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ÄKTA system Training Guide

Purifier 100 / 10

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ÄKTApurifier

Buffers
Injector

Box

Monitor On-line
Conductivity
pH
Filter

UV Detector Outlet Valve

Pumps Mixer

Pump A
Buffer switching
Pump B valve
ÄKTApurifier 10 / 100
In-line UV 3 Wavelength
filter
detector

Injection
valve (V1) Cond. Conductivity
1 7 monitor
2 6
3 4 5

Mixer pH
pH detector
System
pumps

Fraction
A B collector
Frac-950

1 8
7
2 6
Outlet
3 5
4 valve (V4)

ÄKTApurifier + Sample Pump + Sample Valve


In-line
filter

Injection UV 3 Wavelength
valve (V1) detector
1 7
2 6
3 45 Cond Conductivity
Mixer monitor
System
pumps
pH pH detector
A B

Sample
pump Fraction
1 87 collector
2 6
345 Frac-950

1 87
2 6 Outlet
345
valve (V4)

Sample selection
valve (V5)
ÄKTApurifier 100 Air
In-line UV 3 Wavelength
filter
detector

Injection
valve (V1) Cond. Conductivity
1 7 monitor
2 6
3 4 5

Mixer pH
pH detector
System
pumps

Fraction
A B collector
Frac-950

Air
sensor

1 8
7
2 6
Outlet
3 5
4 valve (V4)

ÄKTApurifier + Buffer Valve


In-line UV 3 Wavelength
filter
detector

Injection
valve (V1) Cond. Conductivity
1 7 monitor
2 6
3 4 5

Mixer pH
pH detector
System
pumps

Fraction
A B collector
Frac-950

1 8
7
2 6
Outlet
3 5
4 valve (V4)

1 87
2 6 Buffer
345 select
valve (V6)
ÄKTApurifier 10 XT

In-line UV 3 Wavelength
filter
detector

Injection
valve (V1) Cond. Conductivity
1 7 monitor
2 6
3 4 5

Mixer pH
pH detector
System
pumps

Fraction
A B collector
Frac-950

1 8
7
2 6
Outlet
3 5
4 valve (V4)
PUMP P-900

Mixer

Pump A Pump B

Proportion
Valve
A2 A1 B2 B1

Acid /Alkali Buffer Salt Water

GRADIENT MIXER
ÄKTA 10 mls/min <6 mls/min 0.6 ml mixer
6 -14 mls/min 2 ml mixer
ÄKTA 100 mls/min <30 mls/min 2 mls mixer
30 - 50 mls/min 5 mls mixer
50 - 100 mls/min 12 mls mixer
PUMP P-900 Inlets

Mixer

Pump A Pump B

Switching
Valves

Buffer Select A2 B1 B2
A1
Valve IV-908

1 8 Water Salt
2 7
6
3 5
4

A12 A14 A16 A18


A11 A13 A15 A17
PUMP P-900 Manually Priming Buffer Inlet Lines

Pump A Pump B

Common inlet A2 A1 Common inlet B2 B1

Manual Flowpath Manual Flowpath

A1 or A2 BufferValveA B1 or B2 BufferValveB

Open

Draw A
ir
out of
Buffer
Line
PUMP P-900 Back - Wash Lines

Pump Back-Wash
Lines

Syringe fitting
Wash OUT

IN-LINE
one-way Buffer
valve OUT
Ball outlet
High pressure check valve
Piston seal

Piston

20% Ethanol : Water

Backwash pump
diaphragm
Ball inlet
check valve

Wash Buffer
IN IN
ON-LINE FILTERS

P-903 ( 10 ml/min Pump )


Low Flow HPLC Filter

P-901 ( 100 ml/min Pump )


High Flow Fibre Filter

Polymer Support

LOAD (sample) INJECT (sample) LOAD (wash loop)


Column Column
Column 1
1
1
2 7 Pump
2 7 Pump
2 7 Pump
3 6
3 6
3 6 5
5 4
5 4
4

PV-908 PV-908
PV-908 1 8
1 8
1 8
7
7
7 2
2
2

3 6
3 6
3 6 4 5
4 5
4 5

Sample Waste Sample Waste


Sample Waste
INJECTION VALVE INV- 907
1
COLUMN
2 7
PUMP
LOAD 3
SAMPLE 6
4 5

Waste Waste

LOAD
1
COLUMN
2 7
PUMP
3
6

4 5

Waste Waste

INJECT

1
COLUMN
7
2 PUMP
3 6
4 5

Waste Waste

WASTE
SAMPLE LOADING METHOD

1
COLUMN
2 7 PUMP
LOAD
SAMPLE 3
6

4 5

Needle Port

Waste Waste

Always tighten by hand


with needle inserted
Do Not Overtighten

1
COLUMN

LOAD 2 7 PUMP
SAMPLE
3
6
4 5

Luer Lock
Adapter

Waste Waste Superloop


SUPERLOOPS

10 ml
50 ml
Buffer
150 ml
Plunger BUFFER
FLUSH
SAMPLE
LOAD
SAMPLE
INJECT
SAMPLE
FLUSH

Sample

Etched
Groove

PUMP or
WASTE COLUMN COLUMN
SYRINGE

Volume Max. allowed column Code No.


pressure

10 ml 4 Mpa 18-1113-81
50 ml 4 Mpa 18-1113-82
150 ml 2 Mpa 18-1023-85
INV-907 Injection Valve - Injection Port

PEEK Valve Body

Injection Needle Port

Valve Seal

Ferrule
Needle

Needle
Adapter
NeedleTube
INV-907 Injection Valve - Luer Adapter

PEEK
PEEKInjection
InjectionValve
ValveBody
Body

PEEK
PEEKInjection
InjectionLuer
LuerAdaptor
Adaptor

Valve
ValveSeal
Seal

Valve
ValveChannel
ChannelPlate
Plate
Fluid Dynamics

Flow
PEEK Tubing
High Resistance
to Flow

Low Resistance
to Flow

End Beginning
Fluid Dynamics
Flow - Injecting a Loop

The way we imagine it Flows

The way it Actually Flows

Bubble Segmentation

100ul Loop
Waste 50ul Load

60ul Load

100ul Load

200ul Load

• Partial Loading Loop = No more than 1/2 Loop Volume


• Total Loading Loop = Inject 2X Loop Volume
LOADING A SAMPLE LOOP

Injection valve
INV-907
1
Column
2 7
Pump
Load 3 6
Inject 4 5

Sample Sample
pump
P-950 Waste
LOADING A SUPER LOOP

Injection valve
INV-907
1
Column
7
2 Pump
Load 3
6
Inject 4 5

Buffer

Sample

Waste

Sample
MULTI-LOADING A SAMPLE LOOP

Injection valve
INV-907
Column 1
7
2 Pump
Load 3 6
Inject
4 5

PV-908

1 8
2 7
I/O
3 6
4 5

Waste
Wash
Samples
MULTI-LOADING A SUPER LOOP

Injection valve
1 INV-907
Column
2 7
Pump
Load 3
6
Inject
4 5

PV-908
1 8
Waste 2
I/O 7
3 6
4 5

Samples

Wash
S3 S4 S5 S6 S8
DIRECT LOADING ON TO COLUMN

1
Column
2 7 Pump
3
6
Inject
4 5
Load

Extra Tube

1/16” or 1/8”
Tubing

Waste

Sample
REVERSE FLOW VALVE ( INV-907 )
1
2 7
Pump
3 6
5
4
Column Extra Tube Stop
Plug

MONITOR

Downward Flow
Position 1

1
2 7
Pump
3 6
5
4
Column Extra Tube
Stop
Plug

MONITOR

Upward Flow
Position 2
OUTLET VALVE PV-908

DETECTOR
To Fraction
Collector
Default

F1 F8
F7
F2 I/O
F3 F6
F5
F4

F7

Waste F4 F8
F5 F6
F3
Fraction Collection
Flowthrough Eluate Peak
Fractionation Fractionation Fractionation

mAU 0%B 100%B %B

500 100

ND
ND OU
OU B
250
NB 50
Inject

U
Start

End

Waste
0 0

A1 A2 A3………………….
HiTrap Connections

1/16” Female

1/16” Male
18-1112-55
1/16” Fingertight Fitting 1/16” Female

M6 Male
18-1112-57
M6 male / 1/16” Female
Phenyl Sepharose

HiTrap Column

M6 Male

M6 Female
18-1112-58

M6 female / 1/16” male


1/16” Male

Flow Cell Inlet


Multiple HiTrap Arrangment ( Series )

ct or
te
De
Phenyl Sepharose Phenyl Sepharose Phenyl Sepharose

e ct or
t
De
Phenyl Sepharose Phenyl Sepharose

ct or
te
De
Phenyl Sepharose
Traditional FPLC fitting to ÄKTA / HPLC

Injector

1/16” Fingertight Fitting


18-1112-55

1/16” FEMALE / M6 FEMALE


10
0u 18-1123-94
l

Flange M6 Fitting

1/16” MALE / M6 FEMALE


18-1112-58

Flow Cell
Tubing Fittings

18-1112-55
TM
1/16” Fingertight Fitting 1/16” PEEK Tubing

18-1112-58

Flange Fitting Adapter FPLC Flange Fitting

18-1112-51

Luer Fitting Adapter Luer Syringe

18-1112-52 18-1112-50
Fingertight Stop Plug 5/16” Stop Plug

Flangless Inlet Connector Flangless Buffer Inlet


3/16” 18-1112-49 Ferrule
1/8” 18-1121-17 3/16” 18-1112-48
1/16” 18-1127-07 1/8” 18-1121-18
1/16” 18-1127-06

18-1112-57
18-1017-98
M6 male / 1/16” Female Tubing Connector
M6 male / O - ring

18-3955-01
1/16”female X 1/16”female Union Ti Union + 2 Fingertight Fittings

18-1123-94

M6 female X 1/16”female Union


FPLC Flange - 1/16” Fingertight
COMPRESSION FITTINGS

Untightened

Correctly tightened

Obstructed

Over tightened Tubing Restricted


PEEK Tubing
TM

Gray, 1/16”x 1.0 mm ( 7.85ul / cm )

Green, 1/16”x 0.75 mm ( 4.42ul / cm )

Orange, 1/16”x 0.5 mm ( 1.96ul / cm )

Blue, 1/16”x 0.25 mm ( 0.49ul / cm )

Teflon / Tefzel Tubing

3/16” OD 18-1112-47

1/8” OD 18-1121-16

18-1119-74
1/16” OD
Tubing Kit Changes - ÄKTApurifier 10

GENERAL FLOW (at delivery)

G5
10
0u
l

Injection valve V1

G9

Outlet Valve V4 (7ul)

Accumulator
(40ul)
UV Flow cell

G8

G6 G7
Conductivity
Flow cell (14ul)
0.25 mm i.d
FR-904
Blue PEEK (0.2Mpa, 5ul)
G5 400mm NB: Never use
G6 160mm the FR-904 with
G7 80mm the pH electrode
G8 130mm
G9 500mm pH Flow cell (100ul)

Tubing Kit No Frac Frac 901 Frac 950


AKTApurifier 10 , 0.50 tubing kit 232 382
AKTApurifier 10 , 0.50 tubing kit + pH electrode 354 504
AKTApurifier 10 , 0.25 tubing kit 88 238
Tubing Kit Changes - ÄKTApurifier 10

HIGH FLOW

G5
10
0u
l

Injection valve V1

H9

Outlet Valve V4 (7ul)

Accumulator
(40ul)
UV Flow cell

H8

H6 H7
Conductivity
Flow cell (14ul)

0.50 mm i.d FR-904


(0.2Mpa, 5ul)
Orange PEEK NB: Never use
H6 160mm the FR-904 with
H7 80mm the pH electrode
H8 130mm
H9 500mm
pH Flow cell (100ul)

Tubing Kit No Frac Frac 901 Frac 950


AKTApurifier 10 , 0.50 tubing kit 232 382
AKTApurifier 10 , 0.50 tubing kit + pH electrode 354 504
AKTApurifier 10 , 0.25 tubing kit 88 238
Tubing Kit Changes - ÄKTApurifier 100

HIGH FLOW (at delivery)

G5
10
0u
l

Injection valve V1

G9

Outlet Valve V4 (7ul)

Accumulator
(40ul)
UV Flow cell

G8

G6 G7
Conductivity
Flow cell (14ul)
0.75 mm i.d
FR-904
Green PEEK (0.2Mpa, 5ul)

G6 160mm NB: Never use


G7 80mm the FR-904 with
G8 130mm the pH electrode
G9 500mm
pH Flow cell (100ul)

Tubing Kit No Frac Frac 901 Frac 950


AKTApurifier 100 , 0.50 tubing kit 232 382
AKTApurifier 100 , 0.75 tubing kit 470 620
AKTApurifier 100 , 1.00 tubing kit 769 809
AKTApurifier 100 , 0.50 tubing kit + pH electrode 354 504
AKTApurifier 100 , 0.75 tubing kit + pH electrode 672 862
AKTApurifier 100 , 1.00 tubing kit + pH electrode 955 995

AKTAbasic 100 , 0.75 tubing 290


Tubing Kit Changes - ÄKTApurifier 100

LOW FLOW

L5
10
0u
l

Injection valve V1

L9

Outlet Valve V4 (7ul)

Accumulator
(40ul)
UV Flow cell

L8

0.50 mm i.d L6 L7
Conductivity
Orange PEEK Flow cell (14ul)
L6 160mm FR-904
L7 80mm (0.2Mpa, 5ul)
NB: Never use
L8 130mm
the FR-904 with
L9 500mm the pH electrode

pH Flow cell (100ul)

Tubing Kit No Frac Frac 901 Frac 950


AKTApurifier 100 , 0.50 tubing kit 232 382
AKTApurifier 100 , 0.75 tubing kit 470 620
AKTApurifier 100 , 1.00 tubing kit 769 809
AKTApurifier 100 , 0.50 tubing kit + pH electrode 354 504
AKTApurifier 100 , 0.75 tubing kit + pH electrode 672 862
AKTApurifier 100 , 1.00 tubing kit + pH electrode 955 995
A-900 Autosampler
A-900 INV-907
1
2 1
2 7 PUMP
3 6 3
6
4 5 MANUAL 4 5
SAMPLE
LOAD
LOAD
Waste Waste
COLUMN LOAD
Sample
Syringe

A-900 INV-907
1
2 1
2 7 PUMP
3 6
3 6
4 5 MANUAL 4 5
SAMPLE
LOAD

INJECT
Waste Waste
COLUMN LOAD
Syringe Sample
Needle Height
Standard Vial

Standard Vial
+
Needle Height Insert
volume left
2mm
450ul

1mm 3mm
350ul 12ul

0mm
200ul
A-900 Sample Injection ( Flushed Loop Injection )
INJECT LOAD INJECT INJECT
INJECT LOAD INJECT INJECT

pump pump pump pump

waste waste waste waste

column column column column

Sample needle

sample sample sample Wash sample


Reservoir

InInINJECT,
INJECT,sample
sample InInLOAD
LOADThe Thesample
sample
InInINJECT
INJECTsample
sample Finalinternal
Final internal
flushesthe
flushes thevalve
valve isisdrawn
drawnup upto
to
isiswashed
washedonto
onto needlewash.
needle wash.
channel. completely
completely thecolumn.
column.
channel. the
Min.( (30ul
30ul) )with
with fillthe
fill theloop.
loop. pump
Min.
AirSegment
Air Segment ( (no
noless
lessthan
thantwice
twice waste
Min.( (35ul
35ul) )without
without loopvolume
loop volume
Min. column
Airsegment
segment recommended) )
recommended
Air INJECT
INJECT

sample
OptionalExternal
Optional External
transport
needlewash.
needle wash. fluid
A-900 Sample Injection ( Partial Loop Injection )
INJECT LOAD INJECT INJECT
INJECT LOAD INJECT INJECT
waste
pump pump pump pump

waste waste waste waste


column column column column

Sample needle Sample needle Sample needle

sample sample sample Wash sample


Reservoir

Thesample
sampleisisdrawn
drawn Finalinternal
internal
InInINJECT,
INJECT,sample
sample The InIninject
injectsample
sample Final
flushesthethevalve
valve uptotopartially
partiallyfill
fillthe
the needlewash.
wash.
flushes up isiswashed
washedonto
onto needle
channel.
channel. loopwith
loop with thecolumn.
the column.
Min.( (30ul
Min. 30ul) )with
with programmedvolume.
programmed volume. pump
AirSegment
Air Segment ( (no
nomore
morethan
thanhalf
half waste
Min.( (35ul
Min. 35ul) )without
without loopvolume
loop volume
Airsegment
Air segment recommended) )
recommended column
INJECT
INJECT

OptionalExternal
Optional External sample

needlewash.
needle wash. transport
fluid
A-900 Sample Injection ( ul Pick-up Injection )
INJECT LOAD LOAD INJECT
INJECT LOAD LOAD INJECT

pump pump pump pump

waste
column column column
column

transport Sample needle Sample needle Sample needle


fluid

sample
sample sample sample

InIninject,
inject,valve
valve Thesample
The sampleisis Thesample
The sampleisis InIninject
injectsample
sample
flusheswith
flushes with drawnup
drawn upfrom
from washedinto
washed into isiswashed
washedonto
ontothe
theco
column
lumn
transportfluid
transport fluid thesample
the samplevial
vial theloop
the loopwith
with
transportfluid
transport fluid pump

INJECT pump waste


INJECT
waste
column
INJECT
Finalinternal
internal column INJECT
Final
needlewash.
needle wash.
sample
Wash sample
Optionalneedle
Optional needle
Reservoir wash.
wash. transport
fluid
System Compensation Volume

Equilibration Phase

A
COLUMN TOP

B 0%

A COLUMN TOP

B 20% 8 mls
Effect Command

20%B

0%B

8mls
Gradient Delay Volume
Gradient Phase
A
COLUMN TOP

B 0%

A COLUMN TOP

B 100%
16 mls Command 100%
Effect

100%B

Gradient Delay
Column Pressure ( compensation ) Flow Restrictor

Column Hardware Pressure Limits Column Shatters DANGEROUS


Column Pressures
Column Matrix Pressure Limits Matrix Collapses EXPENSIVE

0.3Reading
0.3 Reading Atmos
Atmos
Press
Press
0.0MPa
0.0 MPa 0.5Reading
Reading
0.5
Pre-column pressure Pump Pre-column pressure Pump
Pump Pump
= 0 MPa 0.3Mpa
Mpa = 0 MPa 0.3Mpa
Mpa
0.3 0.3

0.3 MPa Gel Top Pressure 0.3 MPa

GELPress
PressDrop
Drop ColumnPress
PressDrop
Drop GELPress
PressDrop
Drop ColumnPress
PressDrop
Drop
GEL Column GEL Column
0.3MPa
0.3 MPa 0.3MPa
MPa 0.1MPa
0.1 MPa 0.5MPa
0.5 MPa
0.3
(0.3- -0.0)
0.0) (0.3++0.0)
0.0) (0.3- -0.2)
0.2) (0.5++0.2)
0.2)
(0.3 (0.3 (0.3 (0.5

0.0 MPa Gel Bottom Pressure 0.2 MPa

0.0MPa
0.0 MPa 0.2MPa
MPa
0.2
FlowRestrictor
Flow Restrictor

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