ÄKTA system Training Guide

Transcription

1 ÄKTA system Training Guide Purifier 00 / 0 amersham bioscience amersham bioscience amersham bioscience amersham bioscience

2 ÄKTApurifier Buffers Injector Box Monitor Conductivity ph On-line Filter UV Detector Outlet Valve Pumps Mixer Pump A Pump B Buffer switching valve

3 ÄKTApurifier 0 / 00 In-line filter UV Wavelength detector Injection valve (V) Cond. Conductivity monitor System pumps Mixer ph ph detector A B Fraction collector Frac-90 8 Outlet valve (V) ÄKTApurifier + Sample Pump + Sample Valve In-line filter System pumps A Mixer B Injection valve (V) UV Cond ph Wavelength detector Conductivity monitor ph detector Sample pump 8 Fraction collector Frac-90 8 Outlet valve (V) Sample selection valve (V)

4 ÄKTApurifier 00 Air In-line filter UV Wavelength detector Injection valve (V) Cond. Conductivity monitor System pumps Mixer ph ph detector A B Fraction collector Frac-90 Air sensor 8 Outlet valve (V) ÄKTApurifier + Buffer Valve In-line filter UV Wavelength detector Injection valve (V) Cond. Conductivity monitor System pumps Mixer ph ph detector A B Fraction collector Frac-90 8 Outlet valve (V) 8 Buffer select valve (V)

5 ÄKTApurifier 0 XT In-line filter UV Wavelength detector Injection valve (V) Cond. Conductivity monitor System pumps Mixer ph ph detector A B Fraction collector Frac-90 8 Outlet valve (V)

6 PUMP P-900 Mixer Pump A Pump B Proportion Valve A A B B Buffer Acid /Alkali Salt Water ÄKTA 0 mls/min ÄKTA 00 mls/min GRADIENT < mls/min - mls/min <0 mls/min 0-0 mls/min 0-00 mls/min MIXER 0. ml mixer ml mixer mls mixer mls mixer mls mixer

7 PUMP P-900 Inlets Mixer Pump A Pump B Switching Valves Buffer Select Valve IV-908 A A B B 8 Water Salt A A A A A A A8 A

8 PUMP P-900 Manually Priming Buffer Inlet Lines Pump A Pump B Common inlet A A Common inlet B B Manual Flowpath Manual Flowpath A or A BufferValveA B or B BufferValveB Open Draw Air out of Buffer Line

9 PUMP P-900 Back - Wash Lines Pump Back-Wash Lines Syringe fitting Wash OUT IN-LINE one-way valve High pressure Piston seal Buffer OUT Ball outlet check valve 0% Ethanol : Water Piston Backwash pump diaphragm Wash IN Buffer IN Ball inlet check valve

10 ON-LINE FILTERS P-90 ( 0 ml/min Pump ) Low Flow HPLC Filter P-90 ( 00 ml/min Pump ) High Flow Fibre Filter Polymer Support LOAD () () LOAD (wash loop) Column Pump Column Pump Column Pump 8 PV PV PV-908 Sample Sample Sample

11 ION VALVE INV- 90 COLUMN LOAD SAMPLE PUMP LOAD COLUMN PUMP COLUMN PUMP WASTE

12 SAMPLE LOADING METHOD COLUMN LOAD SAMPLE PUMP Needle Port Always tighten by hand with needle inserted Do Not Overtighten COLUMN LOAD SAMPLE PUMP Luer Lock Adapter Superloop

13 SUPERLOOPS Buffer 0 ml 0 ml 0 ml Plunger BUFFER FLUSH SAMPLE LOAD SAMPLE SAMPLE FLUSH Sample Etched Groove WASTE PUMP or SYRINGE COLUMN COLUMN Volume Max. allowed column pressure Code No. 0 ml Mpa ml Mpa ml Mpa 8-0-8

14 INV-90 Injection Valve - Injection Port PEEK Valve Body Injection Needle Port Valve Seal Ferrule Needle NeedleTube Needle Adapter

15 INV-90 Injection Valve - Luer Adapter PEEK PEEK Injection Valve Valve Body Body PEEK PEEK Injection Luer Luer Adaptor Valve Valve Seal Seal Valve Valve Channel Plate Plate

16 Fluid Dynamics Flow High Resistance to Flow PEEK Tubing Low Resistance to Flow End Beginning

17 Fluid Dynamics Flow - Injecting a Loop The way we imagine it Flows The way it Actually Flows Bubble Segmentation 0ul Load 00ul Loop 0ul Load 00ul Load 00ul Load Partial Loading Loop = No more than / Loop Volume Total Loading Loop = Inject X Loop Volume

18 LOADING A SAMPLE LOOP Column Load Inject Injection valve INV-90 Pump Sample Sample pump P-90

19 LOADING A SUPER LOOP Column Load Inject Injection valve INV-90 Pump Buffer Sample Sample

20 MULTI-LOADING A SAMPLE LOOP Column Load Inject Injection valve INV-90 Pump PV I/O Samples Wash

21 MULTI-LOADING A SUPER LOOP Column Injection valve INV-90 Pump Load Inject PV-908 I/O 8 Samples S S S S Wash S8

22 DIRECT LOADING ON TO COLUMN Column Pump Inject Load Extra Tube / or /8 Tubing Sample

23 REVERSE FLOW VALVE ( INV-90 ) Pump Column Extra Tube Stop Plug MONITOR Downward Flow Position Pump Column Extra Tube Stop Plug MONITOR Upward Flow Position

24 OUTLET VALVE PV-908 DETECTOR Default To Fraction Collector F F F F8 I/O F F F F F F F F F F8

25 Fraction Collection Flowthrough Fractionation Eluate Fractionation Peak Fractionation mau 00 0%B 00%B %B 00 0 Inject UNBOUND Start BOUND End A A A.

26 Phenyl Sepharose HiTrap Connections / Female / Male 8-- / Fingertight Fitting / Female M Male 8-- M male / / Female HiTrap Column M Male M Female 8--8 M female / / male / Male Flow Cell Inlet

27 Phenyl Sepharose Phenyl Sepharose Phenyl Sepharose Phenyl Sepharose Phenyl Sepharose Phenyl Sepharose Multiple HiTrap Arrangment ( Series ) Detector Detector Detector

28 Traditional FPLC fitting to ÄKTA / HPLC Injector / Fingertight Fitting ul / FEMALE / M FEMALE 8--9 Flange M Fitting / MALE / M FEMALE 8--8 Flow Cell

29 Tubing Fittings 8-- / Fingertight Fitting / PEEK TM Tubing 8--8 Flange Fitting Adapter FPLC Flange Fitting 8-- Luer Fitting Adapter Luer Syringe 8-- Fingertight Stop Plug 8--0 / Stop Plug Flangless Inlet Connector / 8--9 /8 8-- / 8--0 Flangless Buffer Inlet Ferrule / 8--8 / / M male / / Female Tubing Connector M male / O - ring / female X / female Union Ti Union + Fingertight Fittings 8--9 M female X / female Union FPLC Flange - / Fingertight

30 COMPRESSION FITTINGS Untightened Correctly tightened Obstructed Over tightened Tubing Restricted

31 PEEK TM Tubing Gray, / x.0 mm (.8ul / cm ) Green, / x 0. mm (.ul / cm ) Orange, / x 0. mm (.9ul / cm ) Blue, / x 0. mm ( 0.9ul / cm ) Teflon / Tefzel Tubing / OD 8-- /8 OD 8-- / OD 8-9-

32 Tubing Kit Changes - ÄKTApurifier 0 GENERAL FLOW (at delivery) G 00 ul Injection valve V G9 Outlet Valve V (ul) UV Flow cell G8 Accumulator (0ul) 0. mm i.d Blue PEEK G 00mm G 0mm G 80mm G8 0mm G9 00mm G Conductivity Flow cell (ul) G ph Flow cell (00ul) FR-90 (0.Mpa, ul) NB: Never use the FR-90 with the ph electrode Tubing Kit No Frac Frac 90 Frac 90 AKTApurifier 0, 0.0 tubing kit 8 AKTApurifier 0, 0.0 tubing kit + ph electrode 0 AKTApurifier 0, 0. tubing kit 88 8

33 Tubing Kit Changes - ÄKTApurifier 0 HIGH FLOW G 00 ul Injection valve V H9 Outlet Valve V (ul) UV Flow cell H8 Accumulator (0ul) H Conductivity Flow cell (ul) H 0.0 mm i.d Orange PEEK H 0mm H 80mm H8 0mm H9 00mm NB: Never use the FR-90 with the ph electrode ph Flow cell (00ul) FR-90 (0.Mpa, ul) Tubing Kit No Frac Frac 90 Frac 90 AKTApurifier 0, 0.0 tubing kit 8 AKTApurifier 0, 0.0 tubing kit + ph electrode 0 AKTApurifier 0, 0. tubing kit 88 8

34 Tubing Kit Changes - ÄKTApurifier 00 HIGH FLOW (at delivery) G 00 ul Injection valve V G9 Outlet Valve V (ul) UV Flow cell G8 Accumulator (0ul) 0. mm i.d Green PEEK G G G8 G9 0mm 80mm 0mm 00mm G Conductivity Flow cell (ul) G ph Flow cell (00ul) FR-90 (0.Mpa, ul) NB: Never use the FR-90 with the ph electrode Tubing Kit No Frac Frac 90 Frac 90 AKTApurifier 00, 0.0 tubing kit 8 AKTApurifier 00, 0. tubing kit 0 0 AKTApurifier 00,.00 tubing kit AKTApurifier 00, 0.0 tubing kit + ph electrode 0 AKTApurifier 00, 0. tubing kit + ph electrode 8 AKTApurifier 00,.00 tubing kit + ph electrode 9 99 AKTAbasic 00, 0. tubing 90

35 Tubing Kit Changes - ÄKTApurifier 00 LOW FLOW L 00 ul Injection valve V L9 Outlet Valve V (ul) UV Flow cell L8 Accumulator (0ul) 0.0 mm i.d Orange PEEK L L L8 L9 0mm 80mm 0mm 00mm L Conductivity Flow cell (ul) NB: Never use the FR-90 with the ph electrode L FR-90 (0.Mpa, ul) ph Flow cell (00ul) Tubing Kit No Frac Frac 90 Frac 90 AKTApurifier 00, 0.0 tubing kit 8 AKTApurifier 00, 0. tubing kit 0 0 AKTApurifier 00,.00 tubing kit AKTApurifier 00, 0.0 tubing kit + ph electrode 0 AKTApurifier 00, 0. tubing kit + ph electrode 8 AKTApurifier 00,.00 tubing kit + ph electrode 9 99

36 A-900 Autor COLUMN PUMP MANUAL SAMPLE LOAD LOAD Syringe Sample INV-90 A-900 COLUMN PUMP MANUAL SAMPLE LOAD Syringe Sample INV-90 A-900 LOAD LOAD

37 Needle Height Standard Vial Needle Height volume left mm 0ul Standard Vial + Insert mm 0ul mm ul 0mm 00ul

38 A-900 Sample Injection ( Flushed Loop Injection ) LOAD LOAD pump pump pump pump waste waste waste waste column column column column Sample needle Wash Reservoir In In,, In In LOAD LOAD The The flushes flushes the the valve valve is is drawn drawn up up to to channel. channel. completely completely Min. Min. ( ( 0ul 0ul ) ) with with fill fill the the loop. loop. Air Air Segment Segment ( ( no no less less than than twice twice Min. Min. ( ( ul ul ) ) without without loop loop volume volume Air Air segment segment recommended recommended ) ) In In is is washed washed onto onto the the column. column. Final Final internal internal needle needle wash. wash. waste pump column Optional Optional External External needle needle wash. wash. transport fluid

39 A-900 Sample Injection ( Partial Loop Injection ) LOAD LOAD waste pump pump pump pump waste column waste column waste column waste column Sample needle Sample needle Sample needle Wash Reservoir In In,, flushes flushes the the valve valve channel. channel. Min. Min. ( ( 0ul 0ul ) ) with with Air Air Segment Segment Min. Min. ( ( ul ul ) ) without without Air Air segment segment The The is is drawn drawn up up to to partially partially fill fill the the loop loop with with programmed programmed volume. volume. ( ( no no more more than than half half loop loop volume volume recommended recommended ) ) In In inject inject is is washed washed onto onto the the column. column. Final Final internal internal needle needle wash. wash. waste pump column Optional Optional External External needle needle wash. wash. transport fluid

40 A-900 Sample Injection ( ul Pick-up Injection ) LOAD LOAD LOAD LOAD pump pump pump pump waste column column column column transport fluid Sample needle Sample needle Sample needle In In inject, inject, valve valve flushes flushes with with transport transport fluid fluid The The is is drawn drawn up up from from the the vial vial The The is is washed washed into into the the loop loop with with transport transport fluid fluid In In inject inject is is washed washed onto onto the the co column lumn pump pump waste Final Final internal internal needle needle wash. wash. waste Wash Reservoir column Optional Optional needle needle wash. wash. transport fluid column

41 System Compensation Volume Equilibration Phase A COLUMN TOP B 0% A COLUMN TOP B 0% Effect 8 mls Command 0%B 0%B 8mls

42 A Gradient Delay Volume Gradient Phase COLUMN TOP B 0% A COLUMN TOP B 00% Effect mls Command 00% 00%B Gradient Delay

43 Column Pressure ( compensation ) Flow Restrictor Column Pressures Column Hardware Pressure Limits Column Matrix Pressure Limits Column Shatters DANGEROUS Matrix Collapses EXPENSIVE Reading Reading Atmos Atmos Press Press MPa MPa Reading Reading Pre-column pressure = 0 MPa Pump Pump Mpa Mpa Pre-column pressure = 0 MPa Pump Pump Mpa Mpa 0. MPa 0. MPa Gel Top Pressure GEL GEL Press Press Drop Drop Column Column Press Press Drop Drop MPa MPa MPa MPa (0. ( ) 0.0) (0. ( ) 0.0) 0.0 MPa Gel Bottom Pressure GEL GEL Press Press Drop Drop MPa MPa (0. ( ) 0.) 0. MPa Colu Colu mn mn Press Press Drop Drop MPa MPa (0. ( ) 0.) MPa MPa MPa MPa Flow Flow Restrictor