Biacore 2000 Instrument Handbook AD Web

INSTRUMENT HANDBOOK
Biacore2000
BIACORE 2000
Instrument Handbook

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_________________________________________________________________________ Contents
Contents
1. Introduction

2. Description
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5. Application Wizards
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7. Maintenance
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A. System specifications
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B. Application guidelines
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C. Chemical resistance and recommended solutions

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Contents _________________________________________________________________________
E. Writing BIACORE 2000 methods

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Index

______________________________________________________________________ Introduction
1. Introduction
1.1 Biomolecular interaction analysis

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1.2 Safety information

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1.3 Conformance with standards

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_______________________________________________________________________ Description
2. Description

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Figure 2-1. BIACORE 2000 system processing unit and controlling

computer.
2.1 BIACORE 2000 processing unit

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Description _______________________________________________________________________

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IFC Connector Autosampler

block
Sensor Chip
Eluent pump Autosampler pump
Waste bottle Buffer bottle
Figure 2-2. The liquid delivery system with two pumps and an autosampler
transports liquid to the connector block where samples and buffers are
injected to the IFC.
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Figure 2-3. The autosampler compartment.
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2.1.3 Connector block

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Figure 2-4. Side view of the connector block, showing the injection port,
wash station and recovery cup.
2.1.4 Integrated -Fluidic Cartridge (IFC)

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Figure 2-5. Diagram of the channels and valves in the IFC as seen from
above. The inset shows a side view of how the flow cells are formed by
pressing the sensor chip against the IFC. Not to scale.
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Description _______________________________________________________________________
Figure 2-6a. Schematic illustration showing the flow paths during direct
injection. The symbols o and represent open and closed valves
respectively. Not to scale.
During preparation, the leading sample is flushed to waste to ensure
minimum dispersion at the front of the injected sample plug. The continuous
flow pump maintains a constant flow of buffer through the flow cell. The
enlargement shows two air segments separating the sample from buffer
which prevent mixing in the autosampler tubing and IFC channels.
During sample injection, the sample is driven directly through the flow cell by
the autosampler pump.
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_______________________________________________________________________ Description
Figure 2-6b. Schematic illustration showing the flow paths during loop
injection. The symbols o and represent open and closed valves
respectively. Not to scale.
During loop loading, the continuous flow pump maintains a constant flow of
buffer through the flow cell.
During sample injection, buffer from the continuous flow pump pushes the
sample out of the sample loop over the sensor surface in the opposite
direction from loading.

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Description _______________________________________________________________________
Prism
Opto-interface
Prism
Sensor chip
Integrated -Fluidic Cartridge with flow cell block Flow cells
Figure 2-7. Precision-cast channels in the surface of the IFC define four
flow cells on the sensor chip surface. These are matched by ridges of
transparent polymer on the opto-interface, ensuring good optical contact
between the components of the system. Not to scale.
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Figure 2-8. Simplified diagrammatic illustration of sample flow through two

flow cells. The symbols o and represent open and closed valves
respectively. Valves and channels not used in other flow path arrangements
are not included. Not to scale.
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_______________________________________________________________________ Description
2.1.5 Sensor chips

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Surface matrix
Linker layer
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Figure 2-9. Sensor Chip CM5 is a glass slide coated with a thin gold film to
which the carboxymethyl dextran surface matrix is covalently bonded. The
matrix surface forms one wall of the detector flow cell.
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Figure 2-10. The detection unit houses the optical unit with light source and
detector, the opto-interface and the removable sensor chip. Flow cells on
the sensor chip are connected to the liquid handling system through the
Integrated -Fluidic Cartridge. The broken line shows the boundaries of the
thermally insulated box.
2.1.7 Temperature control

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Description _______________________________________________________________________
2.2 BIACORE 2000 system control

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2.5 BIAsimulation software

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3. Installation

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4. Operation
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4.1.1 General Windows techniques

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4.1.3 BIACORE 2000 software screen regions

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Operation ________________________________________________________________________
Menu/Command Toolbar button Shortcut key

File
Open Ctrl-O
Save Ctrl-S
Print Ctrl-P
Edit (sensorgram)
Copy Graph Ctrl-H
Add Report Point
Edit (method)
Undo Ctrl-Z
Cut Ctrl-X
Copy Ctrl-C
Paste Ctrl-V
Find Ctrl-F
Replace Ctrl-R
View
Unzoom Ctrl-U
Reference Line F8
Baseline F9
Notebook
Wizard Results Ctrl-R

Command
Inject/Stop Inject
Transfer
Mix
Flow/Stop Flow
Run
Run/Stop Sensorgram
Stop Sensorgram
Tools
Evaluation Ctrl-E

________________________________________________________________________ Operation
4.2 Preparing the system

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1. Place the sensor chip cassette on the slide with the label facing upwards
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Command:Define Rack Base !! !% &&

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The autosampler compartment viewed from the front, showing the position
R2F2 used for BIAnormalizing solution. Thermo-rack type B is shown in both
base positions.
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4.2.6 Setting the temperature

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Operation ________________________________________________________________________
4.4.2 Data collection rate

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4.4.3 Starting a manual sensorgram

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Command Comments Injected Sample Max flow

volume consumption rate
INJECT General purpose inject. 5-325 l Injected vol +30 l 100 l/min
KINJECT Low dispersion inject for 10-250 l Injected vol +40 l 100 l/min
kinetics and for samples
sensitive to dilution with
running buffer. Controlled
dissociation time after
sample injection.
QUICKINJECT Low sample consumption. 5-325 l Injected vol +10 l 100 l/min
Less preparation time than
INJECT but more sample
dispersion.
COINJECT Injection of second sample Sample 1: Sample 1: 100 l/min
immediately following the 10-100 l injected vol +40 l
first (e.g. for dissociation in
Sample 2: Sample 2:
the presence of ligand).
15-250 l injected vol +40 l
Variable contact times for
first sample only. Recovery
of either first or second
sample, but not both.
BIGINJECT Large volume inject. 326-750 l Injected vol +52 l 50 l/min*
Direct inject up to 325 l,
sample loop between 326
and 425 l, direct between
426 and 750 l.
MANUAL INJECT Manually controlled inject. 1-100 l / Loaded vol +30 l 100 l/min
See Section 4.3.9. loading
*BIGINJECT cannot be performed at higher flow rates because the auto-
sampler tubing must be refilled during injection from the sample loop for
samples larger than 425 l.
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________________________________________________________________________ Operation
Inject Injected Preparation Finishing No. of air

command volume (l) time (s) time (s) segments
INJECT 20 60 40 2
100 70 45
QUICKINJECT 20 40 25 1
100 30 30
KINJECT 20 80 40 3
100 90 40
COINJECT 20, 20 185 80 3
100, 100 250 80
BIGINJECT 350 345 90 3
750 340 95
MANUAL Preparation time for loop 85 2
INJECT loading 100 l: 80 s
Approximate times for preparation and completion of various inject
commands at a flow rate of 20 l/min. Preparation time is the time between
the inject command and the actual start of sample injection. Finishing time is
the time between the end of sample injection and completion of the
command, during which the flow system is washed. Finishing times for
KINJECT do not include the dissociation time specified in the command.
Predip Needle adds about 8 s to the preparation time. Extra Cleanup adds
about 30-40 s to the finishing times for INJECT, QUICKINJECT and
KINJECT and about 70 s to the finishing times for COINJECT and
BIGINJECT.. Recover of 25 l adds about 210 s to the finishing times.
4.4.7 Stopping an injection

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Inject ! #F
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________________________________________________________________________ Operation
* %!!&"! &&'
define aprog practice

flow 30
dilute r1a1 r1a2 r1a3 50
inject r1a3 25
end
main
detection 1
aprog practice
end
! &&!! 7
9!
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FLOW 30
DILUTE r1a1 r1a2 r1a3 50
INJECT r1a3 25
END
MAIN
DETECTION 1
APROG practice
END
SecondsMinutes F %

# %
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INJECT

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Operation ________________________________________________________________________
&&& MAIN

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________________________________________________________________________ Operation
4.7 Closing down the system
4.7.1 BIACORE 2000 Processing Unit

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________________________________________________________________________ Operation

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9. BIACORE Control Software

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9.1 Starting BIACORE Control Software
9.1.1 Starting from the Windows Start menu

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8

_______________________________________________________________System specifications
A. System specifications
A.1 System performance
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B. Application guidelines
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flow rates.
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8

________________________________________________________________ Chemical resistance
C. Chemical resistance and

recommended solutions
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_______________________________________________________Status and event log messages
D. Status and event log messages
D.1 Status window messages

!"## !!
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Not Connected to Instrument

( ( ! &
($ ( ( !*
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Status and event log messages _______________________________________________________
D.2 Event log messages

%# !" )
&- # &
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! !
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4 (
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(
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# (; $ (
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# (# # &
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(# # .;
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8

Low data quality

! $ " ( #((!
& "#" % ) &
Mix: End
# (%# &
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(%# &% )
## .& & Mix: Start 120 l R1A10&
Normal data quality

$ " (!
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Run aborted
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Set temperature
$ # $
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Temperature
#
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# &
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# &
Terminate run
! (
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Timestamp
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Transfer: End
# ( (# &
/

Transfer: Start
( (# &) (
# .& & Transfer:
Start 80 l R1A1->R1A20&
Wash: <item>
(!# & !
.& &Wash: Recovery R1A1 )"#
!! (# 0&
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(!# &

=

9

______________________________________________________ Writing BIACORE 2000 methods
E. Writing BIACORE 2000

methods

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Writing BIACORE 2000 methods _____________________________________________________
E.1 Basic method programming

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The DEFINE LOOP block

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TIMES 2
Sample1 r1a1 10
Sample2 r1a2 15
Sample3 r1a3 10
Sample4 r1a4 20
END

PARAM %sample %position %volume
KEYWORD sample %sample
FLOW 20
* INJECT %position %volume
-0:15 RPOINT baseline -b
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5:00 INJECT r2a1 10 ! regenerate
END
MAIN
DETECTION 1
LOOP cycle STEP
APROG analyse %sample %position %volume
ENDLOOP
END
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TIMES 2 ! do the following rows twice
Sample1 r1a1 10
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TIMES 1
1 10
2 20
3 30
4 40
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APROG analyse %volume
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LPARAM %s2 %p2
A r1a1
B r1a2
C r1a3
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FLOW 20
LOOP cycle1 STEP
LOOP cycle2 STEP
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APROG regenerate
ENDLOOP
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The DEFINE ALERT and DEFINE MESSAGE blocks
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DEFINE ALERT insert_sample

TITLE Unstable sample
BEEP
LTEXT
LTEXT Continuation of this method requires
LTEXT sample in position r2a1. Make sure that
LTEXT the sample is completely thawed before
LTEXT placing it in the autosampler rack.
LTEXT
LTEXT Close the door and click on Continue
LTEXT when you are ready.
GO Continue
STOP Cancel
END
MAIN
RACK 1 thermo_A
RACK 2 thermo_B
ALERT insert_sample
APROG cycle1
...
END
E.3.5 Additional data curves

The CURVE command

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CURVE bufflow ! use default axis
FLOW 20
...
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E.3.6 Closing down

The APPEND command
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APROG analyse
APROG regenerate
APPEND standby
END
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E.4 Method techniques

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E.4.1 Detection mode and flow path
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DETECTION
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APROG'
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MAIN
DETECTION 1 ! single channel mode
APROG single
DETECTION 1,2,3,4 ! multichannel mode
APROG multi
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MAIN
DETECTION 1,2,3,4
APROG epitope
END
DEFINE APROG epitope

PARAM %pos1 %pos2 %pos3 %pos5 %pos5
FLOW 5
FLOWPATH 1,2,3,4
INJECT %pos1 5 ! first Mab
INJECT r1c1 5 ! analyte
...
...
FLOWPATH 1
INJECT %pos2 5 ! second Mab 1
...
FLOWPATH 2
...
FLOWPATH 3
...
FLOWPATH 4
...
FLOWPATH 1,2,3,4
INJECT r1d3 15 ! regenerate
...
END
%
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%
DETECTION '
'

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&

FLOWPATH
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$%.

'

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'

$$
MAIN
DETECTION 4 ! single channel detection in Fc
4
APROG missing_data
END
DEFINE APROG missing_data

FLOW 20
FLOWPATH 1,2,3 ! sample flow through Fc 1-2-3
! detection only in Fc 4
...
END
%
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MAIN.*

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0 '
(B B B (B
(BG(: B:G B:G (BG(:
(B:(BG(B B:G B:G (B:(BG(B


)
'&

MAIN
DETECTION 2-1,4-3 ! single channel mode
APROG subtract_reference
END
E.4.2 Sample injection types

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DEFINE APROG smooth_inject

FLOW 35
10:00 WASH n
FLOW 35 -f ! refill pump
KINJECT r1a1 140 240
END
*INJECT''

'7
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sample injection.
dispersion.
Sample 2: Sample 2:
426 and 750 l.
E.4.3 Variable contact time injections

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35,25,25,10
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3
MAIN
DETECTION 1,2,3,4 ! All four flow cells in flow
path
APROG immob
END
DEFINE APROG immob

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...
INJECT r1a1 35,25 ! Flow cells 1 and 2 for INJECT
... ! Back to all four flow cells
END

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DETECTION 1,2,3,4
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E.4.5 The EXTRACLEAN command

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DEFINE
APROG''* )
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APORG '
APROG#$
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56
7
83
DEFINE APROG test

FLOW 20
>> Unrecognized command
RACK 1 Thermo_a
>> ^
! RACK cannot be used in APROG
MAIN
>> Unrecognized command
APORG test
>> ^
! APORG should be APROG
-
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*

#MAIN.3
DEFINE APROG test

FLOW 20
INJECT r1a1 10
! END missing
>> Expecting keyword END

MAIN
>>^
DETECTION 2
>> Characters outside DEFINE-END or MAIN-END blocks
APROG test
>> ^
END

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PARAM %flow %pos %vol
>> Expecting an integer value, min=0 max=100
FLOW %folw
>> ^
! variable name should be %flow
INJECT %pos %vol

END
DEFINE LOOP cycle

LPARAM %flow %pos %vol
5 r1a1 10
10 r1a1 10
END
MAIN
DETECTION
LOOP cycle STEP
APROG test %flow %pos %volume
>> ^
! variable name should be %vol
ENDLOOP
END

E.5.4 Incorrect parameter values

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E.5.5 Incorrect parameter ordering

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PARAM %vol %pos
FLOW 20
INJECT %pos %vol
END
MAIN
DETECTION 1
>> Expecting a value value, min=5 max=325
APROG test r1a1 10
>> ^
! APROG test is defined with volume before position
END

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DEFINE APROG test

FLOW %flow
INJECT %pos %vol
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LPARAM %vol %pos %flow
10 r1a1 5
20 r1a1 10
END
MAIN
DETECTION 1
LOOP
APROG test %vol %pos %flow
ENDLOOP
END
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>> ^
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8''
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DEFINE APROG test

PARAM %sample %mix %vol
FLOW 20
! TRANSFER %sample %mix %vol
! MIX %mix 100
INJECT %mix 10
END
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DEFINE APROG test

FLOW %flow
INJECT %pos %vol
END
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DETECTION 1
>> Missing parameter
APROG test 25 r1a1
>> ^
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! No value given for %vol
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E.5.7 Prerun error messages

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__________________________________________________________ Method language reference
F. Method language reference

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GO 1 $ !0 2!
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RTEXT 4 , / /,
STOP 1 $ !0 2!
TITLE 3 , ! ALERT,

Method language reference __________________________________________________________

BIGINJECT 6/. 7 8 9:;
<
CAPTION 3 !/.
COINJECT 1 7
CURVE & . !
DILUTE 1 %
DIPNEEDLE & .
EXTRACLEAN , %! 7
FLOW 3 ! %
FLOWPATH 1!! %
FRACTIONS . / /!!! %
INJECT 7
KEYWORD 1!
KINJECT 6%7
MIX =,
MODE 3
PARAM 1 . APROG
QUICKINJECT >7
RECOVER .7 /
RPOINT 3
SCALE 3 ! !/ .
TRANSFER ! % .
WAIT 4 !
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LPARAM 1 . DEFINE LOOP
TIMES 3 ! !.

BEEP 3
CLOCK 1 $ %
CTEXT 4 , 5 $,
LIFETIME 3% !%/ $
LTEXT 4 , ! /,
PARAM 1 . MESSAGE
RTEXT 4 , / /,
TITLE 3 , ! MESSAGE ,


ALERT 1 $ALERT,
APPEND !
APROG $/( APROG+
BEEP 3
DETECTION 3 ! %
DILUTE 1 %
ENDLOOP !
FLOW 3 ! %
FLUSH %/ Flush
LOOP
MESSAGE 1 $MESSAGE,
MIX =,
NORMALIZE %/ Normalize
PRIME %/ Prime
RACK 3 $
RINSE %/ Rinse
TEMP 3
TRANSFER ! % .
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WAIT 4 !
WASH 4 ' /$
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8

ALERT name [parameter ...]
1 $ALERT,"
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& ALERT,"
& ALERT ' $

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4 LOOP - $

. . "D
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, B DEFINE ALERT check_solution

PARAM %pos
LTEXT Place test solution in position %pos
GO OK
END
MAIN
ALERT check_solution r1a1
END
@

APPEND standby
%/ Standby! "
A/BMAIN "
& B
C! ! "
- $ Standby "
APPEND MAIN "
C B %/ Continue...

! ! %
Standby"% $- Continue
$ % APPEND"
, B MAIN
DETECTION 1
APROG immob
APROG analyse ...
APROG regenerate
APPEND standby
END

;

APROG name [parameter ...]
$/( APROG+"
A/BMAIN "
& B C! /! DEFINE

APROG"
& APROG"
& APROG ' $

. PARAM DEFINE APROG"
4 LOOP - $

. . "D
. "
, B DEFINE APROG injection

FLOW 20
END
MAIN
DETECTION 1
END
9

[*] [time] BIGINJECT position volume
6/. 7 (8 9:;

< +"
A/BDEFINE APROG "
& B ! ! B==B33-

===B33333" (E+!
! .
5 !!'
! "
! "
D 7 - ! !

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< ?"
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RECOVERFRACTIONS % BIGINJECT"
3 7 . F; < "
, B DEFINE APROG large_vol

FLOW 50
*0:30 BIGINJECT r1a1 500
4:00 RPOINT response !rpoint 4 min
!after injection start
END

:

BEEP
3! "
A/BDEFINE ALERT-DEFINE MESSAGEMAIN "
& B C"
, B MAIN
APROG immob
BEEP
APROG analyse
END
DEFINE MESSAGE box

BEEP
BEEP
LTEXT Don't miss this message!
END
CAPTION text
3 ! /"
A/BDEFINE APROG "
& B , "
, B DEFINE APROG temp_check

CAPTION Temperature check
CURVE temp
FLOW 10
INJECT r1a1 10
END
G

CLOCK time [option]
1 $ % "
A/BDEFINE MESSAGE "
& B 3 / ! " B==B33-

===B33333"
B 1 $ $"
, B DEFINE MESSAGE countdown

RTEXT minutes remaining
CLOCK 5:00 -m
END

H

[*] [time] COINJECT position1 volume1 position2 volume2
6%7 % % ! %/7 -"/"!
/ // "
A/BDEFINE APROG "
& B ! ! B==B33-

===B33333" (E+!
! .
5 !!'
! "
! "
D ! - ! !

. $!.
7 (3 "@"8+"
/

< "
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D ! -/; ;

< "
3 7 ! "3 7

7 "D $!!
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FRACTIONS % COINJECT"RECOVER!

"
3 7 . F@

< !
"
, B DEFINE APROG double_inject

FLOW 50
*0:30 COINJECT r1a1 50 r1a2 150
0:50 RPOINT resp1 !rpoint 1st inject
3:50 RPOINT resp2 !rpoint 2nd inject
END

CTEXT [text]
4 , ALERTMESSAGE,"
A/BDEFINE ALERTDEFINE MESSAGE "
& B , / @

"
MESSAGE,!
"
, B DEFINE MESSAGE demo

TITLE DEMONSTRATION BOX
LTEXT This text is left-adjusted
CTEXT This text is centered
RTEXT This text is right-adjusted
END

CURVE item [option ...]
& . ! /%%"
A/BDEFINE APROG "
& B C! B

!!! % (
! %+

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,"
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9 $ % @ / $ %
: % ; / %
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8
!!

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, B DEFINE APROG temp_check

CURVE temp -o
FLOW 10
INJECT r1a1 10
END

DEFINE ALERT
1! /, /% 0 20 2

"
A/B ! "
3$ ,B DEFINE ALERT name

[PARAM variable ...]
other commands
END
GOSTOP . $ DEFINE

ALERT !$ , 0 20 2
ALERT,"
! STOPGO- ,%

0 2 % , Continue0 2
% , Abort"! STOPGO
- ,%
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% $ /,% $
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%

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" / 0 2
$"

8

DEFINE APROG
1!! $/"
A/B ! "
3$ ,B DEFINE APROG name

FLOW flowrate
other commands
END
APROG FLOW"FLOW

! APROG" !!
/( CAPTION-MODE-CURVE+ ! FLOW
"
DEFINE LOOP
3 !. ! . $"
A/B ! "
3$ ,B DEFINE LOOP name

LPARAM variable ...
[TIMES count]
value ...
... ...
END
33 "8"8! ! ! "
@

DEFINE MESSAGE
1!/,"
A/B ! "
3$ ,B DEFINE MESSAGE name

[PARAM variable ...]
other commands
END
/, $! ! "/

$ % MESSAGE- ALERT
, $ "
1 $//,

( DEFINE ALERT+"
/, . $/

"!$% !/ % /$
-DEFINE ALERT GO
"

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3 ! % "
A/BMAIN "
& B = /"

D B %"
DETECTION -! %
! / ! %/ B
!
. 1 %

8 8 8
@ @ @
- - -
- -
- -8-@ - -8-@ - -8-@
-@8 - -8-@ - -8-@ -@8
-8-@ - -8-@ - -8-@ -8-@
!! %

. "
! % ( ! +

' $/% FLOWPATH
APROG"INJECT% $ ! %
!'"33 "@"! "
DETECTION FLOWCELL .

! ! %"FLOWCELL ! $%
% ."
, B MAIN
DETECTION 1,2,3,4
APROG immob
APROG analyse
END
9

[*] [time] DILUTE position1 position2 mixpos percent
=, % "A! /,/"
A/BDEFINE APROGMAIN "
& B ! ! B==B33-

===B33333" (E+!
! .
5 !!'
! "
! "
! "
! , "
/ ! "

/;H;I"
! . !, !,
< " DILUTE

r1a1 r1a2 r1a3 20 @
< !9
< !
, 8"
DILUTE $'. % TRANSFER

! %$ MIX"DILUTE%. 5
! !% !,! . !
< "
DILUTE MAIN % . /"
$$! % /,

./! $!! . "
, B DEFINE APROG incubation

FLOW 25
*0:30 DILUTE r1a1 r1a2 r1b1 50
5:00 INJECT r1b1 50 ! inject 5 min after
! mixing sample
END

:

DIPNEEDLE position
& !. $.!! "
A/BDEFINE APROG "
& B & % "

< !
"A %
/! % !!/ $
!!!! .,-- / "
, B DEFINE APROG predip

FLOW 25
DIPNEEDLE r1a2 !sample buffer
INJECT r1a1 50
END
END
1! ! "
A/B ! "
& B C"
G

ENDLOOP
1! ! MAIN "
A/BMAIN "
& B C"
EXTRACLEAN
&!, /! "
A/BDEFINE APROG "
& B C"
4-EXTRACLEAN %$ $!

7 % !"J%.
RECOVERFRACTIONS % INJECT
EXTRACLEAN" /%
, $! 7 "

FLOW 20
INJECT r1a1 30
EXTRACLEAN !extra clean-up after injection
END

H

[*] [time] FLOW flowrate [option ...]
3 ! % "
& B ! ! B==B33-

===B33333" (E+!
! .
5 !!'
! "
% < ?"/

< ?"
B ! ! %! /

! %"
FLOW / $ DEFINE APROG -$
$$ CAPTION-CURVE-KEYWORD-MODE,
PARAMSCALE"
! $ FLOW MAIN
$ ! FLOW APROG"
%APROG MAIN - ! %

< ?
! "AFLOW MAIN !!! ! %
'"
, B DEFINE APROG sample_inject

FLOW 10
INJECT r1a1 10
END

[*] [time] FLOWPATH fc
3 !!! % "
A/BDEFINE APROG "
& B ! ! B==B33-

===B33333" (E+!
! .
5 !!'
! "
% "D

KK-K K-K8K-K@K-K- K-K- -8K
K- -8-@K"
FLOWPATH !!! % / !! %

!! "A DETECTION MAIN
! % "

FLOWPATH $ DETECTION
MAIN"/ - ! %
% "33 "@"!
"
% / APROG"4 APROG

- ! % /
! APROG"
! % / $.$.

INJECT"! %
' ! INJECT! !
7 (3 "@"+"
, B DEFINE APROG analytes

FLOW 25
FLOWPATH 1
INJECT r1a1 50 !from r1a1 to Fc1
FLOWPATH 2
FLOWPATH 3
END
MAIN
DETECTION 1,2,3,4 !multichannel mode
APROG analytes
END

FLUSH
%/ Flush(3 :" "+"
A/BMAIN "
& B C"

FRACTIONS startpos volume no
. / /!!! %" $

/ "
A/BDEFINE APROG "
& B ! ! ! "
. "/;8 ;< "
C!! "/H9"
%$! % $! ! 7
INJECT-KINJECTQUICKINJECT"J%.
EXTRACLEAN % INJECT
FRACTIONS"FRACTIONS !
BIGINJECTCOINJECT"
! (;! ?+"!

' ! 5 . .
- ! 5%
! % ", -' /;< !
! % !
< ?% '

! ?B
! 5%
< "
! ! ! ! !

/ ! ! - ! %
/!! %
% "
, B DEFINE APROG collect

FLOW 20
INJECT r1a1 50
FRACTIONS r2a1 20 5 !collect
5 fractions
!of 20
l
EXTRACLEAN
END

8

GO name
1 $ !0 2! ALERT,"
A/BDEFINE ALERT "
& B , !

%%
$ ! "
, B DEFINE ALERT demo

GO OK
STOP CANCEL
END
@

[*] [time] INJECT position volume
L 7 "
A/BDEFINE APROG "
& B ! ! B==B33-

===B33333" (E+!
! .
5 !!'
! "
! "
D 7 - ! !
. $!.
7 (3 "@"8+"
/;8 ;< "
3 7 . F8

< "
, B DEFINE APROG immob

FLOW 5
DILUTE r1a5 r1a6 r1b1 50 !NHS/EDC
INJECT r1b1 35 !NHS/EDC
* INJECT r2a1 35 !ligand
5:00 INJECT r1c1 35 !ethanolamine
END

;

KEYWORD header value
1! $% "
A/BDEFINE APROG "
& B , ! /

"
-!.
, . "
$ . "
!. "
, B DEFINE APROG report

PARAM %conc
KEYWORD Antibody Mab-451
KEYWORD Conc %conc
FLOW 10
* INJECT r1a1 10
-15 RPOINT baseline -b
30 RPOINT plateau
END
9

[*][time] KINJECT position volume disstime
6% 7 ! "
A/BDEFINE APROG "
& B ! ! B==B33-

===B33333" (E+!
! .
5 !!'
! "
! "
D 7 - ! !
. $!.
7 (3 "@"8+"
/
;
< "
1 (!!!! % /
! % +"/
"
KINJECT / /

!!! / 5
.7 !"
!! 7 -/% /%
$ %/ ! %/ 7 "
3 7 . F@

< "
, B DEFINE APROG kinetics 1

FLOW 10
* KINJECT r1a1 30 240 !4 min dissociation
-20 RPOINT baseline
INJECT r1a3 5 !Regeneration
END

:

LIFETIME
1 $ !%/, ."
A/BDEFINE MESSAGE "
& B C"
!. $" $

/. ! CLOCK"
, B DEFINE MESSAGE develop

LTEXT This process has been running for
LIFETIME
END
G

LOOP name order

command
[...]
ENDLOOP
'!-/

!DEFINE LOOP"
A/BMAIN "
& B C! LOOP! DEFINE

LOOP"
! $%STEP-ORDER
RANDOM" !
%% DEFINE LOOP%
( 3 "8"8+B
STEP / .
/ TIMES
"
ORDER ! .
/ TIMES-
"
RANDOM % / .
"
$%. %
MAIN "
% LOOP '

!!. " MAIN
/ DEFINE LOOP"
, B DEFINE LOOP cycle

TIMES 3
Sample1 r1a1 10
Sample2 r1a2 15
Sample3 r1a3 10
END
MAIN
DETECTION 1
LOOP cycle RANDOM
APROG analyse %sample %pos %vol
ENDLOOP
END

H

LPARAM variable ...
1 . DEFINE LOOP"
A/BDEFINE LOOP "
& B
C! . DEFINE
LOOP "33 "" !.
/ "
LPARAM %$ ! DEFINE

LOOP -$ "

TIMES 3
Sample1 r1a1 10
Sample2 r1a2 15
Sample3 r1a3 10
END
MAIN
DETECTION 1
LOOP cycle RANDOM
ENDLOOP
END
LTEXT [text]
4 ! / , ALERTMESSAGE,"
& B , / @

"
MESSAGE,!
"

END
8

MAIN
1! /!APROG "
A/B ! "
3$ ,B MAIN
other commands
END
$MAIN "
MAIN /

. /"3/. // . $
/, ! APROG% ! MAIN "

8

MESSAGE [name] [parameter ...]
1 $MESSAGE,"
A/BMAIN "
& B C! MESSAGE,!

DEFINE MESSAGE"
D ! $ MESSAGE,"
& MESSAGE ' $

. PARAM DEFINE MESSAGE"
4 LOOP - $

. . "D
. "
/, %/,

, $ " MESSAGE%
% $, //,! "
, B DEFINE MESSAGE no_break

LTEXT Do not interrupt this cycle!
END
MAIN
MESSAGE no_break
APROG unbroken
MESSAGE !clear message box
...
END
8

[*][time] MIX position volume
=, . "
& B ! ! B==B33-

===B33333" (E+!
! .
5 !!'
! "
!,/"
D < ,"/;@;
< "
MIX MAIN % . /"
, B DEFINE APROG sample_mixing

FLOW 10
TRANSFER r1a1 r1a2 60
TRANSFER B r1a2 30
MIX r1a2 75
INJECT r1a2 40
END

88

MODE option [...]
3 !APROG"
A/BDEFINE APROG "
& B C"
B / . !/

"/
"M
"
=,!
/ . (
3 H"G+"/
M;
"1!

"
!MODE APROG$ /!% Run:Data

collection rate."! MODE -
/!% $"A/ MODE
//! APROG% /
! "
MODE$ $$ CAPTION-PARAM-

SCALEKEYWORD"
, B DEFINE APROG low_resolution

MODE -d10 ! reading rate 1 point/10 s
FLOW 10
INJECT r1a1 10
END
8@

NORMALIZE position
%/ Normalize(3 :" "+"
A/BMAIN "
& B ! / $

( 5/ +"
! 5 - % "
PARAM variable ...
1 . ALERT-APROGMESSAGE "
A/BDEFINE ALERT-DEFINE APROG-DEFINE MESSAGE "
& B
C! . "33 "" !
. / "
$ $ PARAM'"

PARAM $ "
. DEFINE LOOP -LPARAM"

FLOW 5
END
MAIN
DETECTION 1
END

8;

PRIME
%/ Prime(3 :" "+"
A/BMAIN "
& B C"
[*] [time] QUICKINJECT position volume
7 % % "
A/BDEFINE APROG "
& B ! ! B==B33-

===B33333" (E+!
! .
5 !!'
! "
! "
D 7 - ! !

. $!.
7 (3 "@"8+"
/;8 ;< "
7 / / / !

!! 7
"1 QUICKINJECT %.
! "
3 7 . F

< "
, B DEFINE APROG quickie

FLOW 20
QUICKINJECT r1a1 500
END
89

RACK base type
3 $"
A/BMAIN "
& B
N !
N/ N $

N $
N $
N RACK!
"! RACK
- / /
Command:Define Rack Base !(
3 @" "@+" /. !
"
RACK ! .
" % %.
!
", -% RACK 1 THERMO_ARACK
1 MICRO- !
. (!
,
+"
!RACK- !
!("/"! MAIN $ RACK 1
THERMO_A- % . +"
! /%

/' RACK-
% ! // Define Rack Base /,
! !(3 @" "@+"!$
Cancel! /,- % "
. $ /B
$ /% /,
! RACK"
RACK$ $$ ALERT
% MAIN "$ / / %
APROG "
, B MAIN
RACK 1 thermo_A
RACK 2 thermo_B
APROG analysis
...
END

8:

RECOVER position volume [option]
.7 / " $

! 7 "
A/BDEFINE APROG "
& B % %

."
D ."/;8;

<
(
8;
! COINJECT+"
O ;< ! .
. "
B 3 .(COINJECT $+"

& . !(! +
( +"1! . "
RECOVER$ $% COINJECT-INJECT-KINJECT

QUICKINJECT"J%. EXTRACLEAN
% INJECT RECOVER"RECOVER
! BIGINJECT"
RECOVER $! INJECT

% "!$% !./
7 -!$
< . !
7 "
4 COINJECT- $ !$.$!

7 " . % INJECT
"! !% COINJECT- !
."
RECOVERFRACTIONS$ % INJECT

"
. !!RECOVER . %%

! .$" $ 7
. B"/"! .$% !!
, %- .. !
/. 7 . "
, B DEFINE APROG recovery

FLOW 10
TRANSFER r1b1 R 200 !prime recovery cup
INJECT r1a1 100
RECOVER r2a1 310
END
8G

RINSE
%/ Rinse(3 :" "+"
A/BMAIN "
& B C"

8H

time RPOINT id [option ...]
3 "
A/BDEFINE APROG "
& B ! ! B==B33-

===B33333"
! ( 8
-
+"
!
"
B "3'

% . "
% %%! ! -
. "D !
B==B33-/
HB
"1! .

B
B
;(;+"
3 .".
!$ !
B
N
B/
NB! .
N B .
"
%%!
/. % . !/
"/ ! %%
! "
/ "
, B DEFINE APROG report

KEYWORD Mab-451
FLOW 10
* INJECT r1a1 10
-15 RPOINT baseline -b
30 RPOINT plateau
END
@

RTEXT [text]
4 / / , ALERTMESSAGE,"
& B , / @

"
,!
"

END

@

SCALE axis min max [option ...]
3 ! !/."
A/BDEFINE APROG "
& B , "& . B

,
$ !
/ ( +
=. ,"
=,. ,"
B A ,"

3 . "
"
$ % SCALE- /!

! / % ! "
! // - /
Method View:Scale /,"
$ % SCALE - ! / Auto.
/ ( + !. . %

AD"
, B DEFINE APROG fix_scale

SCALE y 10000 20000
FLOW 10
INJECT r1a1 10
END
@

STOP name
1 $ !0 2! ALERT,"
A/BDEFINE ALERT "
& B , !

%%
$ ! "

GO OK
STOP Cancel
END

@8

TEMP degrees
3 ! % "
A/BMAIN "
& B ' (P+"/@@

P"
TEMP %

5 ' . " $ $
!/!;P" / $' !
MAIN ( % APROG+"!!! %
< ?
/ 5 "
1 %

P % "
/ .
/
P-' / %
! % " /
5 -$ /$ (3
@"@"@+"
TEMP !!

"3 !!! $'
! % // / "
, B , LOOP

$ ! "
DEFINE LOOP temp_series

LPARAM %degrees
TIMES 1
10
15
20
25
END
DEFINE APROG analysis

...
END
MAIN
LOOP temp_series STEP
TEMP %degrees
NORMALIZE r2f2
APROG analysis
ENDLOOP
END
@@

TIMES count
3 ! !. "
A/BDEFINE LOOP "
& B "D ;
(! +"
' %. %

DEFINE LOOP % " $
$ ' DEFINE LOOP"
3 / !8 ' %%

"

TIMES 3
Sample1 r1a1 10
Sample2 r1a2 15
Sample3 r1a3 10
END
MAIN
DETECTION 1
LOOP cycle RANDOM
ENDLOOP
END
TITLE text
3 , ! ALERTMESSAGE ,"
& B , / @

"
$ ! "

GO OK
STOP CANCEL
END

@;

[*][time] TRANSFER from-pos to-pos volume
! % . "
& B ! ! B==B33-

===B33333" (E+!
! .
5 !!'
! "
!!"
! "
D < !"/;@;
< "
. ! ! '!
. "
. ! ! '

.$"
TRANSFER MAIN % . /"
, B DEFINE APROG recovery

FLOW 10
TRANSFER r1b1 R 200 !prime recovery cup
INJECT r1a1 100
RECOVER r2a1 310
END
@9

UNCLOG
%/ Unclogging(3 :" "+"
A/BMAIN "
& B C"
WAIT time
4 !! "
& B 4 ! B==B33-

==B33333"
% /% "
4 LOOP - $

. . "D
. "
, B DEFINE LOOP wait_time

LPARAM %delay
TIMES 2
5:00
10:00
MAIN
DETECTION 1
WAIT 2:00 !2 minute delay
LOOP wait_time STEP
WAIT %w !vary wait time
APROG regenerate
ENDLOOP
END

@:

[time] WASH item
4 "
& B ! ! B==B33-

===B33333"
%B

N! %
N
N
N
N
WASH fWASH i'5! % "

, % ! % 5
"
, B DEFINE APROG immob

FLOW 5
DILUTE r1a5 r1a6 r1b1 50
TRANSFER r1a6 r1b1 70
MIX r1b1 115
INJECT r1b1 35
WASH n ! extra needle wash
INJECT r2a1 35
INJECT r1c1 35
END
@G

WASHREC [option]
4.$"4 %/

'!. "
3!$ !%/ "
! ! .$% %%

!!"
Example: DEFINE APROG recovery

FLOW 25
2:00 WASHREC -pr2a1
INJECT r1a1 50
RECOVER r2b1 55
END

@H

;

____________________________________________________________________________Index
Index

Add Report Point !"!
#$ %"
& & !
Adjust Method"'
Adjust Scale
& (#)* ""
+),))"
) "
)
)-+)- '
)#+ '
+ '%
&'
#+)-))'
,&)'*
) '
%
++ . "
)$#)'*
ALERT) "
ALERT #(
)"
)/&0APROG
)/ ""
,""
&&
APPEND #"*(
&&)
)"
&)'
/&
-, "
"
&&) -,
&&) -, &)
&&) -, "
"
)/
), & "
) +)%

)#)&'

"
&)&

+&+ "*
+&&
APROG)

Index ____________________________________________________________________________
$!

APROG/-
APROG #(!

$
%
+# '
&& '
) "
&) %
) &) "%
&) '*
)+ '!
1&) "
& )''
&& %
%
& "' *
*
&))*"
& )''

+&) "
*"" *
) "
# %
&
) *

)
+& & !"!

#+ '
Baseline
) ) /
BEEP #"('

/ & "

)2+ -0 )+ -

) "%
*"
) "
BIAsimulation
Biginject "'%"
BIGINJECT #(*
)/
& "
"%

)
#,*"
) "

____________________________________________________________________________Index
)+"

)
#+
3+) *
++
)++ "
))) &)/"

CAPTION #'('
/"
-)//)""

-++)"
-+)-))"
-. "
#+)-& #"

# # & '"
#) "
)(#)*"
CLOCK #"(

Close""
Close- )*
)

--""
)+ - ""
)- ""*
) #$ %%
Coinject "'%"
COINJECT # ("
)) "

)) + %%0+ ))
)
,$!%
Command
Define Rack Base%
Dilute%
Dock%'
Flow%*
Inject%
Mix%
Set Temperature%'
Stop Flow%*
Stop Inject%%
Transfer%
Undock%
Wash%!
$
%"
) #$ %%
%"
) %

Index ____________________________________________________________________________
%
# %
%%
# %"
#$
&& #$ %%
-- %
$-- %

,&&) !*
))

,$!'
#+ %
#%
- # # '"
& & %

" "% "
)
)
,$!'!"%
+- #+ / "
+ )#
)
)'
&)*"
$ ) #+

#%
"'
- ##,*"
Continue- )Standby
+)-&&
)1& + )/
)+ - "
) ""
)) %
) &) '"
"
%

)+ -&)'"
)+
) '
) # &)& ''
Copy Graph '"!
Copy"*
- (
- # %!"
CTEXT #"(""

) '
%
) %
Curve) )
CURVE #"!("

____________________________________________________________________________Index
,&&) !"
$&)/!%
!*
)!"%
+$!
!'
& /)+1!""
1&)!"
+)-))!
+)-& #!
If-Then!"%
/-!"
1!"
& & !"
- !"
)! !!
Vary By Cycle!!
,$!
)!%
Cut"*
1 "*
/) %!
Cycle) )
)) "

Data Collection Rate %
$) /'
%
)!
DEFINEALERT #("%
DEFINEAPROG #("
DEFINELOOP #("
DEFINEMESSAGE #("
DEFINERACKBASE '%
+ ,$!
+ *%
)/ -) #)'!
Delete"'
) + #$ %
) & & "*
Desorb- )* *

&&)""
"
DETECTION #("!

""'
#
,$!'
&+/ # "'
/ "

Index ____________________________________________________________________________
)1'
)1
#"
Dilute "%%
DILUTE #("*
) )) !

DIPNEEDLE #("'
.
+ *!'
&"!
&)/
&)/"
Dock !%'
&)'
##& %'
) % "" %!
#"
/
"
Edit
Add Report Point"!
Adjust Method"'
Copy"*
Copy Graph"!
Cut"*
Delete"'
Find
Paste"'
REPLACE
Select All
Undo"*
#$ %
*
& & *
/ & '
& /)+1!""
& /+)-))%
& / #/#) *%%
END/-
END #("'
ENDLOOP #("(
,/ &"
& &&&" "
)
#) #
'

& "

____________________________________________________________________________Index
)
"%
/ #*
Evaluation
) '
&)/
1 '
+ )) !
*
4
-- !
*
++ %%
1 APROG
Exit"
Export"
1& '"
1& "
1 ) ''
Extended Event Log! *
Extra Cleanup
%
EXTRACLEAN # %("
+) !
+) 5-1&)"
File
Close""
Exit"
Export"
New Method*"
Open*"
Print"%
Save""
Save As"
Show Info"%
+)) #&& "* "
Find
+ "

Fit To Page"
+)-
,$!*
+)-))
,$!
+)-)) '
)
)!
!
Flow "!%*
FLOW #!(
+)-& # "%'

#

Index ____________________________________________________________________________
,$!*!

) . %
) %*
+)- "!%*
++ &)&!
!
FLOWCELL #
+) . %
Flowcell%*
FLOWPATH #"( "

FLUSH #(

Flush- )***
+ )) %
&)& !
!
- #Stop Inject
Fractions%
FRACTIONS #( %
+%
#+ #$ %

GO #"(
+%
#-'
#,"%
2++
Help
#
)
## /"
)1'

++& & !
+/ !
(
/)& '
))+ '
()'
If-Then!'
,$!"%
), & "
),)

&) +)-& # %

&)& *
+) '
& *

____________________________________________________________________________Index
& ) *

) !
)$)// !
Inject "'%"
,$!*
INJECT # (
. &)0&).
.
. +'*
. &
)'
Injection Port )*"
. #$'

)+))""
#&
#$ %%
)) %"
)) )+ -%
++) ""
&)'
6+) 0(
)& ''
#++
KEYWORD #"

( !
/-
,$!*!"
#+

& & )"
Keywords '

)/" "%
)
)/-, %
) ) /
)1&
) '

Kinject "'%"
KINJECT # ( *
)).
)/
#+
) # )'
) # # "
34 ""
LIFETIME #( '
)""
),
)# "

1

Index ____________________________________________________________________________
)+)- '
) )1& %"
)$)//
&%
) #&))& %
) % "" %!
#"
) #) "
LOOP) ""
LOOP #""(
)&"%
)& ""
)&.
)&"
Lost Data Found
)- $) /'
'
'
LPARAM #""(%
LTEXT #"(%
MAIN(%"
MAIN)
MAIN #(%"
+%
) %

)*"
+ /$ *"
*"
) )%
) ) + ""
Manual Inject "' %"
) %
&& #.
) %
).
)) "
)
& '
) "
&& "
) *

& & ) *
Markers *
+ )1& %
& "'
1++ ' "
+$/ "
/++ '
1

____________________________________________________________________________Index

MESSAGE) "
MESSAGE #(%
#
. "'
- #
+)+ "
& %!
&
) "
-/) %

"'
)
#)
#
ALERT"(
APPEND"*(
APROG(!
BEEP"('
BIGINJECT(*
CAPTION'('
CLOCK"(
COINJECT("
%

%
CTEXT"(""
CURVE"!("
DEFINEALERT("%
DEFINEAPROG("
DEFINELOOP("
DEFINEMESSAGE("
DETECTION("!
DILUTE("*
DIPNEEDLE("'
END("'
ENDLOOP("(
EXTRACLEAN %("
FLOW!(
FLOWPATH"( "
FLUSH(
FRACTIONS( %
GO"(
$ %"
INJECT(
KEYWORD"
( !
KINJECT( *
LIFETIME( '
LOOP""(
LPARAM""(%
LTEXT"(%
& ("

1

Index ____________________________________________________________________________
PARAM*(%
&
PRIME(%!
QUICKINJECT(%!
RACK(%*
RECOVER(%'
RINSE(%
RPOINT"
(
RTEXT"( "
SCALE(
STOP"( %
TEMP(
TIMES""(
TITLE(
TRANSFER( !
UNCLOG( *
WAIT( *
)%
WASH( '
WASHREC(
# )%
#+
%

$ )
)1"
/ 1
1 1"
1 )/
#+ )"
#
# #/ 1 %
- #"
#)

1 )/ "'
'
--*
Method
# %
)- #*
&) %
,)&)'*
,&)&
1(%%
,$!'!"
Mix " %
MIX #(%%
MODE #(%
# %"
#+) %
) #)&&)
) #) '
1

____________________________________________________________________________Index
)/ - "'!

) &)" "
) &) + )+ -'"

) &))
) &)--
)%

) %
&)& *"
)& ''
Needle Replacement/Positioning )*"
)&"%
New Method"
New Window

'"
&)) '
+ Desorb*
NORMALIZE #(%
Normalize- )*
),
& )/ '
), #*

&&) -,
+) !
&) *
Notebook !
)"
)#)&'
Open"
&"
& # # *

& )/
& ,)),
&
#("
& +
ORDER)&"
Overlay %
PARAM #*(%

& *
&
#
& ,!!
Paste"'
&
DEFINEALERT("

1

Index ____________________________________________________________________________
DEFINEAPROG(
DEFINELOOP(
DEFINEMESSAGE(
MAIN(%
&++
&) ) %
Position+))1 "'
& # &))*"
&-&%
Predip Needle ""%
&).
Preferences *

&&)+"
&& +
&&&).
&&)
& %*

Prerun Method %
PRIME #(%!
Prime- )*!
& #/&
& #/ !
Print"%
&
#+ "
+ "
& ,"
& &&) -, &) *
& &)& *
& "%
& '
& "
& /%%
& #
&&+)) "*%*
'%
&& !
&&
/'!
)+ '
+)) "*
&)/ &*"
Quickinject "'%"
QUICKINJECT # (%!
%
+ '%
&&) -,
1

____________________________________________________________________________Index
RACK #(%*

& ''
/& * '
RANDOM)&"
random '

) &
# ''
) ""
&&) ""
) "
RECOVER #(%'
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Biacore is a registered trademark of Biacore AB. Copyright Biacore 2006 BR-1002-31
INSTRUMENT HANDBOOK

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INSTRUMENT HANDBOOK

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Figure 2-1. BIACORE 2000 system processing unit and controlling

2.1 BIACORE 2000 processing unit

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IFC Connector Autosampler

Eluent pump Autosampler pump

Waste bottle Buffer bottle

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Figure 2-3. The autosampler compartment.

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