Agilent Technologies 1260 Infinity User manual

Agilent Technologies
Agilent 1260 Infinity
Purification Solution
Method Developer's
Quick Start Guide

Purification Solution - Developer's Guide
Notices
© Agilent Technologies, Inc. 2017
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Manual Part Number
M8368-90011 Rev. D
Edition
11/2017
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CAUTION
A CAUTION notice denotes a
hazard. It calls attention to an
operating procedure, practice, or
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Software Revisions
This guide is valid for revision A.01.06 of
the Automated Purification Software in
combination with revision C.01.08 of the
OpenLAB CDS ChemStation Edition.

Purification Solution - Developer's Guide 3
In This Book
In This Book
This manual contains instructions for Method Developers on how to use the
Automated Purification software.
1 The Role of the Method Developer
This chapter describes the role of the user of the Automated Purification
Software in Expert mode.
2 Preparing Default Purification Methods
This chapter describes the three default Purification methods that can be used
as a basis for the development of specific Purification methods.
3 Setting Up an Analytical and Preparative System
This chapter gives step-by-step instructions on setting up the analytical
system and the preparative system in the Automated Purification software.
4 Setting Up and Running a Purification Task
This chapter gives instructions for the most important steps for setting up and
running a Purification Task.
5 Reviewing Purification Results
This chapter describes how to review the results of a Purification Task.
6 Calibration Procedures
This chapter gives step-by-step instructions for the important calibration
procedures for the Purification system.
7 Checkout Procedure
This chapter gives step-by-step instructions on how to carry out a checkout
procedure to confirm the correct operation of your Purification system.

4 Purification Solution - Developer's Guide
Contents
Contents
1 The Role of the Method Developer 7
2 Preparing Default Purification Methods 9
General Method Settings 11
Analytical Method Settings 16
Preparative Method Settings 19
Agilent Active Splitter (G1968D/E/F) 23
Critical Method Parameters 24
3 Setting Up an Analytical and Preparative System 27
Setting up an analytical system 28
Setting up a preparative system 30
4 Setting Up and Running a Purification Task 33
Setting up a purification task 34
Configuring a new task 35
System Suitability Tests 38
Running a task 39
Processing partial data and cloning tasks 40
Reviewing analytical results 42
5 Reviewing Purification Results 43
6 Calibration Procedures 45
Characterizing the delay volumes: Mixing point to UV detector and Column to UV
detector 48
Characterizing the column volume 63
System Configuration and Delay Time Calibration 70
7 Checkout Procedure 83
Acquire an analytical run using the Chemstation sequence 85
UV-only Workflow: Focused gradient in the Purification software 89

Purification Solution - Developer's Guide 5
Contents
UV/MS Workflow: Focused gradient in Purification software 92

6 Purification Solution - Developer's Guide
Contents

7
Purification Solution - Developer's Guide
Agilent Technologies
1
The Role of the Method Developer
This chapter describes the role of the user of the Automated Purification
Software in Expert mode.
Purification software Method Developers work in Expert mode. They are
responsible for establishing the software work environment such that
Operators (working in Easy Prep mode) can use the purification software to
purify their samples.
This comprises the following tasks:
•Creating the (analytical and) preparative base methods that are used in the
instrument runs (see “Preparing Default Purification Methods” on page 9).
•Creating the analytical and preparative system parameter sets for all
analytical and preparative instruments and operating conditions that are
used by the Operators (see “Setting Up an Analytical and Preparative
System” on page 27). These systems should be named such that Operators
can easily identify their system. To retrieve system parameters, a set of
calibration procedures has to be executed, see “Calibration Procedures” on
page 45.
•Creating a set of purification task templates that are optimized for
purifying the sample types that are provided by the Operators (see “Setting
Up and Running a Purification Task” on page 33). We recommend that you
provide a task template for each combination of analytical and preparative
system that applies. Operators are expected to create their purification
tasks based on a Method Developer’s task template that matches their
system combination and separation needs.
•Creating a system suitability test task (see “System Suitability Tests” on
page 38) to verify that the instrument is still operating as expected. System
suitability tests can be executed by operators and method developers.
However, only method developers can accept a failed system suitability test
to release a blocked instrument.
•Instructing Operators on the work environment and how to set up and run
purification tasks.

8 Purification Solution - Developer's Guide
1 The Role of the Method Developer
In This Book
•Fixing problems (for example, adjusting parameters or target
identification) of Operators' tasks, to which the Operators do not have
access.
Method Developers have full access to all features of the purification software
and to the ChemStation while working within the main purification software
user interface (the purification task screen).

9
Purification Solution - Developer's Guide
Agilent Technologies
2
Preparing Default Purification Methods
General Method Settings 11
Analytical Method Settings 16
Preparative Method Settings 19
Agilent Active Splitter (G1968D/E/F) 23
Critical Method Parameters 24
This chapter describes the three default Purification methods that can be used
as a basis for the development of specific Purification methods.
This description summarizes the recommended purification method settings
for purification-related modules. Most of these method settings can be freely
modified according to your needs, with limitations noted in “Critical Method
Parameters” on page 24. For the alternative modules, which are not listed
here, use the applicable settings of the default modules.
Start with a new method:
• Method > New Method.
Methods settings are accessible from Instrument > Setup Instrument Method or
Method > Edit Entire Method.
NOTE
It is important for Purification software to mark the Save Method with Data check box in
Method > Runtime Check List.
NOTE
Switch off all pumps while you are setting up the methods.

10 Purification Solution - Developer's Guide
2 Preparing Default Purification Methods
In This Book
NOTE
It is highly recommended that the pumps are not switched off in a pre- or post-run
command of the method.
NOTE
Methods rely on the instrument configuration and driver revision. If you change the
instrument configuration or upgrade the driver revision for which you originally set up your
purification (base) method, you must at least save the method for the changed instrument
configuration under a new name. However, we recommend that, for a changed instrument
configuration or upgraded driver revision, you create a new method from scratch.
Otherwise, method resolution will try to adapt the methods from the old
configuration/driver revision with every method load (that is, for every sample run),
leading to a method changed state, and preventing the run going forward.
When creating a new task based on a task template with a different instrument
configuration, make sure that you choose an appropriate preparative base method for the
current instrument configuration and driver revision.
We recommend that you choose different task root folders for different instrument
configurations so that you do not mix different configurations in the same task root folder.

Purification Solution - Developer's Guide 11
Preparing Default Purification Methods
2
General Method Settings
General Method Settings
In all modules, set the Stoptime to As Injector/No Limit (infinite run time). The
actual stoptime will be specified when the method is used.
Isocratic Pump (G1310B)
Make-up pump for splitter used with separated preparative MSD instrument.
Quaternary Pump (G1311B)
Make-up pump for splitter and analytical pump for combined analytical and
preparative MSD instrument.
Prep Pump Cluster (G1361A and G1391A)
Stoptime Select As Injector/No limit
Pressure Limits Consider Active Splitter pressure limit (69 bar).
Stoptime Select As Injector/No limit
Pressure Limits • Because of the 400-bar pressure limit of the preparative
system, set the upper limit for analytical scouting runs to 400
bar or lower.
•Consider Active Splitter pressure limit (69 bar) when used as
make-up pump.
Solvent
description texts
•Channel A: Water
•Channel B: Acetonitrile
Solvent B 2%
Pressure Limits Set Max. limit. Find the pressure limit of the used columns (for
example, 21.2 mm guard system seals finger-tight up to 135 bar;
21.2 mm prep column without the guard up to 350 bar).
Stoptime Select As Injector/No limit

12 Purification Solution - Developer's Guide
2 Preparing Default Purification Methods
General Method Settings
Prep Pump (G7161A)
Prep Pump (G7161B)
Advanced Channel A Set Compressibility to 46 (Water)
Advanced Channel B Set Compressibility to 115 (Acetonitrile)
Solvents A
Select Aqueous and type the description text
Water .
B
Mark the check box, enter
2 % , select Acetonitrile, and type description text
Acetonitrile .
Pressure Limits Set Max limit: find the pressure limit of the columns used (for
example, 21.2 mm guard system seals finger-tight up to 135 bar;
21.2 mm prep column without the guard up to 350 bar).
Advanced In Compressibility, keep the check box Use Solvent Types
marked
Solvents A
Select solvent line 1 or 2, select Aqueous and type the
description text
Water .
B
Mark the check box, select solvent line 1 or 2, enter
2 % , select Acetonitrile, and type description text
Acetonitrile .
Pressure Limits Set Max limit: find the pressure limit of the columns used (for
example, 21.2 mm guard system seals finger-tight up to 135 bar;
21.2 mm prep column without the guard up to 350 bar).
Advanced In Compressibility, keep the check box Use Solvent Types
marked

Purification Solution - Developer's Guide 13
Preparing Default Purification Methods
2
General Method Settings
Dual Loop Autosampler (G2258A)
Prep Sampler (G7157A)
Valve 2/10 (G1170A and G4730A)
Selection of a flow path with a certain column.
Other Valves
Needle Wash Enable Needle Wash with Mode: Flush Port and Time15 s (to
avoid contamination of injection seat, do not use less than 10 s).
The wash procedure requires needle wash solution.
Stoptime Select As Pump/No limit
Advanced In Auxiliary, mark Vial/Well bottom sensing only if the vessels
used are suitable for this function, such as flat-bottomed glass
vials; see the Dual Loop Autosampler manual for more
information.
Needle wash Set Time15 s. The wash procedure requires needle wash
solution.
Stoptime Select As Pump/No Limit.
Position Select Use valve position
Position switch at end of run Select Do not switch
Position Names Enter description of preparative and analytical positions. For the
combined setup, set Position 1: Analytical, Position 2:
Preparative.
Position Select Use valve position (if applicable)

14 Purification Solution - Developer's Guide
2 Preparing Default Purification Methods
General Method Settings
UV (DAD, MWD,VWD)
UIB II (G1390B)
•If an auxiliary detector is connected via the UIB II box, set all related
parameters in the UIB II method.
•If there is no detector connected via UIB II box, clear both Analog In check
boxes in Advanced > Analog In Port Settings
•Set Stoptime to As Pump/Injector
Fraction Collectors (G1364B, G1364E, G7159B, G7166A and FCC*)
*
FCC is a Fraction Collector Cluster of two or more modules.
•G1364E, G7159B, G7166A and FCC: Set Collection Behavior to Disable Fraction
Collection
•Set Stoptime to As Pump/Injector
Wavelength DAD and MWD
Set Signal AWavelength to 263 nm, Bandwidth: 4 nm, clear
Reference wavelength.
VWD
Set Signal to 263 nm.
Peakwidth DAD and MWD
20 Hz (> 0.013 min)
VWD
40 Hz (> 0.013 min)
Advanced > Spectrum
(optional for DAD)
Store: All, Range from: 200 nm to 400 nm, Step: 2 nm
Autobalance Mark Prerun.
Lamps on required for
acquisition
(DAD and MWD)
Mark UV Lamp.
Miscellaneous
VWD
Mark Lamps on required for acquisition.
Stoptime Select As Pump/Injector

Purification Solution - Developer's Guide 15
Preparing Default Purification Methods
2
General Method Settings
Flow Modulator (G7170B)
MSD
Access by Method > Edit entire method or by right click on MSD icon
Save the method as
General_Purification
• Method > Save Method as
Splitting Operation Select Splitting disabled.
Set Up MSD Signals General
Peakwidth: 0.1 min (this defines the MSD data rate; make sure
that it is sufficient).
Active Signals: Mark 1 and 2 signals and name them, for
example,
Positive and
Negative
MSD Signal Settings
Signal: 1, Mode: Scan, Polarity: Positive, Mass range: 125 – 725,
Step size: 0.1
Signal: 2, Mode: Scan, Polarity: Negative, Mass range: 125 –
725, Step size: 0.1
MSD type-specific settings •MSD G6120B/G6125B
If the actual cycle time is above 0.9 s, consider decreasing it
by using only one scanning polarity (lower cycle time will
require less delay coil volume):
•General > Active Signals > clear Signal 2
•MSD Signal Settings > Signal 1 > specify polarity
•MSD G6130B/G6135B
General > Peakwidth > 0.07 min

16 Purification Solution - Developer's Guide
2 Preparing Default Purification Methods
Analytical Method Settings
Analytical Method Settings
Only for the combined analytical and preparative system.
Load the general settings of the purification method:
General_Purification(“General Method Settings” on page 11)
Save the method as
Analytical_Purification:
• Method > Save Method As.
Quaternary Pump (G1311B)
Prep Pump Cluster (G1361A and G1391A)
Prep. Pump (G7161A)
Prep. Pump (G7161B)
Flow 1.0 mL/min
Advanced Set Maximum Flow Gradient to about 10 times the target flow.
Flow 0mL/min
Flow 0mL/min
Flow 0mL/min

Purification Solution - Developer's Guide 17
Preparing Default Purification Methods
2
Analytical Method Settings
Dual Loop Autosampler (G2258A)
Valve 2/10 (G1170A and G4730A)
UIB II (G1390B)
If the Active Splitter is connected to the external contacts of the UIB II:
• Timetable: Empty
• External Contacts: Contact A: mark Closed
Injection Loop Lower
Injection mode Depends on installed loop volume and injection volume.
•Loop equal to injected volume: set Full loop with overfill
factor 5, disable Plug Settings
•Loop larger than injected volume: set Partial loop filling and
set Plug Settings to maximum Plug volume:
•Mark Draw Plug before and after the sample
•Plug Volume: set the volume later in the method based on
injection volume: Plug volume = ½[(Loop volume) –
(Injection volume)]
•Draw Plug from: select Location, specify vial location
NOTE
The application of the Plug removes up to 27 µL of the
purge solvent from the lower loop that is introduced by the
seat capillary in front of the loop, which is purged after
each run. The purge solvent has typically high elution
strength by design; therefore, the removal of this solvent
from the lower injection loop by the Plug feature is highly
recommended.
Injector Cleaning Rinse volume to 15
Advanced Draw speed and Eject speed to 1000.0 µL/min.
Position Use valve position: set analytical position
Expected ERI Mode No mode check

18 Purification Solution - Developer's Guide
2 Preparing Default Purification Methods
Analytical Method Settings
Save the method changes:
• Method > Save Method.

Purification Solution - Developer's Guide 19
Preparing Default Purification Methods
2
Preparative Method Settings
Preparative Method Settings
Load the general settings of the purification method:
General_Purification(“General Method Settings” on page 11)
Save the method as
Prep_Purification:
• Method > Save Method As.
Isocratic Pump (G1310B)
Separated preparative MSD system — make-up pump
Quaternary Pump (G1311B)
Combined analytical and preparative MSD system — as make-up pump:
Prep Pump and Prep Pump Cluster (G7161A/B, G1361A and G1391A)
Flow 1.5 mL/min
Advanced Set Maximum Flow Gradient to about 10 times the target flow.
Channel D 100 %
Flow 1.5 mL/min
Advanced Set Maximum Flow Gradient to about 10 times the target flow.
Flow 20 mL/min (scale-up of 4.6 × 50 mm to 21.2 × 50 mm columns
with 5 µm particle size).
Advanced Channel A and B In Maximum Flow Gradient, set both Flow ramp up and Flow
ramp down to about 10 times the target preparative flow.

20 Purification Solution - Developer's Guide
2 Preparing Default Purification Methods
Preparative Method Settings
Dual Loop Autosampler (G2258A)
Prep Sampler (G7157A)
Valve 2/10 (G1170A and G4730A)
UV (DAD, MWD, VWD)
•For instruments with Agilent active splitter or if using the slope-based
collection mode, set Peakwidth to:
•for DAD and MWD: > 0.1 min (2 s response time) (2.5 Hz)
•for VWD: > 0.2 min (4 s response time) (2.5 Hz)
UIB II (G1390B)
If an MSD is configured:
•G1364B: Expected ERI Mode: MSD
•G7159B: No mode check
If the Agilent Active Splitter (G1968D) is connected to the external contacts of
the UIB II:
• Timetable:
•0, Change contacts, Contact A state: Closed
•0.01, Change contacts, Contact A state: Open.
• External Contacts: Contact A: mark Closed
Injection Loop Upper
Injection mode Partial Loop Filling
Injector Cleaning Rinse volume: 1 (increase to 5 for injections below 200 µL)
Advanced Draw speed and Eject speed: 8000.0 µL/min (decrease to 3000.0
for injections below 200 µL)
Advanced Set Draw Speed to 3000.00 µL/min.
Position Use valve position: set the preparative position