Waters nanoACQUITY UPLC Quick Start Manual
nanoACQUITY UPLC
System
Quick Start Guide
71500097503/Revision D
CORPORATION
Copyright © Waters Corporation 2006.
All rights reserved.
Copyright notice
© 2006 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF
AMERICA AND 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
Corporat io n assumes no re spo n sibility fo r an y errors that may app ea r in t h is
document. This document is believed to be complete and accurate at the time
of publicatio n. In no eve n t s h all Wate rs Corpor ation be liable fo r in ciden t al or
consequential damages in connection with, or arising from, the use of this
document.
Trademarks
Millennium and Waters are registered trademarks, and ACQUITY UPLC,
Atlantis, BEH Technology, Empower, MassLynx, MassPREP, nanoACQUITY
UPLC, and Symmetry are trademarks of Waters Corporation.
CTC PAL is a trademark of CTC Analytics AG.
Optima is a registered trademark of Fisher Scientific Company, L.L.C.
Teflon is a registered trademark of E.I. duPont de Nemours and Company.
Windows is a registered trademark of Microsoft Corporation.
Other trademarks or registered trademarks are the sole property of their
respective owners.
Customer comments
Please contact us if you hav e questi ons, s ug gestions for improvements, or f ind
errors in this document. Your comments will help us improve the quality,
accuracy, and organization of our documentation.
You can reach us at tech_comm@waters.com.
Waters Corporation
34 Maple Street
Milford, MA 01757
USA
iii
Operating this device
When operating this device, adhere to standard quality control procedures
and the following equipment guidelines.
Attention: Changes or modifications to this unit not expressly approved by
the party responsible f or compliance c ould void t he user’s a uthority to operate
the equipment.
Important: Toute modification sur cette unité n’ayant pas été expressément
approuvée par l’autorité responsable de la conformité à la réglementation
peut annuler le droit de l’utilisateur à exploiter l’équipement.
Achtung: Jedwede Änderungen oder Modifikationen an dem Gerät ohne die
ausdrückliche Genehmigung der für die ordnungsgemäße Funktionstüchtigkeit verantwortlichen Personen kann zum Entzug der
Bedienungsbefugnis des Systems führen.
Avvertenza: eventuali modifiche o alterazioni apportate a questa unità e
non espressamente approvate da un ente responsabile per la conformità
annulleranno l’autorità dell’utente ad operare l’apparecchiatura.
Atencion: cualquier cambio o modificación efectuado en esta unidad que no
haya sido expresamente aprobado por la parte responsable del cumplimiento
puede anular la autorización del usuario para utilizar el equipo.
iv
Caution: Use caution when working with any polymer tubing under
pressure:
• Always wear eye protection when near pressurized polymer tubing.
• Ext in guish a ll n earby flames.
• Do not use tubing that has been severely stressed or kinked.
• Do not use nonmetallic tubing with tetrahydrofuran (THF) or concentrated
nitric or sulfuric acids.
• Be aware that methylene chloride and dimethyl sulfoxide cause
nonmetallic tubing to swell, which greatly reduces the rupture pressure of
the tubing.
Attention: Manipulez les tubes en polymère sous pression avec precaution:
• Portez systématiquement des lunettes de protection lorsque vous vous
trouvez à prox im ité de tub e s e n polymère pressu ri sé s.
• Eteignez toute flamme se trouvant à proximité de l’instrument.
• Evitez d'utiliser des tubes sévèrement déformés ou endommagés.
• Evitez d'utiliser des tubes non métalliques avec du tétrahydrofurane
(THF) ou de l'acide sulfurique ou nitrique concentré.
• Sachez que le chlorure de méthylène et le diméthylesulfoxyde entraînent le
gonflement des tuyaux non métalliques, ce qui réduit considérablement
leur pression de rupt u re.
Vorsicht: Bei der Arbeit mit Polymerschläuchen unter Druck ist besondere
Vorsicht ange bracht:
• In der Nähe von unter Druck stehenden Polymerschläuchen stets
Schutzbrille tragen.
• Alle offenen Flammen in der Nähe löschen.
• Keine Schläuche verwenden, die stark geknickt oder überbeansprucht
sind.
• Nicht metallische Sch läu che nicht fü r Tetrahydrofura n (TH F) oder
konzentrierte Salpeter- oder Schwefelsäure verwenden.
• Durch Methylenchlorid und Dimethylsulfoxid können nichtmetallische
Schläuche quellen; dadurch wird der Berstdruck des Schlauches erheblich
reduziert.
v
Attenzione: prestare attenzione durante l’utilizzo dei tubi di polimero
pressurizzati:
• Indossare sempre occhiali da lavoro protett ivi nei p ressi di tubi di polimero
pressurizzati.
• Estinguere ogni fonte di ignizione circostante.
• Non utilizzare tubi soggetti che hanno subito sollecitazioni eccessive o son
stati incurva ti.
• Non utilizzare tubi non metallici con tetraidrofurano (THF) o acido
solforico o nitrico concentrato.
• Tenere presente che il cloruro di metilene e il dimetilsolfossido provocano
rigonfiamento nei tubi non metallici, riducendo notevolmente la resi stenza
alla rottura dei tubi stessi.
Advertencia: se recomienda precaución cuando se trabaje con tubos de
polímero sometidos a presión:
• El usuario deberá protegerse siempre los ojos cuando trabaje cerca de
tubos de polímero sometidos a presión.
• Si hubiera alguna llama las proximidades.
• No se deb e tra ba ja r co n t u bo s que se h ay an do bl ado o sometido a al ta s
presiones.
• Es necesario utilizar tubos de metal cuando se trabaje con
tetrahidrofurano (THF) o ácidos nítrico o sulfúrico concentrados.
• Hay que tener en cuenta que el cloruro de metileno y el sulfóxido de
dimetilo dilatan los tubos no metálicos, lo que reduce la presión de ruptura
de los tubos.
vi
vii
Caution: The user shall be made aware that if the equipment is used in a
manner not specified by the manufacturer, the protection provided by the
equipment may be impaired.
Attention: L’utilisateur doit être informé que si le matériel est utilisé d’une
façon non spécifiée par le fabricant, la protection assurée par le matériel
risque d’être défectueuses.
Vorsicht: Der Benutzer wird darauf aufmerksam gemacht, dass bei unsachgemäßer Verwenddung des Gerätes unter Umständen nicht ordnungsgemäß
funktionieren.
Attenzione: l’utente deve essere al corrente del fatto che, se l’apparecchiatura viene usta in un modo specificato dal produttore, la protezione fornita
dall’a pparecchiat u ra potrà essere invalidata.
Advertencia: el usuario deberá saber que si el equipo se utiliza de forma
distinta a la espe c ifi cada por el fabr ica nt e , las medidas de prote cción del
equipo podrían ser insuficientes.
viii
Caution: To protect against fire hazard, repl ace f us es wi th those of the s a me
type and ra t ing .
Attention: Remplacez toujours les fus ib les pa r d’autres du mêm e type et de
la même puissance afin d’éviter tout risque d’incendie.
Vorsicht: Zum Schutz gegen Feuergefahr die Sicherungen nur mit
Sicherungen des gleichen Typs und Nennwertes ersetzen.
Attenzione: per una buona protezione contro i rischi di incendio, sostituire i
fusib ili con altri dello s tes so tipo e amperaggio.
Advertencia: sustituya los fusibles por otros del mismo tipo y características
para evitar el ri es go de incendio.
ix
Caution: To avoid possible electrical shock, disconnect the powe r cord befo re
servicing the instrument.
Attention: Afin d’éviter toute possibilité de commotion électrique,
débra n ch e z le co rdon d’al imentat io n de la prise avant d’effectuer la maint e nance de l’instrument.
Vorsicht: Zur Vermeidung von Stromschlägen sollte das Gerät vor der
Wartung vom Netz getrennt werden.
Attenzione: per evitare il rischio di scossa elettrica, scollegare il cavo di
alimentazione prima di svolgere la manutenzione dello strumento.
Precaución: para evitar descargas eléctricas, desenchufe el cable de alimentación de l in st ru mento antes de re alizar cualquier reparación.
x
Observing safety precautions
Observe all safety precautions while servicing, repairing, installing, and
operating the instrument. Failing to do so violates safety standards and
intende d u se of the ins tr ument. Waters C o rpo ration assumes n o liabilit y fo r
failure to comply with these precautions.
Precautions can be of these two types:
• Warnings that indicate risk of injury or death
• Cautions that indicate risk of damage to the system or equipment
Accompanying the word “Warning” or “Caution,” these are the safety
precaution symbols you might encounter on instruments and/or in documents :
Warning: Indicates a potential health or safety hazard. Refer to the
manual.
Warning: Indicates hazardous voltages can ex ist .
Warning: Indicates hot surfaces or high temperatures can exist.
Warning: Indicates danger from needle-stick punctures.
Warning: Indicates danger fro m ultraviolet ra diation.
Warning: Indicates danger from corrosive substances.
Warning: Indicates danger from contaminatio n by a biolog ical agent.
Warning: Indicates danger from toxic substances.
Warning: Indicates danger from flammable substances.
Warning: Indicates danger from laser radiat ion .
xi
Using Waters equipment
In addition to warning symbols, you might encounter the following symbols and
labels on p ackagi n g, in s t ru ments , an d / o r in documen ts.
Direct current
Alternating current
Protective conductor terminal
Frame or chassis terminal
Fuse
Electrical power on
l
Electrical power off
xii
Keep upright
Keep dry
Fragile, handle contents with care
Safety and electromagnetic equipment compatibility
United States – FCC rules
This device complies with Part 15 of the FCC Rules. Operation is subject to
the following two conditions: (1) this device may not cause harmful
interfer e nc e , a n d (2) this device must ac ce pt an y interfere n ce re ceived,
including interference that may cause undesired operation.
Changes or modifications to this unit not expressly approved by the party
responsible for compliance could void the user’s authority to operate the
equipment.
Rationale: This equipment has been tested and found to comply with the
limits for a Class B digital device, pursuant to Part 15 of the FCC Rules.
These limits are designed to provide reasonable protection against harmful
inter fe ren ce in a res i den tia l inst al lat io n. This equi pme nt ge ner at es, uses, and
can radiate radio frequency energy and, if not installed and used in
accordance with the instructions, may cause harmful interference to radio
communications. However, there is no guarantee that interference will not
occur in a particular installation. If this equipment does cause harmful
interference to radio or t e le vision reception, whic h can be determined b y
turning the equipment off and on, the user is encouraged to try to correct the
interference by one or more of the following measures:
• Reorient or relocate the receiving antenna.
• Increase the separation between the equipment and receiver.
• Connect the equipment into an outlet on a circuit different from that to
which the receiver is connected.
• Consult the dealer or an experienced radio TV technician for help.
Shielded cables must be used with this unit to ensure compliance with the
Class B FCC limits.
United States – safety requirements
Waters products meet the safety requirements for laboratory instruments set
forth by the Occupational Safety and Health Administration (OSHA). All
products are evaluated b y an OSHA-appr oved, Na tionally Recognized Testing
Laboratory (NRTL) to ensure they meet applicable safety standards. NRTLs
perform safety testing on instruments to ensure the safety of the operator.
xiii
Waters pr o du ct s carry a safe ty la be l from an NRTL to show co mplianc e . T h e
particular safety standard with which Waters complies is UL 61010A-1:
Electrical equipment for laboratory use; Part 1: General Requirements.
Canada – spectrum management
This Class B digital apparatus complies with Canadian ICES-003.
Cet appareil numérique de la classe B est conforme à la norme NMB-003.
Waters products meet the safety requirements for laboratory instruments set
forth by the Standards Council of Canada. All products are evaluated by an
approved laboratory to meet Canada’s safety requirements. Waters
instrume n t s carry a safety label from an approved testi n g laborato ry to show
compliance. The particular Canadian safety standard with which Waters
complies is CAN/CAS-C22.2 No. 1010.1: Safety requirements for electrical
equipmen t fo r measurem ent, control and labo ratory us e, Part 1: Gene ral
Requirements.
Europe – safety and electromagnetic compatibility
Waters products have been tested to meet the safety and electromagnetic
requirements of the European community. Display of the CE mark indicates
compliance to these requirements. The safety requirements are set forth via
the standard EN61010: Safety requirements for electrical equipment for
measurement, con t ro l , a n d laboratory use – Part 1: Ge n e ral require ments.
The EMC requirements are supported in the standard EN61326: Electrical
equipment for the measurement, control, and laboratory use – EMC
requirements. Compliance to the EN61010 standard ensures the safety of the
operator from any hazardous situations that could have been caused by the
instrument. Adherence to the EMC standard guarantees that the instrument
will not cause interference to adjacent electronic products nor will other
electronic units interfere with its operation.
Australia – emissions requirements
Australian aut h o riti e s re qu ir e tha t in st ru ments do not excee d specified
radiat io n limits. The se radiation limits are given in the standard A S/NZS
2064: Limits and methods of measurement of electronic disturbance
charac teristics o f in d u strial, sc ie n t ific and medical (ISM) radio frequ en cy
equipment. Conformance to this standard is shown by displaying the
Australian C-tick mark.
xiv
nanoACQUITY UPLC system information
Intended use
Use the Waters® nanoACQUIT Y UPLC system for analyzing compounds an d
introducing separated sample components into a mass spectrometer.
The Waters nanoACQUITY UPLC system is not intended for use in
diagnostic procedures.
When you develop methods, follow the “Protocol for the Adoption of Analytical
Methods in the Clinical Chemistry Laboratory,” American Journal of Medical
Technology, 44, 1, pages 30–37 (1978). Th is protocol covers good operati n g
procedures and techniques necessary to validate system and method
performance.
Biological hazard
When you analyze physiological fluids, take all necessary precautions and
treat all s pe cime ns a s pot en tia lly i nf ect iou s. P rec aut io ns are out li ned in “C DC
Guidelines on Specimen Handling,” CDC – NIH Manual, 1984.
Calibration
Follow acce ptable met h o ds o f calibratio n with pu re st an dards to calibrate
methods. Use a minimum of five standards to generate a standard curve. The
concentration range should cover the entire range of quality-control samples,
typica l sp e cimens , and atyp ical spec imens.
Quality control
It is recommended that you routinely run three quality-control samples.
Quality-control samples should represent subnormal, normal, and
above-no rmal leve ls of a compou n d . E n su re t h at qu ality-con t ro l sample
results are within an acceptable range, and evaluate precision from day to day
and run to run. Data collected when quality-control samples are out of range
might not be valid. Do not report this data until you ensure that
chromatographic system performance is acceptable.
xv
xvi
Table of Contents
Operating this device .......................................................................................... iv
Observing safety precautions ........................................................................... xi
Using Waters equipment ................................................................................... xii
Safety and electromagnetic equipment compatibility ............................. xiii
United States – FCC rules.............................................................................. xiii
United States – safety requirements.............................................................. xiii
Canada – spectrum management.................................................................... xiv
Europe – safety and electromagnetic compatibility........................................ xiv
Australia – emissions requirements................................................................ xiv
nanoACQUITY UPLC system information .................................................... xv
Intended use....................................................................................................... xv
Biological hazard................................................................................................ xv
Calibration ......................................................................................................... xv
Quality control................................................................................................... xv
1 System Overview .................................................................................... 1-1
Instruments and components ........................................................................ 1-1
nanoACQUITY operating modes ................................................................... 1-3
Direct injection ................................................................................................. 1-4
Single-pump trapping...................................................................................... 1-4
Two-pump trapping......................................................................................... 1-5
Two-dimensional liquid chromatography (2D-LC)......................................... 1-5
nanoACQUITY binary solvent manager ...................................................... 1-6
How the binary solvent manager works......................................................... 1-6
Flow control modules....................................................................................... 1-6
Auxiliary solvent manager ............................................................................. 1-7
Flow control module......................................................................................... 1-7
Table of Contents xvii
Sample manager ................................................................................................ 1-7
Sample consumption guidelines...................................................................... 1-8
Heating and trapping module ........................................................................ 1-9
TUV optical detector ...................................................................................... 1-10
Mass detectors ................................................................................................. 1-10
Q-Tof Premier™............................................................................................. 1-10
LCT Premier™............................................................................................... 1-11
MassLynx data system ................................................................................... 1-11
nanoACQUITY UPLC Console ..................................................................... 1-11
2 Preparing System Hardware ............................................................... 2-1
Powering-on the system .................................................................................. 2-1
Monitoring startup tests ................................................................................. 2-2
Monitoring the LEDs of system instruments ............................................. 2-2
Power LED....................................................................................................... 2-2
Status LEDs..................................................................................................... 2-3
Preparing the auxiliary solvent manager ................................................... 2-4
Priming the seal wash..................................................................................... 2-5
Preparing the binary solvent manager ....................................................... 2-8
Priming the seal wash..................................................................................... 2-8
Priming the binary solvent manager............................................................ 2-10
Preparing the sample manager ................................................................... 2-12
Selecting weak and strong wash solvents.................................................... 2-12
Priming the sample manager........................................................................ 2-14
Washing the sample manager needle........................................................... 2-15
Characterizing the needle seal...................................................................... 2-17
Characterizing the needle and sample loop volumes................................... 2-18
Loading sample plates in the sample manager............................................ 2-19
Preparing the detector .................................................................................. 2-21
Preparing the TUV detector.......................................................................... 2-21
Preparing the mass spectrometer................................................................. 2-22
xviii Table of Contents
Conditioning the column .............................................................................. 2-23
Shutting down the system ............................................................................ 2-24
Between analyses........................................................................................... 2-24
Overnight or weekends.................................................................................. 2-24
More than 72 hours (long-term).................................................................... 2-25
3 Configuring System Software ............................................................. 3-1
Configuring MassLynx ..................................................................................... 3-1
Starting the nanoACQUITY UPLC Console from MassLynx .................. 3-5
Configuring events ........................................................................................... 3-6
4 Verifying System Operation ......................................... ....................... 4-1
Required materials ........................................................................................... 4-1
Preparing the mobile phases ......................................................................... 4-2
Preparing the sample ....................................................................................... 4-3
Preparing the system ....................................................................................... 4-4
Creating the test methods ............................................................................... 4-6
Creating the instrument method.................................................................... 4-6
Performing the test ........................................................................................ 4-10
Table of Contents xix
xx Table of Contents
1 System Overview
Contents:
Topic Page
Instruments and components 1-1
nanoACQUITY operating modes 1-3
nanoACQUITY binary solvent manager 1-6
Auxili ary solve n t manage r 1-7
Sample manager 1-7
Heating and t rapping modu le 1-9
TUV optical detector 1-10
Mass detectors 1-10
MassLynx data system 1-11
nanoACQUITY UPLC Console 1-11
Instruments and components
The Wate rs® nanoACQUITY UPLC™ system is designed for capillary-tonano-scale sep ara t ion s. Its se n si tivi t y, re so lut ion, and reprod ucibility well
suit it for biomark e r discov e ry and pr ot e o mics applicatio n s , in cluding protein
identification and characterization.
The system consists of these modules and components:
• Bina ry solvent manager with flow control mod u les
• Auxiliary solvent manager (for NanoLockSpray™ lock-mass addition
and two-pump trapping ) with flow control module
• Sample manager and heating and trapping module, which holds the
analytical column
1-1
• Waters Ma ssLynx™ ch romatography an d mass spectrometry software
• nanoACQUITY UPLC Console, which provides control, status, and
advan ce d diagnos ti c in fo rmatio n in a grap h ical display
Waters nanoACQUITY UPLC system:
Solvent tray
Heating and
trapping module
(HTM)
Column heater
(“away” position)
Sample manager
Binary solvent
manager (BSM)
Auxiliary solvent
manager (ASM)
The system is optimized for high-resolution separations at precise nanoflow
rates. With clo sed loop co n tro l, t h ose rates rang e be t we e n 0. 20 and
5.00 µL/min. With open loop control and nanoACQUITY UPLC columns of
internal diameters ranging from 75 µm to 1 mm, the nanoflow rates can
extend to 100 µL/min. The column hardware and the matched outlet tubing
can withst an d up to 69,000 kPa (690 bar, 10,000 psi). The column dimen s io n s
allow optimal MS-co mpatible fl ow rates, and matched outl et tubing minimizes
the effect of extra-column volume.
1-2 System Overview
Flow control modules
Smaller-column diameters require lower flow rates, which can encourage
extra-column bandspreading. The system counters this with precise injection
volume capacity and gradient formation, optional detector flow cell volume,
and low-volume connection tubing.
Waters offers nanoA C QUITY UP LC co lumns p acked with 1.7-µm, bridge d,
ethane-silicon (BEH), hybrid particles as well as conventional reversed phase
packing materials, typically 3- to 5-micron particle sizes. Compared with
traditio nal HPLC columns, nan oACQU ITY UPLC colum ns delive r s u perior
resolut ion and sensiti vity in th e same r un time, or gr e ater sensitiv it y and
faster run times with equivalent resolution.
nanoACQUITY operating modes
The nanoACQUITY system can operate in direct i njec ti on mode or any of four
trapping m o de s. Trapping im pro v e s system perfor ma n ce in se veral ways:
• Removes salts
•Cleans samples
• Concen t rat e s larger sam p l e vo lu mes
• Decre as e s sample lo ading time
Each mode requires a different configuration of solvent managers and
columns:
• Direct injection mode uses the binary solvent manager with an
analytical column.
• Single-pump trapping uses the binary solvent manager with a trap
column and an analytical column.
• Two-pump trapping uses the auxiliary solvent manager and
nanoACQUITY binary solvent manager with a trap column and an
analytical column.
• Onlin e t wo -dimensiona l liquid ch ro matogr ap h y (2D-L C ) t rapping, w it h
salt plugs, use s the auxiliary solv e n t manager an d bi n ary solven t
manage r with an ion ex ch ange column, trap colum n , and analyt i c al
column.
• Off-line 2D-LC trapping requires the CTC PAL MALDI Spotter
Microfraction Collector.
1-3
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