nanoACQUITY UPLC System Operator’s Guide...Attenzione: l’utente deve essere al corrente del fatto che, se l’apparecchia-tura viene usta in un modo specificato dal produttore,

nanoACQUITY UPLC System

Operator’s Guide

71500097502 / Revision D

Copyright © Waters Corporation 2006.All rights reserved.

C O R P O R A T I O N

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 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, the use of this document.

TrademarksMillennium 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.

PEEK is a trademark of Victrex Corporation.

Phillips is a registered trademark of Phillips Screw Company.

Teflon is a registered trademark of E.I. duPont de Nemours and Company.

TORX is a registered trademark of Textron Corporation.

Windows is a registered trademark of Microsoft Corporation.

Other trademarks or registered trademarks are the sole property of their respective owners.

Customer commentsPlease contact us if you have questions, suggestions for improvements, or find errors in this document. Your comments will help us improve the quality, accuracy, and organization of our documentation.

You can reach us at [email protected].

Waters Corporation34 Maple StreetMilford, MA 01757USA

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 for compliance could void the user’s authority 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 Funktion-stüchtigkeit 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.• Extinguish all nearby 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 proximité de tubes en polymère pressurisés.• 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 rupture.

Vorsicht: Bei der Arbeit mit Polymerschläuchen unter Druck ist besondere Vorsicht angebracht:• 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.• Nichtmetallische Schläuche nicht für Tetrahydrofuran (THF) 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 protettivi nei pressi di tubi di polimero

pressurizzati.• Estinguere ogni fonte di ignizione circostante.• Non utilizzare tubi soggetti che hanno subito sollecitazioni eccessive o son

stati incurvati.• 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 resistenza 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 debe trabajar con tubos que se hayan doblado o sometido a altas

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.

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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 unsach-gemäßer Verwenddung des Gerätes unter Umständen nicht ordnungsgemäß funktionieren.

Attenzione: l’utente deve essere al corrente del fatto che, se l’apparecchia-tura viene usta in un modo specificato dal produttore, la protezione fornita dall’apparecchiatura potrà essere invalidata.

Advertencia: el usuario deberá saber que si el equipo se utiliza de forma distinta a la especificada por el fabricante, las medidas de protección del equipo podrían ser insuficientes.

viii

Caution: To protect against fire hazard, replace fuses with those of the same type and rating.

Attention: Remplacez toujours les fusibles par d’autres du même 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 fusibili con altri dello stesso tipo e amperaggio.

Advertencia: sustituya los fusibles por otros del mismo tipo y características para evitar el riesgo de incendio.

ix

Caution: To avoid possible electrical shock, disconnect the power cord before servicing the instrument.

Attention: Afin d’éviter toute possibilité de commotion électrique, débranchez le cordon d’alimentation de la prise avant d’effectuer la mainte-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 alimen-tación del instrumento antes de realizar 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 intended use of the instrument. Waters Corporation assumes no liability for 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 exist.

Warning: Indicates hot surfaces or high temperatures can exist.

Warning: Indicates danger from needle-stick punctures.

Warning: Indicates danger from ultraviolet radiation.

Warning: Indicates danger from corrosive substances.

Warning: Indicates danger from contamination by a biological agent.

Warning: Indicates danger from toxic substances.

Warning: Indicates danger from flammable substances.

Warning: Indicates danger from laser radiation.

xi

Using Waters equipmentIn addition to warning symbols, you might encounter the following symbols and labels on packaging, instruments, and/or in documents.

Direct current

Alternating current

Protective conductor terminal

Frame or chassis terminal

Fuse

Electrical power on

Electrical power off

Keep upright

Keep dry

Fragile, handle contents with care

l

xii

Safety and electromagnetic equipment compatibility

United States – FCC rulesThis device complies with Part 15 of the FCC Rules. Operation is subject to the following two conditions: (1) this device may not cause harmful interference, and (2) this device must accept any interference received, 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 interference in a residential installation. This equipment generates, 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 television reception, which can be determined by 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 requirementsWaters products meet the safety requirements for laboratory instruments set forth by the Occupational Safety and Health Administration (OSHA). All products are evaluated by an OSHA-approved, Nationally 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 products carry a safety label from an NRTL to show compliance. The particular safety standard with which Waters complies is UL 61010A-1: Electrical equipment for laboratory use; Part 1: General Requirements.

Canada – spectrum managementThis 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 instruments carry a safety label from an approved testing laboratory to show compliance. The particular Canadian safety standard with which Waters complies is CAN/CAS-C22.2 No. 1010.1: Safety requirements for electrical equipment for measurement, control and laboratory use, Part 1: General Requirements.

Europe – safety and electromagnetic compatibilityWaters 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, control, and laboratory use – Part 1: General requirements. 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 requirementsAustralian authorities require that instruments do not exceed specified radiation limits. These radiation limits are given in the standard AS/NZS 2064: Limits and methods of measurement of electronic disturbance characteristics of industrial, scientific and medical (ISM) radio frequency equipment. Conformance to this standard is shown by displaying the Australian C-tick mark.

xiv

nanoACQUITY UPLC system information

Intended useUse the Waters® nanoACQUITY UPLC system for analyzing compounds and 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). This protocol covers good operating procedures and techniques necessary to validate system and method performance.

Biological hazardWhen you analyze physiological fluids, take all necessary precautions and treat all specimens as potentially infectious. Precautions are outlined in “CDC Guidelines on Specimen Handling,” CDC – NIH Manual, 1984.

CalibrationFollow acceptable methods of calibration with pure standards 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, typical specimens, and atypical specimens.

Quality controlIt is recommended that you routinely run three quality-control samples. Quality-control samples should represent subnormal, normal, and above-normal levels of a compound. Ensure that quality-control 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 System Setup ........................................................................................... 2-1

Installing the system ........................................................................................ 2-1

Before you begin installing the system ........................................................ 2-3 Tools.................................................................................................................. 2-4 Stacking the modules ...................................................................................... 2-4

Making Ethernet and signal connections ................................................... 2-7 Ethernet connections ....................................................................................... 2-7 Signal connections ........................................................................................... 2-8 Sample manager I/O signal connectors .......................................................... 2-9 Generating the injection start signal............................................................ 2-10

Connecting to the electrical source ............................................................ 2-11

Connecting the solvent supply plumbing ................................................. 2-12

Connecting capillary tubing ........................................................................ 2-15 Installation recommendations for fittings.................................................... 2-15 Plumbing the binary solvent manager and sample manager ..................... 2-18 Plumbing the auxiliary solvent manager ..................................................... 2-19 Plumbing the heating and trapping module ................................................ 2-20 Plumbing the mass spectrometer and optional TUV detector .................... 2-21

Calibrating the XYZ mechanism using the teach block ......................... 2-23

xviii Table of Contents

3 Preparing System Hardware ............................................................... 3-1

Powering-on the system .................................................................................. 3-1

Monitoring startup tests ................................................................................. 3-2

Monitoring the LEDs of system instruments ............................................. 3-2 Power LED ....................................................................................................... 3-3 Status LEDs ..................................................................................................... 3-3

Preparing the auxiliary solvent manager ................................................... 3-4

Preparing the binary solvent manager ....................................................... 3-8 Priming the seal wash ..................................................................................... 3-8 Priming the binary solvent manager............................................................ 3-10

Preparing the sample manager ................................................................... 3-12 Selecting weak and strong wash solvents .................................................... 3-12 Priming the sample manager........................................................................ 3-14 Washing the sample manager needle ........................................................... 3-15 Characterizing the needle seal...................................................................... 3-17 Characterizing the needle and sample loop volumes................................... 3-18 Loading sample plates in the sample manager............................................ 3-19

Preparing the detector .................................................................................. 3-21 Preparing the TUV detector.......................................................................... 3-21 Preparing the mass spectrometer ................................................................. 3-22

Conditioning the column .............................................................................. 3-23

Shutting down the system ............................................................................ 3-24 Between analyses........................................................................................... 3-24 Overnight or weekends.................................................................................. 3-24 More than 72 hours (long-term).................................................................... 3-25

4 Configuring System Software ............................................................. 4-1

Configuring MassLynx ..................................................................................... 4-1

Starting the nanoACQUITY UPLC Console from MassLynx .................. 4-5

Configuring events ........................................................................................... 4-6

Table of Contents xix

5 Verifying System Operation ................................................................ 5-1

Required materials ........................................................................................... 5-1

Preparing the mobile phases ......................................................................... 5-2

Preparing the sample ....................................................................................... 5-3

Preparing the system ....................................................................................... 5-4

Creating the test methods ............................................................................... 5-6

Performing the test ........................................................................................ 5-10

6 Maintaining the System ........................................................................ 6-1

Contacting Waters technical service ............................................................ 6-2

Maintenance considerations .......................................................................... 6-3 Safety and handling......................................................................................... 6-3 Proper operating procedures ........................................................................... 6-3 Spare parts ....................................................................................................... 6-4

Configuring maintenance thresholds .......................................................... 6-4

Maintaining the solvent managers ............................................................... 6-6 Replacing the filters......................................................................................... 6-6 Replacing a check valve................................................................................... 6-8 Replacing solvent bottle filters...................................................................... 6-10 Cleaning the air filters in the solvent manager door................................... 6-11 Replacing the air filters in the solvent manager door ................................. 6-11 Replacing the solvent manager head seals................................................... 6-12 Replacing the solvent manager plungers ..................................................... 6-16 Replacing the vent valve cartridge ............................................................... 6-20 Replacing the fuses ........................................................................................ 6-22 Cleaning the module’s exterior ..................................................................... 6-23

Maintaining the sample manager ............................................................... 6-24 Defrosting the sample compartment ............................................................ 6-24 Replacing the sample needle assembly ........................................................ 6-25 Characterizing the needle seal...................................................................... 6-33 Characterizing the needle and loop volumes................................................ 6-34

xx Table of Contents

Replacing the puncture needle...................................................................... 6-35 Replacing the sample loop............................................................................. 6-37 Replacing the metering syringe .................................................................... 6-40 Replacing the wash syringes ......................................................................... 6-42 Modifying syringe configuration parameters ............................................... 6-44 Replacing the injection valve cartridge ........................................................ 6-45 Replacing the fuses ........................................................................................ 6-47 Cleaning the module’s exterior ..................................................................... 6-49

Maintaining the heating and trapping module ....................................... 6-49 Replacing the trap valve cartridge................................................................ 6-49 Cleaning the module’s exterior ..................................................................... 6-50

Maintaining the TUV detector ..................................................................... 6-51 Maintaining the flow cell............................................................................... 6-51 Flushing the flow cell..................................................................................... 6-52 Reverse flushing the flow cell........................................................................ 6-53 Replacing the flow cell ................................................................................... 6-53 Replacing the lamp ........................................................................................ 6-55 Replacing the fuses ........................................................................................ 6-58 Cleaning the detector’s exterior .................................................................... 6-59

Testing the system’s fluidic integrity ......................................................... 6-60 Performing the dynamic leak test................................................................. 6-60 Performing the static decay test ................................................................... 6-62 Auto zeroing the flow control module ........................................................... 6-64 Performing the set pressure diagnostic ........................................................ 6-66 Performing the sample syringe leak test...................................................... 6-70 Performing the wash syringe leak test......................................................... 6-71 Performing the needle seal leak test ............................................................ 6-71 Repairing leaks in the sample manager....................................................... 6-72

Table of Contents xxi

A Specifications ........................................................................................ A-1

Auxiliary solvent manager specifications ................................................... A-1

Binary solvent manager specifications ....................................................... A-5

Sample manager and heating and trapping module specifications ... A-10

Optional TUV detector specifications ........................................................ A-13

B Spare Parts ............................................................................................ B-1

Solvent manager spare parts ........................................................................ B-1

Sample manager spare parts ........................................................................ B-3

TUV detector spare parts .............................................................................. B-4

nanoACQUITY columns .................................................................................. B-4

C Solvent Considerations ....................................................................... C-1

Introduction ...................................................................................................... C-1

Preventing contamination of mass spectrometry backgrounds .......... C-2

Operating procedures ..................................................................................... C-3

Clean solvents ................................................................................................... C-3

Solvent quality .................................................................................................. C-3

Solvent preparation ........................................................................................ C-4 Optional degassing .......................................................................................... C-4

Water ................................................................................................................... C-5

Using buffers ..................................................................................................... C-5

Index ..................................................................................................... Index-1

xxii Table of Contents

1 System Overview

Instruments and components

The Waters® nanoACQUITY UPLC™ system is designed for capillary-to- nano-scale separations. Its sensitivity, resolution, and reproducibility well suit it for biomarker discovery and proteomics applications, including protein identification and characterization.

The system consists of these modules and components:

• Binary solvent manager with flow control modules

• 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

Contents:

Topic Page

Instruments and components 1-1

nanoACQUITY operating modes 1-3

nanoACQUITY binary solvent manager 1-6

Auxiliary solvent manager 1-7

Sample manager 1-7

Heating and trapping module 1-9

TUV optical detector 1-10

Mass detectors 1-10

MassLynx data system 1-11

nanoACQUITY UPLC Console 1-11

1-1

• Waters MassLynx™ chromatography and mass spectrometry software

• nanoACQUITY UPLC Console, which provides control, status, and advanced diagnostic information in a graphical display

Waters nanoACQUITY UPLC system:

The system is optimized for high-resolution separations at precise nanoflow rates. With closed loop control, those rates range between 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 withstand up to 69,000 kPa (690 bar, 10,000 psi). The column dimensions allow optimal MS-compatible flow rates, and matched outlet tubing minimizes the effect of extra-column volume.

Solvent tray

Sample manager

Auxiliary solvent manager (ASM)

Binary solvent manager (BSM)

Heating and trapping module (HTM)

Flow control modules

Column heater (“away” position)

1-2 System Overview

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 nanoACQUITY UPLC columns packed with 1.7-µm, bridged, ethane-silicon (BEH), hybrid particles as well as conventional reversed phase packing materials, typically 3- to 5-micron particle sizes. Compared with traditional HPLC columns, nanoACQUITY UPLC columns deliver superior resolution and sensitivity in the same run time, or greater sensitivity and faster run times with equivalent resolution.

nanoACQUITY operating modes

The nanoACQUITY system can operate in direct injection mode or any of four trapping modes. Trapping improves system performance in several ways:

• Removes salts

• Cleans samples

• Concentrates larger sample volumes

• Decreases sample loading 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.

• Online two-dimensional liquid chromatography (2D-LC) trapping, with salt plugs, uses the auxiliary solvent manager and binary solvent manager with an ion exchange column, trap column, and analytical column.

• Off-line 2D-LC trapping requires the CTC PAL MALDI Spotter Microfraction Collector.

1-3

For typical operating conditions, use the following table as a starting point.

Direct injectionThe direct injection mode is used to inject sample directly onto an analytical column. The trap valve stays in the “elute” position, which routes all flow through the analytical column. The sample manager post-injection mode can be programmed in either of two ways:

• To keep the sample loop in the flow path throughout the run (most common).

• To remove the loop from the flow path after time that the user enters.

Single-pump trappingAt nL/min flow rates, samples larger than 2 µL are slow to load onto a small-bore column. Using single-pump trapping improves sample loading time by loading samples onto a separate trap column at a higher flow rate while excess solvent, salts, and impurities elute to waste. After loading, the trap column is connected to the flow path, and a gradient elutes the sample from the trap column onto the analytical column, usually at a slower rate.

nanoACQUITY UPLC typical operating conditions:

ModeAnalytical Column ID

(µm)

Analytical Flow Rate (µL/min)

Trap Column (ID, µm ×

length, mm)

Trapping Flow Rate (µL/min)

Injection Volume (µL)

Direct 75 0.20—0.40 n/a n/a 1—5

Direct 100 0.40—0.60 n/a n/a 1—5

Direct 150 0.80—1.20 n/a n/a 1—10

Direct 300 4.00—5.00 n/a n/a 1—10

1-pump trapping

75 0.20—0.40 180 × 20 4.00 1—5

1-pump trapping

100 0.40—0.60 180 × 20 4.00 1—5

2-pump trapping

100 0.40—0.60 180 × 20 4.00 (up to 50)

1—5

2D-LC 75 0.20—0.40 180 × 20 3.00—5.00 5—10

1-4 System Overview

Two-pump trappingWith two-pump trapping, the sample manager loads the sample into the sample loop. A dedicated trapping pump in the auxiliary solvent manager (pump A) then loads the sample onto the trapping column. With the HTM (heating and trapping module) valve in the waste position, unwanted solutes flush through the trapping column and elute to waste while the column retains the analytes. When trapping is finished, the valve then closes the waste pathway and opens the flow path. Gradient elution proceeds as the binary solvent manager pumps solvents through the trapping and analytical columns and out to the detector or mass spectrometer.

Two-dimensional liquid chromatography (2D-LC) 2D-LC can separate and characterize complex biological compounds, for example, as an adjunct to protein digestion and polyacrylamide gel electrophoresis (PAGE) techniques.

Pump A of the auxiliary solvent manager serves as a sample loading pump, injecting the sample onto an ion exchange column. Salt plugs of increasing strength are prepared separately and injected from vials in the sample manager, to elute various portions of the sample off the ion exchange column and onto a trap column. At that point, separation and analysis proceeds as in two-pump trapping.

Two-dimensional liquid chromatography can increase the amount of information from complex proteomics samples, reducing their complexity and dynamic range prior to mass spectrometry (MS) analysis.

• To further improve the identification and increase the sequence coverage of high and low abundance proteins from complex samples, the system can perform fully automated online 2D-LC separations of complex protein digest samples using an ion exchange column in the first dimension and a reversed phase UPLC (nanoACQUITY columns with BEH Technology™) in the second dimension.

• For some of the most complex proteomics samples, off-line 2D-LC separation using the CTC PAL MALDI Spotter/Fraction Collector is available to minimize band broadening and thus preserve high peak capacity nanoscale and capillary separations. To minimize sample complexity and dynamic range, the CTC PAL™ microfraction collection platform first collects discrete fractions from a gradient separation onto MALDI targets or into microtiter plates for LC/electrospray ionization (ESI) analysis, followed by MALDI or LC/ESI MS/MS analysis.

1-5

nanoACQUITY binary solvent manager

The nanoACQUITY binary solvent manager is a high-pressure pump that moves solvent through the system. It provides steady (pulse-free) solvent flow at flow rates ranging from 0.20 to 5.00 µL/min (under closed loop control) or to 100.0 µL/min (under open loop control) at 69,000 kPa (690 bar, 10,000 psi). The solvent manager can generate high-pressure gradients with minimal gradient delay.

How the binary solvent manager worksEach of the solvent manager’s two independent pump systems, pump A (left) and pump B (right), contains two linear-drive actuators. Each left and right 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 mixing tee. From there, the solvent mixture flows to the sample manager.

To create gradients and mixtures, 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 solvent manager, whose firmware measures pump head pressures during the pumping cycle. Thus the solvent manager adjusts the precompression to ensure consistent solvent delivery and minimize pump-induced detector baseline disturbances.

Flow control modulesThe flow control modules allow the binary solvent manager to perform nano-flow chromatography by monitoring the solvent flow in each solvent channel. Flow control is calibrated for water, acetonitrile, and methanol.

The flow control modules’ two mass flow sensors measure the flow from each pump separately, before they are mixed. The binary solvent manager monitors the output and adjusts the solvent flow accordingly for precise flow from 0.20 to 5.00 µL/min. Solvents are mixed in the flow control module tee outlet.

1-6 System Overview

Auxiliary solvent manager

The nanoACQUITY auxiliary solvent manager incorporates two isocratic, high-pressure pumps that move solvent through the system and provide steady (pulse-free) solvent flow.

• Pump A is for sample loading.

• Pump B is for NanoLockSpray lock-mass addition, two-pump trapping, and 2D online separation.

In the NanoLockSpray lock-mass addition mode, the pump operates at flow rates from 0.10 to 5.00 µL/min.

Flow control moduleThe flow control module allows the auxiliary solvent manager to perform nano-flow chromatography by monitoring the solvent flow in the solvent channel. Flow control is calibrated for water, acetonitrile, and methanol.

The flow control module’s mass flow sensor measures the flow from the pump. The auxiliary solvent manager monitors the output and adjusts the solvent flow accordingly for precise flow from 0.20 to 5.00 µL/min.

Sample manager

The sample manager injects the samples it draws from microtiter plates, or vials, into the chromatographic flow stream. In maximum throughput mode, the sample manager can perform an injection in approximately 45 seconds, including a single default wash, or it can do so in less than 60 seconds, including a dual default wash.

The sample manager accepts standard footprint plates, 5.03 ±0.02 inch × 3.365 ±0.02 inch, that conform to SBS/ANSI-compliant plates (maximum height of 2.2 inches, including covers). You may program any combination of plates and vial holders for automated sample processing. Samples are loaded into the sample manager via the front door. The sample manager can maintain samples at any temperature between 4 and 40 °C (39 and 104 °F) in ambient conditions of 25 °C (77 °F) or less.

1-7

Sample consumption guidelinesSample consumption varies depending on your system configuration and injection mode.

Sample consumption guidelines

System Configuration

Injection ModeRequested Sample Size (µL)

Sample Consumed (µL)

Maximum Injection Volume (µL)a

Loop: 2 µLNeedle: 15 µL

Partial loop 1 1 1.9

Partial loop with needle overfill

Not applicable Not applicable Not applicable

Full loop

Auto 2 4 2

1 × Overfill 2 2 2

2 × Overfill 2 4 2

3 × Overfill 2 6 2




2 6 3.8

Full loop

Auto 5 10 5

1 × Overfill 5 5 5

2 × Overfill 5 10 5

3 × Overfill 5 15 5




5 9 7.5

Full loop

Auto 10 20 10

1 × Overfill 10 10 10

2 × Overfill 10 20 10

3 × Overfill 10 30 10

1-8 System Overview

Heating and trapping module

The heating and trapping module is attached to the sample manager and serves as its top cover. The heating and trapping module’s column tray can accommodate a nano tee and any analytical Waters column up to 300 microns internal diameter and 250 mm length. The column compartment heats to temperatures from 5 °C (9 °F) above ambient to 65 °C (149 °F).

To reduce dispersion associated with dead volume and minimize the length of tubing between system modules, the column tray swings outward to any position between 0° and 180°. In the 0° “home” position, the column tray is directly above the sample manager and can be connected to an optional optical detector. In the 180° “away” position, the analytical column can be plumbed into a mass spectrometer (located on the system’s right).


Partial loop 5 5 19


5 9 15

Full loop

Auto 10 40 20

1 × Overfill 10 20 20

2 × Overfill 10 40 20

3 × Overfill 10 60 20

a. Maximum injection volume is approximately 0.1 µL less than actual sample loop volume. Sampleloop volumes are nominal value only; volumes vary slightly from loop to loop.

Sample consumption guidelines (Continued)

System Configuration

Injection ModeRequested Sample Size (µL)

Sample Consumed (µL)

Maximum Injection Volume (µL)a

1-9

You can select several types of columns for the nanoACQUITY system.

• Analytical columns can have an I.D. of 75 to 300 µm and a length of 10 to 25 cm. A nanoACQUITY BEH C18 column has particles of 1.7 µm. Alternative particle sizes are available with Symmetry® C18 columns (3.5 µm) or Atlantis® dC18 columns (3.0 µm).

• The nanoACQUITY UPLC trap column is 180 µm I.D. × 2 cm long with Symmetry C18, 5 µm particles.

• An ion exchange column of 180 µm I.D. × 2.4 cm length with SCX material can be used in online 2D-LC techniques.

TUV optical detector

The optional TUV (tunable ultraviolet) optical detector can be used as the system’s sole detector or in conjunction with a mass spectrometer. A two-channel, ultraviolet/visible (UV/Vis) absorbance detector, the TUV detector operates from 190 to 700 nm. Its light-guiding flow cell is intended for high sensitivity chromatography with high peak capacity. The detector, controlled by MassLynx software for LC/MS applications, operates as an integral part of the system.

Mass detectors

The nanoACQUITY UPLC™ system acts as a mass spectrometry inlet for nanoflow rate applications such as proteomics. When coupled with a mass spectrometer, the system provides sensitive, robust, and reproducible LC/MS and LC/MS/MS analyses. The system can be configured with an optional Q-Tof Premier or LCT Premier mass detector.

Q-Tof Premier™The Waters Q-Tof Premier mass spectrometer is an exact-mass API/MALDI MS/MS platform for the pharmaceutical, biotechnology, and life science industries. Designed in conjunction with the nanoACQUITY UPLC system, the Q-Tof Premier quantifies, identifies, and characterizes compounds from simple or complex mixtures.

1-10 System Overview

LCT Premier™The Waters LCT Premier is a benchtop mass spectrometer that uses a high resolution, orthogonal acceleration (oa), time-of-flight (ToF) design to enable automated exact mass measurements. The instrument provides information on elemental composition, structural characteristics (through the use of in-source collision-induced dissociation), and specificity for identifying compounds in complex matrices or from a database search.

MassLynx data system

The system runs under MassLynx software control. MassLynx is a 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 Help.

nanoACQUITY UPLC Console

The nanoACQUITY UPLC Console is a software application that replaces the keypads and small display screens traditionally found on the front of system hardware. As such, it provides a convenient way to configure settings, monitor performance, run diagnostic tests, and maintain the system and its modules.

From the software’s Web-like interface, you can quickly navigate to visual representations of each system module and its components. You can also navigate to interactive diagrams, which show module interconnections and provide diagnostic tools for troubleshooting problems.

The nanoACQUITY UPLC Console includes controls for configuring, monitoring, maintaining, and managing system components. For example, graphical status indicators monitor and report the real-time use of components like the detector lamp. The status indicators let you configure usage thresholds that, when reached, display messages and change the status indicator’s color. These alerts can help you schedule routine maintenance before problems occur.

General categories are logically grouped in the menus. The sub-menus, windows, system tree, and task buttons provide access to system and module information and functionality.

1-11

1-12 System Overview

2 System Setup

Installing the system

A Waters engineer can install the system, or you can do it yourself.

However, a Waters engineer must perform the system upgrade procedure.

To install the nanoACQUITY UPLC System (10,000-psi maximum pressure):

Requirement: To avoid particulate contamination and to purge air from the system, flush each tubing assembly as you connect it to the system.

1. Install the solvent supply plumbing and waste lines:

• Binary solvent manager

• Auxiliary solvent manager

• Sample manager

2. Power-on all modules.

3. Characterize the needle and loop volumes.

Contents:

Topic Page

Installing the system 2-1

Before you begin installing the system 2-3

Making Ethernet and signal connections 2-7

Connecting to the electrical source 2-11

Connecting the solvent supply plumbing 2-12

Connecting capillary tubing 2-15

Calibrating the XYZ mechanism using the teach block 2-23

2-1

4. Install the nano tee and capillary tubing.

5. Install the column(s):

• Analytical

• Trap (optional)

• SCX (optional) for 2D-LC methods

6. Connect the TUV detector (optional).

7. Connect the mass spectrometer (optional).

To upgrade the system from 5,000 to 10,000 psi capability, a Waters engineer performs these tasks:

1. Removes all capillary tubing in the system modules.

2. Removes stainless steel tubing in the binary and auxiliary solvent managers.

3. Replaces the flow control module in the BSM and performs autozero calibration.

4. Replaces the BSM plumbing lines and the check valves.

5. Adds a jumper to the BSM personality card.

6. Replaces the injection valve cartridge and the sample loop in the sample manager.

7. Disconnects the wiring assemblies between the heater and the personality card in the sample manager, and then replaces the column heater assembly.

8. Powers-on all modules.

9. Characterizes the needle and loop volumes.

10. Replaces the nano tee and capillary tubing in the auxiliary solvent manager and binary solvent manager.

2-2 System Setup

Before you begin installing the system

Requirements: To install the system, you should know generally how to set up and operate laboratory instruments and computer-controlled devices. You should also know how to handle solvents.

Before installing the system, ensure that

• The site requirements are met.

• The required components are present.

• None of the shipping containers or unpacked items are damaged.

Caution:

• To avoid overheating, and to provide clearance for cable connections, make sure there is at least 16 cm (6 inches) of clearance at the rear of the system.

• To maintain proper drainage and leak control, the nanoACQUITY UPLC System must be within 2° of level.

If you discover any damage or discrepancy when you inspect the contents of the cartons, immediately contact the shipping agent and your local Waters representative.

Customers in the USA and Canada should report damage and discrepancies to Waters Technical Service (800 252-4752). Others should phone their local Waters subsidiary or Waters corporate headquarters in Milford, Massachusetts (USA), or they may visit http://www.waters.com, and click About Waters > Corporate Information > Worldwide Offices.

For complete information on reporting shipping damages and submitting claims, see Waters Licenses, Warranties, and Support Services.


ToolsYou need the following tools to install the system:

Stacking the modulesInstalling the system begins by stacking the system modules on a bench or the optional cart and installing the network switch.

To install the system modules:

1. If your system includes the optional nanoACQUITY cart, lock the casters so that the cart will not roll during system installation.

TORX® driver, T20 Open-end wrench, 5/8-inch

Hex wrench, 2-mm Open-end wrenches, 5/16-inch (2)

Hex wrench, 2.5-mm Open-end wrench, 1/2-inch

Pocket knife Open-end wrench, 3/16-inch

Phillips® screwdriver Open-end wrench, 1/4-inch

Short, slotted screwdriver Small mirror

Nut driver, 9/32-inch Teach block

Warning: To prevent injury and avoid damage, at least two people should install the system modules.

2-4 System Setup

Optional nanoACQUITY cart:

2. If you are installing the system on a bench top, lift the auxiliary solvent manager onto the bench. If you are using the nanoACQUITY cart, place the auxiliary solvent manager on the cart’s lower shelf.

3. If you are installing the system on a bench top, place the binary solvent manager atop the auxiliary solvent manager. If you are using the optional cart, place the binary solvent manager on the cart’s upper shelf.

4. Position the sample manager on top of the binary solvent manager.

5. Open the front access door of the sample manager, and remove the foam block from the sample compartment.

6. Open both the sample manager fluidics tray and the binary solvent manager door, and ensure that the drip management system is properly aligned.

Tip: PEEK™ fittings, sample syringe, and wash syringes inside the sample manager can loosen during shipping. To prevent leaks, ensure all PEEK fittings and syringes are tight.

7. Place the 8-port network switch in the opening in the back of the sample manager.

TP02518

Lower shelf

Upper shelf

Push handlePower connections

Bottle tray

Casters


Alternatives: Install the switch on the right-hand side of the top module near the back, or on the bench top or cart beside the system.

8. If your system includes a mass spectrometer

a. place the nanoACQUITY system stack to the left of the mass spectrometer to minimize the tubing run between the heating and trapping module and the detector inlet.

b. place the solvent tray module atop of the heating and trapping module.

c. refer to the documentation supplied with the mass spectrometer to complete the installation.

9. If your system includes a TUV detector

a. place the TUV detector atop the heating and trapping module, ensuring that the feet are properly positioned in the indentations on the top of the heating and trapping module, aligning the detector's drip tray over the drain routing hole on the top left side of the heating and trapping module.

Proper placement for drip management system:

TP02465

Guides forfeet placement

Drain routing holefor drip management system

Front of heating and trapping module

2-6 System Setup

10. Place the solvent tray module atop the detector.

Making Ethernet and signal connections

Ethernet connections

To make Ethernet connections:

1. Unpack and install the preconfigured nanoACQUITY workstation.

2. Set aside the longest Ethernet cable.

3. Connect one end of one Ethernet cable to the network switch, and then connect the cable’s other end to the Ethernet card on the workstation.

Tip: On preconfigured systems, the Ethernet card is identified as the Instrument LAN card.

4. Connect one end of the longest Ethernet cable to the Ethernet port on the rear of the auxiliary solvent manager, and then connect the cable’s other end to the network switch.

5. Connect one end of one Ethernet cable to the Ethernet port on the rear of the binary solvent manager, and then connect the cable’s other end to the network switch.

6. Connect one end of one Ethernet cable to the Ethernet port on the rear of the sample manager, and then connect the cable’s other end to the network switch.

7. If you are using a TUV detector, connect one end of one Ethernet cable to the Ethernet port on the rear of the detector, and then connect the cable’s other end to the network switch.

Warning: To avoid toppling the modules stacked on the cart, move the cart by pushing the cart handle, not the modules. Also, to avoid spills, remove all solvent reservoirs from the solvent tray before moving the cart.


Signal connectionsThis section describes signal connections between the system modules.

Rule: To meet regulatory requirements for immunity from external electrical disturbances that can affect the performance of this module, do not use cables longer than 3 meters (9.8 feet) when you connect to the I/O connectors. Also, ensure you connect the shield of the cable to ground at one module only.

I/O connector cover

A plastic cover is supplied for each I/O connector. To protect the module from static discharge, attach the I/O connector cover after making connections to the I/O screw terminals.

Requirement: To satisfy safety regulations, attach the cover to each module’s I/O connector, even if there are no signal connections to the module.

To attach the I/O connector cover:

1. Snap the female half of the cover onto the I/O connector.

2. Route the signal cables from the I/O connector through the half-round hole in the back of the cover.

3. Hold the signal cables in place, and snap the male half of the cover onto the female half, making sure that the signal cables are not pinched in the cover.

Attaching I/O connector cover:

Warning: To avoid electric shock, power-off the system modules before making electrical connections.

I/O connector cover

2-8 System Setup

Sample manager I/O signal connectorsFor the mass spectrometer to start collecting data, the Inject Start terminals on the sample manager must be connected to an Event I/P terminal set on the mass spectrometer.

The rear panel of the sample manager includes two removable connectors that hold the screw terminals for I/O signals. These connectors are keyed so that they can receive a signal cable inserted only one way.

Sample manager I/O signal connectors:

Sample manager analog-out/event-in connections:

Signal Connections Description

Inject Start Sends a contact closure signal (at least 2 seconds) to indicate that an injection has started. If your system requires a longer pulse, use one of the switches to configure an event start/stop signal at the start of a run.

Auxiliary In Reserved for future use.

Inject Hold In TTL input signal from other system modules that delays the injection.

0−2V Analog 2 Out Chart output functionality.

Connector I

12345678910

+−

+−

+−

Inject StartInject StartAuxiliary InAuxiliary InGroundInject Hold InInject Hold InGround0-2V Analog Out0-2V Analog Out

12345678910

Switch 1 OutSwitch 1 OutSwitch 2 OutSwitch 2 OutSwitch 3 OutSwitch 3 OutSwitch 4 OutSwitch 4 OutRun Stopped OutRun Stopped Out

Connector II


Generating the injection start signalTo generate the injection start function at the mass spectrometer, when the sample manager begins an injection, connect the Inject Start terminals on the sample manager to an Event I/P terminal set on the mass spectrometer using a signal cable. Without this connection, the mass spectrometer will not start collecting data.

Switch 1 Out Output that allows control of third party instruments.




Run Stopped Out Indicates that the sample manager stopped operating because of an error condition or an operator request (pressing the Stop Flow button, for example).

Connecting the sample manager to a mass spectrometer:

Sample Manager (Connector I) Mass Spectrometer (Event I/P Terminal Strip)

Pin 1 Inject Start (red) + (red)

Pin 2 Inject Start (black) − (black)

Sample manager analog-out/event-in connections: (Continued)

Signal Connections Description

2-10 System Setup

Sample manager connections to a mass spectrometer:

Requirement: In addition to making the physical (wire) connection between the sample manager and the mass spectrometer, you must also configure the event in MassLynx. See Configuring events on page 4-6.

Connecting to the electrical source

Each system module requires a separate, grounded power outlet. The ground connection in all these outlets must be common and connected near the system.

To connect to the electrical source:

Recommendation: Use a line conditioner or an uninterruptible power supply (UPS) for optimum long-term input voltage stability.

1. Connect the female end of the power cord to the receptacle on the rear panel of each module.

2. Connect the male end of the power cord to a suitable wall outlet.

Alternative: If your system includes the optional nanoACQUITY cart, connect the modules’ power cords to the power strips on the back of the cart, and then connect each power strip to separate wall outlets running on separate circuits.

123456789

10

+−

+−

+−

Inject StartInject StartAuxiliary InAuxiliary In

GroundInject Hold InInject Hold In

Ground0−2V Analog Out0−2V Analog Out

Sample Manager Connector I

Mass spectrometer Event I/P connector

Red

Black

Signal cable


Connecting the solvent supply plumbing

Connect the solvent supply plumbing before you connect the capillary (fused silica) tubing. Doing so lets you flush the capillary tubing as you connect it.

See also: Solvent Considerations

Caution: The nanoACQUITY UPLC System should not be run with high pH mobile phases. Alkaline solutions such as ammonium hydroxide (pH 10) may etch glass solvent bottles and the silica capillary tubing, resulting in an elevated chemical background being detected by mass spectrometry.

Recommendation: Flush the system with the appropriate solvents before passing eluent into the column, optical detector, and/or mass spectrometer.

For optimal mass spectrometry results

You may want to passivate solvent bottles or reservoirs to thoroughly clean them before adding solvents.

To passivate the bottles:

1. Sonicate the bottles in 20 to 30% nitric acid for approximately 30 minutes.

2. Rinse the bottles thoroughly with MilliQ water.

Caution: To avoid contamination, avoid washing nanoACQUITY solvent bottles as follows:

• In a dishwasher

• With other glassware

• Using detergent

These cleaning methods can cause contamination resulting in high mass spectrometry backgrounds.

Requirement: To prevent contamination, always use powder-free nitrile gloves when handling components of the system.

Warning: Observe safe laboratory practices when you handle solvents. See the Material Safety Data Sheets for the solvents you use.

2-12 System Setup

Tip: Remove protective fittings from ports before connecting the fluid lines. Remove protective O-rings from tubing before installation. The fittings and O-rings protect the parts and keep them assembled during shipping.

To plumb the solvent supplies:

1. Prepare the solvent reservoirs.

a. Choose solvent reservoirs that snugly fit the reservoir caps supplied in the startup kit. Waters recommends 500-mL reservoirs.

b. Fill the solvent reservoirs with the appropriate MS-grade solvents.

Requirements: Solvent A must be aqueous (for example, water with 0.1% formic acid) and solvent B organic (for example, acetonitrile with 0.1% formic acid).

c. Place the solvent supply bottles in the solvent tray, on top of the system stack, to maintain adequate solvent head pressure and ensure proper solvent delivery.

2. Unroll the factory-plumbed solvent inlet lines and needle wash lines, and feed them through the retaining clips at the right, front corner of the modules. Place them in their respective reservoirs.

Tip: Remove protective O-rings from tubing before installation.

3. Connect the weak needle wash (WNW) and strong needle wash (SNW) lines from the sample manager to the in-line degasser in the binary solvent manager. Install each line in its port, and then finger-tighten the knurled nuts.

Tip: To remove any contaminants or particles, always prime all lines as you install them.

4. Plumb the system drain waste line to a waste container.

5. If the nanoACQUITY UPLC system stack is positioned to the left of a mass spectrometer, swing the door of the heating and trapping module to the “away” position, on the right. Then route the lines in front of the door hinge. Place the ends in the solvent tray, for later installation in reservoirs.

Exception: If your system does not have a mass spectrometer positioned to the right of the system stack, route the tubing between the heating and trapping module door and hinge, through the detector's clips, and then into the solvent tray.


Tip: Remove protective fittings from ports before connecting the lines.

6. Install the waste lines as follows.

a. Attach a waste line to the barbed fitting of the drain located at the bottom, right corner of the auxiliary solvent manager, and route it to a suitable waste container.

b. Attach a waste line to the barbed fitting of the drain located at the bottom, right corner of the binary solvent manager, and route it to a suitable waste container.

Caution: To ensure that waste fluid drains properly

• push the waste line fully onto the barbed fitting. The tubing must completely cover the fitting.

• place the waste container below the system stack.

• ensure that the drain tubes do not crimp or bend. A crimp or bend may prevent adequate flow to the waste container.

• ensure the exit of the drain tube is not covered by waste solvent, and if necessary shorten the waste tube so that no portion of it drops below the top of the waste container.

Waste fluid drainage by gravity

c. Route a waste line from the barbed fitting on the rear of the solvent tray module to a suitable waste container.

TP01807Correct

End of waste tube is above solvent

Waste tube should not have any crimps or bends

Incorrect

Waste container has sufficient size

2-14 System Setup

7. Insert the solvent tubing into the solvent bottles in the solvent tray.

Solvent tubing in bottles:

Connecting capillary tubing

When the solvent supply plumbing is connected, make the capillary tubing connections. The appropriate fittings and tubing are already assembled but must be properly set.

See also: Installing the nanoACQUITY Upgrade Kit, nanoACQUITY UPLC Plumbing, and Tubing Installation (in www.waters.com > Services and Support > Support Center > Connections Elite > Systems > nanoACQUITY) to upgrade a nanoACQUITY UPLC system from 5,000-psi maximum pressure to 10,000-psi maximum pressure.

Installation recommendations for fittingsCaution: Failure to follow these guidelines can result in leaks or broken tubing and may introduce particulates into the fluid paths.

Warning: To avoid exposure to solvent vapors, route the in-line degasser exhaust tubing to a fume hood or other suitable exhaust system.

Warning: To avoid spills, remove all solvent reservoirs from the solvent tray before moving the cart.

Solvent tray

Solvent bottles

Solvent tubing

Movable bars


The system tubing assemblies are composed of fused silica capillary tubing with the appropriate fittings attached. Each assembly is a specific, pre-assembled component of the fluid path. You need only install the tubing assembly in the correct location and tighten the fittings.

Caution: To ensure accuracy and cleanliness, use only Waters nanoACQUITY pre-cut tubing assemblies (see Spare Parts).

Tightening the capillary tubing connections

Use care when tightening tubing connections. Fused silica leaks when undertightened and fractures when overtightened.

2-16 System Setup

Guidelines:

• If the M-detail fitting is new and has never been installed, tighten the fitting snug (finger-tight) plus 180° with a 3/16-inch wrench.

nanoACQUITY M-detail fitting:

• If the V-detail fitting is new and has never been installed, tighten the fitting snug (finger-tight) plus one full turn (360°) using the 1/4-inch open-end wrench.

nanoACQUITY V-detail fitting:

• If either type of fitting has been installed and removed once or more, tighten it snug (finger-tight) plus 1/6-turn (60°). Proper tightening techniques are the same for both types of fittings if previously used.

To tighten both types of tubing assemblies:

Caution: Take care not to cross-thread the fitting during installation.

1. Start inserting the fitting into the port.

2. Grasp the fused silica tubing and gently push it into the fitting as you continue to tighten. Bottoming the fused silica tubing into the port minimizes system dead volume.

3. Tighten the fitting as recommended on page 2-16.

Capillary tubing

Capillary tubing

Two-piece ferrule

2-micron frit

Gold-plated compression screw

Crimp sleeve


Plumbing the binary solvent manager and sample manager

To plumb the binary solvent manager and sample manager:

1. Seat the fused silica tubing into the outlet port on the flow control module of the binary solvent manager, and tighten the fitting as recommended on page 2-16.

2. In the nanoACQUITY UPLC Console, set the system to deliver 10 µL/min of 50% A/50% B, and then flush the tubing until the system pressure stabilizes.

3. Open the sample manager fluidics tray, and seat the other end of the flow control module outlet tube into port 5 on the sample manager’s injection valve. Tighten the compression fitting using the 1/4-inch open-end wrench.

Injection valve connections:

4. Seat the injection valve outlet tube and ferrule into port 6 of the injection valve. Tighten the compression fitting using a 1/4-inch open-end wrench.

5. Flush the tubing at 10.0 µL/min of 50% A/50% B until the system pressure stabilizes.

Injection valve outlet tubing into port 6

Flow control module outlet tubing into port 5

2-18 System Setup

Plumbing the auxiliary solvent managerAll modes of operation use pump B of the auxiliary solvent manager for nanoLockSpray with a mass spectrometer.

Plumbing of pump A depends on the mode: direct injection, single-pump trapping, or two-pump trapping.

To plumb the B side of the auxiliary solvent manager:

1. Seat the fused silica tubing into the outlet port on the flow control module of the auxiliary solvent manager, and tighten the fitting as recommended on page 2-16.

2. In the nanoACQUITY UPLC Console, set the lock spray pump to deliver 5 µL/min, and then flush until the system pressure stabilizes.

See also: nanoACQUITY UPLC Online Help and the documentation for the mass spectrometer.

For 2D-LC online trapping or two-pump trapping

Optional: You can perform 2D-LC online trapping or two-pump trapping. Each mode requires specific connections to pump A of the auxiliary solvent manager.

See also: Documentation provided with the 2D-LC Online Trapping Kit or the Two-Pump Trapping Kit.

Installing the trap column

To install the optional trap column:

1. Insert the trap column inlet tubing into port 6 of the injection valve, and then tighten the fitting as recommended on page 2-16.

Tip: The arrow on the column tag, located on the inlet side of the trap column, indicates the direction of flow.

2. Snap the trap column into the bracket. The magnets on the bracket secure it to the heating and trapping module.

3. Insert the trap column outlet tubing into the nano tee, and then tighten the fitting as recommended on page 2-16.


Plumbing the heating and trapping moduleThe heating and trapping module’s column heater may run in the closed configuration when using an optical detector or in the open position, at a 180º angle, to interface more closely with a mass spectrometer.

Caution: Once the column heater has been adjusted into either the open or closed position, do not move the hinge. This can crimp or break the fluidic connection to the column.

To install the nano tee in the heating and trapping module:

1. Insert the tubing from port 6 of the injection valve or the trap column outlet tubing (if you installed a trap column) into the nano tee port, and then gently slide the tubing into the port until it bottoms. Hold the tubing in position and tighten the fitting as recommended on page 2-16.

2. Flush the tubing at 10.0 µL/min of 50% A and 50% B until the system pressure stabilizes.

3. Insert the trap valve tubing into the side port of the nano tee, and then gently slide the tubing into the port until it presses inside the tee. Hold the tubing in position, and tighten the fitting as recommended on page 2-16.

4. Set the system to trapping mode (trap valve open).

5. Flush the tubing at 4.0 µL/min until the system pressure stabilizes.

6. Insert the analytical column inlet tubing into the opposing nano tee port, and then gently slide the tubing until it presses inside the tee. Hold the tubing in position, and tighten the fitting as recommended on page 2-16.

7. Set the system to analytical mode, and flush the tubing at an analytical flow rate until the system pressure stabilizes. Allow at least 5 minutes.

8. Open the heating and trapping module door, remove the inside panel, and tilt the column tray downward. You may need to lift the door slightly to disengage it from the retaining clip.

9. Route the injection valve outlet tube through the notch on the left-hand side of the column tray, and then place the nano tee in the column tray. The magnetic tee attaches itself to the tray.

10. Route the trap valve tubing through the notch at the top of the tray.

2-20 System Setup

11. Route the column outlet tubing through the channel on the right side of the column tray and the notch on the heating and trapping module door.

Installing the nano tee:

12. Tilt the column tray upward to the closed position, and refit the panel. The panel snaps into place.

Plumbing the mass spectrometer and optional TUV detectorPlumbing a detector involves connecting the column outlet tubing to the detector or mass spectrometer inlet.

To plumb a mass spectrometer:

1. If not already done, swing the heating and trapping module door fully to the right, the “Away” position. You can then connect the mass spectrometer’s inlet without increasing tubing length.

2. Route the column outlet tubing to the mass spectrometer’s inlet. Consult the instrument’s documentation for more information about connecting to the inlet.

Alternative: If your system includes both a TUV detector and a mass spectrometer, route the outlet tubing from the detector to the mass spectrometer inlet.

When connecting the tubing to the TUV inlet, use the zero-dead-volume union kit to ensure that the capillary tubing is inserted the correct depth into the union. Correct tubing installation minimizes system dead volume within the system.

TP02516

nano tee

To trap valve

To detectorFrom sample manageror trap column

Tubing notch


To plumb a TUV detector:

1. Open the front panel door of the TUV detector, and install the 10-nL flow cell so that the three thumbscrews align with their holes in the bulkhead.

2. Hand tighten the thumbscrews.

TUV detector flow cell:

3. Remove the protective cover from the flow cell inlet tubing.

4. Connect the column outlet tubing to the flow cell inlet tubing using the zero-dead-volume union supplied with the TUV detector:

a. Insert the capillary sleeve tubing into the zero-volume depth gauge until it bottoms.

b. Slide the sleeve tubing through the union body, and tighten the depth gauge into the union.

c. Slide a 6-40 nano fitting, knurled end first, onto the flow cell inlet tube.

d. Slide the flow cell inlet tube into the sleeve tube, which is in the union, until the inlet tube bottoms.

Flow cell assembly

Outlet tubing

Inlet tubing

Captive screw

2-22 System Setup

e. Hold the flow cell inlet tube so that it stays bottomed in the union, and then tighten the 6-40 nano fitting into the union.

f. Unscrew the zero-volume depth gauge and remove it from the union.

g. Slide a 6-40 nano fitting onto the column outlet tube, knurled end first.

h. Slide the column outlet tube into the sleeve tube in the union until the inlet tube bottoms.

i. Hold the column outlet tube so that it stays bottomed in the union, and then tighten the 6-40 nano fitting into the union.

5. Route the detector outlet tubing into a suitable waste container.

6. Close the sample manager door, sample manager fluidics tray, auxiliary solvent manager door, and binary solvent manager door.

7. In the nanoACQUITY UPLC Console, set the system to deliver 0.4 µL/min of 50% A/50% B, and then flush the tubing until the system pressure stabilizes.

Calibrating the XYZ mechanism using the teach block

Before performing any chromatography, you must verify that the XYZ mechanism positions the needle properly in the sample chamber.

Tip: If your system includes the optional sample organizer, complete its installation first, before calibrating the XYZ mechanism. Otherwise, you must repeat the calibration after installing both the sample organizer and sample manager.

Warning: To avoid puncture wounds keep hands or loose clothing clear of the needle assembly mechanism while it is moving. The sample manager beeps three times whenever the door is open and the needle assembly mechanism is about to move.


To calibrate the XYZ mechanism:

1. Open the sample manager door, and then remove the sample plate from the sample tray.

2. Loosen the screw holding the sample tray holder in place by turning it counterclockwise a quarter-turn, and then slide the tray holder out. If your system does not include a sample organizer, you must remove two sample tray holders from the sample chamber.

3. With your finger, remove the teach block from the sample manager chassis floor.

Tip: If the teach block is not in its proper location, it might have slipped down or moved around.

4. Insert the teach block into the locating holes.

Teach block in position:

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

6. Click Troubleshoot > Calibrate XY and Zp axes.

Teach blockin position

2-24 System Setup

Calibrate XY and Zp Axes dialog box:

7. Click Start. A warning appears, prompting you to keep clear of the sample chamber. Confirm that all plates and sample tray holders are removed, and ensure the teach block is installed.

8. Click OK. The XYZ mechanism positions the puncture needle above the teach block.

Positioning the puncture needle:

9. Examine the location of the puncture needle relative to the needle hole in the teach block.

Teach block

Puncture needle

Needle hole

XYZ mechanism foot


10. Select the 1.0-millimeter displacement increment, and then click the +Z button to position the XYZ mechanism foot close to the top of the teach block. Each click of the mouse moves the needle 1 millimeter.

Alternative: You can use the keyboard arrow keys instead of clicking the on-screen buttons to adjust the needle position. Doing so lets you adjust the position without taking your eyes off the needle.

Caution: Avoid holding down an arrow key. Doing so stores keystrokes and causes the needle to continue moving, even after you release the key.

11. Select the 0.1 millimeter displacement increment, and then fine tune the adjustment so that the XYZ mechanism foot is just above the teach block, but not touching it.

Caution: To avoid triggering the top-of-plate sensor prematurely, do not allow the mechanism foot to touch the teach block.

12. To laterally position the puncture needle, use the +X and −X buttons to move it left or right, respectively.

13. With the aid of a small mirror, use the +Y and −Y buttons to adjust the front-to-back location of the puncture needle so that it is centered over the needle hole.

Determining the front-to-back position:

14. Using the 0.1 millimeter displacement increment, lower the needle into the teach block hole to confirm an accurate X and Y alignment.

2-26 System Setup

15. Ensure no lateral deflection of the puncture needle: grasp the needle mounting block, and move it back and forth.

16. Click Calibrate. A warning appears, prompting you to keep clear of the sample chamber.

17. Click OK. The puncture needle fully lowers into the teach block to calibrate the top-of-plate sensor.

18. Click Save. When the confirmation window appears, click Yes.

19. Remove the teach block, place it in the storage location in the floor of the sample compartment, and re-insert the sample tray holder(s).

20. Click Maintain > Calibrate needle Z axis.

Calibrate Needle Z Axis dialog box:

21. Click Start, and then click OK in the confirmation window.

22. Use the +Z button to drive the sample needle down to within 1 millimeter of the tray surface.

23. Switch the displacement increment to 0.1 millimeter, and lower the sample needle until it almost touches the surface of the sample tray holder.

Tip: To easily and efficiently achieve the correct needle setting, slide a business card under the needle. Then lower the needle until it touches the card lightly but does not restrict the card's free movement.

24. Click Save, and then click Yes in the confirmation window.


2-28 System Setup

3 Preparing System Hardware

Powering-on the system

Powering-on the system entails starting each system module, the nanoACQUITY workstation, and the MassLynx software.

1. Press the power switch on the top, left-hand side of each module’s door. Each module beeps three times and runs a series of startup tests. Full initialization usually requires about seven minutes.

Contents:

Topic Page

Powering-on the system 3-1

Monitoring startup tests 3-2

Monitoring the LEDs of system instruments 3-2

Preparing the auxiliary solvent manager 3-4

Preparing the binary solvent manager 3-8

Preparing the sample manager 3-12

Preparing the detector 3-21

Conditioning the column 3-23

Shutting down the system 3-24

3-1

The power and status LEDs change as follows:

• Each LED shows red for a few seconds, except for the power LEDs.

• During initialization, these LEDs flash green:

– Auxiliary solvent manager’s flow LED

– Binary solvent manager’s flow LED

– Sample manager’s run LED

– Detector’s lamp LED

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

2. Power-on the workstation. You can monitor the nanoACQUITY UPLC Console for messages and visual signals.

3. Start MassLynx.

Monitoring startup tests

These startup tests run when you power-on the workstation.

• CPU board

• Memory (RAM and ROM)

• External communication system (Ethernet)

• Clock

If the startup tests indicate a malfunction, see Troubleshooting, in the nanoACQUITY Online Help.

Monitoring the LEDs of system instruments

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

3-2 Preparing System Hardware

Power LEDThe power LED, on the left side of a module’s front panel, indicates when the module is powered-on or powered-off.

Status LEDs

Flow LED (Auxiliary and binary solvent managers)

The flow LED, to the right of the power LED on the front panels of the auxiliary and binary solvent managers, indicates the flow status.

Run LED (Sample manager)

The run LED, to the right of the power LED on the sample manager’s front panel, indicates the run status.

Lamp LED (Detector)

The lamp LED, to the right of the power LED on the detector’s front panel, indicates the lamp status.

Status LED indications:

LED mode and color Description

Unlit Indicates the module is currently idle.

Constant green Auxiliary and binary solvent managers— Indicates the solvent manager is operating normally and solvent is flowing.

Sample manager—Indicates the sample manager is operating normally, attempting to complete any outstanding samples or diagnostic requests. When sample and diagnostic requests are finished, the LED reverts to the unlit mode.

Detector—Indicates the detector lamp is on and is operating normally.

3-3

Preparing the auxiliary solvent manager

For optimal performance of the nanoACQUITY UPLC system, you must prepare the solvent manager for operation. Preparing the auxiliary solvent manager includes

• priming the seal wash.

• priming the auxiliary solvent manager.

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.

See also: Solvent Considerations.

Flashing green Sample manager—Indicates the system is waiting for at least one module to become operable. Detector lamp warm-up and column temperature equilibration times typically cause such a delay.

Detector—Indicates the detector is initializing or calibrating.

Flashing red Indicates an error has stopped the module. Look at the nanoACQUITY UPLC Console for information on the error that caused the failure.

Constant red Indicates a module failure that prevents further operation. Power-off the module, and then power-on. If the LED is still constant red, contact your Waters service representative.


Status LED indications: (Continued)

LED mode and color Description


Requirements:

• To maintain the efficiency of the auxiliary solvent manager, and to obtain accurate, reproducible chromatograms, use only HPLC-grade (or higher) quality solvents, water, and additives. For details, see Solvent Considerations.

• Solvent A must be aqueous and solvent B must be organic (acetonitrile or methanol). The flow control module supports only the solvents shown in the Solvent drop-down list of the solvent manager’s instrument method dialog box.

• The nanoACQUITY UPLC System should not be run with high pH mobile phases. Alkaline solutions such as ammonium hydroxide (pH 10) can etch glass solvent bottles and the silica capillary tubing, resulting in an elevated chemical background being detected by mass spectrometry.

For high sensitivity applications, Waters recommends MS-grade eluents. In our laboratories, Waters has had success with Baker water and Fisher Optima™ acetonitrile. It is important to flush the system with the appropriate solvents before passing eluent into the column, optical detector, and/or mass spectrometer.

Your system is configured with the degassers removed from the fluidic pathway (bypassed). Only the weak and strong wash solvents are degassed.

Priming the seal washPrime the seal wash in the auxiliary solvent manager to lubricate the plungers and flush away solvent and/or any precipitated salts that have seeped past the plunger seals from the high-pressure side of the piston chambers.

Prime the plunger seal wash under these conditions:

• After using buffered mobile phase

• When the solvent manager has been inactive for a few hours or longer

• When the solvent manager is dry

• When troubleshooting a low-pressure error

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.

Rule: To prevent contamination, do not recycle seal wash.

3-5

Recommendations:

• Seal wash should contain no more than 10% organic solvent.

• Before priming the plunger seals, ensure the solvent reservoir contains sufficient solvent for priming and use.

Required materials

• Tubing adapter (startup kit)

• 30-mL syringe (startup kit)

• Seal-wash solution

• Powder-free nitrile gloves

To prime the seal wash:

1. Ensure the seal-wash waste line is in a suitable waste container. Never recycle seal wash.

Caution: To prevent contamination, wear powder-free nitrile gloves when handling the solvent filter. Skin oils can contaminate the filter.

2. If the system is dry

a. remove the seal-wash inlet tube from the solvent reservoir, and disconnect the inlet filter.

b. connect the tubing adapter to the syringe.

c. fill the syringe with seal-wash solution, and then connect the syringe assembly to the seal-wash inlet tube.

3. In the nanoACQUITY UPLC Console, select Auxiliary Solvent Manager from the system tree.

4. Click Control > Prime seal wash, and then click Start.

5. If you connected a syringe, push on the syringe plunger to force seal-wash solvent through the system.

6. Click Control > Prime seal wash when the seal-wash solvent flows from the seal-wash tube to stop the priming process.

7. Remove the syringe and adapter, reconnect the filter, and place the seal-wash inlet tube into the seal-wash solvent reservoir.


Priming the auxiliary solvent manager

Priming, a timed operation, replaces solvent in the path from the reservoir to the auxiliary solvent manager. During priming, the vent valve moves to Vent position to ensure minimal backpressure.

Prime the auxiliary solvent manager when performing these tasks:

• Changing reservoirs or solvents

• Preparing a new system or auxiliary solvent manager for use

• Running the system after it has been idle for more than four hours

To prime the auxiliary solvent manager:

1. In the nanoACQUITY UPLC Console, select Auxiliary Solvent Manager from the system tree.

2. Click Control > Prime A/B Solvents.

Prime A/B Solvents dialog box:

3. Select solvent B, and then select B1.

4. In the Time box, specify the number of minutes from 0.10 through 999.99. The default value is 1.0 minute.

Recommendation: 2.0 minutes.

5. Click Start.

6. When finished, repeat as needed for B1 and for all solvents in use.

3-7

Preparing the binary solvent manager

For optimal performance of the nanoACQUITY UPLC system, you must prepare the solvent manager for operation. Preparing the solvent manager includes these tasks:

• Priming the seal wash

• Priming the binary solvent manager

Requirements:

• To maintain the efficiency of the binary solvent manager, and to obtain accurate, reproducible chromatograms, use only HPLC-grade (or higher) quality solvents, water, and additives. For details, see Solvent Considerations.

• Solvent A must be aqueous and solvent B must be organic (acetonitrile or methanol).

• The flow control module supports only the solvents shown in the Solvent drop-down list of the solvent manager’s instrument method dialog box.

Priming the seal washPrime the seal wash in the binary solvent manager to lubricate the plungers and flush away solvent and/or any precipitated salts that have seeped past the plunger seals from the high-pressure side of the piston chambers.

Prime the plunger seal wash under these conditions:

• 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

• When troubleshooting a low-pressure error


Rule: To prevent contamination, do not recycle seal wash.



Recommendations:

• Seal wash should contain no more than 10% organic solvent.

• Before priming the plunger seals, ensure the solvent reservoir contains sufficient solvent for priming and use.

Required materials

• Tubing adapter (startup kit)

• 30-mL syringe (startup kit)

• Seal-wash solution


To prime the seal wash:

1. Ensure the seal-wash waste line is in a suitable waste container. Never recycle seal wash.

Caution: To prevent contamination, wear powder-free nitrile gloves when handling the solvent filter. Skin oils can contaminate the filter.

2. If the system is dry

a. remove the seal-wash inlet tube from the solvent reservoir, and disconnect the inlet filter.

b. connect the tubing adapter to the syringe.

c. fill the syringe with seal-wash solution, and then connect the syringe assembly to the seal-wash inlet tube.

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

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

5. If you connected a priming syringe, push on the syringe plunger to force seal-wash solvent through the system.

6. When the seal-wash solvent flows from the seal-wash waste tube, click Control > Prime seal wash to stop the priming process.

7. Remove the syringe and adapter, reconnect the filter, and place the seal-wash inlet tube into the seal-wash solvent reservoir.

3-9

Priming the binary solvent managerPriming, a timed operation, replaces solvent in the path from the reservoirs to the vent valve, not including the flow control module. During priming, the vent valve moves to Vent position to ensure minimal backpressure. The flow rate during priming is 8 mL/min total (4 mL/min each for pumps A and B).

Prime the binary solvent manager when performing these tasks:

• Changing reservoirs or solvents

• Preparing a new system or binary solvent manager for use

• Running the system after it has been idle for more than four hours

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.

Recommendations:

• Whenever you change solvents, always purge and autozero the flow control module (see page 6-64).

• Ensure the solvent reservoirs contain enough solvent for adequate priming and use, and ensure 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, so priming both solvents for 5 minutes requires approximately 20 mL of each solvent.

To prime the binary solvent manager:

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

2. Click Control > Prime A/B Solvents.


Prime A/B Solvents dialog box:

3. Select solvent A1 or B1.

4. In the Time (min) box, specify the number of minutes from 0.10 through 999.99. The default value is 1.0 minute.

Recommendation: 2.0 minutes.

5. Click Start.

6. When finished, repeat as needed for B1 and for all solvents in use.

3-11

Preparing the sample manager

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

• Priming the sample and wash syringes

• Characterizing the seal

• Characterizing the needle and sample loop volumes

• Loading sample plates

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

Tip: For venting purposes, the sample manager and sample organizer fans are always operating.

Selecting weak and strong wash solventsTip: For best performance, follow these guidelines when selecting wash solvents. Otherwise, performance can be reduced, specifically Area/Height RSD and Linearity. This does not mean that all other solvent combinations are prohibited. 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 and buffers are miscible and soluble.

Caution: The nanoACQUITY UPLC System should not be run with high pH mobile phases. Alkaline solutions such as ammonium hydroxide (pH 10) can etch glass solvent bottles and the silica capillary tubing, resulting in an elevated chemical background being detected by mass spectrometry.

Warning: To avoid electric shock, power-off and unplug the sample manager before performing any maintenance operation such as replacing fuses.


Suggested wash solvents:

• Strong wash solvent—50 to 100% acetonitrile/water or methanol/acetonitrile (with 0.1% formic acid)

• Weak wash solvent—100% water, or 0 to 25% acetonitrile or methanol (with 0.1% formic acid); initial conditions of the gradient or isocratic conditions. High sample concentrations can require other weak wash solvents. For best results, weak wash solvent should be equivalent to the

– mobile phase composition (for isocratic separations).

– initial gradient condition (for gradient separations).


Tip: For best performance, the weak wash solvent should 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 should include the same organic species. This might not always be practicable, especially in the case of “sticky” samples. You can, however, use a 100% organic strong wash solvent.

Solvent composition

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

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

Concentration of strong solvent

Strong solvent should be no stronger than the concentration needed to reduce carryover to an acceptable level.

Solubility of analyte and sample

The matrix must be soluble in both the weak and strong wash solvents.Caution: Proteins (in plasma, for example) are not soluble in solvents with less than 40% organic content.


Priming the sample managerDuring the priming process the sample needle fills with solvent, the solvent changes, and/or air is purged from the lines. You prime the sample needle and/or sample syringe to accomplish these tasks:

• Prepare the sample manager for operation

• Rinse the internal needle, the external piercing needle, and the injection port

• Remove bubbles from the lines

Guidelines: Ensure that the priming solvent is correctly composed and that it is high in quality and miscible with the other solvents. Use filters in all solvent reservoirs. Ensure the volumes of solvents are sufficient for priming.

Caution: Allow the priming sequence to finish. Stopping the priming sequence can leave strong solvent in the needle, which can affect the chromatography.

Requirement: The sample manager must be primed before you attempt to characterize the seal.

Sample diluent The weak wash solvent can contact the sample, so match the weak wash solvent and sample matrix as closely as possible. To offset adverse effects on peak shape caused by the matrix’s composition, adjust the weak wash composition when using the module in partial loop mode.

Wash volume ratio (weak to strong)

Within a method, this should be about 3:1, weak wash to strong wash, sufficient to ensure the weak wash flushes the strong from the needle and sample loop.

Cycle times Higher viscosity wash solvents lengthen wash cycles. For high-throughput work (cycle times < 1 min), adjust cycle times accordingly to accommodate the longer wash cycles.

Wash solvent effects: (Continued)

Property Effect


To prime the sample manager:


2. Click Control > Prime syringes.

Alternative: Right-click in the MassLynx sample manager control panel, and then click Prime syringes.

Prime Syringes dialog box:

3. Select Sample syringe and wash syringes.

4. Type the number of primes in the “Number of cycles” text box. The default value is 1.

Recommendation: Waters recommends 5 to 7 cycles when you are changing solvents.

5. Click OK.

Tip: Each prime takes approximately 2 to 4 minutes.

6. When the system status is Idle, priming is finished. Click Close.

Washing the sample manager needleWashing the needle is an optional procedure that flushes strong and/or weak wash solvent through the needle and injection port. Washing the sample manager needle removes contaminants from its inner and outer surfaces and from the external piercing needle and 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.


Tip: Priming the system washes the sample needle, so whenever you prime the system, you can omit this procedure.

Recommendations:

• Do not use buffered solvents as wash solvents.

• Match the types of organic species in the wash solvents to those in the chromatographic application.

Example: If the weak wash solvent is 30% acetonitrile and 70% water, the strong wash solvent should contain a greater concentration of acetonitrile in water.

• To ensure that the strong wash solvent is completely removed, the system washes the needle with 200 µL of weak wash solvent after you use strong wash solvent. You can increase, but not decrease, the default value of 200 µL.

• Do not interrupt the priming sequence; wait until it finishes.

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.

To wash the sample manager needle:


2. Click Control > Wash Needle.

Wash Needle dialog box:

3. In the Strong Wash box, specify the volume for the strong wash solvent. The default value is 0.0 µL. To omit strong wash solvent enter 0 in the Strong Wash box, or leave it blank.


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.

4. In the Weak Wash box, specify the volume for the weak wash solvent. The default value is 200.0 µL.

Caution: If you do not use a sufficient quantity of weak wash solvent, the strong wash solvent can contact the sample and contaminate it.

5. Click OK. The needle wash begins.

6. When washing is complete, the status returns to Idle. Click Close.

To stop a needle wash routine before it finishes:

From the sample manager information window, click Control > Reset SM.

Characterizing the needle sealThe seal calibration 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 priming the sample manager or after you replace and/or adjust these items:

• The needle

• Any part of the needle assembly

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

• A home or top-of-plate sensor

• The seal mechanism

• The NVRam battery on the CPU2000


To characterize the needle seal:


2. Click Maintain > Characterize > Needle seal.

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

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

5. Click Results to view the needle seal characterization results.

6. Click Close.

Characterizing the needle and sample loop volumesWhenever you replace the sample loop and/or the sample needle, you must instruct 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.

• Characterizing the loop volume compares the loop’s nominal volume to its measured volume.

• Characterizing the needle volume compares the needle’s nominal volume, 15.0 µL, to its measured volume.

Tip: Characterizing the system volume is critical to acceptable sample manager performance.

Requirements:

• Before characterizing the volumes, prime the sample manager, and then characterize the seal.

• Perform a method setup (MassLynx) with any method that has the same air gap and sample draw rate that you will be using.

Tip: This procedure takes approximately 15 minutes.

To characterize the needle and sample loop volumes:



2. Click Maintain > Characterize > Needle and loop volumes.

3. In the Characterize Needle and Loop Volumes dialog box, click Start.

4. Click Results.

• If the needle fails the test, check all fittings for leaks. Also, check the needle to see if it is bent, broken, or blocked.

• If the sample loop fails the test, the syringe draw (aspiration) rate used for the test might be too high. Also examine all fittings for leaks, or determine whether the loop is blocked or leaking.

• Check the configuration and make sure the needle and loop sizes are correct.

5. Click Close.

Loading sample plates in the sample managerThe sample manager holds up to two ANSI/SBS plates, which you load through the front door. The left plate is referred to as position 1, the right one as position 2.

The nanoACQUITY UPLC Sample Manager (SM) supports ANSI sample plates and vial holders only. The Sample Manager (SM) does not support vial plates with cap mats or vials without pre-slit septa.

To load a sample plate:

1. Open the sample manager door.

2. Squeeze the tray button as you pull the tray toward you.

3. Load the plate onto the tray so that position A,1 is at the right-rear corner, and the forward edge of the plate is behind the spring inside the front of the carrier.


Positioning the sample plate:

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

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

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

TP02389

A-1 well position

Button

Sample plate

Plate tray


Preparing the detector

If your system includes a TUV detector or mass spectrometer, prepare it for operation by following the procedures in this section.

See also: the documentation included with your mass spectrometer.

Preparing the TUV detectorPreparing the TUV detector involves two steps: starting and verifying.

Starting the detector

Use only thoroughly degassed HPLC-grade solvents. Gas in the mobile phase can form bubbles in the flow cell, causing the detector to fail the power-up diagnostic tests.

To start the TUV detector:

1. Ensure the detector flow cell is filled with transparent solvent (methanol or water) that it is free of air bubbles, and that the door is closed firmly.

Tip: The detector might not initialize correctly if the flow cell contains air.

2. Press the power switch on the detector door. The detector beeps three times and runs a series of startup tests while the lamp LED blinks.

Initialization requires approximately 2 minutes, and lamp warm-up requires approximately 3 minutes.

3. When the lamp LED shows constant green, start MassLynx. You can monitor the nanoACQUITY UPLC Console for messages and visual signals. For best results, allow 30 minutes for the baseline to stabilize.

Tip: The absorbance value appears in the nanoACQUITY UPLC Console and also in the MassLynx 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 instead of absorbance values.

4. Configure the detector according to the instructions in the MassLynx Online Help.


Recording sample and reference energies

After you install the detector or perform maintenance operations, 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.

2. Flush the system tubing with filtered, degassed, HPLC-grade methanol or acetonitrile.

3. Pump mobile phase for 15 minutes or more at 2 µL/min.

Caution: The maximum pressure drop across the flow cell is 1,000 psi. If the solvent is viscous (methanol/water, for example), you might need to decrease the maximum flow rate to prevent breaking the cell.

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

Tip: The detector might not initialize correctly if air is present in the cell.

5. When both LEDs show constant green, initialization is complete.

6. Start the MassLynx software.

7. To determine baseline values on the detector for future reference, and to monitor lamp aging (for decreased lamp output energy), record the baseline sample and reference energies.

Requirement: Perform this procedure each time you change the lamp.

8. Set the wavelength to 230 nm.

9. Flush the flow cell for 15 minutes or more with HPLC-grade methanol at 2 µL/min.

10. Record the sample and reference energies at 230 nm.

Preparing the mass spectrometerPrepare the optional mass spectrometer for operation after you prepare the sample manager. For specific steps, consult the mass spectrometer’s accompanying documentation.

To configure MassLynx, see page 4-1 and MassLynx Online Help.


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 should equal the gradient table run time.

Tips:

• Flush new columns with high organic (up to 85%) for 15 to 20 minutes at your typical analytical flow rate.

• To ensure best performance, flush with higher organic at the end of each run.

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—typically 10 column-volumes—and out to waste before connecting the column to the detector.

To condition the column:

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

2. In MassLynx, open the Sample Set window, and select an inlet method that includes the chromatographic conditions you want to use.

3. In the Samples table, add an inlet prerun field.

a. In the Run Samples page, select Samples > Format > Customize.

b. In the Custom Field Display window, select Inlet prerun.

c. To save the column as part of the window, save the sample set format.

4. Select method setup in the Sample Set window as a pre-inlet method, and select a method for the inlet file (these methods can be the same).

5. Run the sample set line. The system runs the condition column method and then runs the separation method.


Shutting down the system

Caution: Buffers left in the system can precipitate, damaging module components.

You might want to shut down the system

• between analyses

• overnight

• for a weekend

• for 72 hours or more

Tip: Set system shutdown parameters in the MassLynx Shutdown Editor. Consult the MassLynx Online Help for more information.

Between analyses

To shut down the system between analyses:

1. Between analyses, continue to pump the initial mobile phase mixture through the column to maintain the column equilibrium necessary for good retention-time reproducibility.

2. If a few hours will pass before the next injection, slow the flow rate in the interim to a few tenths of a µL/min to conserve solvent.

Tip: Ensure that Auto-Shutdown for your shutdown method is deactivated.

3. Keep the detector operating, and the heating and trapping module at operating temperature, during this period.

Overnight or weekends

To shut down the system overnight or over a weekend:

1. Flush the column with a 1:1 mixture of methanol/acetonitrile. This keeps the column bed in an active, wetted state.

Requirement: If you are using buffers, you must first flush the column with a high-water-content mobile phase (90% water). Then stop the pump flow.

2. Power-off the detector to lengthen lamp life.


3. The heating and trapping module can operate overnight but should be shut down over the weekend.

More than 72 hours (long-term)

To shut down the system long-term or indefinitely:

1. Follow the preceding procedure for overnight or weekend shutdown.

2. After flushing the column and letting it cool to ambient temperature, disconnect the inlet and outlet tubes, and join them with a union.

Recommendation: You should flush high-ionic buffers not only from the column but also from the system. Flushing the column with methanol/acetonitrile leaves that solvent in the fluid lines of the solvent manager, sample manager, and detector, eliminating the risk of solute precipitation.

3. Pump water through the system for 10 to 20 minutes at 10 µL/min, followed by isopropyl alcohol for another 10 to 20 minutes. Then turn the pump off, leaving isopropyl alcohol in the fluid lines.

Caution: If any system modules are to be used for another type of analysis, ensure that the liquids pumped initially through the system are miscible with methanol, water, methanol/acetonitrile, or isopropyl alcohol. Likewise, before restarting the system, ensure that any residual material not miscible with the initial methanol/water mobile phase has been flushed thoroughly from the system with an appropriate intermediate solvent.

Warning: To prevent injury, set the power switch to off, and then unplug its power cord from the AC outlet to completely interrupt power to a system module. The power switch on each system module controls the basic operational state of that module. Nevertheless, a portion of the module remains powered-on after the module is switched off.

Shutting down the system 3-25


4 Configuring System Software

Configuring MassLynx

Requirements:

• MassLynx software must be installed.

• You must be assigned administrator privileges to configure the Instrument Control Option Pack (ICOP).

You must start the application and select system modules to configure MassLynx:

To start MassLynx:

1. Select Start > All Programs > MassLynx > MassLynx V4.1.

Alternative: You can also double-click the MassLynx desktop shortcut.

If MassLynx Security is not enabled, MassLynx starts and the MassLynx window appears. If MassLynx Security is enabled, the MassLynx Login dialog box appears.

2. In the MassLynx Login dialog box, type your user name and password, and select your domain.

3. Click OK.

Contents:

Topic Page

Configuring MassLynx 4-1

Starting the nanoACQUITY UPLC Console from MassLynx 4-5

Configuring events 4-6

4-1

To select modules in the nanoACQUITY system:

1. In the MassLynx window, click Inlet Method.

Inlet Method window:

2. Select Tools > Instrument Configuration.

4-2 Configuring System Software

Inlet Configuration window:

3. Click Configure.

4. In the Welcome screen of the Inlet Configuration wizard, click Next.

5. In the Select Pump dialog box, select Waters Acquity, and then click Next.

6. In the Select Auto Sampler dialog box, select Waters Acquity, and then click Next.

7. In the Select Detector dialog box, select Waters Acquity TUV as the detector if present, and then click Next.

8. Click Next > Finish > Finish.

To install the Instrument Control Option Pack:

1. Click OK to start the Instrument Control Option Pack installation.

2. Ensure that “Install new instrument software, or upgrade existing installation(s)” is selected, and then click Next.

4-3

Instrument Control Option Pack dialog box (Select window):

3. Select ACQUITY Binary Solvent Manager, ACQUITY Sample Manager, and ACQUITY TUV Detector, and then click Next. A progress bar appears at the bottom of the dialog box.

Requirement: You must select ACQUITY TUV Detector even if your system does not include a TUV detector. If you do not do so, the control will be blank when you open the nanoACQUITY UPLC Console.

4. In the Results screen of the Instrument Control Option Pack dialog box, click Finish. Result: The Inlet Method window appears.


Starting the nanoACQUITY UPLC Console from MassLynx

The nanoACQUITY UPLC Console is a software application that replaces the keypads and small display screens traditionally found on the fronts of system instruments. It provides a convenient way to configure settings, monitor performance, run diagnostic tests, and maintain the system.

To start the nanoACQUITY UPLC Console from MassLynx:


2. Click the ACQUITY Additional Status tab.

ACQUITY Additional Status tab:

3. Click Display console .

Result: The nanoACQUITY UPLC Console window appears.

Starting the nanoACQUITY UPLC Console from MassLynx 4-5

Configuring events

Besides making the signal connections you made in “Making Ethernet and signal connections” on page 2-7, you must configure the events in MassLynx.

To configure events:


2. Select Tools > Instrument Configuration.

3. Click Events & Triggering, and then click Next.

4. Select the check boxes that correspond to the event in and event out connections you made to the mass spectrometer.

Example: If you connected the inject start terminals on the sample manager connector to the number 1 Event I/P connector on the mass spectrometer, select box 1 in the Event In section.

Choose Events dialog box:

5. Click Next. In the Configuration Successful window, click Finish.


5 Verifying System Operation

This chapter explains how to run a gradient performance test to verify that the system is operating properly. The sample you use to verify the system is included in the system startup kit.

Before you begin this procedure, the system must be set up and configured as described in this guide’s System Setup, Preparing System Hardware, and Configuring System Software sections.

Required materials

Make sure these materials are on hand before you begin the verification test:

• MS-grade water

• acetonitrile (Waters recommends Fisher Scientific’s Optima® brand)

• MS-grade formic acid

• MassPREP™ Peptide Standard (system startup kit)

• nanoACQUITY BEH C18 analytical column (1.7 µm, 100 µm × 100 mm)

• nanoACQUITY Symmetry C18 trap column (5 µm, 180 µm × 20 mm)

• powder-free nitrile gloves

Contents:

Topic Page

Required materials 5-1

Preparing the mobile phases 5-2

Preparing the sample 5-3

Preparing the system 5-4

Creating the test methods 5-6

Performing the test 5-10

5-1

See also: For optimal mass spectrometry results on page 2-12.

Requirement: To prevent contamination, always use powder-free nitrile gloves when handling components of the nanoACQUITY system.

Caution: Never change directly between immiscible eluents or between buffered solutions and organic eluents. Immiscible eluents form emulsions in the flow path. Combining buffered solutions and organic eluents 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 page 3-10.

Preparing the mobile phases

The verification test requires two mobile phases: 0.1% formic acid/water for solvent A and 0.1% formic acid/acetonitrile for solvent B. The test also requires weak wash and strong wash.

Requirement: All solvents must be HPLC-grade or better.

To prepare solvent A, 0.1% formic acid/water:

1. Measure 100 mL of HPLC-grade water in a 100-mL graduated cylinder.

2. Carefully transfer the water to a 250-mL reservoir bottle.

3. Pipette 100 µL of formic acid into the reservoir bottle.

4. Cap the reservoir bottle and mix well.

5. Label the reservoir bottle as 0.1% formic acid/water.

6. Submerge lines A1, Seal Wash, Weak Wash, and Strong Wash in the solvent A reservoir bottle with 0.1% formic acid/water.

7. Place the solvent A reservoir bottle in the solvent tray.

To prepare solvent B, 0.1% formic acid/acetonitrile:

1. Measure 100 mL of acetonitrile in a 100-mL graduated cylinder.

2. Carefully transfer the acetonitrile to a 250-mL reservoir bottle.

3. Pipette 100 µL of formic acid into the reservoir bottle.

4. Cap the reservoir bottle and mix well.

5-2 Verifying System Operation

5. Label the reservoir bottle as 0.1% formic acid/acetonitrile.

6. Submerge the B1 line in the solvent B reservoir bottle with 0.1% formic acid/acetonitrile.

7. Place the solvent B reservoir bottle in the solvent tray.

Preparing the sample

The verification test uses the MassPREP Peptide Standard to verify that your system is operating correctly. The mixture includes nine peptides with a broad range of polarities and isoelectric points. In addition, there is a void volume marker. The following table lists the components of the peptide mixture, in elution order, and their molecular weights.

Tip: Because Enolase T35, Enolase T37, and Melitin are weaker ionizers in ESI, they might not be detected.

The peptide mixture contains approximately 1.5 µg (~1 nmole) of each peptide. When reconstituted in 1 mL of solvent (95:5 water/acetonitrile with 0.1% formic acid), the final sample concentration is ~1 pmole/µL.

MassPREP peptides mixture components:

PeptideMW, monoisotopic (g/mol)

M + 2H M + 3H

Allantoin (V0 marker) 158.0440

RASG-1 (not trapped) 1000.4938

Angiotensin frag. 1-7 898.4661 450.2401 300.4957

Bradykinin 1059.5613 530.7877 354.1941

Angiotensin II 1045.5345 523.7743 349.5185

Angiotensin I 1295.6775 648.8458 432.8995

Renin substrate 1757.9253 879.9697 586.9821

Enolase T35 1871.9604 936.9872 624.9938

Enolase T37 2827.2806 1414.6473 943.4339

Melitin 2845.7381 1423.8761 949.5864

5-3

To prepare the sample:

1. Pipette 1.0 mL (0.95 mL of 0.1% formic acid/water you mixed in the previous section and 0.050 mL of 0.1% formic acid/acetonitrile mobile phase) into the vial containing the peptides mixture.

2. Cap the sample vial, and vortex-mix the sample for 10 seconds.

3. Pipette 1.0 mL of 0.1% formic acid/water into an empty vial, and then cap the vial. The mobile phase blank will be injected from this vial.

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

The peptides solution is stable for one week when stored at 8 ºC. You can extend stability by freezing the sample at -20 ºC. However, for best results, you should run the verification test immediately after preparing the sample.

Caution: The sample degrades rapidly when contaminated with endopeptidase or exopeptidase enzymes.

Preparing the system

Recommendation: Flush the system with the appropriate solvents before passing eluent into the column, optical detector, and/or mass spectrometer.

To prepare the system for verification:

1. Connect and install the trap column (see page 2-19).

2. Install the analytical column in the heating and trapping module, and then connect the column outlet to a suitable waste container (see page 2-20).

3. In the nanoACQUITY UPLC Console, perform these tasks if you have not already done so:

a. Prime the A1 and B1 solvent lines for 5 minutes.

b. Prime the seal wash pump for one minute.

Warning: Always observe safe 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.


c. Prime the sample and wash syringes 20 cycles.

d. Characterize the system volume.

Tip: Characterizing the system volume is critical to acceptable sample manager performance.

4. If either the trap or analytical column are new, flush them as follows.

5. Connect the analytical column outlet to the mass spectrometer inlet.

Exception: If your system includes a TUV detector, connect the column outlet to the TUV flow cell inlet, and then connect the TUV flow cell outlet to the mass spectrometer inlet.

6. Equilibrate the system with initial starting conditions of the gradient (99% A, 1% B) until the detector baseline is stable.

Tip: To maintain proper drainage and leak control in the nanoACQUITY UPLC system, the SM Fluidics tray must be fully closed during routine operation.

Duration (minutes)

Flow rate (µL/min)

%B solventTrap valve position

20 10 85 Trapping

20 0.4 85 Analytical

10 0.4 50 Analytical

10 0.4 1 Analytical

5-5

Creating the test methods

Follow the steps below to create the methods, setting the parameter values to match those reflected in the accompanying screen representations. This method is designed as a rapid test procedure implementing a 150-µm ID column at high flow rate.

Creating the instrument method

To create the instrument method:

1. Create an instrument method with these binary solvent manager parameters.

Exception: If you use the 75 µm ID × 100 mm column, in the gradient table, enter 0.300 µL/min as the flow rate.

Binary solvent manager instrument parameters (General tab):


Binary solvent manager instrument parameters (Trapping tab):

Tip: The instrument Comment field is limited to 500 characters. This ensures correct spacing when generating reports.

Recommended trapping parameters (with a 5-microliter loop):

• Flow rate: 15 µL/min

• %A: 99.0, %B: 1.0

• Sample loading time (duration): 1 minute

• High pressure limit: 5000 psi

• The system uses the binary solvent manager as the trapping pump for one minute of the gradient performance test.

20.00

1.00

15.00099.0

1.0


2. Set these parameters in the sample manager instrument method.

Sample manager instrument parameters:


3. If your system includes a TUV detector, set these parameters in the TUV detector instrument method.

4. Save the instrument method.

5. Create a sample list composed of six 1-µL injections of the peptides mixture and one 1-µL injection of the blank mobile phase.

Tip: The typical column back pressure at 0.400 µL/min is approximately 2300 psi.


Performing the test

When the system is prepared and the test methods are created, you are ready to perform the test.

To perform the test:

1. At the MassLynx main page, select Run > Start.

2. Verify the settings for the test, and then click OK.

3. Verify these options, afterward clicking OK for operations, quantify, and LIMS export.

4. When the sample list is complete, enter the appropriate results in the table, below.

Retention Time Reproducibility (Six injections):

Peak Component

Peak Retention Time (six injections) Mean

ValueRSD

1 2 3 4 5 6

1 Allantoin (V0 marker)

2 RASG-1

3 Angiotensin frag. 1-7

4 Bradykinin

5 Angiotensin II

6 Angiotensin I

7 Renin substrate

8 Enolase T35

9 Enolase T37

10 Melitin


5. Review the gradient performance report. The system verification 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, to determine this.)

• The peak retention times show a relative standard deviation (RSD) of less than 15.0 seconds (0.25 minutes). Use the table you completed to determine this.

Sample system verification test chromatogram (MS detection):

Example: This is a representative chromatogram. The results from your system can vary slightly.

Angiotension frag 1-7

Bradykinin

Angiotensin II

Angiotensin I

Performing the test 5-11


6 Maintaining the System

Waters recommends that you perform the following routine maintenance on the nanoACQUITY system to ensure reliable operation and accurate results. When using the system throughout the day (and on nights and weekends), or when using aggressive solvents such as buffers, perform these maintenance tasks more frequently.

Contents:

Topic Page

Contacting Waters technical service 6-2

Maintenance considerations 6-3

Configuring maintenance thresholds 6-4

Maintaining the solvent managers 6-6

Maintaining the sample manager 6-24

Maintaining the heating and trapping module 6-49

Maintaining the TUV detector 6-51

Testing the system’s fluidic integrity 6-60

Recommended routine maintenance schedule:

Maintenance procedure

Frequency For information...

Replace solvent bottle filters.

At regularly scheduled preventive maintenance interval

See page 6-6.

Replace solvent manager air filters.

At regularly scheduled preventive maintenance

See page 6-11.

6-1

Contacting Waters technical service

Customers in the USA and Canada should report maintenance problems they cannot resolve to Waters Technical Service (800 252-4752). Others should phone their local Waters subsidiary or Waters corporate headquarters in Milford, Massachusetts (USA), or visit http://www.waters.com, and click About Waters > Corporate Information > Worldwide Offices.

When you phone Waters Technical Service, be ready to provide this information:

• Nature of the symptom

• Instrument serial numbers (see Troubleshooting in the nanoACQUITY Online Help)

• Flow rate

• Operating pressure

• Solvent(s)

• Detector settings (sensitivity and wavelength)

• Type and serial number of column(s)

• Sample type

• MassLynx software version and serial number

• nanoACQUITY workstation model and operating system version

For complete information on reporting shipping damages and submitting claims, see Waters Licenses, Warranties, and Support Services.

Clean the system modules with a soft, lint-free cloth, or paper dampened with mild soap and water.

As needed See page 6-23, page 6-49, page 6-50, and page 6-59.

Recommended routine maintenance schedule: (Continued)

Maintenance procedure

Frequency For information...

6-2 Maintaining the System

Maintenance considerations

Safety and handlingObserve these warnings and cautions when you perform maintenance procedures on your system.

Caution:

• To avoid damaging electrical parts, never mechanically disconnect an electrical assembly while it remains connected to the electrical source. To disconnect an assembly from the electrical source, set the module’s On/Off switch to Off, and then unplug the power cord from the AC outlet. After power is removed, wait 10 seconds after unplugging the power cord before you disconnect the assembly.

• To prevent circuit damage due to static charges, do not touch integrated circuit chips or other system modules that do not require manual adjustment.

Requirement: To prevent contamination, wear powder-free nitrile gloves when performing maintenance procedures.

See also:

• The section titled, For optimal mass spectrometry results on page 2-12

• Appendix C, Solvent Considerations

Proper operating proceduresTo keep your system running smoothly, follow the operating procedures and guidelines in Preparing System Hardware and Verifying System Operation.

Warning: To prevent injury, always observe good laboratory practices when you handle solvents, change tubing, or operate the system. Know the physical and chemical properties of the solvents you use. See the Material Safety Data Sheets for the solvents in use.

Warning: To avoid electric shock, do not open the power supply cover. The power supply does not contain user-serviceable parts.


Spare partsSee Appendix B, Spare Parts for spare part information. Parts not included in Appendix B are not recommended for replacement by the customer.

Configuring maintenance thresholds

Maintenance counters prompt you to perform system maintenance when a performance threshold is exceeded. Maintenance thresholds can be set for these conditions:

• Solvent manager pumped volume—The binary solvent manager requires regular maintenance, the frequency of which depends on the volume of fluid it pumps. You can set a threshold to alert you when the pumped fluid reaches a specified volume (pumped fluid volume threshold).

• Sample manager injection cycles—The sample manager requires regular maintenance, the frequency of which depends on how many injections it makes. You can set a threshold to alert you when the injection count reaches a specified count (injections threshold).

• Detector lamp life—The detector’s lamp requires regular replacement, the frequency of which depends on its operating time. You can set a threshold to alert you when the lamp has operated for a specified number of hours (time threshold).

Guidelines:

• The module operates regardless of whether you specify a maintenance threshold. In either case, the status bar displays the value until you reset it.

• If a maintenance bar terminates in a curve, no threshold is specified.

• If a maintenance threshold is exceeded, these events take place:

– A text message in the nanoACQUITY UPLC Console informs you of the condition.

– The status bar, which displays the threshold and actual values, changes from green to yellow.

– The LED in the associated control panel in the MassLynx Additional Status tab changes from green to yellow.


• Exceeding a threshold does not affect the performance of methods, sample sets, or scheduled sample runs underway, and it does not prevent their continued performance.

See also: nanoACQUITY Online Help.

To configure maintenance thresholds:

1. In the nanoACQUITY UPLC Console, select Maintenance Counters from the system tree.

Predictive maintenance indicators panel:

2. Right-click a maintenance bar.

3. Click the “Enable warning level” box.

4. Specify a maintenance threshold value.

5. Click OK .


Maintaining the solvent managers

The maintenance procedures in this section apply to the auxiliary solvent manager and the binary solvent manager.

Perform these procedures when you discover a problem with a solvent manager component or during preventive maintenance. For information about isolating problems in a solvent manager, see Troubleshooting in the nanoACQUITY Online Help.

Recommendation: Whenever you replace seals, always wet their surfaces with water, methanol, or a compatible mobile phase.

Replacing the filtersThe auxiliary solvent manager has one solvent filter, and the binary solvent manager has two. The procedure for replacing the filters is identical.

Required materials

• 1/4-inch open-end wrench

• 5/8-inch open-end wrench

• Solvent filters

To replace a filter:

1. Stop the solvent flow.

2. Use the 5/8-inch open-end wrench to hold the filter in place.

3. Use the 1/4-inch open-end wrench to disconnect the outlet compression fitting.

Warning: To prevent injury, always observe good laboratory practices when you handle solvents, change tubes, or operate the solvent manager. Know the physical and chemical properties of the solvents you use. Refer to the Material Safety Data Sheets for the solvents in use.

Warning: To prevent injury, always wear eye protection and gloves when handling solvents.


Disconnecting the outlet compression fitting:

4. Again using the 5/8-inch open-end wrench to hold the filter in place, disconnect the inlet compression fittings with the 1/4-inch wrench.

Disconnecting the inlet compression fitting:

5. Remove the old filter from the bracket.

6. Carefully remove the new filter from the packaging.

7. Insert the new filter into the bracket.

8. Reattach the compression fittings to the filter, and tighten the compression fittings finger-tight with an additional 1/6-turn.

Filter

Outlet compression fitting

Inlet compression fitting Filter


Replacing a check valveThe procedure for replacing a check valve is identical for all solvent manager heads in the auxiliary solvent manager and binary solvent manager.

Required materials

• 1/4-inch, 5/16-inch, and 1/2-inch open-end wrenches

• Check valve assembly

To replace the check valve:

1. Power-off the solvent manager.

2. Move the solvent bottles to a location below the solvent manager head.

Use the 5/16-inch open-end wrench to hold the check valve cartridge in place.

3. Use the 1/4-inch open-end wrench to disconnect the tubing connection from the check valve.

Disconnecting the tubing from the check valve:

4. Use the 1/2-inch open-end wrench to loosen the check valve, and remove the check valve assembly from the solvent manager head.

Warning: To avoid spills, move the solvent bottles to a location below the solvent manager head.

Check valve

Tubing connection

5/16-inch open-end wrench



Disconnecting the check valve from the solvent manager head:

Caution: When you remove the check valve, make sure you also remove the PEEK washer.

5. Carefully remove the new check valve and washer from the packaging.

6. Examine the check valve.

• Primary check valves have a shorter housing and retainer.

• Accumulator check valves have a longer housing and retainer, with a visible groove or retaining ring.

Inserting PEEK washer into check valve:

7. Insert the new PEEK washer into the new check valve. Make sure the chamfered edge of the washer faces upward.

Pressure transducer

Pressure transducer cable

Check valve

Solvent manager head


PEEK washer with chamfered edge facing up

Shorter retainer

Longer retainer with visible groove or retaining ring

Primary check valve Accumulator check valve


8. Insert the check valve assembly into the solvent manager head, and tighten the check valve nut finger-tight plus an additional 1/8-turn.

9. Reattach the fitting to the check valve, and tighten the compression screw finger-tight, plus an additional 1/4-turn for existing stainless steel tubing or 3/4-turn for new stainless steel tubing. Make sure to hold the check valve cartridge in place with the 5/16-inch wrench when tightening the tube compression screw to the check valve.

Replacing solvent bottle filtersReplace a solvent bottle filter if the flow of solvent from the reservoir becomes restricted. You need only replace the filter disc, not the entire filter assembly.

Because a clogged filter can indicate contaminated solvent or a dirty solvent bottle, Waters recommends you clean the solvent bottle when you replace the filter (see also page 2-12).

Recommendation: Use only Waters bottle stoppers.

Requirement: To help prevent contamination, wear powder-free nitrile gloves when performing this procedure.

To replace a solvent bottle filter:

1. Remove the cap and the solvent tubing from the solvent bottle.

2. Grasp the solvent tubing, and pull off the solvent filter assembly.

Replacing the solvent bottle filter:

Filter discSolvent tubing

TP02517

Filter body Filter cap

Marked side


3. Grasp the top portion of the filter body between your thumb and forefinger. Then unscrew the filter cap by turning it counterclockwise.

4. Remove the filter disc from the filter cap.

5. Insert the new filter disc into the filter cap so that the marked side of the filter disc faces away from the filter body.

6. Screw the filter cap onto the filter body until the cap fits snugly.

7. Ensure the marking on the filter disk is visible at the bottom of the filter assembly. If it is not, remove the filter cap, reverse the disc, and then replace the filter cap.

8. Push the filter assembly onto the solvent tubing.

9. Insert the solvent tubing and filter assembly into the solvent bottle, and replace the bottle cap.

Cleaning the air filters in the solvent manager doorThe procedure for cleaning the air filters is identical for the auxiliary solvent manager and binary solvent manager.

To clean the air filters:

Use a vacuum cleaner to clean the air filters located inside the binary solvent manager door.

Replacing the air filters in the solvent manager doorIf the air filters cannot be cleaned by vacuuming, replace them with new filters. The procedure for replacing the air filters is identical for the auxiliary solvent manager and binary solvent manager.

Required materials

• T10 TORX driver

• Solvent manager air filters

To replace the air filters:

1. Using the T10 TORX driver, remove the eight screws that secure the air filter frames and air filters to the inside of the solvent manager door.


Air filters inside the solvent manager door:

2. Remove the old air filters from the air filter frames and discard the filters.

3. Align the new air filters with the air filter frames.

4. Using the T10 TORX driver, attach the air filters and frames to the inside of the solvent manager door with the eight screws.

Replacing the solvent manager head sealsThe procedure for replacing the solvent manager head seals is identical for the auxiliary solvent manager and binary solvent manager.

See also: Troubleshooting in the nanoACQUITY Online Help to help determine whether you need to replace the solvent manager pump head seals.

Required materials

• High-pressure seal rebuild kit

• T27 TORX driver (startup kit)

• 1/4-inch and 5/16-inch open-end wrenches

• Water, methanol, or compatible mobile phase

• Dental pick

TP02429

Air filter

Frame

Air filter

Frame


To remove the solvent manager head:

1. Flush the solvent manager with appropriate solvent.

2. In the nanoACQUITY UPLC Console, select Binary Solvent Manager or Auxiliary Solvent Manager from the system tree.

3. Click Maintain > Heads.

Head maintenance dialog box:

4. Select the head (Primary or Accumulator) from the solvent manager (pump A or pump B) you plan to perform maintenance on.

5. Click Move Backward, and then wait for the plunger to stop.


7. Remove the seal wash tubes secured to the seal wash housing by two barbed fittings.

8. Use the 1/4-inch open-end wrench to disconnect the outlet tube from the transducer.

9. Use the 5/16-inch open-end wrench to hold the check valve cartridge in place, and then use the 1/4-inch open-end wrench to disconnect the tubing connection from the check valve.

10. Disconnect the pressure transducer cable from the bulkhead.



11. Using the T27 TORX driver, remove the two head bolts, which are accessible from the front of the pressure transducer.

Removing the head bolts securing the solvent manager head:

The bolts remain captive behind the mounted transducer.

12. Gently pull the head off the assembly, making sure not to tilt the head during the extraction.

Removing the solvent manager head:

Caution: To avoid damaging the plunger, support the solvent manager head from below as you remove the head.

TP02435


To remove the solvent manager head seals:

1. Stand the solvent manager head upright on a clean surface.

2. Remove the old seal wash seal and discard it.

Solvent manager head seals:

3. Invert the solvent manager head so that the seal wash housing separates from the head.

4. Using the dental pick, carefully pry the high pressure seal out of the solvent manager head. Take care not to scratch any surfaces.

5. Using the dental pick, carefully remove the PTFE O-ring.

6. Press the new PTFE O-ring into its seat with your thumbs.

7. Lubricate the new high pressure seal and place it loosely in the seat.

8. Center the seal backup ring over the high pressure seal so that the cross-side faces upward.

9. Orient the seal wash housing so that the holes on its side are aligned with the holes on the side of the solvent manager head.

10. Reinstall the seal wash housing into the solvent manager head, pushing down until you hear the high pressure seal snap into place.

11. Lubricate the seal wash seal, and slide it onto the plunger. Slide the seal back until it contacts the support plate.


High pressure seal

PTFE O-ring

Seal backup ring

Seal wash housing

Seal wash seal


To reattach the solvent manager head:

1. Moisten the seals and plunger with methanol.

2. Carefully slide the head assembly over the sapphire plunger.

3. Align the mounting holes in the support plate with the two 5-mm screws that are captive in the head assembly.

4. With the head assembly secure against the support plate, use the T27 TORX driver to tighten the head screws.

Replacing the solvent manager plungersThe procedure for replacing the solvent manager plungers is identical for the auxiliary and binary solvent managers.

See also: Troubleshooting in the nanoACQUITY Online Help to determine whether you need to replace the plungers.

Required materials

• 1/4-inch, 5/16-inch, and 1/2-inch open-end wrenches

• TORX driver, T27

• Replacement plunger

To remove the solvent manager head:

1. Flush the solvent manager with appropriate solvent.



4. Select the head (Primary or Accumulator) of the pump (A or B) in the solvent manager you plan to perform maintenance on.





7. Remove the seal wash tubes secured to the seal wash housing by two barbed fittings.

8. Remove the fittings attached to the solvent manager head and inlet port.

9. Use the 5/16-inch open-end wrench to hold the check valve cartridge in place.

10. Use the 1/4-inch open-end wrench to disconnect the tubing connection from the check valve.

11. Disconnect the pressure transducer cable from the bulkhead.

12. Using the T27 TORX driver, remove the two support plate bolts.

Removing the solvent manager head and support plate bolts:

13. Gently pull the solvent manager head and support plate off the actuator housing, making sure not to tilt the head during the extraction.

Bolt

Bolt


Removing the solvent manager head and support plate:

Caution: To avoid damaging the plunger, support the solvent manager head from below as you remove the head.

To replace the plunger:

1. Stand the solvent manager head assembly upright on a clean surface, and place it aside if you are not replacing any seals as part of this procedure. If you are replacing seals, see page 6-12 for specific instructions.

Recommendation: Waters strongly recommends that you replace the solvent manager head seals when you replace the plunger.



4. In the Head Maintenance dialog box, select the head (Primary or Accumulator) from the solvent manager (pump A or B) you plan to perform maintenance on.

5. Click Move Forward, and then wait for the plunger movement to finish.

6. Use your fingers or the T27 TORX driver, apply pressure on the release collar to disengage the old plunger from the mechanism.

Plunger


Support plate


Replacing the plunger:

7. Grasp the new plunger with a clean, lint-free cloth, and insert the plunger while applying pressure to the release collar.

To reattach the solvent manager head:

1. Lubricate the plunger with methanol.



4. In the Head Maintenance dialog box, select the head (Primary or Accumulator) from the solvent manager (pump A or B) you plan to perform maintenance on.


6. Place the solvent manager head assembly in position on the mechanism. Insert the two T27 TORX screws into the holes in the support plate, and tighten the screws.

7. Connect the pressure transducer cable to the bulkhead.

8. Reattach all fittings. Make sure to hold the check valve cartridge in place with the 5/16-inch wrench when tightening the tube compression screw to the check valve.

9. Click Close.

T27 driver

Plunger

Spring-loaded release collar

Piston


Replacing the vent valve cartridgeThe procedure for replacing the vent valve cartridge is similar for the auxiliary solvent manager and binary solvent manager.

Required materials

• Hex wrench, 2-mm

• Vent valve cartridge

To replace the vent valve cartridge:


2. Click Interactive Display.

3. Click Control , and then ensure the Vent Valve is set to Vent.

Interactive display showing vent valve setting:

4. Remove the fittings attached to the vent valve cartridge.

Vent valve setting


Solvent manager vent valve cartridges with fittings:

5. Use the 2-mm hex wrench to remove the hex screw at the 10 o’clock position on the vent valve cartridge.

6. Remove the vent valve cartridge from the vent valve assembly.

7. Carefully remove the replacement vent valve cartridge from the packaging.

8. Ensure that the groove in the cartridge housing and the groove on the drive clamp are aligned. If they are not, turn the drive clamp until the grooves align.

Aligning vent valve cartridge grooves:

9. Insert the new vent valve cartridge into the vent valve cartridge chamber.

BSM: Pump B Vent/WasteASM: Pump B (Lockmass) Vent/Waste

From Pump A

Hex screw in10 o’clock position

From Pump B

Pump BTo System

Pump A Vent/Waste

BSM: Pump A To SystemASM: Flow Control Module to Mass Spectrometer 12

3

4 5

6

Drive clamp

Aligned grooves


Caution: The vent valve cartridge must slide fully into the vent valve assembly. If it does not, contact your Waters service representative.

10. Insert the 2-mm hex screw at the 10 o’clock position on the vent valve cartridge, and then use the 2-mm hex wrench to tighten it.

11. Reattach all fittings.


13. Prime the solvents:

a. Click Control > Prime A/B Solvents.

b. Select the solvent line(s) you want to prime (A or B).

c. Choose solvent A1 for line A and solvent B1 for line B.

d. In the Time (min) box, type the number of minutes to prime the solvent manager.

e. Click Start. The vent valve switches to the Vent position, one or both pumps begin priming at specified flow rates, and the solvent manager status reads “Pump Priming.” When priming is complete, the status returns to Idle.

f. Click Close.

Replacing the fuses

The procedure for replacing the fuses is identical for the auxiliary solvent manager and binary solvent manager. Each solvent manager requires two 5-A, 250 V, 5 × 20 mm, slow-blow, IEC type fuses.

Suspect an open or otherwise defective fuse when any of these conditions apply:

• Solvent manager fails to power-on.

• Solvent manager status LEDs are unlit.

• Fan does not operate.

Warning: To avoid electric shock, power-off and unplug the solvent manager before examining the fuses. For continued protection against fire, replace fuses only with fuses of the same type and rating.


To replace the fuses:

Requirement: Replace both fuses, even when only one is defective.

1. Power-off the solvent manager.

2. Disconnect the power cord from the power entry module.

3. Use a flat-blade screwdriver to open the fuse holder door, which is located above the power entry module on the rear panel.

Removing the fuseholders:

4. With minimum pressure, pull on each spring-loaded fuse holder to remove it.

5. Discard the fuses.

6. Insert the new fuses into the holders and the holders into the power entry module.

7. Reconnect the power cord to the power entry module.

Cleaning the module’s exteriorUse a soft cloth, dampened with water, to clean the outside of the solvent manager.

Caution: Do not use solvent to clean the module’s exterior.

Warning: For continued protection against fire, replace fuses with the appropriate type and rating.

Fuse holders

Fuse holder door

Fuse

Power entry module


Maintaining the sample manager

Defrosting the sample compartmentDefrost the sample compartment whenever the sample manager is unable to reach a setpoint of 10 °C or lower.

Tips: To prevent the sample compartment from freezing

• keep its temperature at 8 °C (46.4 °F) or higher.

• open its door only when necessary. (Opening the door admits humid air into the sample compartment, which causes condensation and freezing.) Waters recommends loading multiple samples into a well plate, rather than using an individual vial for each sample to minimize the need to open the door frequently.

To defrost the sample compartment:

1. Open the sample compartment door and remove any samples.

Caution: To prevent heavy condensation from forming inside the sample compartment and damaging it, the sample compartment door must remain open throughout the defrost procedure.


3. Click Maintain > Defrost.

Defrost dialog box:

4. Click Start. The sample compartment defrosts until its inside temperature reaches 40 °C (104 °F).


5. Click Close.

6. Insert the samples in the sample compartment, and then close the door.

Replacing the sample needle assembly

Caution: To avoid operational problems, ensure the sample needle is properly installed.

Replace the needle assembly when the

• sample manager is unable to reach sample transfer pressure.

• needle is bent.

• needle tip is damaged.

• needle is plugged.

Required material

Needle assembly

To remove the needle assembly:

1. In the nanoACQUITY UPLC Console, click Sample Manager, in the left pane.

2. Select Maintain > Change needle. A message tells you to remove the right-hand sample plate from the sample manager compartment.


4. Pull out the right-hand tray and remove the sample plate, if one is loaded.

5. After removing the sample plate from the tray, click OK.

6. Unscrew the needle assembly’s one-piece fitting from injection valve port 2.

Warning: To avoid puncture wounds, keep hands or loose clothing clear of the needle assembly mechanism while it is moving. The sample manager beeps three times whenever the door is open and the needle assembly mechanism is about to move.


injection valve:

7. Remove the one-piece fitting from the needle.

8. In the sample compartment, loosen the red thumbscrew on the needle mounting bracket.

Injector Port 2 fitting


Needle assembly:

9. Push the needle latch back to release the needle mounting cylinder from its mounting cavity.

Red thumbscrew

Needle latch


Releasing the needle from the mounting cylinder:

10. Lift the needle tip out of the white plastic guide at the bottom of the XYZZ mechanism.

Removing the needle from the guide:

LatchMounting cavity

Mounting cylinder

Needle tip

White plastic guide


11. Remove the needle assembly from the sample compartment.

ACQUITY UPLC sample needle assembly:

To install the sample needle assembly:

1. Insert the needle tip into the guide tube next to the injection valve.

Threading the needle assembly through the needle guide tube:

2. Gently push the needle assembly into the sample compartment.

Warning: To avoid puncture wounds or damage to the end of the needle, do not touch or press the end of the sample needle.

Needle tip

Fitting

Protective cap

Mounting cylinderClamping sleeve

ID sleeve

Needle guide tube


3. From inside the sample compartment, take hold of the needle assembly tubing as it enters the chamber from above.

4. Remove the protective cap from the needle tip.

5. Hold the needle by the mounting cylinder, and with the needle tip pointing downward, insert it into the white plastic guide at the bottom of the XYZZ mechanism.

Inserting the needle tip into the needle guide:

6. Insert the needle mounting cylinder into the mounting cavity.

Needle tip

Needle guide


Inserting the needle cylinder into the mounting cavity:

7. Pull the needle latch forward to secure the needle assembly.

8. Form a loose loop so that the portion of the tubing with the black clamping sleeve can be secured by the thumbscrew. Be sure that the loop is below the top of the Z-flag, as shown in the figure above.

9. Align the tubing so that the portion just to the left of the clamping sleeve fits into the notch behind the thumbscrew.

10. Tighten the thumbscrew so that it secures the portion of the needle with the black sleeve.

Latch

Mounting cylinder

Mounting cavity

Loose loop of tubing

Flag


Needle latched with loop:

11. Ensure that the needle tubing is fully inserted into port 2 on the injection valve. Then thread the one-piece fitting into the port, tightening the fitting until it is finger-tight.

12. Close the sample manager door.

13. Prime the syringes:

a. In the nanoACQUITY UPLC Console, click Control > Prime syringe.

Prime Syringes dialog box:

Thumbscrew

Loose loop of tubing

Needle assembly


b. Select Sample syringe and wash syringes.

c. Type 1 in the “Number of cycles” text box.

d. Click OK. The sample manager status displays “Priming.” When priming is complete, the status returns to “Idle.”

e. Click Close.

14. Calibrate the XYZ mechanism as described on page 2-23.

Characterizing the needle sealTip: Characterizing the needle seal is critical to acceptable sample manager performance.

The seal characterization function finds the position at which the needle achieves a seal on the wash station block.

Requirement: Make sure the sample manager is primed before calibrating the needle seal.

To characterize the needle seal:

1. Click Maintain > Characterize > Needle seal.

Characterize Needle Seal dialog box:

2. Click Start. The calibrate seal operation begins, and the sample manager status displays “Calibrating seal.”

3. When needle seal calibration is complete, the Results pane appears. If the characterization is successful, click Close. Continue with Characterizing the needle and loop volumes on page 6-34.

4. If characterization is unsuccessful, examine the needle to ensure you installed it properly. Make any necessary adjustments, and then


calibrate the needle seal again. If unsuccessful again, see Troubleshooting in the nanoACQUITY Online Help.

Characterizing the needle and loop volumesPerform this procedure whenever you change

• mobile phases

• wash solvents

• sample loop

• needle

• syringes

Whenever you replace the sample loop and/or sample needle, you must characterize the volume of the replacement parts to accommodate the differently sized parts and to ensure optimal chromatographic results. Do this regardless of whether the sizes of the replacement parts equal those of the original parts or differ from them.

Changes in the composition of the weak wash solvent can affect viscosity, surface tension, and/or polarity. During sample injection, the weak wash solvent both precedes and follows the sample in the fluidic lines, so the sample is directly affected by the weak wash solvent.

Requirements:

• Specify the sizes of the sample needle, loop, and syringe in the Volumes dialog box before you characterize their volumes.

• Prime the sample manager before characterizing the volumes.

• Perform a method setup in Empower or MassLynx with any method that has the same air gap and sample draw rate that you will be using.


To characterize the needle and loop volumes:

1. Click Maintain > Characterize > Needle and loop volumes.

Characterize Needle and Loop Volumes dialog box:

2. Click Start. The characterization begins.

3. When needle and loop volume characterization is complete, the Results pane appears. If characterization is successful, click Close.

4. If the characterization is unsuccessful, ensure the system is properly primed, and then attempt to characterize the needle and loop volumes again. If still unsuccessful, see Troubleshooting in the nanoACQUITY Online Help.

Replacing the puncture needleReplace the puncture needle if it becomes damaged.

Required materials

• nanoACQUITY UPLC puncture needle (see Spare Parts)

• Needle-nose pliers

• TORX driver, T6

• Clean sheet of paper

Warning: For protection against injury, do not remove the protective cover from the puncture needle until the replacement is complete. Touching or pressing the end of the needle can cause a puncture wound.


To replace the puncture needle:


2. Select Maintain > Replace > Needle. A message tells you to remove the right-hand sample plate from the sample manager compartment.


4. Pull out the right-hand tray, and remove the sample plate, if one is loaded.

5. Use a small, thin coin to turn the screw on the bottom of the tray 1/4 turn counterclockwise, releasing the tray.

6. Remove the well plate tray from the sample manager compartment.

7. In the nanoACQUITY UPLC Console, click OK to move the needle to the maintenance position.

Puncture needle:

8. Place a sheet of paper on the floor of the sample manager compartment, beneath the puncture needle. This will catch the needle if it falls during replacement.

9. Loosen the T6 setscrew that secures the puncture needle. The needle should fall out of the housing. If it does not, grasp the needle with the

T6 setscrew

Puncture needle Foot (modified to show puncture needle detail)


needle-nose pliers, and carefully pull downward. If the needle will not come out of the housing, loosen the setscrew until the needle pulls free.

10. Grasp the new puncture needle by its protective plastic cover. Exert a gentle pressure so as not to deform it.

11. Slide the puncture needle over the sample needle and into the housing. Orient the needle so that the longest part of the tip faces the back of the sample manager compartment.

12. Hold the puncture needle in position while you tighten the setscrew with the T6 TORX driver.

13. Carefully remove the protective cover from the puncture needle.

14. Remove the paper from the sample manager compartment.

15. Replace the tray and turn the screw 1/4 turn clockwise to secure the tray.

16. Close the door, and then select Control > Reset SM.

Replacing the sample loopReplace the sample loop when it clogs or when its capacity does not suit your chromatographic needs.

Required materials

• nanoACQUITY UPLC sample loop (see Spare Parts)

• Open-end wrench, 1/4-inch

To replace the sample loop:

1. Slide out the sample manager fluidics tray.

2. Hold the sample loop, and pull the injection valve out toward you.

Caution: To avoid kinks, do not pull on the volume detection device (port 3) or sample needle (port 2) tubing.

3. Use the wrench to remove the fittings on ports 1 and 4 of the injection valve.


Sample loop fittings on the injection valve:

4. Remove the sample loop and its fittings.

5. Carefully remove the replacement sample loop and fittings from the packaging.

Sample loop (assembled):

6. Remove the O-rings from the sample loop.

7. Slide a two-piece ferrule and compression screw onto one end of the sample loop, and then seat the end into injection valve port 1.

Port 1 fitting

Port 4 fitting

Sample loop

Inlet from pump

Outlet to column

To Ports 1 and 4 on injection valve


Caution:

• To prevent dead volume in the system, ensure that the ends of the loop are fully inserted into the ports in the injection valve.

• Use only Waters-approved nanoACQUITY UPLC loops with nanoACQUITY fittings. Each loop should be fitted to its injection valve.

8. Tighten the fitting with the wrench until snug.

9. Remove the fitting and inspect the ferrule to ensure it hasn’t moved.

Ferrule installation:

10. Reinstall the fitting in injection valve port 1.

Tip: To prevent bandspreading, hold the tube while you tighten the fitting to keep it snug.

11. Repeat steps 7 through 10 for the other end of the sample loop at injection valve port 4.

12. Push the injection valve back to the closed position.

Caution: To avoid tubing damage, do not push on the tubing when sliding the injection valve into the closed position.

13. Slide the fluidics tray into the closed position.

Caution: Be careful not to crush the volume detection device line.

14. Ensure the sample manager compartment door is closed.

15. Characterize the new system volumes:

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

b. Click Maintain > Calibrate System Volume to characterize the new system volumes.

c. When finished, click Close.

Correct IncorrectTP02506 TP02507


Replacing the metering syringeTip: Air bubbles in the syringe adversely affect system pressure, baseline, volume, and peak area. Remove air bubbles by gently tapping on the syringe as it is drawing upwards.

To ensure accurate results, use only syringes approved by Waters for use in the nanoACQUITY UPLC system (see Spare Parts).

Replace the metering syringe when either of these conditions arise:

• The syringe plunger tip becomes worn or discolored.

• You want to change to the other syringe size.

Required materials

• Replacement metering syringe (see Spare Parts)

• Degassed, weak wash solvent

To replace the metering syringe:


2. Click Maintain > Replace > Sample syringe. The syringe moves to the down position.

3. Remove the knurled screw that holds the syringe barrel to the syringe mounting bracket.

Warning: To avoid injury, ensure that no injection is in progress or pending before you remove the metering syringe.


Metering syringe assembly components:

4. Unscrew the metering syringe counterclockwise until it separates from the metering syringe valve.

5. Depress the syringe barrel to clear the top mounting bracket, and remove the syringe.

Removing the metering syringe:

6. Carefully remove the replacement metering syringe from the packaging.

Metering syringe valve

Metering syringe

Knurled screw

Metering syringe valve

Mounting bracket

Threaded holder

Syringe plunger


7. Partially fill the new metering syringe (by hand) with weak wash solvent (to help remove air bubbles). Make sure all air bubbles in the syringe are removed.

8. Pull the syringe plunger down so that the plunger end slides over the threaded post on the syringe guide mounting bracket.

9. Screw the new metering syringe partially into the metering syringe valve.

10. Finger-tighten the metering syringe.

11. Install and finger-tighten the knurled screw that holds the metering syringe plunger to the mounting bracket.

12. Run the Prime Metering Syringe Only option until you see no air bubbles in the metering syringe.

Replacing the wash syringesReplace the wash syringes when the syringe plunger’s tip leaks or becomes worn or discolored.

The only wash syringe size available is 2.5 mL. To ensure accurate results, use only syringes that are approved by Waters for use in the nanoACQUITY UPLC system (see Spare Parts).

Required materials

• Replacement wash syringes (see Spare Parts)

• Degassed wash solvents

To replace the wash syringes:

1. Open the sample manager door, and then slide out the sample manager fluidics tray.

Warning: To avoid injury, ensure that no injection is in progress or pending before you remove the metering syringe.


Wash syringe assembly components:

2. Remove the knurled screws that secure the wash syringe plungers to the wash syringe mounting brackets.

3. Unscrew each wash syringe counterclockwise until it separates from the mounting bracket.

4. Depress the syringe barrel to clear the top mounting bracket, and remove the syringe.

Strong wash syringe

Knurledscrews

Weak wash syringe

Mounting bracket

Mounting bracket


Removing a wash syringe:

5. Carefully remove each replacement wash syringe from the packaging.

6. Partially fill each new wash syringe (by hand) with weak or strong wash solvent (to help remove air bubbles).

7. Pull each wash syringe plunger down so that the plunger end slides over the threaded post on the syringe guide mounting bracket.

8. Screw each new wash syringe partially into the mounting bracket. Do not tighten.

9. Finger tighten each wash syringe.

10. Install and finger tighten the knurled screw that holds each wash syringe barrel to the mounting bracket.

11. Slide the fluidics tray closed.

12. Run the “Sample syringe and wash syringes” prime option three times to remove air from the syringe.

Modifying syringe configuration parametersYou can configure the system for a syringe size that is different from the one currently fitted.

Mounting bracket

Wash syringe plunger


To modify syringe configuration parameters:


2. Select Configure > Volumes.

Volume Configuration dialog box:

3. In the Loop Size text box, type the size of the loop that will be used with the syringe.

4. Select the appropriate sample syringe size from the list, and then click OK.

Tip: When you reconfigure the syringe size, you might need to change the syringe draw rate in the separations method.

Replacing the injection valve cartridge

Required materials


• Injection valve cartridge (see Spare Parts)

To replace the injection valve cartridge:

1. Remove the fittings attached to the injection valve.

2. Hold the sample loop, and pull the injection valve out toward you.


injection valve cartridge with fittings:

3. Use the 2-mm hex wrench to remove the screw at the 10 o’clock position on the injection valve cartridge.

4. Remove the injection valve cartridge from the injection valve assembly.

5. Carefully remove the replacement injection valve cartridge from the packaging.

6. Ensure that the groove in the cartridge housing and the groove on the drive clamp are aligned. If they are not, turn the drive clamp until the grooves line up.

Aligning injection valve cartridge grooves:

7. Insert the new injection valve cartridge into the injection valve assembly.

FittingsFittings

Drive clamp

Aligned grooves


Caution: If the injection valve cartridge does not slide fully into the injection valve assembly, contact your Waters service representative.

8. Insert the 2-mm hex screw at the 10 o’clock position on the injection valve cartridge, and use the 2-mm hex wrench to tighten it.

9. Depress the metal clip, and slide the injection valve in.


11. Power-on the sample manager.

12. Prime the syringes.

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

b. Click Control > Prime syringes.

c. Select sample syringe and wash syringes.

d. Ensure the Number of Cycles field is set to 1.

e. Click OK. The sample manager status displays “Priming.” When priming is complete, the status returns to Idle.

f. Click Close.

13. Characterize the needle and loop volumes.

a. Click Maintain > Characterize > Needle and loop volumes.

b. Click Start. The test results appear in the Loop and Needle areas of the screen.

c. Click Close.

Replacing the fuses

The sample manager requires two 10-A fuses.

Warning: To avoid electrical shock, power-off and unplug the sample manager before examining fuses. For continued protection against fire, replace fuses only with those of the same type and rating.


Suspect an open or defective fuse when any of these conditions apply:

• The sample manager or heating and trapping module fails to power-on.

• The sample manager or heating and trapping module status LEDs are unlit.

• The fan does not operate.


Requirement: Replace both fuses, even when only one is open or defective.

1. Power-off the sample manager.

2. Disconnect the power cord from the power entry module.

3. Use a flat-blade screwdriver to open the fuse holder door, which is located above the power entry module on the rear panel.

Removing the fuseholders:

4. With minimum pressure, pull on each spring-loaded fuse holder and remove it.

5. Remove and discard the fuses.

6. Insert the new fuses into the holders and the holders into the power entry module.


8. Power-on the sample manager.

Warning: For continued protection against fire, replace fuses with the appropriate type and rating.

Fuse holders

Fuse holder door

Fuse

Power entry module


Cleaning the module’s exteriorUse a soft cloth, dampened with water, to clean the outside of the sample manager.

Caution: Do not use solvent to clean the module’s exterior.

Maintaining the heating and trapping module

The heating and trapping module, which contains the analytical column, is powered by the sample manager. If the module loses power, the fuse in the sample manager might need replacing (see page 6-47).

Replacing the trap valve cartridge

Required materials


• Trap valve cartridge (see Spare Parts)

To replace the trap valve cartridge:


2. Remove the fittings attached to the trap valve cartridge.

Heating and trapping module trap valve cartridge with fittings:

Tip: This figure depicts the layout for single-pump trapping. Your layout might be different if you are running a different application.

Port 1From nano tee

Hex screw in10 o’clock position

Port 6To waste


3. Use the 2-mm hex wrench to remove the hex screw at the 10 o’clock position on the trap valve cartridge.

4. Remove the trap valve cartridge from the trap valve assembly.

5. Carefully remove the replacement trap valve cartridge from the packaging.

6. Ensure that the groove in the cartridge housing and the groove on the drive clamp are aligned. If they are not, turn the drive clamp until the grooves align.

Aligning trap valve cartridge grooves:

7. Insert the new trap valve cartridge into the trap valve cartridge chamber.

Caution: The trap valve cartridge must slide fully into the trap valve assembly. If it does not, contact your Waters service representative.

8. Insert the 2-mm hex screw at the 10 o’clock position on the trap valve cartridge, using the 2-mm hex wrench to tighten it.


Cleaning the module’s exteriorUse a soft cloth, dampened with water, to clean the outside of the heating and trapping module.

Caution: Do not use solvent to clean the exterior.

If residue from solvent leaks accumulates in the column tray, remove the column, and wipe the tray clean with a soft cloth.

Drive clamp

Aligned grooves


Maintaining the TUV detector

Tip: If you open the door, close it firmly before resuming normal operation.

The detector requires minimal routine maintenance. To achieve optimal performance, follow these recommendations:

• Filter and degas solvents to prolong column life, reduce pressure fluctuations, and decrease baseline noise.

• To conserve lamp life, extinguish the lamp while leaving the detector running but idle. Note, however, that you should do so only when you plan to leave the lamp unlit for more than 4 hours.

• Invoke the lamp optimization software routine at least once a week by powering-off the detector, waiting 10 seconds, and then powering-on the detector.

• If you use buffered mobile phase, flush it from the detector before powering-off to prevent

– plugging of the solvent lines and flow cell

– damage to the module components

– microbial growth

Maintaining the flow cellFlush the flow cell when it becomes contaminated with the residues of previous runs and after each detector shutdown. A dirty flow cell can cause baseline noise, decreased sample energy levels, calibration failure, and other problems. Always flush and purge the flow cell as your initial attempt to correct these problems. If the problems persist, reverse flush the flow cell. If reverse flushing also fails, replace the flow cell.

Precautions

Observe these precautions when handling, removing, or replacing a flow cell:

• To prevent contamination, use powder-free nitrile gloves.

• Take care to avoid scratching the flow cell.

Warning: To prevent injury, do not remove the detector’s top cover. No user-serviceable parts are inside.


Caution: To avoid damaging the flow cell, handle it with care. Do not disassemble the flow cell. To avoid damaging your column, remove it before you flush the system.

Required materials

• Wrench, suitable for removing and replacing the column

• Stainless steel union and tubing

• A solvent that is miscible in both the mobile phase and water


• HPLC-quality water

• HPLC-quality methanol

• Separate container for acid waste

Flushing the flow cellCaution: Do not connect any tubing or device that can create backpressure exceeding the flow cell’s maximum rating of 6900 kPa (69 bar, 1000 psi).

To flush the flow cell:


2. Disconnect the column inlet and flow cell inlet tubing.

Tip: For more efficient flushing, replace the column with tubing.

3. Connect the column inlet tubing to the flow cell inlet.

4. If another module is downstream of the flow cell outlet, break the connection at the other module, and route the outlet tubing to waste while flushing.

5. Flush the system with 100% HPLC-quality water for 10 minutes at 2 µL/min.

6. Flush the system with a solution of 90:10 methanol/water for 10 minutes at 2 µL/min.

7. Pump 100% methanol through the flow cell to clean it internally.

8. Reattach the column.


9. If the mobile phase is not miscible in water, use an intermediary solvent.

10. Resume pumping of the mobile phase.

Reverse flushing the flow cellIf directly flushing the flow cell does not improve flow cell performance, reverse flush it.

To reverse flush the flow cell:

1. Reverse the inlet and outlet tubing connections to the flow cell.

2. Flush the flow cell for approximately 15 minutes. Decreasing system pressure indicates the flow cell is clean.

3. If the flow cell remains dirty or blocked, remove and replace it.

See also: Contacting Waters technical service.

Replacing the flow cell

To replace the flow cell:

1. Power-off the detector.

2. Open the door by gently pulling its right edge toward you.


TUV detector flow cell:

3. Disconnect the detector’s inlet and outlet tubing from the main column connection.

4. Remove the flow cell:

• Using a 1/4-inch, flat-blade screwdriver, loosen the three captive screws on the flow cell assembly’s front plate.

• Grasp the handle and gently pull the assembly toward you.

5. Unpack and inspect the new flow cell. If you are replacing the flow cell with a different type, you must change the flow cell inlet tubing (see “Plumbing the mass spectrometer and optional TUV detector” on page 2-21).

Caution: To avoid damaging the capillary tubing, touch the bare tubing as little as possible. During normal handling, the shield protects the capillary tubing.

Flow cell assembly

Outlet tubing

Inlet tubing

Captive screw

Captive screw

Captive screw

Handle


Top view of the flow cell assembly:

6. Insert the new flow cell assembly into the detector, and then tighten the captive screws.

7. Connect the inlet tubing to the main column connection and flow cell inlet, and connect the outlet tubing to the flow cell outlet.

8. Close the door, purge the cell, and then power-on the detector.

Caution: To ensure the detector is properly aligned and calibrated, the flow cell must be filled with solvent before you power-on the detector. An empty flow cell will cause a calibration error.

Replacing the lampChange the lamp when it repeatedly fails to ignite or when the detector fails to calibrate.

Requirement: Record the new lamp’s serial number in the nanoACQUITY UPLC Console. Otherwise, the date of the previous lamp installation remains in the detector’s memory, voiding the new lamp’s warranty.

Waters warrants 2000 hours of lamp life, or one year of operation, whichever comes first.

Warning: To prevent burn injuries, allow the lamp to cool for 30 minutes before removing it. The lamp housing gets extremely hot during operation.

Shield

Capillary tubing

Flow cell body

Front of flow cell assembly


To remove the lamp:

1. Power-off the lamp:

• To power-off the lamp manually, click TUV Detector in the left pane of the nanoACQUITY UPLC Console, and then click . The green Lamp LED on the nanoACQUITY UPLC Console darkens, as does the Lamp LED on the door.

• To power-off the lamp using a timed event, see the instructions in the MassLynx online Help.

2. Power-off the detector.

3. Allow the lamp to cool for 30 minutes, and then open the door.

4. Detach the lamp power connector from the detector.

Removing the lamp:

5. Loosen the two captive screws in the lamp base.

Warning: To avoid eye injury from ultraviolet radiation exposure• power-off the detector before changing the lamp.• wear eye protection that filters ultraviolet light.• keep the lamp in the housing during operation.

Lamp power connector

Lamp base

Alignment pinCaptive screw

Captive screw


6. Pull the lamp assembly out of the lamp housing, and then gently withdraw the lamp.

Caution: Do not touch the glass bulb of the new lamp. Dirt or fingerprints adversely affect detector operation. If the bulb needs cleaning, gently rub it with ethanol and lens tissue. Do not use abrasive tissue. Do not apply excessive pressure.

To install the lamp:

1. Unpack the new lamp from its packing material without touching the bulb.

2. Inspect the new lamp and lamp housing.

3. Position the lamp so that the cut-out on the lamp base plate is at the 1 o’clock position, in line with the alignment pin on the lamp housing. Then gently push the lamp forward until it bottoms into position. Ensure the lamp is flush against the optics bench.

4. Tighten the two captive screws, and then reconnect the lamp power connector.

5. Close the door, power-on the detector, and then wait about 30 minutes for the lamp to warm and the baseline to stabilize before resuming operations.

Tip: Cycling power to the detector (that is, powering-off and then powering-on the module) initiates the verification procedures.

6. In the nanoACQUITY UPLC Console, select Maintain > Change Lamp.

Warning: Lamp gas is under slight negative pressure. To prevent shattering the glass, use care when disposing of the lamp.


Change Lamp dialog box:

7. Click New Lamp.

8. Type the serial number for the new lamp, and then click OK.

9. Click Close.

Replacing the fuses

The TUV detector requires two 100 to 240 VAC, 50 to 60 Hz, F 3.15-A, 250-V FAST BLO, 5 × 20 mm (IEC) fuses.

Suspect a fuse is open or defective when

• the detector fails to power-on.

• the fan does not operate.

Warning: To avoid electrical shock, power-off and unplug the TUV detector before examining the fuses. For continued protection against fire, replace fuses only with those of the same type and rating.



Requirement: Replace both fuses, even when only one is open or defective.

1. Power-off the detector and disconnect the power cord from the power entry module.

2. Pinch the sides of the spring-loaded fuse holder, which is above the power entry module on the rear panel of the detector. With minimum pressure, withdraw the spring-loaded fuse holder.

Removing the fuseholder:

3. Remove and discard the fuses.

4. Make sure that the new fuses are properly rated for your requirements, and then insert the new fuses into the holder and the holder into the power entry module, gently pushing until it locks into position.


Cleaning the detector’s exteriorUse a soft cloth, dampened with water, to clean the outside of the TUV detector.

Caution: Do not use solvent to clean the exterior.

Fuses

Fuse holder

Power entry module


Testing the system’s fluidic integrity

These procedures can help you examine the solvent flow path through the modules of the nanoACQUITY UPLC System.

To locate leaks in the binary or auxiliary solvent manager:

• Performing the dynamic leak test

• Performing the static decay test (optional)

To test the flow through the solvent path:

• Auto zeroing the flow control module (for both solvent managers)

• Performing the set pressure diagnostic (for binary solvent manager only)

To locate leaks in the sample manager:

• Performing the sample syringe leak test

• Performing the wash syringe leak test

• Performing the needle seal leak test

• Repairing leaks in the sample manager

Tip: You can also perform the set pressure diagnostic with the fluidic path fully plumbed, making sure the injection valve is blocked (see page 6-66).

See also: nanoACQUITY UPLC Online Help.

Performing the dynamic leak testUse the dynamic leak test to assess the flow path through the binary solvent manager or auxiliary solvent manager.

The dynamic leak test performs a pressure ramp-up, and then employs feedback control to maintain constant pressure within the solvent manager. By monitoring plunger travel during the constant pressure phase, it calculates the leak rates of the primary and accumulator actuators. These leak rates can indicate whether the check valves, tubes, fittings, or plunger seals are faulty.

Perform the dynamic leak test whenever you

• suspect leakage in the check valves or plunger seals.

• perform maintenance on the binary or auxiliary solvent manager.

• replace seals or fittings in the fluid path.


Requirement: If you replaced the seals, run the solvent manager at 69,000 kPa (690 bar or 10,000 psi) for 30 minutes before performing the dynamic leak test. Neglecting to properly break-in the seals can cause testing failure.

To perform the dynamic leak test:

1. Make sure that all fittings on the solvent manager are tight.

2. In the nanoACQUITY UPLC Console, select Binary or Auxiliary Solvent Manager from the system tree.

3. Click Maintain > Dynamic leak test.

4. In the Test Parameters area, select a module (pump) to test.

5. In the Accumulator text box, specify your typical operating pressure.

6. Click Start. The test time appears in the Run Time bar graph.

7. When the test ends, the Results pane appears.

The main test criterion is the leak rate. A rate below 0.150 µL/min for each actuator indicates a satisfactory test result—a “passed” test. Other aspects of the test that determine pass/fail status include percent of stroke needed to attain pressure and plunger travel limits.

Example: If the primary actuator must intake solvent to maintain constant pressure while the accumulator is under test, an accumulator failure is triggered, the result of excessive back-leakage through the outlet check valve.

To repair leaks:

1. Inspect all fittings.

2. Inspect the check valves and pump seals.

3. Review the messages in the Results pane of the test. These messages might help identify the source of the problem.

4. Failure of the accumulator test suggests the following elements should be examined:

• Outlet check valve

• Accumulator plunger and high pressure pump seal

• Tubing between the accumulator and accumulator pressure transducer


• Tubing between the accumulator pressure transducer and vent valve

• Vent valve rotor

5. Failure of the primary test suggests the following elements should be examined:

• Inlet check valve

• Primary plunger and high pressure pump seal

• Tubing between the primary and primary pressure transducer

• Tubing between the primary pressure transducer and outlet check valve

6. If both the left (primary) and right (accumulator) heads of pump A or B fail the test with a similar decay rate, the leak is likely common to both solvent manager heads. Therefore, examine these elements:

• Tubing between the outlet check valve and vent valve

• Fittings between the outlet check valve and vent valve


7. If you are unable to correct for a dynamic leak test failure, contact Waters Technical Service.

Performing the static decay testRequirement: If you replaced the seals, run the solvent manager for 30 minutes before performing the static decay test. Failure to properly break-in the seals before performing the static decay test can cause the test to fail. Set a flow rate that causes a pressure reading of at least 20,700 kPa (207 bar, 3000 psi), preferably at your typical operating pressure (up to 10,000 psi).

The static decay test performs a pressure ramp-up, and then monitors the pressure decay in the solvent manager to determine whether the check valves, tubes, fittings, or plunger seals are faulty.

Perform the static decay test whenever you

• suspect leakage in the check valves or plunger seals.

• perform maintenance on the binary or auxiliary solvent manager.

• replace fittings in the fluid path.


To perform the static decay test:

1. Make sure that all the fittings on the solvent manager are tight.

2. Prime the A and B solvents for at least 3 minutes.

See also: Solvent Considerations.


4. Click Maintain > Static decay test.

Static Decay Test dialog box:

5. In the Test Parameters area, select a module (pump A or B) to test.

6. In the Accumulator text box, specify a pressure between 20,700 and 69,000 kPa (between 207 and 690 bar, or 3000 and 10,000 psi).


8. When the test ends, the Results pane appears.

The main test criterion is the decay rate. A rate of 500 psi/min for each actuator indicates a satisfactory test result—a “passed” test. Other aspects of the static decay test that determine pass/fail status include leak rate, percent of stroke, and final pressure.

Example: Excessive primary pressure while the accumulator is compressed can trigger an accumulator failure, the result of excessive valve leakage.

9. Click Close.


To repair leaks:

1. Inspect all fittings.

2. Inspect the check valves and pump seals.

3. If the Accumulator test fails, examine the elements from the outlet check valve to the vent valve (including the rotor).

4. If the Primary test fails, examine the elements from the inlet check valve to the outlet check valve.

5. If both the left (primary) and right (accumulator) heads of pump A or B fail the decay test with a similar decay rate, the leak is likely common to both solvent manager heads. Therefore, examine these elements:

• Tubing between the outlet check valve and vent valve

• Fittings between the outlet check valve and vent valve


6. If you are unable to correct for a static decay test failure, perform the dynamic leak test on page 6-60.

Auto zeroing the flow control module

Use the auto zero procedure to set the flow transducer's internal calibration at zero flow. You can auto zero the flow control module under these conditions:

• While the entire fluidic path from binary or auxiliary solvent manager, sample manager, current column configuration, to mass spectrometer or detector, is connected.

• While the flow control module is in isolation. This requires a pin plug at the outlet mixing tee.

Requirements:

• If you just restarted the solvent manager, allow 60 minutes for it to warm up. The system pressure plot stabilizes at approximately 0 psi, and the BSM Measured Flow Rates A and B plots reach an average steady-state value.

• Prime both flow controllers with the appropriate solvents.


Purging the flow control module

To purge the flow control module:

1. In the nanoBSM page, select the current flow rate. The Set Flow dialog box opens.

2. Enter your typical operating analytical flow rate, change the composition to 50% A and 50% B, and then click OK.

3. Make sure the System Pressure, Measured Flow Rate - A, and Measured Flow Rate - B plots appear, and then, in the Plot Properties dialog box, change the plot time to 1 minute.

4. Purge for 5 to 10 minutes.

5. Select Stop Flow.

• If the flow control outlet is already plumbed through the sample manager, do not disconnect any fittings.

• However, if the flow control outlet is not connected to the system, place a pin plug in the flow control outlet port of the solvent manager, and secure the plug tightly.

Auto zeroing the flow control module

To auto zero the flow control module:

1. In the nanoACQUITY UPLC Console, select Binary Solvent Manager > Troubleshoot > Auto zero flow transducers.

2. Select Pump A and Pump B for test parameters, and then click Start. The Run Time appears on the bar. The test requires 7 to 15 minutes.

3. When the Results and the “test is complete” messages appear, click Close.

4. If the flow control module has a pin plug, remove it.

5. Enter your typical operating analytical flow rate, change the composition to 50% A and 50% B, and then click OK.

6. Purge for 5.0 minutes.


Performing the set pressure diagnosticThe set pressure diagnostic is a tool that allows you to measure the effective leak rate, under constant pressure, of the system from the binary solvent manager’s flow control module to the trap valve and nano tee. It can determine the integrity of the flow path or of individual components.

To perform this test, you should have a good understanding of the nanoACQUITY plumbing scheme (tubing and valve positions) and how to isolate the flow path under test conditions.

See also: nanoACQUITY UPLC Online Help (“Selecting an injection technique” and “Selecting a sample trapping technique”) for diagrams and details.

With this diagnostic you specify a constant pressure, and the binary solvent manager adjusts the flow delivery to maintain the pressure as the flow transducers measure the effective leak rate.

Restriction: The set pressure diagnostic is not available for the auxiliary solvent manager.

Requirements:

• Ensure that you thoroughly purged the flow control module (see page 6-65).

• Verify that the plots of the BSM Measured Flow Rates A and B read 0 µL/min (within ± 0.007 µL/min). If not, perform the auto zero procedure on page 6-64.

• The recommended procedure might require you to insert a pin plug into a port. See Table titled “Test summary table:” on page 6-67 for locations of components that require pin plugs during the test.

Possible test sequences

The recommended diagnostic test procedure takes this systematic, iterative approach:

• If the system is operational (unbroken flow path), you can start at test B in the Test summary table.

• If the system is starting up for the first time, perform all tests, starting with test A.

After performing a test (A, B, C1, C2, or D), go to Diagnosis on page 6-67 to determine the next step.


Diagnosis

• Test A - If results are significantly above the leak rate criteria, call Waters Technical Service.

• Test B - If results are significantly above the leak rate criteria, perform test A. If flow control module is acceptable, tighten or replace the capillary tubing from the flow control module to the injection valve.

• Test C1 - If results are significantly above the leak rate criteria, perform test B. If test B passes, the trap column or the nano tee might need replacement. If test B fails, perform test A.

Test summary table:

TestComponents under test

Leak rate criteria at 10,000 psi (Measured)

Location to plug

Position of injection valve

Position of trapping valve

A Flow control module

0.015 µL/min Outlet mixing tee

N/A N/A

B Flow control module, injection valve

0.015 µL/min N/A Blocked N/A

C1 Flow control module, injection valve, sample loop off-line, trap column with tee

0.018 µL/min Nano tee (two PEEK plugs)

Load N/A

C2 Flow control module, injection valve, sample loop inline, and trap column with tee

0.018 µL/min Nano tee (two PEEK plugs)

Inject N/A

D Flow control module, injection valve, trap column with tee, and trap (HTM) valve

0.025 µL/min Nano tee (one PEEK plug; vent line installed)

Inject Analyt-ical


• Test C2 - If C1 passes and C2 fails, check the sample loop.

• Test D - If results are significantly above the leak rate criteria, perform test B. If test B passes, perform test C1. If test C1 passes, the capillary tubing from the nano tee to the trap valve might need fittings tightened or replaced, or the trap valve might need replacement.

To perform the set pressure diagnostic:

1. In the nanoACQUITY UPLC Console, select Binary Solvent Manager from the system tree and then select Troubleshoot > Set pressure diagnostic.

Set Pressure Diagnostic dialog box:


Tip: If the valve settings are unavailable, make sure the sample manager is powered-on, and then close the Set Pressure Diagnostic dialog box. In the MassLynx inlet method, right-click in the ACQUITY Sample Manager control panel, select Reset Communications, and then repeat step 1.

2. Select Start Flow. This interrupts whatever flow the binary solvent manager is generating; pump A drives the pressure in the system down to 0 psi, while pump B holds zero flow (default settings).

Tip: The pump set to operate in “zero-flow” mode acts as a virtual pin plug at the outlet mixing tee, while the pump set to operate in “const pressure” mode acts as a constant pressure source. We recommend operating with only one pump in constant-pressure mode, typically the aqueous solvent pump, pump A.

3. Wait for the pressure to decrease until it is close to 0 psi by monitoring the BSM system pressure measured value and the plot, and then select Stop Flow. All flow stops.

4. Monitor the A and B Measured flow readings. If peak-to-peak readings exceed ± 0.007 µL/min, auto zero the flow control module (see page 6-64).

5. In the Pressure Control area, enter the pressure you wish to maintain in Set. The allowed range is 0 to 10,000 psi (690 bar, 69,000 kPa). The recommended value is 10,000 psi. However, if this is the initial test, first enter a smaller value such as 1,000 psi and allow it to stabilize before entering a higher value such as 10,000 psi.

Tip: You can raise or lower the pressure setting at any time.

6. In the Flow area, set A1 to Constant pressure, set B1 to Zero Flow, and then click Start Flow. Allow a few minutes for the pressure and flows to stabilize.

Caution: If the entire system is plumbed with columns, start with test B (see Table titled “Test summary table:” on page 6-67).

7. Set the plot time to 1 minute. If needed, change the Plot Properties (Plot tab) to display the System Pressure, Measured Flow Rate - A, and Measured Flow Rate - B plots (see Figure titled “Set Pressure Diagnostic dialog box:” on page 6-68).

8. When the system reaches the set pressure, monitor the measured flow of pump A (i.e., the pump in Const pressure mode). The instantaneous flow


readings on pumps A and B follow a cyclical trend. The typical value is the average between the minimum and maximum cycle peaks.

• The Commanded flow is the net flow needed to maintain constant pressure (for the pump set in constant pressure mode).

• The Measured flow is the effective leak rate for the pump operating under constant pressure. For example, when pump A operates under constant pressure mode during the test, this value indicates pump A’s effective flow after the flow sensor. When pump B operates under zero flow, this value should be close to zero.

• The difference between the Commanded flow and the Measured flow represents the pump’s internal leakage, upstream of the flow sensor. If this difference is greater than 0.150 µL/min, then perform a dynamic leak test (see page 6-60) to test the pump’s internal leakage, which might indicate a problem with the check valves and high-pressure plunger seal.

9. When testing is finished, click Stop Flow > Close.

Tips:

• You can press Close without first pressing Stop Flow.

• The Stop Flow button stops all pump flows, but the binary solvent manager does not actively depressurize the system pressure. This button is useful, for example, when you want to temporarily interrupt the flow while setting a pin plug.

• The Close button exits the set pressure diagnostic. When you select Close, the binary solvent manager actively depressurizes the system pressure to 0 psi, and then stops all flows.

Performing the sample syringe leak testThe sample syringe leak test checks for leaks in the path between the sample syringe and the injection valve.

Recommendation: Prime the sample syringe three times before starting this test.

To perform the sample syringe leak test:



2. Click Maintain > Leak Test > Sample syringe (static).


4. When the test ends, the Results pane appears. Click Close.

Performing the wash syringe leak testThe wash syringe leak test checks for leaks in the path between the weak and strong wash syringes and the injection valve.

Recommendation: Prime the wash syringe before starting this test.

To perform the wash syringe leak test:


2. Click Maintain > Leak Test > Wash syringe (static).



Performing the needle seal leak testThe needle seal leak test checks for leaks in the path that includes the weak wash syringe, the wash block, the injection valve, and the sample syringe.

Recommendation: Prime before starting the needle seal leak test.

To perform the needle seal leak test:


2. Click Maintain > Leak Test > Needle seal (static).




Repairing leaks in the sample manager

To repair leaks:

1. When a leak test fails in the sample manager, inspect the sample manager for leaks, and tighten fittings or syringes where leaks are seen.

2. If tightening does not fix the leak, replace any failed or leaky fittings, tubes, or syringes.

3. If the leak test still fails, contact Waters Technical Service.


A Specifications

Auxiliary solvent manager specifications

Contents:

Topic Page

Auxiliary solvent manager specifications A-1

Binary solvent manager specifications A-5

Sample manager and heating and trapping module specifications A-10

Optional TUV detector specifications A-13

Auxiliary solvent manager physical specifications

Attribute Specification

Height 22.86 cm (9 inches)

Depth (without cables connected at rear; includes heads and tubes at front)

66.04 cm (26 inches)

Width 41.91 cm (16.5 inches)

Weight 26.31 kg (58 pounds)

Auxiliary solvent manager environmental specifications


Operating temperature 15 to 28 °C (59 to 82 °F)

Operating humidity 20 to 80%, noncondensing

Shipping and storage temperature −40 to 70 °C (−40 to 158 °F)

Shipping and storage humidity 10 to 90%, noncondensing

A-1

Acoustic noise (instrument generated) <65 dBA

Internal cooling DC-powered fans provide internal cooling by pulling air through the unit and exhausting it from the rear of the unit.

Airflow requirements Air enters the front of the system allowing side-by-side placement of systems.

Auxiliary solvent manager electrical specifications


Protection classa Class I

Overvoltage categoryb II

Pollution degreec 2

Moisture protectiond Normal (IPXO)

Line voltages, nominal Grounded AC

Line voltage 100 to 240 VAC

Frequency 50 to 60 Hz

Fuse 5 A, 250 V, 5 × 20 mm, slow-blow, IEC type

Power consumption 360 VA

Contact closure outputs (SW1 to SW3)

Maximum voltage: 30 VDCMaximum current: 0.5 AMaximum VA rating: 10 WContact resistance (nominal): 0.2 ohmsScrew terminal connector

Auxiliary solvent manager environmental specifications (Continued)


A-2

Run stopped output Maximum voltage: 30 VDCMaximum current: 0.5 AMaximum VA rating: 10 WContact resistance (nominal): 0.2 ohmsScrew terminal connectorBehavior: If an error message exists, switch is closed then opened when error is cleared

Stop flow input TTL signal or contact closure:Input voltage range: ±30 VDCLogic High: ≥3.0 VDCLogic Low: ≤1.9 VDCMinimum pulse width: 100 msecScrew terminal connector

Start gradient input Same as stop flow input

Auxiliary input 1 Same as stop flow input


Chart outputs (1 and 2) 0 to 2 volts full scale, screw terminal (A/D range is actually -0.1 to 2.1 to allow for offsets)

RS232 (development and manufacturing use)

9-pin subminiature D connector (socket type)

Ethernet RJ45 connector

a. Protection Class I – The insulating scheme used in the instrument to protect from electrical shock. Class I identifies a single level of insulation between live parts (wires) and exposed conductive parts (metal panels), in which the exposed conductive parts are connected to a grounding system. In turn, this grounding system is connected to the third pin (ground pin) on the electrical power cord plug.

b. Overvoltage Category II – Pertains to instruments that receive their electrical power from a local level such as an electrical wall outlet.

c. Pollution Degree 2 – A measure of pollution on electrical circuits, which can produce a reduction of dielectric strength or surface resistivity. Degree 2 refers only to normally nonconductive pollution. Occasionally, however, expect a temporary conductivity caused by condensation.

Auxiliary solvent manager electrical specifications (Continued)


A-3

d. Moisture Protection – Normal (IPXO) – IPXO means that no Ingress Protection against any type of dripping or sprayed water exists. The X is a placeholder that identifies protection against dust, if applicable.

Auxiliary solvent manager performance specifications

Item Specification

Priming Able to pump and wet prime bottles at the level of the pump or higher. Syringe (startup kit) is required for dry priming.

Vent valve Automated vent valve for priming pump and automated leak test.

Number of solvents Two (A1, B1)

Solvent and seal wash containers Customer selectable, located in separate bottle tray.

Most common solvents and modifiers

Reverse phase: water, methanol, acetonitrilepH Range: 1 to 12Modifiers: TFA, phosphate, formic acid, ammonium acetate, acedic, bicarbonate, ammonium hydroxide, acetic acid

Pump seal wash (1 pump) Pump and seals required to wash the rear of the high pressure seal (pulse type flow, about 20 µL per pulse)

Flow rate, pressure operating envelope

See Figure titled “Pressure flow envelope for B side pump” on page A-5.

Flow rate increment Settable to 0.001 µL (1 nL)/minute

Leak management Drip trays manage all leaks from the pump into a drain that is directed to the front or rear of the module.“Pass-through” drain system collects leakage and waste from the sample manager.

Flow ramping Not required

A-4

Pressure flow envelope for B side pump

Binary solvent manager specifications

Binary solvent manager physical specifications


Height 22.86 cm (9 inches)

Depth (without cables connected at rear; includes heads and tubes at front)

66.04 cm (26 inches)



Binary solvent manager environmental specifications





Pre

ssur

e (p

si)

Flow Rate (µL/minute)

= Open Loop Control= Closed Loop Control

0

2000

10,000

8000

6000

4000

5 100

A-5


Acoustic noise (instrument generated)

<65 dBA

Internal cooling DC-powered fans provide internal cooling by pulling air through the unit and exhausting it from the rear of the unit.

Airflow requirements Air enters the front of the system allowing side-by-side placement of systems.

Binary solvent manager electrical specifications




Pollution degreec 2



Line voltage 100 to 240 VAC


Fuse 5 A, 250 V, 5 × 20 mm, slow-blow, IEC type


Contact closure outputs (SW1 to SW3)

Maximum voltage: 30 VDCMaximum current: 0.5 AMaximum VA rating: 10 WContact resistance (nominal): 0.2 ohmsScrew terminal connector

Binary solvent manager environmental specifications (Continued)


A-6

Run stopped output Maximum voltage: 30 VDCMaximum current: 0.5 AMaximum VA rating: 10 WContact resistance (nominal): 0.2 ohmsScrew terminal connectorBehavior: If an error message exists, switch is closed then opened when error is cleared

Stop flow input TTL signal or contact closure:Input voltage range: ±30 VDCLogic High: ≥3.0 VDCLogic Low: ≤1.9 VDCMinimum pulse width: 100 msecScrew terminal connector

Start gradient input Same as stop flow input



Chart outputs (1 and 2) 0 to 2 volts full scale, screw terminal (A/D range is actually -0.1 to 2.1 to allow for offsets)

RS232 (development and manufacturing use)

9-pin subminiature D connector (socket type)

Ethernet RJ45 connector




Binary solvent manager electrical specifications (Continued)


A-7


Binary solvent manager performance specifications

Item Specification

General High pressure mixing, binary gradient

Priming Able to pump and wet prime bottles at the level of the pump or higher. Syringe (startup kit) is required for dry priming.

Vent valve Automated vent valve for priming pump and automated leak test.

Number of solvents Two (A1, B1)

Solvent and seal wash containers Customer selectable, located in separate bottle tray.

Solvent degassing (needle wash solvents only)

Built-in solvent degassing module, 2 channels for sample manager needle wash degassing. Chambers use Teflon AF tubing.

Most common solvents and modifiers

Reverse phase: water, methanol, acetonitrilepH Range: 1 to 12Modifiers: TFA, phosphate, formic acid, ammonium acetate, acedic, bicarbonate, ammonium hydroxide, acetic acid

Pump seal wash (1 pump) Pump and seals required to wash the rear of the high pressure seal (pulse type flow, about 20 µL per pulse)

Flow rate, pressure operating envelope

See Figure titled “Pressure flow envelope for B side pump” on page A-5.

Flow rate increment Settable to 0.001 µL (1 nL)/minute

A-8

Gradient precision Retention time reproducibility <0.25 min standard deviation for retained peptides, based on 6 repeat injections. Test conditions are as follows:Flow rate: 400 nL/minSample: Waters peptide standard mixture (part number 186002337)Injection mode: partial loop in 5-µL loopInjection volume: 1 µLColumn: Waters BEH 1.7 µM, 100 µM I.D. × 100 mmColumn temperature: 35 °CEluent A: water/0.1% formic acidEluent B: acetonitrile/0.1% formic acidGradient: linear from 1-50% B over 30 minutesDetection: UV at 214 nmAmbient temperature variation: ≤2 °C per hour

Gradient composition curves Eleven (11) gradient curves (including linear step [2], concave [4], and convex [4]

Leak management Drip trays manage all leaks from the pump into a drain that is directed to the front or rear of the module.“Pass-through” drain system collects leakage and waste from the sample manager.

Flow ramping Not required

Binary solvent manager performance specifications (Continued)

Item Specification

A-9

Sample manager and heating and trapping module specifications

Sample manager physical specifications


Height 27.31 cm (10.75 inches)

Depth 71.12 cm (28 inches)



Heating and trapping module physical specifications



Depth 71.12 cm (28 inches)



Sample manager environmental specifications






Heating and trapping module environmental specifications





A-10


Sample manager electrical specifications


Protection classa


Class I

Overvoltage categoryb


II

Pollution degreec


2

Moisture protectiond


Normal (IPXO)


Voltage range 100 to 240 VAC


Fuse 10 A


Heating and trapping module electrical specifications




Heating and trapping module environmental specifications (Continued)


A-11

Pollution degreec 2





Maximum VA input 750 VA





Sample manager performance specifications

Item Specification

Injection cycle time (5 µL partial loop with air gaps)

~45 seconds (or less) with a single 200 µL weak wash – standard mode~60 seconds (or less) with default dual wash (100 µL strong and 500 µL weak)

40 seconds (with default dual wash)

Injection volume 0.5 to 50 µL, in 0.1 µL increments

Sample carryover 0.005% (or 1.25 nL) with dual wash and UV detection

Audible noise 65 DB

Heating and trapping module electrical specifications (Continued)


A-12

Optional TUV detector specifications

Residual volume 3 µL with maximum recovery vial and needle (p/n 186000327)

Binary solvent manager degasser Two channels for sample manager needle wash degassing

S/C heater/cooler 4 to 40 °C (39 to 104 °F), ±3 °C (±5.4 °F) in 25 °C (77 °F) ambientProgrammable in 1 °C (1.8 °F) increments

Heating and trapping module performance specifications

Item Specification

Heating range 5 °C above ambient to 65 °C (149 °F)

Temperature accuracy ±1.0 °C (1.8 °F)

Temperature stability ±0.1 °C (0.2 °F)

TUV detector physical specifications



Depth 50.8 cm (20.0 inches)

Width 28.4 cm (11.2 inches);34.3 cm (13.5 inches) with fluid conduit and side filler panel

Weight 9.3 kg (20.5 pounds)

Sample manager performance specifications (Continued)

Item Specification

A-13

TUV detector environmental specifications






TUV detector electrical specifications




Pollution degreec 2




Line frequency 50 to 60 Hz

Fuse Two fuses, 100 to 240 VAC, 50 to 60 HzF 3.15-A, 250-V FAST BLO, 5 × 20 mm (IEC)


Two attenuated analog outputchannels:2 VFS / 0 mVFSTwo event outputs

Four event inputs

Attenuation range: 0.0001 to 4.000 AU2 V output range: −0.1 to +2.1 V10 mV output range: −0.5 to 10.5 mVType: contact closureVoltage: +30VCurrent: 1 AInput voltage: +30 V maximum; 100 ms minimum period



A-14



TUV detector operational specificationsa

Item Specification

Wavelength range 190 to 700 nm

Bandwidth 5 nm

Wavelength accuracy ±1 nm

Wavelength repeatability

±0.25 nm

Baseline noise, single wavelengthb

< ±6.00 × 10-6 AU, shunt in place, 230 nm, 1.0 second filter time constant

Baseline noise, dual wavelengthb

< ±5.0 × 10-5 AU, shunt in place, 230 nm and 280 nm, 2.0 second filter time constant

Linearityb ≤5% at 2.5 AU, propyl paraben, 257 nm

Drift 5.0 × 10-4 AU/hour/°C, shunt in place, at 230 nm (after 1 hour warm-up)

Maximum data rate 80 Hz (points/1.0 second peak)

Sensitivity setting range

0.0001 to 4,0000 AUFS

Filter setting range Single wavelength:• 0.1 to 5.0 seconds, Hamming (default)• 1 to 99 seconds, RCDual wavelength:• 1 to 50 seconds, Hamming (default)• 1 to 99 seconds, RC

Optical Component Specifications

Lamp source Deuterium arc lamp

Flow cell nanoACQUITY light-guiding flow cell

A-15

Path length 25 mm

Cell volume 2.40 µL (2400 nL)

Pressure limit 6900 kPa (69 bar, or 1000 psi)

Materials 316 stainless steel, fused silica, PEEK, Teflon AF2400

a. All specifications derive from instrument performance after a 1-hour warm-up time.b. ASTM Standard E1657-94.

TUV detector optical specifications

Item Specification

Monochromator Fastie Ebert configuration

Grating Plane holographic 1800 grooves/mm

Optical bandwidth 5 nm

Lamp power 30 W

TUV detector operational specificationsa (Continued)

Item Specification

A-16

B Spare Parts

This appendix lists recommended spare parts and options for the nanoACQUITY UPLC system. Unlisted parts are not recommended for customer replacement.

Solvent manager spare parts

The spare parts in the following table can be installed in nanoACQUITY UPLC systems that can operate up to 10,000 psi.

Contents:

Topic Page

Solvent manager spare parts B-1

Sample manager spare parts B-3

TUV detector spare parts B-4

nanoACQUITY columns B-4

Recommended spare parts for the solvent managers (10,000 psi maximum pressure)

Item Part Number

Primary check valve assembly (2) 700002596

Accumulator check valve assembly (2) 700002968

Primary inlet filter kit TBD

Replacement filter pack (2) TBD

Wash seal (2) 700002598

Head seal and seal wash spacer 700002599

Plunger (2) 700002600

Teflon O-ring WAT076152

B-1

In-line filter, 1/2-micron 700002696

In-line filter and housing 289002111

Vent valve cartridge 700002660

Tube, solvent select valve to primary inlet filter 430001470

Primary inlet filter assembly 289002472

Tube, “B” transducer to vent valve P6 430001535

Tube, “A” transducer to vent valve P3 430001534

Tube, vent valve port 5 to filter 430001512

Tube, vent valve port 2 to filter 430001511

Tube, vent valve port 1 to waste 430001209

Tube, vent valve port 4 to waste 430001210

Tube, filter to flow control inlet B 430001569

Tube, filter to flow control inlet A 430001568

Tube, transducer inlet 430001151

Tube, degasser port B2 to solvent select valve B 430001113

Tube, degasser port B1 to solvent select valve B 430001114

Tube, degasser port A1 to solvent select valve A 430001115

Tube, degasser port A2 to solvent select valve A 430001116

Tube, transducer to check valve 430001121

Tube, ASM to MS, 25 µm × 60” 430001572

Tube, solvent inlet system tube set 700002713

Ferrule set, two-piece swagelock 700002635

Compression screws, gold (10) 700002645

Fuse, 5A, 250V, 5mm × 20mm, slo blo 700002604

Pin plug, solid TBD

Recommended spare parts for the solvent managers (10,000 psi maximum pressure) (Continued)

Item Part Number

B-2

Sample manager spare parts

Recommended spare parts for the sample manager (10,000 psi max. pressure)

Item Part Number

Syringe, 2.5 mL 700002569

Syringe, 100 µL, HP 700002570

Needle seal O-ring 700002572

Sample needle, 15 µL 700002708

Sample loop, 2 µL 430001264




Nano tee, M detail 289002576

Injector cartridge 700002907

Trap valve cartridge 700002660

Fitting, one-piece, PEEK, M detail 405005068

Fitting, plug, M detail 410001400

Tube, fused silica, 25 µM, flow sensor to injection valve 430001564

Tube, fused silica, 25 µM × 10”, injection valve to nano tee 430001570

Tube, fused silica, 40 µM × 16”, nano tee to trap valve 430001571

Fuse, 0.25A, 250V SMD 4000A INTERR 242.25 700002576

Fuse, 10A, 5mm × 20mm, slo blo 700002577

Mounting block, trap column 289002802

2D/2-pump trapping kit 205000398

Tube, BSM to trap valve, 2D 430001575

Tube, ASM to injection valve, 2D 430001576

Tube, injection valve to trap valve, two-pump trapping 430001629

Tube, injection valve to ion exchange column, 2D 430001577

B-3

TUV detector spare parts

nanoACQUITY columns

The columns in the following table can be installed in nanoACQUITY UPLC systems that can operate up to 10,000 psi.

Recommended spare parts for the TUV detector

Item Part Number

nanoACQUITY TUV flow cella

a. Intended for TUV models with serial number J05UPT700N and later.

205015013

Flow cell, 250 nLa 205000158

Flow cell, 10 nLb

b. Intended for TUV models with serial number J05UPT699N and earlier.

205000159

Fuse, 3.15A, 250V, 5mm × 20mm, fast-acting (5) WAT055634

nanoACQUITY columns (10,000 psi maximum system pressure)

Part Number

Column style MaterialParticle Size (micron)

I.D. (micron)

Length (cm)

186003491 nanoACQUITY Symmetry C18 3.5 75 10








186003499 nanoACQUITY Atlantis dC18 3 75 10



B-4






186003507 nanoACQUITY SCX 5 180 2.35

186003542 nanoACQUITY BEH C18 1.7 75 10






186003513 nanoACQUITY CUSTOM

186003514 nanoACQUITYTrap column

Symmetry C18 5 180 2.0

nanoACQUITY columns (5,000 psi maximum system pressure)

Part Number


I.D. (micron)

Length (cm)




nanoACQUITY columns (10,000 psi maximum system pressure) (Continued)

Part Number


I.D. (micron)

Length (cm)

B-5

















186002840 nanoACQUITY CUSTOM

186002841 nanoACQUITYTrap column

Symmetry C18 5 180 2

nanoACQUITY columns (5,000 psi maximum system pressure) (Continued)

Part Number


I.D. (micron)

Length (cm)

B-6

C Solvent Considerations

Introduction

Solvents tested with nanoACQUITY UPLC Systems include acetonitrile, water, trifluoroacetic acid, formic acid, and methanol.

Caution: The recommendations below are based upon experiences in Waters’ research and development and applications labs. Where specific brands are mentioned, Waters did not receive any compensation for those recommendations. We offer them as guidance only.

Contents:

Topic Page

Introduction C-1

Preventing contamination of mass spectrometry backgrounds C-2

Operating procedures C-3

Clean solvents C-3

Solvent quality C-3

Solvent preparation C-4

Water C-5

Using buffers C-5

Warning: To avoid chemical hazards, always observe safe laboratory practices when operating your system. To prevent injury, always observe good laboratory practices when you handle solvents, change tubing, or operate the system. Know the physical and chemical properties of the solvents you use. See the Material Safety Data Sheets for the solvents in use.

C-1

Caution: The nanoACQUITY UPLC System should not be run with high pH mobile phases. Alkaline solutions such as ammonium hydroxide (pH 10) can damage the silica capillary tubing, resulting in an elevated chemical background being detected by mass spectrometry.

Requirement: For optimal performance, solvent A must be aqueous (water) and solvent B must be organic (acetonitrile or methanol).

Preventing contamination of mass spectrometry backgrounds

For optimal results in mass spectrometry

You might want to thoroughly clean the solvent bottles or reservoirs by passivation before adding solvents to the containers.

To passivate the bottles:

1. Sonicate the bottles in 20 to 30% nitric acid for approximately 30 minutes.

2. Rinse the bottles thoroughly with Millipore® Milli-Q™ water.

The quality and cleanliness of vials, vial inserts, well plates and solvent bottles are equally important. Rinse with the mobile phase only.

Caution: To avoid contamination, avoid washing nanoACQUITY solvent bottles as follows:

• In a dishwasher

• With other glassware

• Using detergent

These cleaning methods can cause contamination resulting in high mass spectrometry backgrounds.

Requirement: To prevent contamination, always use powder-free nitrile gloves when handling components of the system.


C-2

Operating procedures

Your operating procedures must be consistent with operating in extreme high-sensitivity mode.

When adjusting fittings or replacing a column on the system, wear powder-free nitrile gloves to protect the surfaces from contamination with keratin and associated grooming products. If you have lotion on your hands when you adjust a fitting, the fitting can become contaminated. If your gloves have powder on them, your fittings will as well. Should this type of contamination occur, sonicate the fittings in methanol or a mixture of water:methanol (1:1, v/v) for 30 minutes.

Clean solvents

Clean solvents provide reproducible results and permit you to operate with minimal module maintenance.

A dirty solvent can cause baseline noise and drift and loss of sensitivity in the mass spectrometer. It can also block the solvent reservoir and inlet filters with particulate matter.

Solvent quality

The source of your solvents is extremely important. Use MS-grade solvents, if available, for the best possible results. These solvents are normally pre-filtered. Waters does not recommend further filtering as this can introduce contamination, particularly for LC/MS operations.

Many vendors produce HPLC-grade solvents, specifically acetonitrile and methanol. These solvents are analyzed for UV-absorbing organic contaminants and might not be suitable for high-sensitivity MS or MS/MS use. Checking the baseline noise of the solvent by direct infusion into the MS is a good way to detect impurities in the solvent and avoid contamination of the column, sample, or system. Waters has analyzed several leading manufacturers’ solvents with the Q-Tof mass spectrometer.

As the baseline sensitivity requirements for your MS detector become more critical, you might require targeted impurity levels below ppb that will place a further burden on solvent suppliers to meet your specific application needs. As a good starting point, Waters recommends using solvents that meet or exceed

Operating procedures C-3

the specifications of the Fisher® Optima grade of acids and solvents. We have found that Fisher Optima has reduced levels of non-UV absorbing contaminants such as polyethylene glycol (PEG) and inorganic iron.

Many reversed phase separations use a mobile phase modifier, typically trifluoroacetic acid (TFA), formic acid, and/or acetic acid. These modifiers increase separation efficiency by improving peak shape. The quality of these reagents is important because they can contribute to background issues either directly or upon trace enrichment during column re-equilibration. Waters recommends using formic acid that meets or exceeds the specification of EM Scientific, part number FX0440-11. Waters recommends using TFA in 1 mL ampoules supplied by Pierce, part number 28904.

Solvent preparation

Proper solvent preparation can prevent many pumping problems. The most common problem is bubble formation, which can affect flow rate consistency. Aside from leaky fittings, the problem of bubble formation arises from two sources: solvent outgassing and cavitation.

Optional degassing Waters performs system-level evaluation for peptide separations with the degassers removed from the fluidic pathway (bypassed). Your system is configured with the degassers bypassed.

Recruiting the degassers can change the composition of pre-mixed water: organic eluent mixtures. If you use the degassers, do not use eluents that are mixtures of aqueous and organic solvents. Waters has found that mixtures such as 97% water and 3% acetonitrile (v/v) can provide reduced chromatographic retention time reproducibility over extended periods because the small percentage of organic can be removed in the degassers.

If you wish to use the degassers, Waters can install them in the fluidic pathway. It is also possible that at flow rates less than 1 µL/min the degassers will remove volatile mobile phase modifiers such as formic acid and trifluoroacetic acid. This can affect retention time reproducibility and peak shape.

C-4

Water

In reversed phased gradient separations, sample elution proceeds from high aqueous to high organic conditions. To prepare the column for the next separation, we re-equilibrate it in initial starting conditions, typically 90% water. Organic-based impurities, often expressed as total organic carbon (TOC), in the water can bind to the reversed phase column packing material. This trace enrichment of impurities can dramatically affect your results.

To avoid impurities, use water of the highest purity. We use Millipore® Milli-Q™ water in our labs and routinely change the cartridges. To ensure optimal operation in your lab, Waters recommends that you use water that meets or exceeds Milli-Q water quality.

Using buffers

Caution: Some buffers can be incompatible with mass spectrometers. Consult the documentation shipped with your mass spectrometer for compatible buffers.

When you use aqueous buffers, adjust their pH, filter them to remove insoluble material, and then blend them with organic modifiers, accordingly. After you use a buffer, flush it from the pump by priming with at least five system volumes of HPLC-grade, distilled or deionized, water. For shutdowns lasting more than one day, flush the pump with 20% methanol/water to prevent growth of microorganisms.

Tip: To avoid salt precipitation, nonvolatile buffer concentrations should not exceed 100 mM.

Water C-5

C-6

Index

Aabsorbance values, display of 3-19air bubbles, removing from metering

syringe 6-40air filter, solvent manager 6-11aligning

injector valve cartridge grooves 6-46

trap valve cartridge grooves 6-50vent valve cartridge grooves 6-21

analytical columns 1-9ANSI plates 3-18auto zero flow control module 6-64auxiliary solvent manager

check valve, replacing 6-8flow LED 3-2overview 1-7plumbing 2-17preparing 3-4priming 3-6replacing solvent filter 6-6specifications

electrical A-2environmental A-1performance A-4physical A-1

Bbinary solvent manager

air filters 6-11check valve, replacing 6-8checking for leaks 6-62cleaning

air filters 6-11exterior 6-23

flow LED 3-2fuses, replacing 6-22

head sealsreattaching 6-16removing 6-15

maintaining 6-6overview 1-6plumbing 2-16plunger, replacing 6-18preparing 3-7priming 3-9pumped volume warning 6-4replacing solvent filter 6-6solvent bottle filters, replacing 6-10solvent manager head

reattaching 6-19removing 6-16

solvent manager head, removing 6-13

specificationselectrical A-6environmental A-5performance A-8physical A-5

vent valve cartridge, replacing 6-20bottle filters, replacing 6-10buffers C-5buffers, avoiding damage 3-22

Ccalibrating XYZ mechanism 2-22capillary tubing, connecting 2-15cartridge

injector valve, replacing 6-45trap valve, replacing 6-49vent valve, replacing 6-20

characterizingneedle 3-17needle and loop volumes 6-35

Index-1

needle seal 3-16, 6-33sample loop volume 3-17

check valve, replacing 6-8chemistry technologies 1-3chromatogram, gradient performance

test 5-11cleaning

air filters, solvent manager 6-11sample manager exterior 6-49solvent bottles 2-11, C-2solvent manager exterior 6-23TUV detector exterior 6-59

columncompatibility 1-3conditioning 3-21overview 1-3spare parts B-4, B-5trapping 1-4

columnsanalytical 1-9ion exchange 1-10trap 1-9, 2-18

conditioning column 3-21configuring

events 4-6instruments in MassLynx 4-2maintenance warnings 6-4MassLynx 4-1syringe parameters 6-45

connectionsanalog signal 2-7Ethernet 2-7power 2-11solvent supply 2-12

connector cover, attaching 2-8console

overview 1-11starting from MassLynx 4-5

contacting Waters Technical Service 2-3, 6-2

contamination, avoiding 2-12, C-2creating

instrument method 5-6test methods 5-6

Ddamage, reporting 2-3data system 1-11defrosting sample compartment 6-24degassers, bypassed 3-4detector, TUV

maintaining 6-51overview 1-10preparing 3-19

detector lamp life, maintenance threshold 6-4

detergents 2-12, C-2diagnostic, set pressure 6-66diagnostics, startup 3-19direct injection mode 1-3, 1-4dirty flow cell 6-51drain routing hole 2-6drain tube configuration 2-14drainage, proper 2-13drip management system 2-6drip management system, proper

placement for 2-6dynamic leak test 6-60

Eelectrical specifications

auxiliary solvent manager A-2binary solvent manager A-6heating and trapping module A-11sample manager A-11TUV detector A-14

Index-2

environmental specifications A-10auxiliary solvent manager A-1binary solvent manager A-5heating and trapping module A-10sample manager A-10TUV detector A-14

equipment guidelines vEthernet connections 2-7events, configuring 4-6exhaust hood 2-14

Fferrule installation 6-39filter

air 6-11bottle, replacing 6-10

fittingM-detail 2-15V-detail 2-16

fittingsinstallation recommendations 2-15on binary solvent manager vent

valve 6-21on injection valve 6-38on trap valve 6-49tightening 2-16

flow cell, TUV detectordirty 6-51pictured 2-21, 6-54, 6-55replacing 6-53reverse flushing 6-53

flow control moduleauto zero 6-64purging 6-65

flow control modules 1-6, 1-7flow LED, solvent manager 3-2flow rate

during priming 3-9during shutdown 3-22

I

flushing, TUV detector flow cell 3-20, 6-52

fume hood 2-14fuses, replacing

sample manager 6-48solvent manager 6-22TUV detector 6-59

Ggenerating inject start signal 2-10gradient performance report 5-11gradient performance test

chromatogram 5-11creating test methods 5-6preparing mobile phase for 5-2preparing sample for 5-3

Hhardware, preparing 3-1head seals, solvent manager

reattaching 6-16removing 6-15

heating and trapping module 1-5, 2-18maintaining 6-49overview 1-9specifications


HTM valve 6-49

II/O connector covers 2-8I/O signal connectors

cover, attaching 2-8sample manager 2-8

initialization, system start-up 3-1inject start, generating 2-10

Index-3

injection valve fittings 6-38injections threshold warning 6-4injector valve cartridge

connecting 2-17replacing 6-45

Inlet Configuration window 4-3installing

lamp, TUV detector 6-57needle assembly 6-29system components 2-4

installing the system 2-1Instrument Control Option Pack 4-3instrument method, TUV detector,

channel A 5-9instrument method, creating 5-6instrument parameters

binary solvent manager, general tab 5-6

binary solvent manager, trapping tab 5-7

sample manager, general tab 5-8instruments, cleaning 6-2intended use xviion exchange column 1-10

Llamp LED, TUV detector 3-3lamp, TUV detector

aging 3-20installing 6-57removing 6-56replacing 6-55

LCT Premier 1-10leak test

dynamic 6-60needle seal 6-71sample syringe 6-70static decay 6-62wash syringe 6-71

leaks, repairing 6-61, 6-64, 6-72LED

monitoring 3-2power 3-2

loading sample plates 3-18lock-mass addition 1-7logging in to MassLynx 4-1

Mmaintaining

checking for leaks 6-71solvent manager 6-6system 6-1TUV detector 6-51

maintenancechecking for leaks 6-62, 6-70, 6-71considerations 6-3heating and trapping module 6-49injections threshold warning,

setting 6-4problems, reporting 6-2pumped volume warning, setting

6-4safety considerations 6-3sample manager 6-24schedule 6-1spare parts 6-4warnings, configuring 6-4

mass spectrometerinject start connection 2-10overview 1-10preparing 3-20

MassLynxinlet configuration 4-3logging in 4-1overview 1-11starting 4-1starting console from 4-5

MassPREP peptides mixture 5-3

Index-4

M-detail fitting 2-15metering syringe, replacing 6-40mobile phase for shutdown 3-22mobile phases to avoid 2-11modifying syringe configuration

parameters 6-45monitoring

lamp aging 3-20startup tests 3-2system instrument LEDs 3-2

NnanoACQUITY system

columns B-4, B-5plumbing 2-15

nanoACQUITY UPLC Console 1-11starting 4-5

NanoLockSpray 1-7needle

characterizing 3-17, 6-35guide tube, pictured 6-29installing 6-29puncture, replacing 6-35removing 6-25seal leak test 6-71seal, characterizing 3-16, 6-33volume, characterizing 6-35wash, stopping 3-16washing 3-14

needle seal, characterizing 3-16, 6-33needle seal leak test 6-71needle wash 2-13

Ooff-line 2D-LC trapping 1-3online 2D-LC trapping 1-3operating conditions 1-4operational specifications, TUV

detector A-15

I

optical specifications, TUV detector A-16

optionssample loop sizes B-3spare parts B-1syringe sizes B-3

O-rings 2-12overview

auxiliary solvent manager 1-7binary solvent manager 1-6MassLynx 1-11sample manager 1-7solvent considerations C-1TUV detector 1-10

Ppassivating bottles 2-11, C-2peak retention times 5-11peptides mixture 5-3performance specifications

auxiliary solvent manager A-4binary solvent manager A-8heating and trapping module A-13sample manager A-12

physical specificationsauxiliary solvent manager A-1binary solvent manager A-5heating and trapping module A-10sample manager A-10TUV detector A-13

platesANSI 3-18SBS 3-18

plates, standard 1-7plumbing

auxiliary solvent manager 2-17binary solvent manager 2-16detector 2-20mass spectrometer 2-20

Index-5

sample manager 2-16solvent supply 2-12

plumbing, solvent supply 2-11plunger, solvent manager, replacing

6-18positioning sample plate 3-18power

connections 2-11removing completely 3-23

power LED 3-2powering on 3-1precipitation, preventing 3-4, 3-9, 3-22,

5-2preparing system test mix 5-4pressure flow envelope A-5preventing contamination 2-12priming

auxiliary solvent manager 3-4, 3-6binary solvent manager 3-9sample manager 3-13seal wash 3-5, 3-8

pumped volume maintenance warning 6-4

puncture needlepositioning 2-24replacing 6-35, 6-36

purging flow control module 6-65

QQ-Tof Premier 1-10

Rreattaching

head seals, solvent manager 6-16solvent manager head 6-19

recordingreference energies 3-20sample energies 3-20

reference energies, recording 3-20

regulatory requirements, signal cables 2-7

removingflow cell, TUV detector 6-54head seals, solvent manager 6-15lamp, TUV detector 6-56needle assembly 6-25solvent manager head 6-13, 6-16

repairing leaks 6-61, 6-64, 6-72replacing

air filters, solvent manager 6-11check valve, solvent manager 6-8flow cell, TUV detector 6-53fuses

sample manager 6-48solvent manager 6-22TUV detector 6-59

injector valve cartridge 6-45lamp, TUV detector 6-55metering syringe 6-40plunger, solvent manager 6-18puncture needle 6-35, 6-36sample loop 6-37solvent bottle filters 6-10trap valve cartridge 6-49vent valve cartridge 6-20wash syringes 6-42

reverse flushing, TUV detector flow cell 6-53

routine maintenance schedule 6-1run LED, sample manager 3-3running system test mix 5-10

Ssafety considerations, maintenance 6-3safety precaution symbols xiisafety rules xivsalts, preventing precipitation 3-4, 3-9,

3-22, 5-2

Index-6

samplecompartment, defrosting 6-24loop volume, characterizing 3-17loop, replacing 6-37plates, loading 3-18

sample and reference energies, recording 3-20

sample energies, recording 3-20sample list, peptides test 5-9sample loop

characterizing 6-35installing 6-38replacing 6-37

sample managerchecking for leaks 6-70, 6-71fuses, replacing 6-48generating inject start signal 2-10I/O signal connectors 2-8injections threshold warning 6-4injector valve cartridge 6-45leaks, repairing 6-72loading sample plates 3-18locating leaks 6-60maintaining 6-24metering syringe, replacing 6-40needle assembly, removing 6-25needle assembly, installing 6-29needle seal, characterizing 3-16,

6-33needle, washing 3-15overview 1-7plumbing 2-16preparing 3-11priming 3-13puncture needle, replacing 6-35repairing leaks 6-72run LED 3-3sample compartment, defrosting

6-24

I

sample loop, replacing 6-37signal connectors 2-8spare parts B-3specifications


syringe configuration parameters, modifying 6-45

wash syringes, replacing 6-42washing needle 3-14XYZ mechanism, calibrating 2-22

sample needlecharacterizing 6-35installing 6-29washing 3-14

sample needle assembly 6-27sample plates, loading 3-18sample syringe leak test 6-70SBS plates 3-18seal wash prime, performing 3-5, 3-8seal, characterizing 3-16seals, proper break-in 6-61selecting

system instruments 4-2wash solvents 3-11

service, requesting 6-2set pressure diagnostic 6-66setting maintenance warning

injections threshold 6-4pumped volume 6-4

shutting down the system 3-22signal connections 2-7single-pump trapping 1-3, 1-4solvent

bottle filters, replacing 6-10bottles, cleaning 2-11, C-2reservoirs 2-12

Index-7

supply, connecting 2-12tray 2-12

solvent considerations C-1solvent filter, replacing 6-6solvent manager

headreattaching 6-19removing 6-13, 6-16

locating leaks 6-60replacing fuses 6-23replacing solvent filter 6-6spare parts B-1

solvent supply plumbing 2-11solvents

buffers C-5changing 5-2general considerations C-3preferred 2-12preparation C-4priming 3-6quality guidelines C-3strong wash 3-11water C-5weak wash 3-11

solvents to avoid 2-11spare parts

maintenance 6-4recommended B-1sample manager B-3solvent manager B-1TUV detector B-4

specificationselectrical A-2, A-6, A-11, A-14environmental A-1, A-5, A-10, A-14optical A-16performance A-4, A-8, A-12, A-13physical A-1, A-5, A-10, A-13

specifications, operational A-15

startingconsole 4-5MassLynx 4-1TUV detector 3-19

startup diagnostics 3-19startup tests 3-2static decay test 6-62stopping, needle wash 3-16strong needle wash 2-13strong wash solvent 3-11syringe

configuration parameters, modifying 6-45

metering, replacing 6-40wash, replacing 6-42

systeminformation xvimaintaining 6-1powering on 3-1setup 2-4shutting down 3-22test mix 5-10

system instruments, selecting from MassLynx 4-2

system shut down 3-22system verification test, example 5-11

Tteach block 2-22test methods, creating 5-6tests, startup 3-2tightening fittings 2-16tightening recommendations, fittings

2-15tools, installation 2-4trap column 1-9, 2-18trap valve cartridge fittings 6-49trap valve cartridge, replacing 6-49

Index-8

trappingsingle-pump 1-4two-pump 1-5

trapping column 1-4trapping mode 1-3trapping parameters 5-7trapping tab, BSM instrument

parameters 5-7tubing, connecting 2-15TUV detector

cleaning exterior 6-59flow cell

flushing 6-52replacing 6-53reverse flushing 6-53

fuses, replacing 6-59lamp, replacing 6-55lamp LED 3-3lamp, cooling time 6-56lamp, installing 6-57lamp, removing 6-56maintaining 6-51overview 1-10plumbing 2-20preparing 3-19spare parts B-4specifications

electrical A-14environmental A-14operational A-15optical A-16physical A-13

starting 3-19verifying 3-20

two-pump trapping 1-3, 1-5

Uunion, zero-dead-volume 2-21upgrading the system 2-2

I

VV-detail fitting 2-16vent valve cartridge fittings 6-21vent valve cartridge, replacing 6-20verifying TUV detector 3-20

Wwarnings

configuring 6-4injections threshold 6-4pumped volume maintenance 6-4

wash solvent effects 3-12wash solvents, selecting 3-11wash syringe leak test 6-71wash syringes, replacing 6-42washing

sample manager needle 3-15solvent reservoirs 2-12, 6-10, C-2

waste, proper draining of 2-13water, as solvent C-5Waters Technical Service, contacting

2-3, 6-2weak needle wash 2-13weak wash solvent 3-11

XXYZ mechanism, calibrating 2-22

Zzero-dead-volume union 2-21

Index-9

Index-10

Documents

nanoACQUITY UPLC System Operator’s Guide...Attenzione: l’utente deve essere al corrente del fatto che, se l’apparecchia-tura viene usta in un modo specificato dal produttore,