akta purifier core 100 training

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

SAMPLE APPLICATION

Different ways of applying sample

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

End Beginning

PEEK Tubing

High Resistanceto Flow

Low Resistanceto Flow

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




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 )



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


IV-907

LOAD

Sample

Waste

LOADING SAMPLE LOOP with SAMPLE PUMP ( sample volumes >10 mls )

NOTE: SampleDoes not enterpump This means

Minimal wastage



Sample PumpSample Pump

3


IV-907

LOAD

Sample

Waste

1

7

4

INJECT

Wash volume 2/3 loop vol.6

2

5



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




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




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)



Less Wasted Sample

3

( sample volumes > 10 mls )

1

7

6

2

4 5

Sample



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



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 )

Collecting Peaks

Different ways of collecting Fractions

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

Optional Functionality

akta purifier core 100 training

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