basic Method Editor interface consists of two panes the Method outline and the Phase Properties Text Instructions pane By default the Toolbar Phase Library pane and Gradient pane are also displayed in the Method Editor The display of these panes is however optional Two more panes may be displayed in the Method Editor the Method Navigator and Flow Scheme The illustration below shows the Method Editor with all the optional panes displayed UNICORN 6 1 Method Manual 28 9817 65 AB 15 2 The UNICORN Method Editor 2 1 The Method Editor d Editor UATITLED a EVESEN NC EEE E E E a TK Phase Livary HDL vo 9 X Pise Progen bere ant bretructiones a e nee sari Column paietu Besut Hame G Takami Performa Tend Show by tachrar Afira Gas a Equilibration x pres __ Stet Prot ans Courir ype Ary Mathod N i Bane Cotas Preparer x Grer 1 Column voume 0 100 mi Eiin Coia ash Sample Applicaton Prerpure br precon 20 00 MPa Ng 30 009 m n ao m Presse im delacckmn Z000 MPa O2 2100 eee Ej Center a 7 if Method B
306. y be more suitable to use a time or volume base for individual blocks The table follows lists few examples when different bases should be used the method should be adjusted according to the selected column In this way you do not need to edit the method when changing column size the same volume should be used regardless of which column is used a defined time is required and the volume used Is not critical or if the flow rate is zero 296 UNICORN 6 1 Method Manual 28 9817 65 AB 9 Text edit methods 9 2 Working with methods in the Text Instructions pane 9 2 1 Base instruction Edit settings for a base instruction The following table describes how to edit settings for a base instruction Step Action 1 Select the base instruction for which to edit the settings in the Text Instruc tions area E Hain H E 0 00 Phase Method Settings 0 00 Phase Equilibration 0 00 Base SameAsMain 0 00 Inlet A A1 f Inlet A Result e The settings for the selected Base instruction are displayed in the Param eters for Base area in the Instruction Box Breakpoint Instructions Parameters for Base Other Column Type Base 0 00 cy Block Var Any v Continue Base End_Block y End Cv care Volume 0 100 999999 0 o a Loop Var K 0 100 ml End Loop Message New chromatogram o Edit Variable Import Block e Ifa Base instruction in a phase or block was
307. ystem 154 UNICORN 6 1 Method Manual 28 9817 65 AB 5 Design of Experiments 5 3 Runa scouting created with DoE 5 3 Runascouting created with DoE Introduction This section describes how to view the optimized Scouting scheme generated from DoE and how to print the method including DoE For information about how to start and monitor Scouting runs see AKTA avant and UNICORN 6 1 User Manual View the Scouting scheme generated from DoE When creating a Design of Experiments the final step is the generation of the optimized Scouting scheme The table below describes how to view the Scouting scheme gener ated from DoE Step Action 1 In the Method Editor e Click the Scouting icon re or e Select Tools Scouting Result The following dialog is displayed as a reminder of that a DoE has been created for the method If you change the Scouting scheme the DoE experimental plan is changed and the results may not be reliable for use in the DoE evaluation Method Editor e i 4 Design of Experiments has been created For this method Changes to the scouting scheme may affect the design UNICORN 6 1 Method Manual 28 9817 65 AB 155 5 Design of Experiments 5 3 Runa scouting created with DoE Step Action 2 Click OK Result The Scouting dialog opens displaying the Scouting scheme where it is possible to view the Scouting runs to be performed Scouting Scouting parameters Sample Application Direct s

UNICORN™ 6.1 - GE Healthcare Life Sciences

Contents

Download Pdf Manuals

Related Search

Related Contents

UNICORN&trade; 6.1 - GE Healthcare Life Sciences

Contents

Download Pdf Manuals

Related Search

Related Contents

UNICORN™ 6.1 - GE Healthcare Life Sciences