ACQUITY UPLC System Quick Start Guide Black Box Electric Pencil Sharpener Elite Cabinets Grounding Kit 71500082503re

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ACQUITY UPLC System
Quick Start Guide
71500082503/Revision E

Copyright notice
© 2004−2010 WATERS CORPORATION. PRINTED IN THE UNITED
STATES OF AMERICA AND IN IRELAND. ALL RIGHTS RESERVED. THIS
DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY
FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.
The information in this document is subject to change without notice and
should not be construed as a commitment by Waters Corporation. Waters
Corporation assumes no responsibility for any errors that may appear in this
document. This document is believed to be complete and accurate at the time
of publication. In no event shall Waters Corporation be liable for incidental or
consequential damages in connection with, or arising from, its use.

Trademarks
ACQUITY UPLC, Millennium, UPLC, and Waters are registered trademarks,
and eCord, Empower, MassLynx, nanoACQUITY UPLC, and “THE SCIENCE
OF WHAT’S POSSIBLE” are trademarks of Waters Corporation.
Keps is a registered trademark of Illinois Tool Works, Inc.
PharMed is a registered trademark of Saint-Gobain Ceramics & Plastics, Inc.
Windows is a registered trademark of Microsoft Corporation.
Other trademarks or registered trademarks are the sole property of their
respective owners.

Customer comments
Waters’ Technical Communications department invites you to tell us of any
errors you encounter in this document or to suggest ideas for otherwise
improving it. Please help us better understand what you expect from our
documentation so that we can continuously improve its accuracy and
usability.
We seriously consider every customer comment we receive. You can reach us
at tech_comm@waters.com.

Contacting Waters
®

Contact Waters with enhancement requests or technical questions regarding
the use, transportation, removal, or disposal of any Waters product. You can
reach us via the Internet, telephone, or conventional mail.
Waters contact information
Contacting medium

Information

Internet

The Waters Web site includes contact
information for Waters locations worldwide.
Visit www.waters.com.

Telephone and fax

From the USA or Canada, phone 800
252-HPLC, or fax 508 872 1990.
For other locations worldwide, phone and fax
numbers appear in the Waters Web site.

Conventional mail

Waters Corporation
34 Maple Street
Milford, MA 01757
USA

iii

Safety considerations
Some reagents and samples used with Waters instruments and devices can
pose chemical, biological, and radiological hazards. You must know the
potentially hazardous effects of all substances you work with. Always follow
Good Laboratory Practice, and consult your organization’s safety
representative for guidance.

Considerations specific to ACQUITY UPLC instruments
High voltage hazard
Warning: To avoid electric shock, do not remove the ACQUITY UPLC
instrument’s protective panels. The components they cover are not
user-serviceable.

Safety advisories
Consult Appendix A in the Waters ACQUITY UPLC System Operator’s Guide
for a comprehensive list of warning and caution advisories.

Operating the ACQUITY UPLC instruments
When operating the ACQUITY UPLC instruments, follow standard
quality-control (QC) procedures and the guidelines presented in this section.

Applicable symbols
Symbol

Definition
Authorized representative of the European
Community
Confirms that a manufactured product complies
with all applicable European Community
directives

ABN 49 065 444 751

Australia C-Tick EMC Compliant
Confirms that a manufactured product complies
with all applicable United States and Canadian
safety requirements

Audience and purpose
This guide is intended for personnel who operate ACQUITY UPLC
instruments.

Intended use of the ACQUITY UPLC system
Waters designed the ACQUITY UPLC system to isolate, concentrate,
separate, detect, and measure individual analytes in solution mixtures for
research applications of rapid qualitative analysis, quantitative analysis,
and/or micropreparative purification.

Calibrating
To calibrate LC systems, follow acceptable calibration methods using at least
five standards to generate a standard curve. The concentration range for
standards should include the entire range of QC samples, typical specimens,
and atypical specimens.

Quality-control
Routinely run three QC samples that represent subnormal, normal, and
above-normal levels of a compound. Ensure that QC sample results fall within
an acceptable range, and evaluate precision from day to day and run to run.
Data collected when QC samples are out of range might not be valid. Do not
report these data until you are certain that the instrument performs
satisfactorily.

ISM classification
ISM Classification: ISM Group 1 Class B
This classification has been assigned in accordance with CISPR 11 Industrial
Scientific and Medical (ISM) instruments requirements. Group 1 products
apply to intentionally generated and/or used conductively coupled
radio-frequency energy that is necessary for the internal functioning of the
equipment. Class B products are suitable for use in both commercial and
residential locations and can be directly connected to a low voltage,
power-supply network.

EC Authorized Representative

Waters Corporation (Micromass UK Ltd.)
Floats Road
Wythenshawe
Manchester M23 9LZ
United Kingdom

Telephone:

+44-161-946-2400

Fax:

+44-161-946-2480

Contact:

Quality manager

Table of Contents
Copyright notice ................................................................................................... ii
Trademarks ............................................................................................................ ii
Customer comments ............................................................................................ iii
Contacting Waters ............................................................................................... iii
Safety considerations .......................................................................................... iv
Considerations specific to ACQUITY UPLC instruments................................ iv
Safety advisories ................................................................................................. iv
Operating the ACQUITY UPLC instruments .................................................. v
Applicable symbols .............................................................................................. v
Audience and purpose.......................................................................................... v
Intended use of the ACQUITY UPLC system .................................................... v
Calibrating ........................................................................................................... v
Quality-control .................................................................................................... vi
ISM classification ................................................................................................. vi
ISM Classification: ISM Group 1 Class B ......................................................... vi
EC Authorized Representative ......................................................................... vi

1 System Overview .................................................................................... 1-1
Instruments, components, and data systems ............................................. 1-2
Examples of Waters ACQUITY UPLC systems ............................................. 1-3
UPLC system guidelines .................................................................................. 1-4
ACQUITY UPLC columns calculator.............................................................. 1-5
Binary solvent manager .................................................................................. 1-5
How the binary solvent manager works ......................................................... 1-6
Sample manager ................................................................................................ 1-7
How sample flows ............................................................................................ 1-7

Table of Contents

vii

High temperature column heater ................................................................. 1-8
Column manager ............................................................................................... 1-8
Column heater/cooler ....................................................................................... 1-9
30-cm column heater/cooler ............................................................................ 1-9
Optional sample organizer ............................................................................. 1-9
Detectors ...........................................................................................................
TUV detector ..................................................................................................
PDA detector ..................................................................................................
ELS detector...................................................................................................
FLR detector...................................................................................................
Median baseline filter....................................................................................

1-10
1-10
1-10
1-10
1-11
1-11

Mass spectrometers ........................................................................................ 1-11
SQ detector ..................................................................................................... 1-11
TQ detector..................................................................................................... 1-11
Data systems .................................................................................................... 1-12
Empower software ......................................................................................... 1-12
MassLynx software ........................................................................................ 1-12
Columns ............................................................................................................. 1-12
eCord column chip ......................................................................................... 1-13
FlexCart ............................................................................................................. 1-13
For additional information ........................................................................... 1-14

2 Preparing System Hardware ............................................................... 2-1
Powering-on the system .................................................................................. 2-1
Monitoring startup tests ................................................................................. 2-3
Monitoring system instrument LEDs ........................................................... 2-3
Power LED ....................................................................................................... 2-4
Status LEDs ..................................................................................................... 2-4

viii

Table of Contents

Enabling the leak sensors ............................................................................... 2-6
Preparing the binary solvent manager ....................................................... 2-7
Performing a seal wash prime ........................................................................ 2-7
Priming the binary solvent manager .............................................................. 2-9
Priming a dry binary solvent manager......................................................... 2-10
Priming a wetted binary solvent manager ................................................... 2-12
Preparing the sample manager ...................................................................
Selecting weak wash and strong wash solvents...........................................
Priming the sample manager........................................................................
Washing the sample manager needle ...........................................................
Characterizing the needle seal......................................................................
Characterizing the needle and sample loop volumes...................................
Using the extended puncture needle ............................................................
Loading sample plates in the sample manager............................................
Selecting the optimum sample injection mode.............................................
Installing the optional sample manager shade ............................................

2-15
2-15
2-17
2-19
2-21
2-22
2-23
2-24
2-25
2-28

Preparing the column manager .................................................................. 2-30
Preparing the sample organizer .................................................................
Initiating communications ............................................................................
Loading sample plates ...................................................................................
Displaying sample plate information ...........................................................

2-30
2-30
2-31
2-35

Starting the TUV detector ............................................................................ 2-36
Starting the TUV detector............................................................................. 2-37
Conditioning the column .............................................................................. 2-39
Shutting down the system ............................................................................ 2-40
Shutting down for less than 24 hours........................................................... 2-40
Shutting down for more than 24 hours......................................................... 2-41
Running HPLC methods on an ACQUITY UPLC system ....................... 2-42
System considerations ................................................................................... 2-42
Choosing fittings ............................................................................................ 2-44

Table of Contents

3 Configuring System Software ............................................................. 3-1
Configuring Empower software .................................................................... 3-1
Starting Empower software and logging in.................................................... 3-1
Selecting system instruments ......................................................................... 3-2
About the binary solvent manager control panel........................................... 3-4
About the sample manager control panel....................................................... 3-6
About the TUV detector control panel ............................................................ 3-9
About the column manager control panel .................................................... 3-11
Starting the ACQUITY UPLC Console from Empower software ......... 3-12
Configuring MassLynx software ................................................................. 3-13
Starting the ACQUITY UPLC Console from MassLynx software ........ 3-14

4 Verifying System Operation ................................................................ 4-1
Preparing the system ....................................................................................... 4-2
Creating the test methods ............................................................................... 4-5
Creating the instrument method .................................................................... 4-5
Creating the sample set method ..................................................................... 4-9
Performing the gradient performance test .............................................. 4-10

Table of Contents

System Overview
This section describes the components and features of the ACQUITY
®
UPLC system.
Contents
Topic

Page

Instruments, components, and data systems

1-2

UPLC system guidelines

1-4

Binary solvent manager

1-5

Sample manager

1-7

High temperature column heater

1-8

Column manager

1-8

Column heater/cooler

1-9

30-cm column heater/cooler

1-9

Optional sample organizer

1-9

Detectors

1-10

Mass spectrometers

1-11

Data systems

1-12

Columns

1-12

FlexCart

1-13

For additional information

1-14

1-1

Instruments, components, and data systems
ACQUITY UPLC systems include a binary solvent manager, sample manager,
column heater, detectors (tunable ultraviolet, photodiode array, evaporative
light scattering, fluorescent, or mass spectrometry), and a specialized
ACQUITY UPLC column.
Small-particle chemistries as utilized in UPLC system chromatography
generate narrow peaks. To maintain these narrow peaks, extra
bandspreading must be controlled by lower detector cell volume, minimized
tubing volumes, and specialized fittings. Narrow peak widths sometimes
require higher data rates. The TUV, PDA, ELS, and FLR detectors can sample
up to 80 data points per second. The SQ and TQ mass spectrometers can
sample at fast acquisition speeds suitable for UPLC.
The binary solvent manager and injector can sustain pressures up to
103,421 kPa (1034 bar, 15,000 psi) and can generate high-pressure gradients
with minimal gradient delay. The upper limit of the flow rate range is 2
mL/min.
The sample manager can accommodate two plates in a microtiter plate format
or 2-mL vials in full-height plate format. An optional sample organizer
increases the capacity of the system to as many as 22 microtiter plates (21 in
the sample organizer and one in the sample manager), or eight vial racks
(seven in the sample organizer and 1 in the sample manager).
Waters® Empower™ chromatography software, MassLynx™ mass
spectrometry software, or certain third-party software controls the ACQUITY
UPLC systems.

1-2

System Overview

Examples of Waters ACQUITY UPLC systems
Single detector system with column manager, no mass spectrometer

Solvent tray
Sample organizer
(optional)

UPLC detector (optional)

Column manager (optional)

Sample manager

Binary solvent manager

Instruments, components, and data systems

1-3

Single detector system with a mass spectrometer and column heater
Sample
organizer
(optional)

Solvent tray

HT column heater
Sample
manager

Mass
spectrometer
(optional)

Binary solvent manager

UPLC system guidelines
Tip: ACQUITY UPLC system guidelines differ from standard HPLC practices.
When performing fast analyses, note that a peak of interest can be as narrow
as 0.5 second. Waters recommends a sampling rate of 25 to 50 points across
the peak, which provides good quantitation and peak representation.
Sampling rates faster than 20 points per peak yield higher baseline noise and
filter time constants must be adjusted accordingly.
The optimal ACQUITY UPLC flow rate differs from that of a typical HPLC
column. The table below offers operating guidelines for ACQUITY UPLC
columns under both isocratic and gradient conditions. Note that the values
provided are approximations and that optimum performance for your
molecule or separation can occur at a different flow rate and/or pressure.

1-4

System Overview

Optimal flow rates for molecular weight range
Column size

Molecular weight

Flow rate

2.1 × 50 mm

<500

600 µL/min

2.1 × 50 mm

1000

300 µL/min

2.1 × 50 mm

1500

150 µL/min

2.1 × 50 mm

2000

100 µL/min

ACQUITY UPLC columns calculator
The ACQUITY UPLC columns calculator estimates the plate count (N) of an
isocratic separation or the peak capacity (Pc) of a gradient separation based on
your current HPLC conditions. It then offers you a choice of one or more
ACQUITY UPLC columns that can provide increased resolving power in the
same amount of time or similar resolving power in less time. The
chromatographic conditions provided are a starting point and can be further
optimized based on your particular requirements. After you install the
ACQUITY UPLC software, the ACQUITY UPLC Columns Calculator shortcut
appears on your computer desktop.

Binary solvent manager
The binary solvent manager is a high-pressure pump that moves solvent
through the system. It provides steady (pulse-free) solvent flow at analytical
flow rates. The binary solvent manager delivers solvent at flow rates of 1
mL/min at 103,421 kPa (1034 bar, 15,000 psi) and up to 2 mL/min at reduced
pressures to 62,053 kPa (621 bar, 9000 psi). The binary solvent manager can
pump two solvents simultaneously.

Binary solvent manager

1-5

Pressure flow envelope

Pressure (kPa)

103,421

62,053

2
Flow rate (mL/min)

How the binary solvent manager works
Each of the binary solvent manager’s two independent pump systems, A (on
the left-hand side) and B (on the right-hand side), contains two linear-drive
actuators (left-hand and right-hand). Each left-hand and right-hand actuator
pair comprises a single reciprocating “serial” pump that delivers precise flow
of a single solvent. The two pump systems combine their two solvents at a
filter/tee mixer. From there, the solvent mixture flows to the sample manager.
The chromatography software controls the two solvents’ mixing ratio by
varying the flow of pump A relative to that of pump B. A pressure transducer
in each pump head relays pressure data to the binary solvent manager, whose
firmware measures pump head pressures during the pumping cycle. Thus the
binary solvent manager independently pre-compresses the solvents in both
the A and B portions to ensure consistent solvent delivery and minimize
pump-induced detector baseline disturbances.

1-6

System Overview

Sample manager
The ACQUITY UPLC sample manager injects the samples it draws from
microtiter plates or vials onto the chromatographic column. A locating
mechanism uses a probe to access sample locations and draw sample from
them. In the needle overfill load-ahead mode, the sample manager can
perform an injection in approximately 15 seconds. The first injection requires
additional overhead time.
The sample manager accepts standard ANSI/SBS footprint plates, 5.03 ±0.02
inch × 3.365 ±0.02 inch, that conform to ANSI standards (maximum height =
2.2 inches, including covers). You can program any combination of these
plates and vial holders for automated sample processing. Samples are loaded
into the sample manager via the front door or the optional sample organizer,
which transfers samples back and forth between the two instruments. The
sample manager can maintain samples at any temperature between 4 and
40 ºC (39.2 to 104 ºF) in 25 ºC (77 ºF) or less ambient conditions.

How sample flows
When the default mode, partial loop with needle overfill, is requested, the
sample manager needle carriage moves to the specified well location and
draws in an air gap. A stainless steel puncture needle pierces the well cover
and lowers into the well. The sample needle emerges from within the puncture
needle, protrudes into the sample well, and draws in a sample volume equal to
the specified injection volume plus 15.0 µL (14.0 µL pre-sample volume and
1.0 µL post-sample volume). The sample needle is then removed from the vial
and the sample syringe continues to pull the sample aliquot through the
sample needle and through the injection valve until the pre-sample and
sample injection volume passes through the injection valve. The valve
actuates, switching the sample loop to the load position. The sample is pushed
back toward the needle and the sample volume is then pushed into the sample
loop. The sample loop moves to the injection position and the sample is carried
by the pump to the column.

Sample manager

1-7

High temperature column heater
The high temperature (HT) column heater is modular and its footprint is
identical to that of the sample manager. The column heater’s front
compartment can accommodate any Waters column up to 4.6 mm ID and
150 mm long. The column rests in a U-shaped tray that swivels outward to
receive the column from either side.
To reduce dispersion associated with dead volume and minimize the length of
tubing between system instruments, the column tray swings outward to any
position between 0 and 180 degrees. In the 0-degree, “home”, position, the
column tray is directly above the sample manager and connected to the optical
detector (on top of the column heater). In the 180-degree, “away”, position, the
column heater can be plumbed into a mass spectrometer (located on the
system’s right-hand side).
The high temperature column heater heats the column compartment to any
temperature from 5 ºC (9 ºF) above ambient to 90 ºC (194 ºF). A film element
insulated to minimize power consumption and facilitate thermal stability is
attached to the tray and produces heat. A passive column stabilizer, inside the
tray, reduces sensitivity to ambient temperature swings and minimizes
bandspreading.
A receptacle on the column heater’s right-hand side receives the column's
eCord™ chip. The eCord column chip stores column information that you can
access from the ACQUITY UPLC Console.
The column heater drip tray captures any leakage, routing it to the sample
manager.

Column manager
The optional column manager can regulate the temperature of up to four
columns from 10 to 90 °C (50 to 194 °F). The column manager also offers a
bypass channel and automated, programmable switching between columns for
methods development. ACQUITY UPLC BEH Technology™ columns are
equipped with eCord Information Management Technology, which captures
the history of each column to assist in tracking column usage. Reusable
high-pressure fittings ease replacement of the columns, when needed.

1-8

System Overview

Column heater/cooler
The optional column heater/cooler can maintain four columns in a series from
10 to 90 °C (50 to 194 ºF), but does not have switching valves. One column’s
eCord connects to the top port to track column usage.

30-cm column heater/cooler
The optional 30-cm column heater/cooler can regulate the temperature of
HPLC columns up to 30 cm long, from 4 to 65 °C (39.2 to 149 ºF).

Optional sample organizer
The optional sample organizer stores microtiter or vial plates and transfers
them to and from the sample manager, automating their processing and
increasing throughput.
The sample organizer’s storage shelf compartment can hold a selection of
ANSI plates. Sample plates are loaded into the organizer through a large,
swing-open front door. The shelf compartment is thermally conditioned by
sample organizer heater/coolers that, together with the sample manager
heater/cooler, control the temperature between 4 and 40 ºC (39.2 to 104 ºF) in
21 ºC (69.8 ºF) or less ambient conditions.
Three subassemblies move plates within the sample organizer: the Z-Drive,
the sample organizer transfer shuttle (Y-axis), and the sample manager
transfer shuttle (X-axis). The Z-Drive moves the Y-axis to the target shelf,
where the Y-axis picks the plate. Then the Z-Drive moves the Y-axis to the
same elevation as the X-axis. The Y-axis shuttles the plate into the X-axis,
which transfers the plate into the sample manager for processing. When the
sample manager finishes with the plate, the X-axis pulls it back into the
sample organizer. The process is reversed to return the plate to the shelf it
came from.

Column heater/cooler

1-9

Detectors
The system can be configured with a TUV, PDA, ELS, or FLR detector or a
combination of them.

TUV detector
The TUV (tunable ultraviolet) optical detector is a two-channel,
ultraviolet/visible (tunable UV/Vis) absorbance detector designed for use in
the ACQUITY UPLC system. The detector, controlled by Empower or
MassLynx software for both LC/MS and LC applications, operates as an
integral part of the system.
The detector offers two flow cell options. The analytical flow cell, with a
volume of 500 nanoliters and a pathlength of 10 mm, and the high sensitivity
flow cell, with a volume of 2.4 microliters and a 25 mm pathlength, both
utilize the Waters patented light-guiding flow cell technology.
The TUV detector operates at wavelengths ranging from 190 to 700 nm.

PDA detector
The PDA (photodiode array) optical detector is an ultraviolet/visible light
(UV/Vis) spectrophotometer that operates between 190 and 500 nm.
The detector offers two flow cell options. The analytical flow cell, with a
volume of 500 nanoliters and a pathlength of 10 mm, and the high sensitivity
flow cell, with a volume 2.4 microliters and a 25 mm pathlength, both utilize
the Waters patented light-guiding flow cell technology.

ELS detector
The ACQUITY UPLC ELS detector is an evaporative light scattering detector
designed for use in the ACQUITY UPLC system. This detector can be
controlled by Empower or MassLynx software.
The detector incorporates a flow-type nebulizer that is optimized for
ACQUITY UPLC system performance.

1-10

System Overview

FLR detector
The Waters ACQUITY UPLC FLR detector is a multi-channel,
multi-wavelength fluorescence detector designed for use in the ACQUITY
UPLC system. Optimized for UltraPerformance LC applications, the FLR
detector features a low volume, axially illuminated flow cell (<2 µL), low-noise
electronics, and high-intensity Hg-Xe lamp resulting in a design that
minimizes stray light while maximizing light throughput, thus enhancing the
quality of the fluorescence signal. The detector has an excitation wavelength
range of 200 to 890 nm, an emission wavelength range of 210 to 900 nm,
support for data rates up to 80 Hz, and offers 3D scanning capability for easier
methods development.

Median baseline filter
The median baseline filter is intended to decrease the effects of gradient
separations on the chromatographic baseline. The filter is available for the
TUV, PDA, and ELS detectors but is most applicable in the absorbance
detectors. The median baseline filter enhances the absorbance detector’s
stability by decreasing its curvature, making the development of integration
methods easier.
See also: ACQUITY UPLC Console online Help.

Mass spectrometers
You can configure the system with an SQ, TQ, or other type of mass
spectrometer. If your system has a mass spectrometer other than an SQ or
TQ, refer to the documentation included with it.

SQ detector
The SQ detector is a single-quadrupole, atmospheric pressure ionization (API)
mass spectrometer. Designed for routine ACQUITY UPLC/MS analyses, it can
scan at speeds up to 10,000 Da/s.

TQ detector
The TQ detector is a tandem quadrupole, atmospheric pressure ionization
(API) mass spectrometer. Designed for routine ACQUITY UPLC/MS/MS
analyses in quantitative and qualitative applications, it can operate at fast
acquisition speeds compatible with UltraPerformance LC.
Mass spectrometers

1-11

Data systems
The system can run under Empower, MassLynx, or certain third-party
software control.

Empower software
Empower software provides a graphical, icon-based user interface that
acquires, processes, manages, reports, and stores chromatographic data.
The base version of Empower software supports data from TUV, PDA, ELS,
and FLR detectors, and single quadrupole mass spectrometers. Popular
software options for ACQUITY UPLC system users include System
Suitability, Chemical Structures, and Method Validation Manager.
See also: Empower online Help.

MassLynx software
MassLynx is a high-performance mass spectrometry application that acquires,
analyzes, manages, and distributes UV and mass spectrometry data. It offers
intelligent instrument control and can acquire nominal mass, exact mass,
MS/MS, and exact mass MS/MS data.
See also: MassLynx Getting Started Guide and MassLynx online Help.

Columns
ACQUITY UPLC columns are packed with 1.7-μm, bridged, ethylsiloxane,
hybrid particles that can mechanically endure high-pressure conditions. The
column hardware and the matched outlet tubing can withstand up to
103,421 kPa (1034 bar, 15,000 psi). The column dimensions allow optimal
MS-compatible flow rates, and matched outlet tubing minimizes the effect of
extra-column volume.
Although the system works with any analytical HPLC column, specially
designed ACQUITY UPLC columns maximize its high-pressure capabilities.
Compared with traditional HPLC columns, ACQUITY UPLC columns deliver
superior resolution and sensitivity in the same run time or equivalent
resolution, greater sensitivity, and faster run times.

1-12

System Overview

eCord column chip
ACQUITY UPLC columns include an eCord column chip that tracks the usage
history of the column. The eCord column chip interacts with the system
software, recording information for up to 50 sample queues run on the column.
In regulated environments, the eCord column chip provides documentation of
the column used in the validation method.
In addition to the variable column usage data, the eCord column chip also
stores fixed column manufacturing data, including
•

unique column identification.

•

certificate of analysis.

•

QC test data.

Once the eCord column chip is attached to the receptacle on the column
heater, information is automatically recorded by the system. No user action is
required. This information is stored only in the eCord column chip.

FlexCart
The optional FlexCart provides for the ACQUITY UPLC system a mobile
platform. It can hold the system instruments as well as the PC and monitor
and provides electrical outlets for system instruments and integrated waste
management. Used with a mass spectrometer, the cart’s adjustable height lets
you position the column outlet close to the inlet probe, minimizing system
dead volume.

FlexCart

1-13

For additional information
Refer to the following documents for further information:

1-14

•

ACQUITY UPLC Quick Reference Card (part number 71508250006)

•

ACQUITY UPLC System Operator’s Guide (part number 71500082502)

•

ACQUITY UPLC System Bookshelf Documentation CD (part number
71500082521)
–

ACQUITY UPLC Photodiode Array Detector Getting Started Guide
(part number 71500108703)

–

ACQUITY UPLC Evaporative Light Scattering Detector Getting
Started Guide (part number 71500109303)

–

ACQUITY UPLC Fluorescence Detector Getting Started Guide (part
number 71500142403)

–

Waters SQ Detector Quick Start Guide (part number 71500126603)

–

Waters TQ Detector Quick Start Guide (part number 71500126803)

–

Controlling Contamination in Ultra Performance LC/MS and
HPLC/MS Systems (part number 715001307)

•

ACQUITY UPLC Console online Help

•

ACQUITY UPLC System release notes

System Overview

Preparing System Hardware
Contents
Topic

Page

Powering-on the system

2-1

Monitoring startup tests

2-3

Monitoring system instrument LEDs

2-3

Enabling the leak sensors

2-6

Preparing the binary solvent manager

2-7

Preparing the sample manager

2-15

Preparing the column manager

2-30

Preparing the sample organizer

2-30

Starting the TUV detector

2-36

Conditioning the column

2-39

Shutting down the system

2-40

Running HPLC methods on an ACQUITY UPLC system

2-42

Powering-on the system
®

Powering-on the system entails starting the ACQUITY UPLC system
workstation, system instruments, and Empower or MassLynx operating
software.
To power on the system
1.

Power-on the ACQUITY UPLC system workstation.

You must power-on the HT column heater, column manager, or column
heater/cooler before any of the other instruments because they contain
the internal Ethernet switch that allows the ACQUITY UPLC system
workstation to communicate with all the instruments.

Powering-on the system

2-1

Tip: If your system contains an HT column heater, it is automatically
powered-on when you power on the sample manager.
To power-on a column manager or column heater/cooler, press the power
button on the top, left-hand side of it’s front panel. Each system
instrument beeps 3 times and runs a series of startup tests.
The power and status LEDs change as follows:

•

Each system instrument’s power LED shows steady green. The
column manager’s and column heater/cooler’s run LEDs all show
red for a few seconds.

•

During initialization, each system instrument’s power LED shows
steady green. The column manager’s and column heater/cooler’s run
LEDs all show flashing green.

•

After the instruments are successfully powered-on, each one’s power
LED shows steady green. The column manager’s and column
heater/cooler’s run LEDs are unlit.

Press the power switch on the top, left-hand side of the binary solvent
manager’s, sample organizer’s, and sample manager’s door. Each system
instrument beeps 3 times and runs a series of startup tests.
Requirement: If your system has a sample organizer, you must power it
on before you power on the sample manager.
The power and status LEDs change as follows:

2-2

•

Each system instrument’s power LED shows steady green. The
binary solvent manager’s flow LED and the sample organizer’s and
sample manager’s run LEDs show red for a few seconds.

•

During initialization, each system instrument’s power LED shows
steady green. The binary solvent manager’s flow LED and the
sample organizer’s and sample manager’s run LEDs show flashing
green. Full initialization of the system usually requires about 7
minutes.

•

After the instruments are successfully powered-on, each one’s power
LED shows steady green. The binary solvent manager’s flow LED
and the sample organizer’s and sample manager’s run LEDs are
unlit.

After the binary solvent manager’s, sample organizer’s, and sample
manager’s power LEDs show steady green, press the power switch on
the top, left-hand side of the detector(s).

Preparing System Hardware

The detector’s power and status LEDs change as follows:
•

The detector’s power LED shows steady green and its lamp LED
shows red for a few seconds.

•

During initialization, the detector’s power LED shows steady green
and its lamp LED shows flashing green.

•

After the detector is successfully powered-on, its power LED shows
steady green. The detector’s lamp LED shows steady green,
indicating that the lamp is ignited.

Tip: To prevent initialization errors, only power on the detector(s) when
the flow cell is wet.
5.

Start the Empower or MassLynx operating software. You can monitor
the ACQUITY UPLC Console for messages and LED indications.

Monitoring startup tests
These startup tests run when you power-on the ACQUITY UPLC system
workstation:
•

CPU board

•

Memory (RAM and ROM)

•

External communication system (Ethernet)

•

Clock

If the startup tests indicate a malfunction, consult the ACQUITY UPLC
Console online Help.

Monitoring system instrument LEDs
Light emitting diodes on each system instrument indicate the instrument’s
state of functioning. The LEDs are specific to their instruments, so the
significance of their various colors and modes can differ from one instrument
to another.

Monitoring startup tests

2-3

Power LED
The power LED, on the left-hand side of an instrument’s front panel, indicates
the power-on or power-off status of the instrument. This LED is green when
power is on and unlit when power is off.
Tip: To provide adequate ventilation, the sample manager and sample
organizer fans are always running, even with the power off. These fans switch
off only when the power cable is removed from the back of the instrument.

Status LEDs
Flow LED (binary solvent manager)
The flow LED, on the right-hand side of the power LED on the binary solvent
manager’s front panel, indicates the flow status. A steady green flow LED
indicates that there is a flow through the binary solvent manager.

Run LED (sample manager, column manager, column heater/cooler,
and sample organizer)
The run LED, on the right-hand side of the power LED on the sample
manager’s, column manager’s, column heater/cooler’s, and sample organizer’s
front panel, indicates the run status. A steady green run LED indicates that
injections are being run.

Lamp LED (detector)
The lamp LED, on the right-hand side of the power LED on the detector’s
front panel, indicates the lamp status. A steady green lamp LED indicates
that the lamp is on.
Status LED indications

2-4

LED mode and color

Description

Unlit

• Binary solvent manager, sample manager,
column manager, column heater/cooler, and
sample organizer – Indicates the instrument
is currently idle.
• Detector – Indicates the detector lamp is
extinguished.

Preparing System Hardware

Status LED indications (Continued)
LED mode and color

Description

Steady green

• Binary solvent manager – Indicates solvent is
flowing.
• Sample manager, column manager, column
heater/cooler, and sample organizer –
Indicates the sample manager, column
manager, column heater/cooler, or sample
organizer is operating normally, attempting to
complete any outstanding samples or
diagnostic function requests. When sample
and diagnostic function requests are finished,
the LED reverts to the unlit mode.
• Detector – Indicates the detector lamp is
ignited.

Flashing green

• Binary solvent manager, sample manager,
and sample organizer – Indicates the system
is waiting for at least one instrument to
become operable. Detector lamp warm-up and
column temperature equilibration times
typically cause such a delay.
• Column manager and column heater/cooler –
Indicates the system is waiting for the
instrument to reach the temperature set point
before operating. Column temperature
equilibration times typically cause such a
delay. The LED also flashes green during
initialization or while waiting for
initialization.
• Detector – Indicates the detector is initializing
or calibrating.

Flashing red

Indicates that an error stopped the instrument.
Refer to the ACQUITY UPLC Console for
information regarding the error.

Steady red

Indicates an instrument failure that prevents
further operation. Power-off the instrument, and
then power-on. If the LED is still steady red,
contact your Waters service representative.

Monitoring system instrument LEDs

2-5

Enabling the leak sensors
Rule: When you power-on the system, the leak sensors default to disabled
unless previously enabled.
To enable the leak sensors
1.

In the ACQUITY UPLC Console, select Control > Leak Sensors.
Leak Sensors dialog box

Click to enable
or disable all
instrument
leak sensors

Click to enable or disable individual
instrument leak sensors

2-6

To enable the leak sensor for an individual instrument, click the status
on the left-hand side of the instrument description. Or, to enable all leak
sensors, click Enable All.

Preparing System Hardware

Preparing the binary solvent manager
For optimal performance of the ACQUITY UPLC system, you must prepare
the binary solvent manager for operation.
To prepare the binary solvent manager for operation, you must perform a seal
wash prime and then prime the binary solvent manager.
Warning: Observe Good Laboratory Practices when you handle solvents.
See the Material Safety Data Sheets for the solvents you use.
Requirement: To maintain the efficiency of the binary solvent manager and to
obtain accurate, reproducible chromatograms, use only MS-grade solvents,
water, and additives. For details, see the Waters ACQUITY UPLC System
Operator’s Guide.
Caution: To avoid damaging binary solvent manager components, do
not use chloroform, methylene chloride, ethyl acetate, or toluene.

Performing a seal wash prime
Prime the seal wash in the binary solvent manager to lubricate the plungers,
fill the tubing paths with solvent, and flush away solvent and/or any
precipitated salts that have been dragged past the plunger seals from the
high-pressure side of the piston chambers.
Prime the plunger seal wash
•

after using buffered mobile phase.

•

when the binary solvent manager has been inactive for a few hours or
longer.

•

when the binary solvent manager is dry.
Caution: To avoid damage to the solenoid valve seats and seals in the
solvent path, do not use a nonvolatile buffer as the seal wash solvent.

Tip: The seal wash self-primes, but you can use a syringe to hasten the
process.
Rule: To prevent contamination, do not recycle seal wash.

Preparing the binary solvent manager

2-7

Recommendations:
•

Seal wash must contain 10% organic solvent. This concentration
prevents microbial growth and ensures that the seal wash can solubolize
the mobile phase.

•

Before priming the plunger seals, ensure the volume of seal wash is
adequate for priming.

See also: Controlling Contamination in Ultra Performance LC/MS and
HPLC/MS Systems (part number 715001307) on the ACQUITY UPLC
System Bookshelf CD.

Required materials
•

30-mL syringe (startup kit)

•

Seal wash solution

•

Tubing adapter (startup kit)

To perform a seal wash prime
1.

Ensure the seal wash inlet tube is in the solvent reservoir.

Remove the seal wash outlet tube from the right-hand side of the drip
tray.

Seal wash
outlet tube
Drip tray

2-8

Push the syringe plunger fully into the syringe barrel.

Connect the tubing adapter to the syringe, and then connect the syringe
assembly to the outlet tubing from the seal wash system.

Preparing System Hardware

In the ACQUITY UPLC Console, select Binary Solvent Manager from
the system tree.

Click Control > Prime seal wash, and then click Yes to begin the seal
wash priming process.

Slowly draw back on the syringe plunger to pull seal wash solvent
through the system.

When the seal wash solution begins to flow into the syringe without
major air bubbles, disconnect the tubing and reinstall it on the fitting on
the drip tray.

Click Control > Prime seal wash, and then click Yes to stop the priming
process.

Priming the binary solvent manager
Priming is used to prepare a new system or binary solvent manager for use,
change reservoirs or solvents, and run the system after it has been idle for
more than 4 hours. During priming, the vent valve moves to Vent position to
both ensure minimal backpressure and direct the flow to waste. The flow rate
during priming is 4 mL/min for each pump being primed.
Tip: If you are priming a dry binary solvent manager, using a syringe shortens
the time required to complete priming.
Caution: To prevent salts from precipitating in the system, introduce
an intermediate solvent, such as water, when changing from buffers to
high-organic-content solvents. Be sure to consult the solvent
miscibility tables in the Waters ACQUITY UPLC System Operator’s
Guide.
Recommendation: Ensure the solvent reservoirs have enough solvent for
adequate priming and the waste container has sufficient capacity for used
solvent. The priming flow rate is 4 mL/min for each pump, or 8 mL/min total.
For example, priming both solvents for 5 minutes requires approximately 20
mL of each solvent.
Warning: To avoid spills, empty the waste container at regular intervals.

Preparing the binary solvent manager

2-9

Priming a dry binary solvent manager
To prime a dry binary solvent manager
1.

Open the instrument’s front door.

Locate the appropriate solvent vent line.

In the ACQUITY UPLC Console, select Binary Solvent Manager from
the system tree.

In the binary solvent manager information window, click Control >
Prime A/B Solvents.

In the Prime A/B Solvents dialog box, select solvent A and/or B.

In the Time box, specify the number of minutes from 0.1 through 60.0.
Default: 1.0 minute
Recommendations: Prime the binary solvent manager until a steady
flow exits the vent tube (typically 7 to 10 minutes).

Click Start. When solvent flows out of the vent line without bubbles, the
path is primed.

Repeat step 3 through step 7 to prime the other solvents.
Requirement: There must be solvent in the A1, A2, B1, and B2
reservoirs for the degasser to function correctly.

To prime a dry binary solvent manager using a syringe
1.

Open the instrument’s front door.

Locate the appropriate solvent vent line.
•

2-10

If you are priming solvent A, follow the stainless steel vent line that
is labeled “A-VENT” from port 4 on the vent valve, and lift it out of
the drip tray.

Preparing System Hardware

•

If you are priming solvent B, follow the stainless steel vent line that
is labeled “B-VENT” from port 1 on the vent valve, and lift it out of
the drip tray.

Vent valve

Drip tray

Solvent
vent lines

Push the syringe plunger fully into the syringe barrel.

Connect the tubing adapter to the syringe.

Connect the syringe assembly to the short length of PharMed tubing,
and then connect the short length of PharMed tubing to the solvent vent
line you located in step 2.

In the ACQUITY UPLC Console, select Binary Solvent Manager from
the system tree.

In the binary solvent manager information window, click Control >
Prime A/B Solvents.

In the Prime A/B Solvents dialog box, select solvent A1.

In the Time box, specify the number of minutes from 0.1 through 60.0.
Default: 1.0 minute
Recommendation: Prime the binary solvent manager until a steady
flow exits the vent tube (typically 3 minutes).

10. Click Start.
11. Slowly draw back on the syringe plunger to pull solvent through the
solvent path. When solvent flows out of the vent line without bubbles,
the path is primed.

Preparing the binary solvent manager

2-11

12. Remove the syringe from the vent line, and reconnect the vent line to the
drip tray.
13. Repeat step 2 through step 12 for solvent A2, B1, and B2.
Requirement: The reservoirs and solvent lines for solvents A1, A2, B1,
and B2 must not be empty. Otherwise the degasser does not function
correctly.

Priming a wetted binary solvent manager
Two functions help prepare the system for operation:
•

Refresh system (Sys Prep)

•

Start up

The length of time the system has been idle determines which is the better.

Refreshing the system
Use the Refresh (Sys Prep) function after the system has been idle a short
period of time (a few hours to overnight) and when you plan to use the same
solvents that you used previously.
You can invoke the Sys Prep function from the control panel or by adding it as
a line in a sample set.
Recommendations:
•

Prime the binary solvent manager for 1 minute if the system has been
idle for 4 or more hours and you will use the solvents that are already in
the system.

•

Prime the binary solvent manager for 4 minutes if you will use new
solvents that are of the same composition of what is already in the
system.

To refresh the system
1.

2-12

In the ACQUITY UPLC Console, click Control > Refresh system (Sys
Prep).

Preparing System Hardware

In the Refresh System (Sys Prep) dialog box, review the settings and
select a different option, if needed. The system primes your current
solvent selections (A1 or A2, B1 or B2).
•

Solvent line A only (default)

•

Solvent line B only

•

Both A and B

Click OK.
Result: The system primes the selected solvents, primes the sample
manager with one weak wash prime (using the wash and sample
syringes), and ignites the lamp in the detector.

Starting up the system
Use the Start up function to prime the binary solvent manager after changing
the mobile phase, after changing the sample needle and/or sample loop, or
after the system has been idle a long period of time (overnight or over a
weekend). Before you begin this procedure, ensure that the system is correctly
configured for use.
Recommendation: Prime the binary solvent manager for 5 minutes if you are
changing to solvents whose compositions differ from the compositions of
solvents already in the system.
To start up the system
1.

In the ACQUITY UPLC Console, click Control > Start up.

In the Prime Solvents tab of the System Startup dialog box, review the
settings for the A/B Solvents. In the A/B Solvents area, you can select or
clear any or all of the solvents: A1, A2, B1, or B2. You can change the
length of time to prime solvents A and B by entering a different number
in Duration of Prime. All selected solvents are primed for the same
duration.
Allowed values: 0.1 to 60.0 minutes
Tip: If you want to return settings to their original values on any tab,
click Set Defaults.
Defaults: Solvents A1, A2, B1, and B2 prime for 1.0 minute each.

Select or clear priming of the seal wash, strong wash, weak wash, and/or
sample syringe.

Preparing the binary solvent manager

2-13

Default: The seal wash is primed for 1.0 minute, the weak wash once,
and the sample syringe once.
4.

If necessary, change the number of cycles to prime the syringes by
entering a different whole number in the Cycles field.
Default: 10 cycles for each syringe selected

Select the Equilibrate to Method tab to review the settings for the final
flow rate, mobile phases, composition, temperatures, and lamp state.
Change the values as needed to match your requirements at
equilibration.
Equilibrate to Method tab values
System startup
parameters

Default

Allowed values

Method initial flow rate

0.25
mL/min

0.1 to 2.0 mL/min

Composition of A and B
(sum must be 100%)

A1, 100
B1, 0%

A1, A2; 0 to 100%
B1, B2; 0 to 100%

Column temperature

Off

Off, or 5.0 °C (9 °F) above
ambient to 65.0 °C (149 °F)

Sample temperature

Off

Off, or 4.0 to 40.0 °C (39.2
to 104 °F) in 25 °C (77 °F)
ambient conditions

Lamp

On or off

If you changed the sample needle or loop, in the Configuration area,
click Change. In the Volume Configuration dialog box, select the new
size of loop and/or needle, and then click OK.

If you changed the sample needle, click the Optional: Characterize
Volume tab, and then select “Characterize seal” and “Characterize
needle and loop volumes”.

If you changed the sample loop, in the Optional: Characterize Volume
tab, select Characterize needle and loop volumes.

Click Start.
Result: The lamp in the optical detector ignites, the ACQUITY UPLC
system sets the column sample temperatures, and all priming starts. If

2-14

Preparing System Hardware

you selected the Characterize seal function after priming finishes, the
sample manager finds the position of the needle seal and then logs the
results into the database.
Finally, the system establishes the method flow rate, solvent selections,
and composition. The default settings for the method initial flow are
100% Solvent A1 at .25 mL/min and 0% B1, the column and sample
temperatures are Off, and the detector lamp is ignited.

Preparing the sample manager
Prepare the sample manager for operation after you prepare the binary
solvent manager. Preparing the sample manager involves these steps:
•

Priming

•

Characterizing the seal

•

Characterizing the needle and sample loop volumes

•

Loading sample plates
Warning: To avoid solvent spills and to maintain proper leak
drainage, always close the sample manager fluidics tray before
operating the system.

Selecting weak wash and strong wash solvents
For best performance, follow these guidelines when selecting wash solvents.
Otherwise performance can be reduced, specifically Area/Height RSD and
Linearity. The guidelines do not prohibit all other solvent combinations,
however. Other combinations can be run with lower performance expectations
or by manipulating default injection parameters.
Use a weak wash solvent based on the sample and mobile phase chemistries of
your application, making sure all solutions/buffers are miscible and soluble.
Recommendation: For buffered aqueous, reversed-phase chromatographic
conditions and MS applications, use a weak wash solvent of 100% water or 0%
to 25% methanol or acetonitrile and a strong wash solvent of 50% to 100%
methanol or acetonitrile. High sample concentrations can require other weak
wash solvents. If your separation permits, Waters recommends adding a small
amount of an organic solvent (~10%) to prevent microbial growth.

Preparing the sample manager

2-15

See the Waters ACQUITY UPLC System Operator’s Guide for further
information about solvents.
Caution: To avoid damage to the solenoid valve seats and seals in the
solvent path, do not use a nonvolatile buffer as the weak wash or
strong wash solvent.
Tip: For best performance, the weak wash solvent must be similar or identical
to your isocratic or initial gradient solvent conditions, excluding buffers. Do
not use salt buffers in wash solvents.
Wash solvent effects
Property

Effect

Organic species

As a general principle, strong and weak solvents
must include the same organic species. Note that
this is not always be practicable. You can,
however, use a 100% organic strong wash solvent.

Solvent composition

The weak wash solvent must reflect as closely as
possible the same composition as the initial
gradient mobile phase.

Adjust the pH of strong and weak solvents for best
peak shape and carryover performance.

Concentration of strong Strong solvent must be no stronger than the
solvent
concentration needed to reduce carryover to an
acceptable level.
Solubility of sample

The sample must be soluble in both the weak and
strong wash solvents.
Caution: Proteins (in plasma, for example) do
not dissolve in solvents whose organic component
is greater than 40%.

Sample diluent

2-16

Preparing System Hardware

The weak wash solvent will contact the sample, so
match these as closely as possible. To offset
adverse effects on peak shape caused by the
matrix’s composition, adjust the weak wash
composition, especially when using the instrument
in partial loop mode.

Wash solvent effects (Continued)
Property

Effect

Wash volume ratio
(weak to strong)

Within a method, use a 3:1 ratio, weak wash to
strong–sufficient to ensure the weak wash flushes
the strong from the needle and sample loop before
sampling.

Cycle times

Higher viscosity wash solvents lengthen wash
cycles.

Priming the sample manager
The priming process fills the sample needle with solvent, flushes new solvent
through the injector lines, and/or purges air from the lines. You prime the
sample needle and/or sample syringe to accomplish these tasks:
•

Prepare a new sample manager for operation

•

Prepare a sample manager for operation after it has been idle for an
extended period

•

Change the solvent in the syringes

•

Remove bubbles from the lines

Ensure that the priming solvent is correctly composed and that it is high in
quality and miscible with any other solvents used in your system. Use filters
in all solvent reservoirs, and ensure the volumes of solvents are sufficient for
priming.
Requirement: The sample manager must be primed before you attempt to
characterize the seal.
To prime the sample manager
1.

In the ACQUITY UPLC Console, select Sample Manager from the
system tree.

Preparing the sample manager

2-17

Sample manager information window

Click Control > Prime syringes.
Alternative: Right-click in the Empower or MassLynx sample manager
control panel, and then click Prime syringes.

In the Prime Syringes dialog box, select Sample syringe and both wash
syringes.
Tip: If you want only to remove air bubbles from the sample syringe, but
do not want to prime the wash syringes, select Sample syringe only.
However, do not select this option routinely. Priming all components at
the same time is good practice.

Type the number of primes in the Number of cycles text box.
Default: 1
Recommendation: Waters recommends 5 to 7 primes when you are
changing solvents.
Caution: Do not abort the sample manager priming sequence.
Doing so can leave strong solvent in the sample needle, which can
adversely affect chromatography.

Click OK to start priming. When the system status is “Idle,” priming is
finished.
Tip: Each prime takes approximately 2 to 4 minutes.

2-18

Preparing System Hardware

Washing the sample manager needle
Washing the needle is an optional procedure that flushes strong and/or weak
wash solvent through the needle and injection port. Washing the needle
removes contaminants from the inside and outside of the needle, the external
piercing needle, and the injection port. You can also perform a needle wash to
ascertain proper flow through the waste tubing and to confirm that the needle
wash system is primed and properly operating.
Rule: Do not use buffered solvents as wash solvents.
Tip: Priming the system washes the sample needle, so whenever you prime
the system, you can omit this procedure.

Observing wash solvent recommendations
Waters recommends that you observe these guidelines for washing the needle:
•

To ensure that the strong wash solvent is completely removed, the
system washes the needle with 500 μL of weak wash solvent after you
use strong wash solvent. You can increase, but not decrease, the default
value of 500 μL.

•

The analytes and sample matrix must be soluble in both weak and
strong solvents. Proteins do not dissolve in solvents whose organic
component is greater than 40%. Do not use buffers in any wash solvent.
Example: If the weak wash solvent is 30% acetonitrile and 70% water,
the strong wash solvent must contain a greater concentration of
acetonitrile in water.

•

The weak wash solvent must be the same as the initial eluting solvent,
and the strong wash solvent must at least equal the composition of the
final eluting solvent.

•

Use a weak wash solvent based on the sample and mobile phase
chemistries of your application. Make sure all solutions are miscible and
soluble. For best results, weak wash solvent must match the initial
gradient conditions and mobile phase composition (isocratic). High
sample concentrations can require additional weak wash solvents.

•

For buffered aqueous, reversed-phase chromatography, use weak wash
solvent consisting of 100% water or up to 25% methanol or acetonitrile.
For strong wash solvent, use 50% to 100% methanol or acetonitrile.

Before you begin, ensure that the solvents are compatible with your
application, that their volumes are sufficient, and that the waste reservoir is
large enough to contain the waste solvent.
Preparing the sample manager

2-19

Needle recommendations
Waters offers needles made from several types of materials to accommodate
compounds that may have specific handling requirements or affinities. Users
must choose the needle material best suited to their application. For example,
the stainless steel needle is recommended when working with samples that
are known to be attracted to hydrophobic polymers.
Choose a stainless steel needle or stainless steel/Teflon needle for greater
strength and more robust operation. Stainless steel needles are also
recommended for use with hexane and tetrahydrofuran; PEEK needles are
not recommended for use with either of these solvents.
Choose smaller needles when using partial loop (pressure assist) mode, or
when using the smallest loops. However, using a smaller needle will increase
cycle times.
To wash the sample manager needle
1.

In the ACQUITY UPLC Console, select Sample Manager from the
system tree. The sample manager information window appears.

Click Control > Wash Needle.
Alternative: Right-click in the Empower or MassLynx sample manager
control panel, and then click Wash Needle.

In the Strong Wash box, specify the volume for the strong wash solvent.
Or, to omit strong wash solvent, enter 0 in the Strong Wash box, or leave
it blank.
Range: 0.0 through 99,999 μL
Default: 0.0 μL
Recommendation: 100 through 500 μL
Tip: Using both a weak and strong wash solvent increases the wash time
and solvent consumption because the system must be fully cleansed of
the strong solvent before starting the next injection.
Caution: To avoid strong wash solvent contacting the sample and
contaminating it, use a sufficient quantity of weak wash solvent.

2-20

Preparing System Hardware

In the Weak Wash box, specify the volume for the weak wash solvent.
Range: 1.0 through 99,999 μL
Default: 200.0 μL without strong wash or 500 μL with strong wash
Recommendation: 200 through 500 μL or three times the strong wash
volume.

Click OK. The needle wash begins.

When needle washing is complete, the status returns to Idle.

To stop a needle wash routine before it finishes
From the sample manager information window, click Control > Reset SM.
Alternative: Right-click in the Empower or MassLynx sample manager control
panel, and then click Reset SM.
Caution: Do not abort the sample needle wash sequence. Doing so can
leave strong solvent in the sample needle, which can adversely affect
chromatography.

Characterizing the needle seal
The needle seal characterization procedure finds the position at which the
needle obtains a seal within the wash station block. The sample manager
must be primed before starting this procedure.
Requirements:
•

Perform this procedure before calibrating the needle and sample loop
volumes.

•

Perform this procedure after you replace and/or adjust these items:
•

Needle

•

any part of the needle assembly

•

Needle (Z) or piercing needle (Zp) flags (home and top-of-plate)

•

Home or top-of-plate sensor

•

Inject port seal

•

Wash station

•

NVRam battery on the CPU2000

Preparing the sample manager

2-21

To characterize the needle seal
1.

In the ACQUITY UPLC Console, select Sample Manager from the
system tree.

In the sample manager information window, click Maintain >
Characterize > Needle seal.

In the Characterize Needle Seal dialog box, click Start. The calibrate
seal operation begins, and the sample manager status displays
“Calibrating seal”.

When calibration ends, the sample manager status displays “Idle”.

Click Results to view the results of the needle seal characterization
operation.

Characterizing the needle and sample loop volumes
Whenever you replace the sample loop and/or the sample needle, you must set
the system to characterize the volume of the replacement parts. Do this
regardless of whether the sizes of the replacement parts are nominally the
same as those of the original parts or differ from them. Also perform this
procedure when the composition of the weak wash solvent changes, because
solvent characteristics such as viscosity, surface tension, and polarity can
change. During sample injection, the weak wash solvent precedes and follows
the sample in the fluid-carrying lines, so the sample is directly affected by the
weak wash solvent.
Characterizing the loop volume compares the loop’s nominal volume (in μL) to
its measured volume.
Characterizing the needle volume compares the needle’s nominal volume (in
μL) to its measured volume.
Tip: Characterizing the system volume is critical to good sample manager
performance.
Requirements:

2-22

•

Specify the sizes of the sample needle, loop, and syringe in the Volumes
dialog box before characterizing the volumes.

•

Prime the sample manager and syringes, and characterize the needle
seal before characterizing the volumes.

Preparing System Hardware

•

Create a method (using Empower or MassLynx software) that has the
same air gap and sample draw rate that you will be using.
–

If you are running under Empower software control, click File >
New Method > Instrument Method in the Project window.

–

If you are running under MassLynx software control, click Inlet
Method > Inlet > Autosampler in the Inlet Editor window.

To characterize the needle and sample loop volumes
1.

In the ACQUITY UPLC Console, select Sample Manager from the
system tree.

Click Maintain > Characterize > Needle and loop volumes.

In the Characterize Needle and Loop Volumes dialog box, click Start.
Tip: This procedure takes at least 5 minutes.

Click Results to view the results of the needle and loop volumes
characterization operation.
Result: If the needle fails the test, suspect it is bent, broken, or blocked.
If the sample loop fails the test, suspect that it is blocked or leaking, that
it has a loose fitting, or that the draw rate is too high.

Using the extended puncture needle
When using an extended puncture needle, be aware of the following:
•

The thumbscrew on the needle mounting bracket is grey plastic or
stainless steel instead of red.

•

When used with ACQUITY UPLC System Instrument Driver version
1.30 or higher, the extended feature is automatically activated.

•

The extended puncture needle is compatible with software released prior
to ACQUITY UPLC System Instrument Driver version 1.30, but the
extended puncture needle functionality is not active.

•

Whenever you calibrate the XYZ mechanism, the extended puncture
needle is automatically recognized.

Preparing the sample manager

2-23

Loading sample plates in the sample manager
The ACQUITY UPLC sample manager holds up to two ANSI/SBS plates that
you load through the front door. The left-hand plate is referred to as position
1, the right-hand plate as position 2.
Exception: If the optional sample organizer is installed, you can load only one
plate through the sample manager front door. You must load the plate on the
right-hand tray. In this case, the right-hand tray becomes the number one
position.

Observing vial and plate recommendations
Waters recommends that you observe these guidelines for loading sample
vials and plates in the sample manager:
•

Use only Waters-certified vials.

•

Do not used Waters total recovery vials with the ACQUITY UPLC
system default settings. If the needle depth position is suitably offset,
total recovery vials can be used successfully with PEEK needles.

•

Use only 1860024XX-series vials and cap mats in the ACQUITY UPLC
sample manager and sample organizer.

•

Always measure other vendor’s plates to determine their suitability for
use in the ACQUITY UPLC sample manager and sample organizer.

•

To avoid warping plates, do not centrifuge them.

•

When selecting a new plate supplier, especially for size 384 plates,
always compare the plate size to Waters specifications.

•

When using PEEK needles:
–

Use pre-slit septa to avoid bending the needle.

–

Use solid septa only when necessary.

–

Use heat-sealed plates instead of cap mats.

–

Do not use re-sealable cap mats, because they can cause alignment
errors.

To load a sample plate

2-24

Open the ACQUITY UPLC sample manager door.

Squeeze the tray button while you pull the tray toward you.

Preparing System Hardware

Load the plate onto the tray so that well position A,1 is at the rear,
right-hand corner and the forward edge of the plate is behind the spring
inside the front of the carrier.
Tip: A represents the row number, 1 represents the vial position.

Slide the tray into the sample manager until it clicks into place.

Sample plate
A,1 well
position

Button

TP02389

Plate tray

Caution: The plates must be positioned correctly to avoid damaging
the sample needle.
5.

Close the sample compartment door. A mechanism on the door ensures
the plates are positioned correctly when the door closes.

Selecting the optimum sample injection mode
The sample manager supports three injection modes – partial loop needle
overfill, partial loop, and full loop.
•

Partial loop needle overfill mode – Provides superior partial loop
accuracy, precision and linearity for a wide range of samples, including
strong and weak acids and bases, hydrophilic, and hydrophobic
compounds. This mode is the best general purpose mode for partial loop
injection.

•

Partial loop mode – Use for those situations where analysis time takes
precedence over any other concern, where the sample volume is very
limited, or where the injection volume is very large.

Preparing the sample manager

2-25

•

Full loop mode – Chosen whenever accuracy and precision are the
primary concerns. It is the recommended mode when using 1.0-mm I.D.
columns.

Partial loop needle overfill mode
Partial loop overfill mode provides optimum performance when injection
volumes are maintained within a range of 10% to 75% measured loop volume.
The generally accepted practice for partial loop injection linearity restricts the
injection volume selected to ≤ 50% nominal loop volume. However, you can
increase the usable loop volume to ≤ 75% measured loop volume by selecting
the needle overfill technique. This is based on achieving an
injection-to-injection variability of ≤ 1% across the specified volume range. In
addition, the correlation between specified injection volume and peak area is
R2 > 0.999.
See also: The table titled “Selecting the injection mode and loop volume” on
page 2-28.

Load ahead mode
The first injection of a sample set and injection sets with different methods do
not utilize load ahead mode. The minimum cycle time is the lesser of either
the two run times or sample preparation and wash.
The following figures show a standard ACQUITY UPLC system cycle time and
load-ahead cycle time.

2-26

Preparing System Hardware

Standard ACQUITY UPLC system cycle time definition
Standard injection mode and first injection in load ahead mode
Total ACQUITY UPLC system cycle time

System
setup

Chromatographic run time
SM sample preparation
and positioning
SM wash time
Sample
injection

Start

End

ACQUITY UPLC system load-ahead cycle time definition
Load ahead mode after the first injection
Total ACQUITY UPLC system cycle time

Buffer delay

System
setup and
sample
positioning
into loop

Chromatographic run time

SM wash time

Start

Sample
injection

SM sample preparation

End

Tip: The buffer delay is a “wait time” that compensates for variations in the
time it takes to load a sample.

Preparing the sample manager

2-27

The following table provides the minimum and maximum injection volumes
for each of the commonly used loops.
Selecting the injection mode and loop volume
Sample
injection
volume (μL)

Loop volume
1 μL

2 μL

5 μL

10 μL

20 μL

50 μL

Partial loop
needle overfill
mode injection
range (µL)

0.1 to .8

0.2 to 1.5

0.5 to 3.8

1.0 to 7.5

2.0 to
15.0

5.0 to
37.0

Partial loop
mode injection
range (µL)

Not
recomme
nded

1.0 to 5.0

2.0 to
10.0

5.0 to
25.0

Full loop mode 1
injection range
(µL)

a. The 50-μL loop must be used with a 250-μL sample syringe.

Installing the optional sample manager shade
If your samples are light-sensitive, and you have the optional sample manager
foam shade, be sure to install it over the sample compartment window.
For the sample manager foam shade part number, see the Waters Quality
Parts Locator on the Waters Web site’s Services/Support page.

Required material
Sample manager foam shade
To install the sample manager foam shade
1.

Turn off the sample manager compartment lights.
Tip: To turn the sample manager compartment lights off, right-click in
the sample manager control panel and then select Turn lights on/off.

2-28

Preparing System Hardware

Insert the foam shade over the outside of the sample compartment
window.

Sample compartment
window
Foam shade

Press the foam shade into place over the window.

Foam shade

Preparing the sample manager

2-29

Preparing the column manager
To prepare the column manager, ensure that each column’s eCord is
connected to the correct port.
See also: For more information on checking the eCord status, consult the
ACQUITY Console online Help.

Preparing the sample organizer
If your system includes a sample organizer, prepare it for operation according
to the procedures in this section.

Initiating communications
To initiate communications between the sample manager and sample
organizer
1.

Open the sample manager door, load a plate onto the right-hand tray,
and then close the sample manager door.
Tip: When the system has both a sample manager and a sample
organizer, the right-hand tray of the sample manager becomes position
1, and the left-hand tray is not available. The bottom shelf in the sample
organizer becomes position 2.
Exception: If the system does not include a sample organizer, the
left-hand tray in the sample manager is designated position 1 and the
right-hand tray is position 2.

2-30

In the ACQUITY UPLC Console, select Sample Manager from the
system tree.

In the sample manager information window, click Configure > Sample
Organizer.

In the Sample Organizer Configuration dialog box, select the sample
organizer from the list of serial numbers, and then click OK.

The sample organizer automatically detects which shelves contain
plates and illuminates their corresponding LEDs.

Preparing System Hardware

Loading sample plates
The sample organizer holds up to 21 standard ANSI/SBS footprint plates that
you load through the front door. However, the actual number of plates you can
load depends on their height. When the system has both a sample manager
and sample organizer, the right-hand shelf in the sample manager is referred
to as position 1 and the bottom shelf in the sample organizer as position 2.
To load sample plates
1.

Open the sample organizer door.
Tip: To keep the sample compartment from freezing, open its door only
when necessary. (Opening the door admits humid air into the sample
compartment, which causes condensation and freezing.)

Pull the shelf toward you.

Load the plate onto the shelf so that position A,1 is at the rear,
right-hand corner and the forward edge of the plate is behind the stop at
the front, left-hand corner.

Shelf label

TP02504

Caution: To prevent spillage, use Waters-approved cap mats,
sealing caps, or heat seal film on the samples. Consult the current
ACQUITY UPLC system release notes for a list of approved
sample covers.

Preparing the sample organizer

2-31

Ensure that the plate does not extend beyond the plate stop at the rear
of the shelf.

Caution: To ensure the transfer shuttle moves freely and without
damaging the sample organizer, you must be able to slide a
shelf/plate/vial combination in or out without interfering with
the shelves or plates directly above and below it.
5.

Slide the shelf into the sample organizer until it stops.
Shelf/plate combination and restrictors

Restrictors

Sample
plate
Shelf

Repeat step 2 through step 5 for the remaining plates.
Caution: To avoid jarring the plates from their shelves, do not
slam the sample organizer door closed.

2-32

Close the sample organizer door. A mechanism on the door ensures the
shelves are positioned correctly when the door closes.

Click Configure > Scan and store shelf layout to update and save the
new shelf configuration.

Preparing System Hardware

To remove and replace the same plate on the same shelf
1.

Open the sample organizer door, and then remove plates that have
finished processing.

Pull a shelf toward you, and then insert a plate of the same type and size
on the shelf.
Caution: To prevent spillage, use Waters-approved cap mats,
sealing caps, or heat seal film on the samples. Consult the current
ACQUITY UPLC system release notes for a list of approved
sample covers.

Load a plate onto a shelf so that position A,1 is at the rear, right-hand
corner and the forward edge of the plate is behind the stop at the front,
left-hand corner. Ensure that the plate does not extend beyond the plate
stop at the back of the shelf.
Caution: To ensure the transfer shuttle moves freely and without
damaging the sample organizer, you must be able to slide a
shelf/plate/vial combination in or out without interfering with
the shelves or plates directly above and below it.

Slide the shelf into the sample organizer until it stops.

Repeat step 3 and step 4 until all plates and holders are placed correctly
on the shelves.
Caution: To avoid jarring the plates from their shelves, do not
slam the sample organizer door closed.

Close the sample organizer door. A mechanism on the door ensures the
shelves are positioned correctly when the door closes.

Click Verify. The sample organizer automatically scans the plates and
shelves, senses which shelves contain plates, compares them to the
saved layout, verifies that they match, and illuminates the
corresponding LEDs inside the sample organizer door.

Configure the plates and shelves in the Empower or MassLynx data
application.
Rule: You can load plates and shelves either before or after configuring
them in the data application, but you must configure them before
running samples.

Preparing the sample organizer

2-33

To rearrange the shelves for a different plate configuration
1.

Open the sample organizer door, and then remove plates that have
finished processing.

Add, move, or remove shelves from the sample organizer so that the
shelf configuration suits the plates you intend to run.
Tip: Standard microtiter plates need one slot. Intermediate plates need
two slots, so allow empty slots above the plate or holder. Deep-well
plates and all vial holders need three slots, so allow two empty slots
above the plate or holder.
Caution: To prevent spillage, use Waters-approved cap mats,
sealing caps, or heat seal film on the samples. Consult the current
ACQUITY UPLC system release notes for a list of approved
sample covers.

Load a plate onto a shelf so that position A,1 is at the rear, right-hand
corner and the forward edge of the plate is behind the stop, at the front,
left-hand corner. Ensure that the plate does not extend beyond the plate
stop at the back of the shelf.
Caution: To ensure the transfer shuttle moves freely and without
damaging the sample organizer, you must be able to slide a
shelf/plate/vial combination in or out without interfering with
the shelves or plates directly above and below it.

Slide the shelf into the sample organizer until it stops.

Repeat step 3 and step 4 until all plates and holders are placed correctly
on shelves.
Caution: To avoid jarring the plates from their shelves, do not
slam the sample organizer door closed.

Close the sample organizer door. A mechanism on the door ensures the
shelves are positioned correctly when the door closes.

Click Configure > Scan and store shelf layout.
The sample organizer initializes and scans the shelves. When it detects
a new shelf, it illuminates the LED to the left-hand side of the shelf,
inside the sample organizer door.

2-34

Preparing System Hardware

Tip: In the ACQUITY UPLC Console, a thin, gray bar appears for each
empty shelf. After the plate or shelf is configured using Empower or
MassLynx software, plate identification appears in a thicker bar.
8.

Displaying sample plate information
To display sample plate information
1.

In the ACQUITY UPLC Console, select Sample Organizer from the
system tree.

In the sample organizer information window, click Configure > Scan and
store shelf layout to update and save the configuration of plates on
shelves.
The number designations of shelves that contain sample plates appear
beside information about the plates.

Preparing the sample organizer

2-35

Sample organizer information window

Shelf number

Plate information

Tips:
•

The shelf and plate information appear only after method setup.

•

Move the pointer over a shelf to display the number of samples
remaining to be run from that shelf.

Starting the TUV detector
If your system includes a TUV detector, prepare it for operation by following
the procedures in this section.
If your system includes a PDA detector, see the ACQUITY UPLC Photodiode
Array Detector Getting Started Guide for information on preparing it.
If your system includes an ELS detector, see the ACQUITY UPLC
Evaporative Light Scattering Detector Getting Started Guide for information
on preparing it.
If your system includes an FLR detector, see the ACQUITY UPLC
Fluorescence Detector Getting Started Guide for information on preparing it.
If your system includes a mass spectrometer, see the documentation that
accompanies the instrument for information on preparing it.
2-36

Preparing System Hardware

Starting the TUV detector
Caution: Use only thoroughly degassed HPLC-grade solvents. Gas in
the mobile phase can form bubbles in the flow cell and cause the
detector to fail the startup diagnostic tests.
Tip: To prevent errors on startup, be sure the flow cell contains degassed,
transparent solvent (acetonitrile or water) and the detector door is closed
firmly.
To start the TUV Detector
1.

Ensure the detector flow cell is filled with degassed, transparent solvent
(acetonitrile or water) and free of air bubbles.

Ensure the detector door is closed firmly.

Press the power switch on the door to power-on the detector. The
detector beeps 3 times and runs a series of startup tests while the lamp
LED blinks. The power LED shows steady green.
Tip: Initialization usually requires approximately 2 minutes, and lamp
warm-up requires approximately 3 minutes.

When the lamp LED shows steady green, start the Empower or
MassLynx software. You can monitor the ACQUITY UPLC Console for
messages and LED indications. For best results, allow at least 30
minutes to equilibrate the detector and stabilize the baseline.
Tips:

•

The absorbance value appears in the ACQUITY UPLC Console and
also in Empower’s Run Samples window or MassLynx’s Inlet Editor
window. If the detector is in dual wavelength mode, two absorbance
values appear.

•

Absorbance values have a resolution of 0.0001 AU.

•

When the lamp is extinguished, “Lamp Off” appears in the software
instead of absorbance values.

Configure the detector according to the instructions in the Empower or
MassLynx online Help, or as provided in “Configuring Empower
software” on page 3-1 and “Configuring MassLynx software” on
page 3-13.

Starting the TUV detector

2-37

TUV detector information window

Recording sample and reference energies
After you install the detector or perform maintenance tasks, like changing the
lamp or flow cell, complete the procedures in this section to verify that the
detector optics and electronics work properly.
To record sample and reference energies
1.

Ensure that the detector is connected to the workstation.

Flush the system tubing with filtered, degassed HPLC-grade
acetonitrile.
Caution: The maximum allowable pressure drop across the flow
cell is 6895 kPa (69 bar, 1000 psi). If the solvent is viscous
(methanol-water, for example), decrease the maximum flow rate
to prevent breaking the cell.

2-38

Pump mobile phase for 15 minutes or more at 0.3 mL/min.

Preparing System Hardware

Ensure the detector cell is filled with solvent and free of air bubbles.

When both LEDs show steady green, initialization is complete.

Start the Empower or MassLynx software.

Launch the ACQUITY UPLC Console from the sample manager control
panel.
For additional information, see “Starting the ACQUITY UPLC Console
from Empower software” on page 3-12 and “Starting the ACQUITY
UPLC Console from MassLynx software” on page 3-14.

Select the TUV detector view in the ACQUITY UPLC Console.

Set the wavelength to 230 nm.

10. In the Console, select TUV Detector > Interactive Display from the
system tree.
11. Record the sample and reference energies at 230 nm.

Conditioning the column
Conditioning the column involves running a solvent gradient through it
without injecting samples or running the Events table. The run time for
conditioning the column must equal the gradient table run time.
Caution: To prevent damage to the detector flow cell, ensure that the waste
solvent does not flow through the detector during this procedure. After
installing a new column, flush solvent through it and out to waste before
connecting the column to the detector (for example, 10 column volumes).
To condition the column
1.

Remove the column inlet line from the detector, and place the end in a
small waste container.

If Empower software controls the system, proceed as follows:
a.

In the Samples table, add a row to the method.

Select Equilibrate/Condition Column (Isocratic or Gradient) as the
function in the new row.

Run the separation method.

Conditioning the column

2-39

If MassLynx software controls the system, proceed as follows:
a.

Open the Sample Set window, and select an inlet method that
includes the chromatographic conditions you want to use.

Run the sample set line. The system runs the separation method.

See also: For more information about column conditioning, consult the
Empower or MassLynx online Help.

Shutting down the system
Caution: Buffers left in the detector can precipitate and damage
instrument components.
Tips:
•

If you are using Empower software to control the system, set system
shutdown parameters in the Instrument Method. Consult the Empower
online Help for more information.

•

If you are using MassLynx software to control your system, set system
shutdown parameters in the Shutdown Editor. Consult the MassLynx
online Help for more information.

Shutting down for less than 24 hours
To shut down the system for less than 24 hours
1.

Continue to pump the initial mobile phase mixture through the column.
Doing so maintains the column equilibrium necessary for good retention
time reproducibility.

To lengthen lamp life, extinguish the detector lamp by clicking
(Lamp Off) in the detector control panel.
Tip: If you are running under MassLynx control, ensure that
Auto-Shutdown for your shutdown method is deactivated.

If a few hours will pass before the next injection, slow the flow rate in
the interim to a few tenths of a mL/min to conserve solvent.
Tip: Ensure that the shutdown method is deactivated.

2-40

Keep the detector operating and the column heater at operating
temperature during this period.

Preparing System Hardware

Shutting down for more than 24 hours
To shut down the system for more than 24 hours
1.

To lengthen lamp life, extinguish the detector lamp by clicking
(Lamp Off) in the detector control panel.

Remove buffer salts and additives by flushing with water.

Flush the column and flow cell with 100% pure organic solvent.
See also: Waters ACQUITY UPLC BEH Column Care and Use
Instructions or ACQUITY UPLC HSS Column Care and Use
Instructions.
Warning: Risk of electric shock. The power switch on each system
instrument controls the basic operational state of that instrument.
Nevertheless, some instrument circuits remain live after the
instrument is switched off. To completely interrupt power to a
system instrument, set the power switch to Off, and then unplug
the instrument’s power cord from the AC outlet.

Power-off the system.
Alternative: If you prefer to leave the system powered-on, turn off the
column heater or reduce the column heater temperature to 40 °C
(104 °F).
Caution: Before using any system or instruments that have been
shut down, under the recommended conditions, ensure that the
new mobile phase is miscible with the recommended storage
solvents: water/methanol or water/acetonitrile. If the mobile phase
and solvents for the new analysis are not directly miscible with the
recommended storage solvents, ensure that an intermediate
solvent, one that is miscible with both the storage solvents and
those for the new analysis, is used to flush the storage solvents
from the system and all of its components.

Cap the flow cell inlet and outlet ports.

Shutting down the system

2-41

Running HPLC methods on an ACQUITY UPLC system
For more information on transferring methods, consult the ACQUITY Console
online Help.
The Waters ACQUITY UPLC Columns Calculator (provided with your
ACQUITY UPLC software and also available on the Waters Web site)
automates the scaling calculations required to convert isocratic or gradient
HPLC methods to UPLC methods.

System considerations
Traditional HPLC methods run on an ACQUITY UPLC system can differ in
retention time—and in some cases peak order—from the same methods run on
traditional HPLC equipment. The differences are primarily due to lower
system volume and reduced bandspreading in the ACQUITY UPLC system,
which operates with a maximum injection volume of 50 μL and a maximum
flow rate of 2 mL/min. You can minimize discrepancies between HPLC and
UPLC methods by compensating for the differences in system volume.
The ACQUITY UPLC system uses a loop-based autosampler. The amount of
sample loaded on the column for a given injection volume, and the subsequent
sample recovery, can be different from when you use a traditional HPLC
injector (direct-inject style). So an injection volume of 10 µL on a UPLC
system can yield different area counts from an equivalent volume injected on
an HPLC system. Full loop injections yield better recoveries (lower area-count
losses) than those made using partial loop injection modes. If an appropriate
sample loop is available (one with a characterized loop volume that matches
your desired injection volume), Waters recommends using full loop injections
when initially transferring an HPLC method to an ACQUITY UPLC system.
If necessary, you can adjust injection volumes or change injection modes after
the initial method transfer.
For information on available sample loop sizes, see the Waters Quality Parts
Locator on the Waters Web site’s Services/Support page.
For more information on ACQUITY UPLC injection modes, consult the
ACQUITY Console online Help.

2-42

Preparing System Hardware

Requirements for using an HPLC column on an ACQUITY UPLC
system
Observe the following requirements when fitting an HPLC column on an
ACQUITY UPLC system:
•

When running columns without an eCord, verify that the console
software is configured to ignore the absence of the eCord.
For information on how to enable/disable the eCord column chip
requirement, consult the ACQUITY Console online Help.

•

Install the column stabilizer. For 150-mm columns, use the optional
150-mm column stabilizer.
For detailed instructions on how to install the column stabilizer, see the
Waters ACQUITY UPLC System Operator’s Guide.
Column stabilizer assembly (two styles)
For 150-mm column

For 50-mm or 100-mm column

Reusable fittings

Inlet tubing to injector

TP02874

•

Do not use the ACQUITY UPLC column in-line filter unit with an HPLC
column.

•

Set the high pressure limit of the binary solvent manager to the
maximum pressure recommended for the HPLC column.

Recommendation: To reduce variability from temperature fluctuations, use a
column oven with a thermostat. For columns longer than 150 mm, use the

Running HPLC methods on an ACQUITY UPLC system

2-43

ACQUITY UPLC system 30-cm column heater/cooler or another appropriate
external oven.
Tip: If you observe a discrepancy between the selectivity and resolution of the
HPLC and UPLC methods, ensure that you properly compensated for system
volume differences. If the discrepancy persists, adjust the temperature of the
column heater by +/-2 °C and assess the result.

Choosing fittings
Column inlet
When connecting the column stabilizer to an HPLC column, choose among the
following types of fittings:
•

Use the Waters-supplied, reusable, UPLC fitting.

•

Replace the three-piece Waters fitting with a one-piece, reusable fitting
suitable for your pressure and temperature requirements.

•

Use a stainless-steel (swage) fitting recommended by the HPLC column
manufacturer.

Waters supplies a finger-tight, reusable fitting for connecting the column
stabilizer to the column inlet. The fitting assembly consists of a ferrule
(PEEK), collet, and compression screw. Because the position of the ferrule is
adjustable, you can use this fitting assembly with any style of column
end-fitting.
Column stabilizer reusable fitting
Reusable fitting

TP02846

PEEK ferrule

Outlet tube
Variable
distance

Collet

Note: Older reusable fittings do not have holes in the side of them.
For more information on replacing fittings and installing columns, see the
Waters ACQUITY UPLC System Operator’s Guide.

2-44

Preparing System Hardware

If you prefer the convenience of a single-piece fitting, you can replace the
three-piece Waters fitting with a single-piece fitting that meets your
temperature and pressure requirements.
Caution: To avoid leaks and connection failures, do not exceed the
temperature or pressure limitations of your fittings.
You can also use stainless-steel fittings recommended by your HPLC column’s
manufacturer. For stainless-steel fittings, the ferrule position remains fixed
(swaged) once it is set. The distance from the ferrule to the end of the tubing
when connected to a UPLC column is approximately 3 mm. Your HPLC
column can require a different distance. Ensure that the distance between the
ferrule and the end of the tubing is appropriate for the end-fitting of the
column you are using. Band broadening and leaks can occur if the fittings are
not properly seated.

Column outlet
If your system has a Waters-supplied, wrench-flat, column outlet fitting that
has been used for a UPLC column connection, replace the swage fitting with a
reusable fitting of your choice, or a swage fitting recommended by the HPLC
column manufacturer.
For all fittings, ensure that the distance between the ferrule and the end of
the tubing is appropriate for the end-fitting of the HPLC column you are
using.
Wrench-flat column outlet fitting
Wrench-flat nut
Tubing
Ferrule
Steel backing ring

For information on tubing and fittings for post-column tubing connections, see
the Waters ACQUITY UPLC System Operator’s Guide and the appropriate
detector user documentation.

Running HPLC methods on an ACQUITY UPLC system

2-45

2-46

Preparing System Hardware

Configuring System Software
Contents
Topic

Page

Configuring Empower software

3-1

Starting the ACQUITY UPLC Console from Empower software 3-12
Configuring MassLynx software

3-13

Starting the ACQUITY UPLC Console from MassLynx software 3-14

Configuring Empower software
Perform these tasks to configure Empower software:
•

Start the software and log in

•

Select system instruments

•

Name the system

Starting Empower software and logging in
To start Empower software and log in
1.

Select Start > Programs (for Windows XP, All Programs) > Empower >
Empower Login.
Alternative: Start the Empower software through the Empower desktop
shortcut.

In the Empower Login dialog box, type your user name and password.

Click OK.

Configuring Empower software

3-1

Selecting system instruments
To select system instruments
1.

In the Empower Pro window, click Configure System.

In the Configuration Manager window, click Acquisition Servers,
right-click the node name, and then select Properties.
Tip: If you are using Empower 1154 software to control the system, the
node name is referred to as the acquisition server name.

In the Acquisition Server dialog box, click the Instruments tab. The
system instruments that are successfully communicating with your
system are shown with a Yes in the “OK?” column.
Instruments tab

3-2

Ensure that a binary solvent manager (ACQ-BSM), sample manager
(ACQ-SM), and detector (ACQ-TUV, ACQ-PDA, or ACQ-ELS) appear in
the instrument list and are successfully communicating with your
system, and then click OK.

Right-click Systems, and then select New > Chromatographic System.

In the System Type area of the New Chromatographic System Wizard
dialog box, select Create New System, and then click Next.

Configuring System Software

System Selection dialog box

In the System Selection dialog box, drag the name of the instrument(s)
you want to include in the new system from the Available Instruments
pane to the New System Instruments pane. Click Next.

When the Access Control dialog box appears, click Next.

In the Name Selection dialog box, specify a name for your system. Enter
comments, if any, and then click Finish. A confirmation dialog box
appears.

10. Click Projects, right-click a project, and then select Open.
11. In the Project window, click Run Samples

Alternative: Access the Run Samples window via the Empower
QuickStart menu.
12. On the Run Samples window, you can monitor control panels for the
binary solvent manager, sample manager, detector, and optional column
manager.

Configuring Empower software

3-3

Control panels

About the binary solvent manager control panel
If Empower software controls the system, the binary solvent manager’s
control panel appears at the bottom of the Run Samples window. If MassLynx
software controls the system, the binary solvent manager's control panel
appears on the Additional Status tab of the Inlet Editor window.
Binary solvent manager control panel
Flow LED
Status
System pressure
Total flow rate

Solvent composition
Stop flow

The binary solvent manager control panel displays flow status, system
pressure, total flow rate, and solvent composition parameters.
Rule: You can edit these parameters when the system is idle by clicking on the
underlined value. You cannot edit binary solvent manager parameters while
the system is running samples.

3-4

Configuring System Software

The following table lists the items in the binary solvent manager control
panel.
Binary solvent manager control panel items
Control panel item

Description

Flow LED

Displays the actual flow LED on the
front panel of the binary solvent
manager unless communications
with the binary solvent manager are
lost.

Status

Displays the status of the current
operation.

System Pressure

Displays the binary solvent manager
pressure, in kPa, bar, or psi.
Pressure units can be customized
through the ACQUITY UPLC
Console.

Total Flow Rate

Displays the total flow rate of the
binary solvent manager. Total flow
rate values range from 0.000 to
2.000 mL/min under normal
operation and 0.000 to 8.000 mL/min
when priming.

Solvent Composition

Displays the percentage of solvent (1
and 2) to be drawn from the pumps
(A and B). Composition values range
from 0.0 to 100.0%.

(Stop Flow)

Immediately stops all flow from the
binary solvent manager.

Configuring Empower software

3-5

You can access these additional functions by right-clicking anywhere in the
binary solvent manager control panel:
Additional functions in the binary solvent manager control panel
Control panel function

Description

Refresh system (Sys Prep)

Refreshes the fluid-carrying lines
according to the current method
conditions.
See “Refreshing the system” on
page 2-12.

Startup

Brings the system to operational
conditions after an extended idle
period or when switching to different
solvents.
See “Starting up the system” on
page 2-13.

Prime A/B solvents

Displays the Prime A/B Solvents
dialog box.
See “Priming the binary solvent
manager” on page 2-9.

Prime seal wash

Displays the Prime Seal Wash
dialog box.
See “Performing a seal wash prime”
on page 2-7.

Reset BSM

Resets the binary solvent manager
after an error condition.

Help

Displays the ACQUITY UPLC
Console online Help.

About the sample manager control panel
If Empower software controls the system, the sample manager’s control panel
appears at the bottom of the Run Samples window. If Masslynx software
controls the system, the sample manager's control panel appears on the
Additional Status tab of the Inlet Editor window.

3-6

Configuring System Software

Sample manager control panel
Run LED

Status

Current HT column
heater temperature

Current sample
compartment
temperature

Display ACQUITY UPLC
Console

Sample
compartment
set point

HT column heater
set point

The sample manager control panel displays current sample compartment and
HT column heater temperatures and set points. You can edit these values
when the system is idle by clicking on the underlined value. You cannot edit
sample manager set points while the system is running samples.
Tip: To keep the sample compartment from freezing, open its door only when
necessary. (Opening the door admits humid air into the sample compartment,
which causes condensation and freezing.)
The following table lists the items in the sample manager control panel.
Sample manager control panel items
Control panel item

Description

Run LED

Displays the actual run LED on the
front panel of the sample manager
unless communications with the
sample manager are lost.

Status

Displays the status of the current
operation.

Current Sample Compartment
Temperature

Displays the current sample
compartment temperature to 0.1 °C
resolution. When active temperature
control is disabled, this field
displays “Off”.

Sample Compartment Set Point

Displays the current sample
compartment set point to 0.1 °C
resolution. When active temperature
control is disabled, this field
displays “Off”.

Configuring Empower software

3-7

Sample manager control panel items (Continued)
Control panel item

Description

Current Column Heater
Temperature

Displays the current column heater
temperature to 0.1 °C resolution,
even when active temperature
control is disabled.

Column Heater Set Point

Displays the current column heater
set point to 0.1 °C resolution. When
active temperature control is
disabled, this field displays “Off”.

(Display Console)

Displays the ACQUITY UPLC
Console.

You can access these additional functions by right-clicking anywhere in the
sample manager control panel:
Additional functions in the sample manager control panel

3-8

Control panel function

Description

Run Sys Prep

Primes the sample manager with
one weak wash prime (wash and
sample syringes).
See “Refreshing the system” on
page 2-12.

Prime syringes

Displays the Prime Syringes dialog
box.
See “Priming the sample manager”
on page 2-17.

Wash needle

Displays the Wash Needle dialog
box.
See “Washing the sample manager
needle” on page 2-19.

Turn lights on/off

Turns the sample manager
compartment and optional sample
organizer lights on or off.

Configuring System Software

Additional functions in the sample manager control panel (Continued)
Control panel function

Description

Reset SM

Resets the sample manager after an
error condition.

Help

Displays the ACQUITY UPLC
Console online Help.

About the TUV detector control panel
If Empower software controls the system, the TUV detector’s control panel
appears at the bottom of the Run Samples window. If Masslynx software
controls the system, the detector's control panel appears on the Additional
Status tab of the Inlet Editor window.
TUV detector control panel
Lamp On/Off LED
Status
Absorbance
units
Value of
wavelength A

Turn detector
lamp On/Off

The TUV detector control panel displays absorbance units and wavelength
values. You can edit these parameters when the system is idle by clicking on
the underlined value. You cannot edit detector parameters while the system is
running samples.
If your system includes a PDA detector, see the ACQUITY UPLC Photodiode
Array Detector Getting Started Guide for information on the control panel.
If your system includes an ELS detector, see the ACQUITY UPLC
Evaporative Light Scattering Detector Getting Started Guide for information
on the control panel.
If your system includes an FLR detector, see the ACQUITY UPLC
Fluorescence Detector Getting Started Guide for information on the control
panel.

Configuring Empower software

3-9

The following table lists the items in the TUV detector control panel.
TUV detector control panel items
Control panel item

Description

Lamp On/Off LED

Displays the actual lamp on/off LED
on the front panel of the detector
unless communications with the
detector are lost.

Status

Displays the status of the current
operation.

Displays the absorbance units.

Displays the value of wavelength A,
in nm. If the detector is in dual
wavelength mode, the value of
wavelength B also appears.
(Lamp On)

Ignites the detector lamp.

(Lamp Off)

Extinguishes the detector lamp.

You can access these additional functions by right-clicking anywhere in the
detector control panel:
Additional functions in the detector control panel

3-10

Control panel function

Description

Autozero

Resets the absorbance value to 0

Reset TUV

Resets the detector, when present,
after an error condition

Help

Displays the ACQUITY UPLC
Console online Help

Configuring System Software

About the column manager control panel
If Empower software controls the system, the column manager’s control panel
appears at the bottom of the Run Samples window. If Masslynx software
controls the system, the column manager's control panel appears on the
Additional Status tab of the Inlet Editor window.
Column manager control panel
Run LED
Current temperature
Column currently
in use

Temperature set point

The column manager control panel displays the current column temperature
and set point. You can edit the set point when the system is idle by clicking on
the underlined value. You cannot edit any underlined values (temperature set
point and column selection) while the system is running samples.
The following table lists the items in the column manager control panel.
Column manager control panel items
Control panel item

Description

Run LED

Displays the actual run LED on the
front panel of the column manager
unless communications with the
column manager are lost.

Temperature

Displays the current column
compartment temperature and set
point to 0.1 °C resolution. When
active temperature control is
disabled, this field displays “Off”.

Column

Displays the column that is
currently in use.

Configuring Empower software

3-11

You can access these additional functions by right-clicking anywhere in the
column manager control panel:
Additional functions in the column manager control panel
Control panel function

Description

Reset CM

Resets the column manager, when
present, after an error condition

Help

Displays the ACQUITY UPLC
Console online Help

Starting the ACQUITY UPLC Console from Empower
software
To start the ACQUITY UPLC Console from Empower software
In the Run samples window, click Display console
control panel.
ACQUITY UPLC Console window

3-12

Configuring System Software

in the Sample Manager

Configuring MassLynx software
Perform these tasks to configure MassLynx software:
•

Start the application

•

Select system instruments

To start MassLynx software
1.

Select Start > Programs > MassLynx > MassLynx.
Alternative: Use the MassLynx desktop shortcut.
If MassLynx Security is not enabled, MassLynx software starts, and the
MassLynx window appears. If MassLynx Security is enabled, the
MassLynx Login dialog box appears.

Type your user name and password, and select your domain.

Click OK. The MassLynx window appears.

To select system instruments
1.

In the MassLynx window, click Inlet Method.

In the Inlet Method window, select Instrument Configuration from the
Tools menu of the Inlet Method window.

In the Inlet Configuration window, click Configure and then click Next.

In the Select Pump dialog box, select Waters ACQUITY as the pumping
device, and then click Next.

Select Waters ACQUITY as the autosampler, and then click Next.

Select Waters ACQUITY TUV, Waters ACQUITY PDA, or Waters
ACQUITY ELS as the detection device, and then click Next.

Click Next.

Click Finish.

Click Finish, and then click OK.

10. In the Instrument Control Option Pack dialog box, ensure that “Install
new instrument software or upgrade existing installation(s)” is selected,
and then click Next.

Configuring MassLynx software

3-13

11. Select ACQUITY Binary Solvent Manager, ACQUITY Sample Manager,
ACQUITY Column Manager, and ACQUITY TUV Detector (or
ACQUITY PDA Detector and/or ACQUITY ELS Detector), and then
click Next. A progress bar appears at the bottom of the dialog box.
12. When the instrument control option pack installation is finished, the
Results screen of the Instrument Control Option Pack dialog box
appears.
13. Click Finish. The Inlet Method window appears.

Starting the ACQUITY UPLC Console from MassLynx
software
To start the ACQUITY UPLC Console from MassLynx software

3-14

In the MassLynx window, click Inlet Method.

In the Inlet Method window, click the ACQUITY Additional Status tab.

Click Display console

Configuring System Software

Verifying System Operation
This chapter explains how to run a gradient performance test on a system
with a TUV detector to verify that your system is operating properly. The
sample you use to verify the system is included in the system startup kit.
To ensure you have the latest version of this procedure, visit
http://www.waters.com and click Waters Division > Services & Support.
Contents
Topic

Page

Preparing the system

4-2

Creating the test methods

4-5

Performing the gradient performance test

4-10

Before you begin this procedure, your system must be set up and configured as
described in the Waters ACQUITY UPLC System Operator’s Guide.
Restrictions:
®

•

This gradient performance test is not applicable to ACQUITY UPLC
systems that have a mass spectrometer.

•

There is no gradient performance test for ACQUITY UPLC systems that
have an ELS detector.

4-1

Preparing the system
Preparation is the same whether the system is controlled by the Empower or
MassLynx data system.
Requirements: Because of the increased sensitivity of the ACQUITY UPLC
system and detectors
•

All solvents, including water and additives, must be of the highest
chemical purity (MS-grade). Failure to use MS-grade solvents results in
high background concentration, low signal-to-noise ratios, and loss of
sensitivity.

•

All MS-grade solvents used with the ACQUITY UPLC system must be
properly filtered prior to their use. Waters recommends that the solvent
be filtered through an appropriate .22-μm or smaller membrane filter
using a solvent clarification kit (47-mm all-glass filter holder with 1-L
flask) immediately before use. A general-purpose laboratory vacuum
pump is also required for use with the all-glass solvent filtration
apparatus.

•

Glassware (such as solvent bottles) must not be washed with detergents
or with other general glassware to prevent contamination. The
glassware must be rinsed with the high-purity solvents to be used.
See also: Controlling Contamination in Ultra Performance LC/MS and
HPLC/MS Systems (part number 715001307) on the ACQUITY UPLC
System Bookshelf CD.

For the system verification test, the mobile phases and wash solvents must be
mixed as follows:

4-2

•

Mobile phase A1 – 10:90 acetonitrile/water (for A1, B2, strong and weak
needle-wash, and the plunger-seal wash lines)

•

Mobile phase A2 – 100% acetonitrile (for solvent line B1 and A2)

•

Mobile phase B1 – 100% acetonitrile

•

Mobile phase B2 – 10:90 acetonitrile/water

•

Weak wash – 10:90 acetonitrile/water

•

Strong wash – 10:90 acetonitrile/water

•

Plunger seal wash – 10:90 acetonitrile/water

Verifying System Operation

To prepare the system for verification
Warning: To avoid chemical exposure risk, always observe Good
Laboratory Practices when you use this equipment and when you
work with solvents and test solutions. Know the chemical and
physical properties of the solvents and test solutions you use. See
the Material Safety Data Sheet for each solvent and test solution
in use.
1.

Prepare a 10:90 acetonitrile/water mobile phase:
a.

Measure 100 mL of filtered acetonitrile into a 100-mL graduated
cylinder.

Carefully transfer the acetonitrile to a 1-L reservoir bottle.

Measure 900 mL of filtered HPLC-grade water into a 1000-mL
graduated cylinder.

Carefully transfer the water to the same 1-L reservoir bottle.

Cap the reservoir bottle and mix well.

Label the reservoir bottle as 10:90 acetonitrile/water.

Submerge lines A1, B2, plunger seal wash, strong needle wash, and
weak needle wash in the reservoir bottle containing the 10:90
acetonitrile/water.

Place the reservoir bottle in the solvent tray.

Prepare a mobile phase of 100% acetonitrile:
a.

Pour approximately 1 L of filtered acetonitrile into a 1-L reservoir
bottle.

Label the reservoir bottle as acetonitrile.

Submerge lines A2 and B1 in the acetonitrile reservoir bottle.

Preparing the system

4-3

Place the reservoir bottle in the solvent tray.
Caution: Never change directly between immiscible eluents or
between buffered solutions and organic eluents. Immiscible
eluents form emulsions in the flow path. Buffered solutions and
organic eluents in combination can result in salt precipitation in
the gradient proportioning valves, pump heads, check valves, or
other parts of the system. Confirm that all fluids in the system are
miscible with acetonitrile. If you need additional information
about priming your system, see “Priming the binary solvent
manager” on page 2-9.

Install the ACQUITY UPLC column in the column heater, close the
column tray, and replace the column heater’s front cover. If you need
more information about installing the column, see the Waters ACQUITY
UPLC System Operator’s Guide.

Access the ACQUITY UPLC Console and perform these tasks:
a.

Prime each solvent line of the binary solvent manager for 10
minutes. See “Priming a wetted binary solvent manager” on
page 2-12.

Prime the seal wash of the binary solvent manager for several
minutes. See “Priming a wetted binary solvent manager” on
page 2-12.

Prime the sample manager at least 5 times. See “Priming the
sample manager” on page 2-17.

Perform the seal characterization procedure in the sample manager.
See “Characterizing the needle seal” on page 2-21.

Calibrate the needle and loop volume in the sample manager.

Alternative: Use the Refresh (Sys Prep) function to prime the binary
solvent manager. See “Refreshing the system” on page 2-12.

4-4

Prepare the sample as listed on the sample instructions, using 10:90
acetonitrile/water.

Place the sample in the vial plate, noting the vial position, and put the
plate in position 2 of the sample manager.

Verifying System Operation

Creating the test methods
The gradient performance test method parameters are the same whether
Empower or MassLynx software controls the system. Follow the steps below to
create the methods, setting the parameter values to match those pictured in
the screen representations.
Tip: Click

on the tabs to display online Help.

Creating the instrument method
To create the instrument method
1.

Create an instrument method with the binary solvent manager
parameters shown in the following screen representation.
Binary solvent manager instrument parameters

Creating the test methods

4-5

Tip: The binary solvent manager parameters are the same regardless of
whether your system includes the TUV or PDA detector.
2.

Set instrument method parameters for the sample manager as shown in
the following screen representation.
Sample manager instrument parameters

Tip: The sample manager parameters are the same regardless of
whether your system includes the TUV or PDA detector.
3.

4-6

On the General tab, click Advanced and then set the parameters shown
in the following screen representation.

Verifying System Operation

Sample manager instrument advanced settings

Tip: The sample manager parameters are the same regardless of
whether your system includes the TUV or PDA detector.

Creating the test methods

4-7

TUV detector instrument parameters

If your system includes the TUV detector, set instrument method
parameters as shown in the following screen representation.
See also:

4-8

•

For information on default values, consult the ACQUITY Console
online Help.

•

For more information on detector parameters, consult the
ACQUITY UPLC Console online Help, Empower online Help, or
MassLynx online Help.

•

If your system includes a PDA detector, see the ACQUITY UPLC
Photodiode Array Detector Getting Started Guide for information on
verifying it.

•

If your system includes an ELS detector, see the ACQUITY UPLC
Evaporative Light Scattering Detector Getting Started Guide for
information on performing a run.

Verifying System Operation

•

If your system includes an FLR detector, see the ACQUITY UPLC
Fluorescence Detector Getting Started Guide for information on
verifying it.

•

If your system includes an SQ detector, see the SQ Detector
Operator’s Guide for information on verifying it.

•

If your system includes a TQ detector, see the TQ Detector
Operator’s Guide for information on verifying it.

Save the instrument method.

Creating the sample set method
If Empower software controls the system, you must create a sample set
method. The sample set method parameters (Inj Vol., # of Injections,
Function, Run Time, and Next Injection Delay) are the same whether your
system includes the TUV or PDA detector. However, the method set and
report methods vary. Before acquiring data, ensure that you chose the
appropriate method set.
For more information on creating a sample set method, consult the Empower
online Help.
To create the sample set method
1.

Set these sample set method parameters:
•

Condition column once (run time = 6.0 minutes)

•

Injection volume = 5 μL (10-mm or 25-mm flow cell or ELSD)

•

# of Injections = 3

•

If Empower software controls the system, run time = 4.0 minutes
(with Next Injection Delay = 2.5 minutes). If MassLynx software
controls the system, total run time = 6.5 minutes

Save the sample set method.

Creating the test methods

4-9

Performing the gradient performance test
Restriction: If your ACQUITY UPLC system uses only a mass spectrometer,
the gradient performance test is not applicable.
When the system is prepared and the test methods are created, you are ready
to perform the gradient performance test. The steps for running the test vary
slightly, depending on whether your system uses Empower or MassLynx
software, but the desired results are the same.
To perform the test
1.

Start the run:
•

If the system is controlled by Empower software, open the project in
Run Samples, select the gradient performance test sample set, and
then select Run and Report.

•

If the system is controlled by MassLynx software, access the
MassLynx main page, and select Start from the Run menu.

When the sample set is complete, enter the appropriate results in the
table, below.
Retention time reproducibility (three replicates)
Peak

4-10

Peak
retention time Std dev
mean value

Acceptable
std dev

2-acetylfuran

≤1.5 secs

acetanilide

≤1.5 secs

acetophenone

≤1.5 secs

propiophenone

≤1.5 secs

butylparaben

≤1.5 secs

benzophenone

≤1.5 secs

valerophenone

≤1.5 secs

Verifying System Operation

Review the gradient performance report. The gradient performance test
result is “passing” when these conditions are realized:
•

The peaks are symmetrical, integrated, and identified correctly.
(Compare the chromatogram on the report to the sample
chromatogram, below.)

•

The peak retention times show a standard deviation of ≤ 2.0 s.
(Consult the table you completed.)

0.50

1.00

1.50

valerophenone

butylparaben

proplophenone

acetophenone

2-acetylfuran
acetanilide

0.34
0.32
0.30
0.28
0.26
0.24
0.22
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
-0.02

benzophenone

Sample gradient performance test chromatogram

2.00
Minutes

2.50

3.00

3.50

4.00

Note that this is a representative chromatogram. The results from your
system can vary slightly.
Performing the gradient performance test

4-11

4-12

Verifying System Operation

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