akta purifier core 100 training
TRANSCRIPT
AKTA Purifier Core 100
• System Features– Flowrate: 0.01 – 100 ml/min– System Pressure: 0 – 10 MPa– Conductivity Range: 1uS/cm – 999.9 mS/cm– pH Range: 0 – 14 (+/-0.1 pH unit)– UV Measurement: 190 – 700 nm in 1nm
steps (3 Simultaneous)
ÄKTA purifier Flowpath
BA
Systempumps
Mixer
FractioncollectorFrac-950
In-linefilter
Injectionvalve (V1)
3 Wavelengthdetector
2
4
7
3
16
5
Conductivitymonitor
pH detector
Cond
pH
UV
Sample
ÄKTA System Practical Handling and Maintenance
• Pumps Purging, BackwashFiltersFlow Restrictor and Pressure
• Application of SampleInjection ValveLoop Loading and RulesSuperloopDirect Injection
•Collecting PeaksFlowthrough FractionationElution FractionationPeak FractionationDelay Volume
Wash OUT
WashIN
BufferIN
BufferOUT
Ball outletcheck valve
Ball inletcheck valve
Piston
Backwash pump diaphragm
IN-LINE one-wayvalve
RECIPROCATING DISPLACEMENT PUMP
High pressurePiston seal
• Check for leakage. If there is a sign of liquid
leaking between the pump head and the
housing side panel or increased or decreased
volume of rinsing solution, replace the piston
Seals.
• When changing eluent, it is important to
remove trapped air.
PUMP P-900 Back - Wash Lines
20% Ethanol : Water
Pump Back-WashLines
Wash OUT
WashIN
BufferIN
BufferOUT
Syringe fitting
Change MONTHLY – Book says Weekly
The purge line is recycled back in the resevoir. Make sure that the resevoir lines are secured.
One-way valve
Default Lines
• Pump A – A1• Pump B – B1
GRADIENT MIXERÄKTA 10 mls/min <6 mls/min
6 -14 mls/min0.6 ml mixer2 ml mixer
ÄKTA 100 mls/min <30 mls/min30 - 50 mls/min50 - 100 mls/min
2 mls mixer5 mls mixer12 mls mixer
PUMP P-900
Proportion Valve
Pump A
Mixer
A2 A1 B2 B1
Pump B Any one Buffer passes through both pump heads.
When purging a buffer line both pump heads must be purged.
The Mixer volume is best changed for different flow rates.
B
A
0%
B
A
20%
COLUMN TOP
Command 20%Effect
8 mls*
( 3 to 4 x Mixer Volume )
COLUMN TOP
Mixer, 2mls
When a command for a %B mix is received by the Pump it takes 3-4 X the mixer volume before it is achieved
This appears in the program as “System compensation volume” and “Gradient delay volume”
BufferIN
BufferOUT
• Only manually purge when pump contains large volume of air
• Unscrew valve and the seal moves out of the way.
•Buffer can then be sucked through the Pump. Note the check valves opening.
Seal
Air
Open
Draw Air out ofBuffer Line
Manual Flowpath
A1 or A2 BufferValveA
Manual Flowpath
B1 or B2 BufferValveB
Pump A Pump B
Common inlet A2 A1 B2 B1Common inlet
~Try This
System Control….Manual…………….Flowpath………….PumpA Inlet……...A2………………….
Execute
ON-LINE FILTERS
P-900 PumpHigh Flow Fibre Filter( for Soft Gel Columns )18-1027-11 ( 10/pack)
Polymer Support mesh
18-1112-44 On-line Filter
Change MONTHLY / 3 MONTHLY – Book says Weekly
• Polymer Fibre Filter ( 5um pores )
•Support Mesh ~ Stops fibres from entering the chromatography steam.
Flow Restrictor and Pressure
FR-902 (0.2Mpa, 5ul)
FR-904 (0.4Mpa, 4ul)
NB: Never usethe FR-904 withthe pH electrode
pH Flow cell (100ul)
Conductivity Flow cell (14ul)
FR-902 (0.2Mpa, 5ul)
100 ul
Injection valve V1
Outlet Valve V4 (7ul)
Accumulator (40ul)
1. Suppresses Air Bubbles
2. Stops Siphoning Effect on Buffer
3. Closes Pump Check Valves efficiently
FR-904(0.4MPa, 7ul)
0 1
0.5
0.3
MPa
Column Gel TOP = 0.3 MPa
Column Gel BOTTOM = 0 MPa
TOP ~ Full resistance to flow, by gel bed.BOTTOM ~ No resistance to flow, open to atmosphere.
Pressure gradient down column Pressure difference = (0.3 – 0.0) = 0.3 MPa
FR-9020.2 MPa
Column Gel TOP = 0.5 MPa
Column Gel BOTTOM = 0.2 MPa
Pressure gradient down column Pressure difference = (0.5 – 0.3) = 0.3 MPa
TOP ~ Full resistance reflects the addition of the Flow Restrictor.
BOTTOM ~ Resistance to flow, due to Flow Restrictor.
This means you should ADDthe 0.2MPa to the recommended Column Pressure, ie. HiTrap (0.3 +0.2)= 0.5MPa
Column PressureFR-902
Column Hardware Pressure Limits
Column Matrix Pressure Limits
Column Pressures
Column Shatters DANGEROUS
Matrix Collapses EXPENSIVE
WARNING !However the set pressure must not EXCEED the recommended Column Hardware pressure.
Column Pressure
pH electrode
DUMMY electrode
ElectrodeFlow Cell
pH Electrode and Flow cell ~ Never leave in line
StorageReservoir
1:1 mixtureBuffer pH 4 and1M KNO3
1
7
65
23
4
PUMPLOADSAMPLE
Waste
COLUMN
Waste
LOAD1
7
65
23
4
PUMP
Waste
COLUMN
Waste
INJECT
17
65
23
4
PUMP
Waste
COLUMN
Waste
WASTE
INJECTION VALVE INV- 907
Loop Loading Rules
• Partial Loading Loop = No more than 1/2 Loop Volume• Total Loading Loop = Inject 2X Loop Volume
50ul Load
60ul Load
100ul Load
200ul Load
Waste 100ul Loop
No Waste
Exactly 100ul
Sample Partial Loading Instruction
1. Ensure that Sample loop is Twice the Sample Volume
2. Ensure that the Injection Valve (INV-907) is at ‘LOAD’ position
3. Flush the Sample loop with at least Twice Sample loop volume, do not detach the Syringe
4. Switch (from UNICORN) the Injection Valve (INV-907) to ‘INJECT’ position
5. Detach the Syringe containing Flush Buffer and attached the Syringe containing Sample
6. Switch (from UNICORN) the Injection Valve (INV-907) to ‘LOAD’ position, then Inject your sample into the Sample loop
7. Leave the Sample syringe on the Injection Valve (Do not remove)
8. Proceed with your Run
System PumpSystem Pump
LOAD
SampleLoopSampleLoop
IV-907
Sample
While buffer is pumped through INJECTION VALVE to column
Sample can be pushed ( “LOAD”ed ) into Loop
1
7
6
2
4 5
MANUALY LOADING SAMPLE LOOP ( sample volumes 25ul to 5.0 mls )
Waste
Injection Valve INV-907Injection Valve INV-907
Syringe NeedleAdaptor
3
INJECT
IV-907
INJECTION VALVE changes from LOAD to INJECT
LOAD
Buffer pushes ( “INJECTS” ) sample onto column
1
7
6
2
4 5
Wash volume 2/3 loop vol.
Waste
Injection Valve INV-907Injection Valve INV-907
System PumpSystem Pump
SampleLoopSampleLoop
Sample
3
MANUALY LOADING SAMPLE LOOP ( sample volumes 25ul to 5.0 mls )
SuperLoopSuperLoop
IV-907LOAD1
7
6
2
4 5
INJECT
MANUALLY LOADING SUPERLOOP ( sample volumes 1 ml to 150 mls )
Injection Valve INV-907Injection Valve INV-907
System PumpSystem Pump
Waste
Sample
While buffer is pumped through INJECTION VALVE to column
Syringe Loads Superloop
INJECTION VALVE Changes from LOAD to INJECT
System Pumps (injects) Sample from Superloop to column
Leur SyringeAdaptor
3
0
50
10
20
30
40
1
7
65
2
4 5
SampleLoopSampleLoop
IV-907
LOAD
Sample
Waste
LOADING SAMPLE LOOP with SAMPLE PUMP ( sample volumes >10 mls )
NOTE: SampleDoes not enterpump This means
Minimal wastage
Injection Valve INV-907Injection Valve INV-907
System PumpSystem Pump
Sample PumpSample Pump
3
SampleLoopSampleLoop
IV-907
LOAD
Sample
Waste
1
7
4
INJECT
Wash volume 2/3 loop vol.6
2
5
Injection Valve INV-907Injection Valve INV-907
System PumpSystem Pump
NOTE: SampleDoes not enterpump This means
Minimal wastage Sample PumpSample Pump
3
LOADING SAMPLE LOOP with SAMPLE PUMP ( sample volumes >10 mls )
SuperLoopSuperLoop
IV-907LOAD
Sample
Waste
1
7
6
2
4 5
INJECT
LOADING SUPERLOOP with SAMPLE PUMP ( sample volumes 5 ml to 150 mls )
NOTE: Sample goesthrough the pump and is pushed intosuperloop
NOTE: Buffer pushesSample onto the column
Injection Valve INV-907Injection Valve INV-907
System PumpSystem Pump
Sample PumpSample Pump
3
0
50
10
20
30
40
IV-907LOAD
Sample
Waste
1
7
6
2
4 5
INJECT
DIRECT LOADING SAMPLE with SAMPLE PUMP ( sample volumes > 10 mls )
Extra Tubing
Injection Valve INV-907Injection Valve INV-907
System PumpSystem Pump
Sample PumpSample Pump
3
IV-907LOAD
Sample
1
7
6
2
4 5
INJECT
DIRECT LOADING SAMPLE with SAMPLE PUMP / AIR SENSOR
Waste Air Stops Sample Pump
No need for AccurateVolume
Air Sensor(watch)Air Sensor(watch)
Injection Valve INV-907Injection Valve INV-907
System PumpSystem Pump
Less Wasted Sample
3
( sample volumes > 10 mls )
1
7
6
2
4 5
Sample
System PumpSystem Pump
Injection Valve INV-907Injection Valve INV-907
DIRECT LOADING SAMPLE with SYSTEM PUMP ( sample volumes > 10 mls )
LOAD
ChangeTo Buffer Inlet A1
Inlet A1
Inlet A2Inlet A2Inlet A2
Inlet A1Inlet A1
Buffer
3
1
2
3
4 5
6
7
8
BUFFER A11
A12A13
INLET A18
1
7
6
2
4 5
Sample
System PumpSystem Pump
Injection Valve INV-907Injection Valve INV-907
Buffer SelectionValve IV-908Buffer SelectionValve IV-908
DIRECT LOADING SAMPLE with SYSTEM PUMP and BUFFER VALVE
LOAD
ChangeTo Buffer
3
Inlet A1Inlet A1
( sample volumes > 10 mls )
The Fractionation Options
X1 X2 X3……………
Was
te 0
%B
100
50
0%B 100%BIn
jec
tmAU
0
500
250
FlowthroughFractionation
FlowthroughFractionation
Eluate Fractionation
Eluate Fractionation
En
d
UN
BO
UN
D
Fixed volumeFixed volume
Tube
Outlet valve
UV
Wat
ch
Sta
rt
UV280Conc %B
A1 A2 A3…………
Slope or
Level
PeakFractionation
PeakFractionation
Delay volume
A1 A2 A3 A4 A5
Was
te
A1 A2 A3 A4
ST
AR
T F
RA
C
EN
D F
RA
C
Delay volume continues collecting fractions after end of run until the delay volume is achieved
FRACTION DELAY
Computer ScreenSignal from UV monitor
Fraction Collector
Delay
vol
ume
Fraction Size = 1 mlFraction delay = 1 ml
Was
te