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ÄKTA system Training Guide FPLC

100 ul. ÄKTA system Training Guide FPLC. ÄKTA FPLC with Frac 920 + Outlet valve. ÄKTA FPLC. In-line filter. UV. Filter Wavelength detector. Injection valve (V1). 1. 7. 6. Cond. 2. Conductivity monitor. 5. 3. 4. Mixer. System pumps. pH. pH detector ( electrode optional).

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ÄKTA system Training Guide FPLC

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  1. 100 ul ÄKTA system Training Guide FPLC

  2. ÄKTAFPLCwith Frac 920 + Outlet valve

  3. ÄKTAFPLC In-line filter UV Filter Wavelength detector Injection valve (V1) 1 7 6 Cond. 2 Conductivity monitor 5 3 4 Mixer System pumps pH pH detector ( electrode optional) A B Fraction collector Frac-950

  4. ÄKTAFPLC Monitor UV Conductivity pH Mixer Filter Gate Flow Restrictor Pump A UV Flow cell Injector Pump B

  5. ÄKTAFPLC XL Reverse Flow valve V7 Column switching Valve V2 Injection valve V1 Sample Pump Outlet Valve V4 Column switching Valve V3 Sample Inlet Valve V8 Buffer A Valve V5 Buffer B Valve V6

  6. ÄKTAFPLC pump system BUFFER A BUFFER B Piston change-over valve Piston and Cylinder Piston Back-wash system Pump A Pump B Common outlet to mixer and column Pressure Monitor and joint pump manifold

  7. ÄKTAFPLC In-line filter Filter Wavelength detector UV Injection valve (V1) 1 7 6 2 Cond. Conductivity monitor 5 3 4 Mixer System pumps pH pH detector ( electrode optional) A B Fraction collector Frac-950 ÄKTAFPLC + Reverse Flow Valve Reverse flow valve (V7) 7 1 6 2 5 3 4 In-line filter Filter Wavelength detector UV Injection valve (V1) 1 7 6 2 Cond. Conductivity monitor 5 3 4 Mixer System pumps pH pH detector ( electrode optional) A B Fraction collector Frac-950

  8. ÄKTAFPLC XL Reverse flow valve (V7) Filter UV detector UV Injection valve (V1) Conductivity monitor In-line filter Cond. Column Selection valve (V2) Mixer pH detector ( electrode optional) System pumps pH A B Sample pump 8 8 8 8 8 8 8 8 8 Fraction Collector Frac-950 1 7 1 1 1 1 1 1 1 1 7 7 7 7 7 7 7 7 2 6 6 6 6 6 6 6 6 6 2 2 2 2 2 2 2 2 5 3 5 5 5 5 5 5 5 5 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 Outlet valve (V4) Buffer select valves (V5) Sample valve (V8) Buffer select valves (V6) Column Selection valve (V3) ÄKTAFPLC + Buffer Valve + Sample Pump + Sample Valve + Outlet Valve Filter UV detector UV Injection valve (V1) In-line filter 1 1 1 7 7 7 6 6 6 Conductivity monitor Cond. 2 2 2 5 5 5 3 3 3 4 4 4 System pumps pH detector ( electrode optional) pH A B Sample pump Outlet valve (V4) Fraction collector Frac 950 Sample valve (V8) Buffer select valve (5)

  9. PUMP P-920 Inlets/Outlets A B A B To Injector and Column

  10. PUMP P-920 Multiple Inlets 8 1 8 1 7 2 7 2 6 6 3 3 5 4 5 4 A8 A6 A4 B8 A2 B6 B4 B2 A3 A7 A1 A5 B3 B7 B1 B5 Pump A Pump B Mixer Injector / Column

  11. pH Electrode and Flow cell DUMMY electrode pH electrode Storage Reservoir 1:1 mixture Buffer pH 4 and 1M KNO3 Electrode Flow Cell

  12. INJECTION VALVE INV- 907 1 COLUMN 7 2 PUMP LOAD SAMPLE 3 6 4 5 Waste Waste LOAD 1 COLUMN 7 2 PUMP 3 6 4 5 Waste Waste INJECT 1 COLUMN 7 2 PUMP 3 6 4 5 Waste Waste WASTE

  13. SAMPLE LOADING METHOD 1 COLUMN PUMP 2 7 LOAD SAMPLE 3 6 5 4 Needle Port Waste Waste Always tighten by hand with needle inserted Do Not Overtighten 1 COLUMN 7 PUMP LOAD SAMPLE 2 3 6 5 4 Luer Lock Adapter Superloop Waste Waste

  14. SUPERLOOPS BUFFER FLUSH SAMPLE LOAD SAMPLE INJECT SAMPLE FLUSH 10 ml Buffer 50 ml Plunger 150 ml Sample Etched Groove PUMP or SYRINGE WASTE COLUMN COLUMN

  15. 4 9 2 9 5 8 1 7 8 3 8 6 SUPERLOOPS

  16. INV-907 Injection Valve - Injection Port PEEK Valve Body Injection Needle Port Valve Seal Ferrule Needle Needle Adapter NeedleTube

  17. INV-907 Injection Valve - Luer Adapter PEEK Injection Valve Body PEEK Injection Luer Adaptor Valve Seal Valve Channel Plate

  18. Fluid Dynamics Flow PEEK Tubing High Resistance to Flow Low Resistance to Flow End Beginning

  19. Fluid Dynamics Flow - Injecting a Loop The way we imagine it Flows The way it Actually Flows Bubble Segmentation 100ul Loop 50ul Load Waste 60ul Load 100ul Load 200ul Load • Partial Loading Loop = No more than 1/2 Loop Volume • Total Loading Loop = Inject 2X Loop Volume

  20. Fraction Collection Design + Frac 950 Peak Fractionation Flowthrough Fractionation Eluate Fractionation 100%B 0%B mAU %B 100 500 BOUND UNBOUND 50 250 Inject End Start 0 Waste 0 A1 A2 A3………………….

  21. ÄKTAFPLC - Tubing Kit Changes GENERAL FLOW (at delivery) 7 1 100 ul Injection valve V1 G3 Outlet Valve V4 (7ul) option G5 Accumulator (40ul) UV Flow cell G4 Conductivity Flow cell (14ul) FR-902 (0.2Mpa, 5ul) 0.50 mm i.d Orange PEEK/Clear G3 600mm (350+250) G4 170mm G5 600mm NB: Never use the FR-904 with the pH electrode option pH Flow cell (100ul)

  22. PEEKTM Tubing Gray, 1/16”x 1.0 mm ( 7.85ul / cm ) 18-1115-83, 2M Green, 1/16”x 0.75 mm ( 4.42ul / cm ) 18-1112-53, 2M Orange, 1/16”x 0.5 mm ( 1.96ul / cm ) 18-1113-68, 2M Blue, 1/16”x 0.25 mm ( 0.49ul / cm ) 18-1120-95, 2M Teflon / Tefzel Tubing 3/16” OD 18-1112-47, 3M 1/8” OD 18-1121-16, 3M 1/16” OD 18-1119-74, 2M

  23. ÄKTAFPLC Tubing Specification Fraction Delay ( UV to Frac. Collector )= 237ul

  24. HiTrap Connections 1/16” Female 1/16” Male 18-1112-55 1/16” Female 1/16” Fingertight Fitting M6 Male 18-1112-57 M6 male / 1/16” Female HiTrap Column Phenyl Sepharose M6 Male M6 Female 18-1112-58 M6 female / 1/16” male 1/16” Male Flow Cell Inlet

  25. Multiple HiTrap Arrangment ( Series ) Phenyl Sepharose Phenyl Sepharose Phenyl Sepharose Phenyl Sepharose Phenyl Sepharose Phenyl Sepharose Detector Detector Detector

  26. Traditional FPLC fitting to ÄKTA / HPLC Injector 1/16” Fingertight Fitting 18-1112-55 1/16” FEMALE / M6 FEMALE 18-1123-94 100 ul Flange M6 Fitting 1/16” MALE / M6 FEMALE 18-1112-58 Flow Cell

  27. COMPRESSION FITTINGS Untightened Correctly tightened Obstructed Over tightened Tubing Restricted

  28. Tubing Fittings M6 male (0.5 mm i.d.) PP FPLC™, for 1/16" o.d. tubing 18-3886-01 M6 male (1.5 mm i.d.) (PP) Pump P-6000, for 2.7 mm o.d. tubing 18-4652-01 Pump P-1 Tubing connector (PP) For 1.8 mm o.d. tubing 19-2150-01 Tubing connector flangeless/M6 male (PP) For 1.8 mm o.d. tubing 18-1003-68 Tubing connector flangeless/M6 female PP For 1.8 mm o.d. tubing 18-1017-98 Tubing connector TC-2 (PP) For 1.8 mm o.d. tubing 19-0045-01 Tubing connector TC-3 (PP) For 1.8 mm o.d. tubing 19-0047-01 SRTC-3, M6 female (1.5 mm i.d.) (PEEK)  19-2144-01 SRTC-2, M6 female (1.5 mm i.d.) (PEEK) 2 XK columns in series 19-2143-01 M6 female, SRV-1 ( PP)  19-2145-01 Domed Nut, M6 (PP) Sealing ends of HR and XK columns 18-2450-01

  29. Tubing Fittings 1/16", female / M6 male Adaptor (PEEK) For 1/16" tubing 18-1112-55 Fingertight connector 1/16", male (PEEK) For 1/16" tubing 18-1112-57 1/16" male/Luer female Injection valve INV-907(PEEK) 18-1112-51 Union M6 female/1/16" male (PEEK) To connect columns with M6 connectors to 18-3858-01 Union 5/16" female/1/16" male (PEEK) To connect columns with M6 connectors 18-1142-08 5/16" female/M6 male (PEEK) To connect columns with M6 connectors 18-1127-76 Union Luerlock female/M6 male (PP) Injection valves V-7, IV-7, MV-7 and IMV-7 18-1027-62 Union Luerlock female/M6 female (PP) 18-1027-12 Union 1/16” female/1/16” female (Ti) 18-3855-01 Union 1/16” female/M6 female (PEEK) 18-1123-94

  30. Tubing Fittings 5/16” / 1/16”ID Inlet Connector ( PEEK)  18-1127- 07 5/16” / 1/8”ID Inlet Connector ( PEEK)  18-1121-17 5/16” / 3/16”ID Inlet Connector ( PEEK) 18-1112- 49 1/16“Flangeless Ferrule(PP) 18-1127-06 For use with 18-1127-07 1/8"Flangeless Ferrule (PP) 18-1121-18 For use with 18-1121-17 3/16"Flangeless Ferrule (PP) 18-1112-48 For use with 18-1112-49 Stop plug female,1/16“ (PP) Sealing the bottom of a HiTrap column 11-0004-64 1/16" male Stop plug (PEEK) for use within ÄKTAdesign 18-1112-52 5/16" male Stop plug (PEEK) for use within ÄKTAdesign 18-1112-50 Fingertight stop plug, 1/16'' (PEEK) Sealing the top of a HiTrap column 11-0003-55

  31. System Compensation Volume Equilibration Phase A COLUMN TOP 0% B Mixer A COLUMN TOP 20% B 8 mls* ( 3 to 4 x Mixer Volume ) Command 20% Effect Compensation Volume ( includes “Dead” volume ) 20%B Programmed Step 0%B 8mls *System Compensation is not needed if Lines are primed and 0%B is used for Equilibration

  32. Gradient Delay Volume Gradient Phase A COLUMN TOP 0% B A COLUMN TOP Mixer 100% B 8 mls ( 4 to 6 x Mixer Volume ) Effect Command 100% 100%B Programmed Gradient System “Dead” Volume “Dead” Volume Gradient Delay (includes “Dead” volume ) *Because of the viscosity of 100%B often requires a larger delay volume than system compensation

  33. FRACTION DELAY Delay volume Waste START END A1 A2 A3 A4 A5 Waste A1 A2 A3 A4 A1 A2 A3 A4 A5 Waste A1 A2 A3 A4 A5 Waste A1 A2 A3 A4 A5 Delay volume continues collection after end of run Waste A1 A2 A3 A4 A5

  34. Column Press Drop 0.5 MPa (0.3 + 0.2) 0.5 Reading Column Shatters DANGEROUS Matrix Collapses EXPENSIVE 0.2 MPa Flow Restrictor PUMP PRESSURE 0.2 MPa 0.5 MPa GEL Press Drop 0.3 MPa (0.5 - 0.2) Pre-column pressure = 0 MPa Atmos Press 0.0 MPa Column Pressure ( compensation ) Flow Restrictor Gel Top Pressure Gel Bottom Pressure Column Hardware Pressure Limits Column Matrix Pressure Limits Column Press Drop 0.3 MPa (0.3 + 0.0) 0.3 Reading PUMP PRESSURE 0.0 MPa Column Pressures 0.3 MPa 0.0 MPa Pre-column pressure = 0 MPa GEL Press Drop 0.3 MPa (0.3 - 0.0)

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