Waters 2489 Manual

2489 UV/Visible
Detector
Operator’s Guide
715003800 / Revision A
This operator's guide is intended for use with the 2489 UV/Visible detector,
part number 186248900, as pictured above. For the documentation to support
the 2489 UV/Visible detector, part number 186002489, see the operator's
guide, part number 71500142102.
Note: To locate the part number for your device, see the serial number label on
the back of the instrument.
Copyright © Waters Corporation 2012
All rights reserved

ii November 9, 2012, 715003800 Rev. A

November 9, 2012, 715003800 Rev. A iii
General Information
Copyright notice
© 2007−2012 WATERS CORPORATION. PRINTED IN THE UNITED
STATES OF AMERICA AND IN IRELAND. ALL RIGHTS RESERVED. THIS
DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY
FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.
The information in this document is subject to change without notice and
should not be construed as a commitment by Waters Corporation. Waters
Corporation assumes no responsibility for any errors that may appear in this
document. This document is believed to be complete and accurate at the time
of publication. In no event shall Waters Corporation be liable for incidental or
consequential damages in connection with, or arising from, its use.
Trademarks
PIC and Waters are registered trademarks of Waters Corporation, and
Empower, e-SAT/IN, FractionLynx, MassLynx, TaperSlit, and “THE
SCIENCE OF WHAT’S POSSIBLE.” are trademarks of Waters Corporation.
Tygon is a registered trademark of Saint-Gobain Ceramics & Plastics, Inc.
Other registered trademarks or trademarks are the sole property of their
owners.
Customer comments
Waters’ Technical Communications department invites you to tell us of any
errors you encounter in this document or to suggest ideas for otherwise
improving it. Please help us better understand what you expect from our
documentation so that we can continuously improve its accuracy and
usability.

iv November 9, 2012, 715003800 Rev. A
We seriously consider every customer comment we receive. You can reach us
at [email protected].
Contact Waters
Contact Waters®with enhancement requests or technical questions regarding
the use, transportation, removal, or disposal of any Waters product. You can
reach us via the Internet, telephone, or conventional mail.
Safety considerations
Some reagents and samples used with Waters instruments and devices can
pose chemical, biological, and radiological hazards. You must know the
potentially hazardous effects of all substances you work with. Always follow
Waters contact information:
Contacting medium Information
Internet The Waters Web site includes contact
information for Waters locations worldwide.
Visit www.waters.com.
Telephone and fax From the USA or Canada, phone 800
252-HPLC, or fax [phone].
For other locations worldwide, phone and fax
numbers appear in the Waters Web site.
Conventional mail Waters Corporation
34 Maple Street
Milford, MA 01757
USA

November 9, 2012, 715003800 Rev. A v
Good Laboratory Practice, and consult your organization’s safety
representative for guidance.
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 addresses good operating
procedures and the techniques necessary to validate system and method
performance.
Safety advisories
Consult Appendix A for a comprehensive list of warning and caution
advisories.
FCC radiation emissions notice
Changes or modifications not expressly approved by the party responsible for
compliance, could void the users authority to operate the equipment. This
device complies with Part 15 of the FCC Rules. Operation is subject to the
following two conditions: (1) this device may not cause harmful interference,
and (2) this device must accept any interference received, including
interference that may cause undesired operation.
Canada spectrum management emissions notice
This class A digital product apparatus complies with Canadian ICES-003.
Cet appareil numérique de la classe A est conforme à la norme NMB-003.
Electrical power safety notice
Do not position the instrument so that it is difficult to operate the
disconnecting device.
Safety hazard symbol notice
Documentation needs to be consulted in all cases where the symbol is
used to find out the nature of the potential hazard and any actions which have
to be taken.

vi November 9, 2012, 715003800 Rev. A
Equipment misuse notice
If the equipment is used in a manner not specified by the manufacturer, the
protection provided by the equipment may be impaired.
Operate this instrument
When operating this instrument, follow standard quality-control (QC)
procedures and the guidelines presented in this section.

November 9, 2012, 715003800 Rev. A vii
Applicable symbols
Audience and purpose
This guide is intended for personnel who install, operate, and maintain 2489
UV/Visible detectors.
Intended use of the 2489 UV/Visible detector
Waters designed the 2489 UV/Visible detector to analyze and monitor many
compounds.
Symbol Definition
Manufacturer
Authorized representative of the European
Community
Confirms that a manufactured product complies
with all applicable European Community
directives
Australia C-Tick EMC compliant
Confirms that a manufactured product complies
with all applicable United States and Canadian
safety requirements
Consult instructions for use
Electrical and electronic equipment with this
symbol may contain hazardous substances and
should not be disposed of as general waste.
For compliance with the Waste Electrical and
Electronic Equipment Directive (WEEE)
2012/19/EU, contact Waters Corporation for the
correct disposal and recycling instructions.

viii November 9, 2012, 715003800 Rev. A
Calibrate
To calibrate LC systems, follow acceptable calibration methods using at least
five standards to generate a standard curve. The concentration range for
standards should include the entire range of QC samples, typical specimens,
and atypical specimens.
When calibrating mass spectrometers, consult the calibration section of the
operator’s guide for the instrument you are calibrating. In cases where an
overview and maintenance guide, not operator’s guide, accompanies the
instrument, consult the instrument’s online Help system for calibration
instructions.
Quality-control
Routinely run three QC samples that represent subnormal, normal, and
above-normal levels of a compound. Ensure that QC sample results fall within
an acceptable range, and evaluate precision from day to day and run to run.
Data collected when QC samples are out of range might not be valid. Do not
report these data until you are certain that the instrument performs
satisfactorily.
ISM classification
ISM Classification: ISM Group 1 Class B
This classification has been assigned in accordance with CISPR 11 Industrial
Scientific and Medical (ISM) instruments requirements. Group 1 products
apply to intentionally generated and/or used conductively coupled
radio-frequency energy that is necessary for the internal functioning of the
equipment. Class B products are suitable for use in both commercial and
residential locations and can be directly connected to a low voltage,
power-supply network.

November 9, 2012, 715003800 Rev. A ix
EC Authorized Representative
Waters Corporation (Micromass UK Ltd.)
Floats Road
Wythenshawe
Manchester M23 9LZ
United Kingdom
Telephone: [phone]
Fax: [phone]
Contact: Quality manager

x November 9, 2012, 715003800 Rev. A

November 9, 2012, 715003800 Rev. A xi
General Information .................................................................................... iii
Copyright notice .................................................................................................. iii
Trademarks ........................................................................................................... iii
Customer comments ............................................................................................ iii
Contact Waters ..................................................................................................... iv
Safety considerations .......................................................................................... iv
Safety advisories.................................................................................................. v
FCC radiation emissions notice .......................................................................... v
Canada spectrum management emissions notice .............................................. v
Electrical power safety notice ............................................................................. v
Safety hazard symbol notice................................................................................ v
Equipment misuse notice ................................................................................... vi
Operate this instrument ..................................................................................... vi
Applicable symbols ........................................................................................... vii
Audience and purpose....................................................................................... vii
Intended use of the 2489 UV/Visible detector................................................. vii
Calibrate........................................................................................................... viii
Quality-control ................................................................................................. viii
ISM classification .............................................................................................. viii
ISM Classification: ISM Group 1 Class B ...................................................... viii
EC Authorized Representative ......................................................................... ix
1 Theory and Principles of Operation ................................................... 17
Detector description ......................................................................................... 17
Principles of operation ..................................................................................... 19
Detector optics.................................................................................................... 19
Wavelength verification and test...................................................................... 24
Table of Contents

xii November 9, 2012, 715003800 Rev. A
Operational modes ............................................................................................. 25
Single-wavelength mode.................................................................................... 25
Dual-wavelength mode...................................................................................... 27
Spectrum scanning ............................................................................................ 28
Cuvette operations............................................................................................. 29
RatioPlot............................................................................................................. 29
MaxPlot .............................................................................................................. 30
Thermal-wander management.......................................................................... 30
2 Install the Detector ................................................................................. 31
Prepare to install the detector ....................................................................... 32
Site selection and power requirements ........................................................ 33
Site selection ...................................................................................................... 33
Power requirements........................................................................................... 35
Unpack and inspect ........................................................................................... 35
Make plumbing connections ........................................................................... 36
Make electrical power connections ............................................................... 38
Make signal connections from rear panel and Ethernet connectors to the
detector......................................................................................................... 40
Make Ethernet connections............................................................................... 43
Connect to other devices .................................................................................. 49
Connect the detector to Empower using an e-SAT/IN module........................ 50
Connect the detector to a 745/745B/746 data module...................................... 53
Connect the detector to a fraction collector...................................................... 55
3 Prepare the Detector .............................................................................. 57
Initialize the detector ....................................................................................... 57
Idle mode ............................................................................................................ 59
Use the operator interface ............................................................................... 59
Use the display................................................................................................... 59
Using the keypad ............................................................................................... 63
Navigate the user interface............................................................................... 68
Primary and secondary functions ..................................................................... 70

November 9, 2012, 715003800 Rev. A xiii
Operate other detector functions ...................................................................... 78
Operate the detector .......................................................................................... 82
Verify that the detector operates properly ....................................................... 83
Wavelength calibration ..................................................................................... 86
Operate the detector in single-wavelength mode............................................. 86
Operate the detector in dual-wavelength mode ............................................... 87
Program timed events, threshold events, and methods .................................. 90
Scan spectra ........................................................................................................ 97
Before you begin................................................................................................. 98
Scan new spectra ............................................................................................. 100
Store a spectrum .............................................................................................. 107
Review a stored spectrum ............................................................................... 109
Subtract a spectrum ........................................................................................ 109
Replay a spectrum ........................................................................................... 110
Scan using the cuvette..................................................................................... 110
Scan using a flow cell and a syringe ............................................................... 112
Conserve lamp life ........................................................................................... 113
Shutting down the detector............................................................................. 115
4 Maintaining the Detector .................................................................... 117
Contact Waters technical service ................................................................. 117
Maintenance considerations ......................................................................... 118
Safety and handling......................................................................................... 118
Spare parts....................................................................................................... 119
Proper operating procedures ........................................................................ 119
Routine maintenance....................................................................................... 119
Maintain the flow cell ..................................................................................... 120
Flush the flow cell............................................................................................ 120
Remove and clean the flow cell ....................................................................... 122
Disassemble and reassemble the flow cell...................................................... 122
Replace the lamp .............................................................................................. 129
Removing the lamp .......................................................................................... 131
Install the new lamp........................................................................................ 134

xiv November 9, 2012, 715003800 Rev. A
Record the new lamp serial number............................................................... 136
Set the lamp threshold .................................................................................... 138
Replace fuses ..................................................................................................... 138
5 Error Messages, Diagnostic Tests, and Troubleshooting ............ 141
Error messages ................................................................................................. 141
Startup error messages ................................................................................... 141
Error messages preventing operation............................................................. 144
User-selected diagnostic tests ....................................................................... 148
Overview........................................................................................................... 148
Use the diagnostic tests................................................................................... 150
Service diagnostic tests ................................................................................... 157
Troubleshoot ..................................................................................................... 157
When you contact Waters................................................................................ 158
Diagnostic tests................................................................................................ 158
Power surges .................................................................................................... 158
Hardware troubleshooting .............................................................................. 158
A Safety Advisories .................................................................................. 161
Warning symbols .............................................................................................. 162
Task-specific hazard warnings........................................................................ 162
Specific warnings ............................................................................................. 163
Caution advisory .............................................................................................. 166
Warnings that apply to all Waters instruments ........................................ 167
Warnings that address the replacing of fuses ........................................... 172
Electrical symbols ............................................................................................ 173
Handling symbols ............................................................................................ 174

November 9, 2012, 715003800 Rev. A xv
B Specifications ........................................................................................ 175
Operational specifications ............................................................................. 176
Optional Waters TaperSlit flow cell specifications ................................. 178
C Solvent Considerations ....................................................................... 181
Introduction ...................................................................................................... 181
Preventing contamination............................................................................... 181
Clean solvents .................................................................................................. 181
Solvent quality ................................................................................................. 182
Preparation checklist....................................................................................... 182
Water ................................................................................................................ 182
Using buffers.................................................................................................... 182
Tetrahydrofuran .............................................................................................. 182
Solvent miscibility ........................................................................................... 183
How to use miscibility numbers...................................................................... 185
Buffered solvents ............................................................................................. 186
Head height ....................................................................................................... 186
Solvent viscosity ............................................................................................... 186
Mobile phase solvent degassing ................................................................... 186
Gas solubility ................................................................................................... 187
Solvent degassing methods ............................................................................ 187
Sparging ........................................................................................................... 188
Vacuum degassing ........................................................................................... 188
Solvent degassing considerations ................................................................... 188
Wavelength selection ...................................................................................... 189
UV cutoffs for common solvents...................................................................... 189
Mixed mobile phases........................................................................................ 190
Wavelength selection for chromophore detection........................................... 191

xvi November 9, 2012, 715003800 Rev. A

Detector description
November 9, 2012, 715003800 Rev. A 17
1Theory and Principles of
Operation
This chapter summarizes the Waters®2489 UV/Visible Detector
features and describes the theory and principles of operation.
See also: Appendix B for system specifications and Appendix C for
information on high-performance liquid chromatography (HPLC)
solvent considerations.
Detector description
The Waters 2489 UV/Visible Detector is a two-channel ultraviolet/visible
(UV/Vis) detector designed for high-performance liquid chromatography
(HPLC) applications.
Contents:
Topic Page
Detector description......................................................................... 17
Principles of operation ..................................................................... 19
Operational modes ........................................................................... 25

1 Theory and Principles of Operation
18 November 9, 2012, 715003800 Rev. A
Waters 2489 UV/Visible Detector:
The detector can operate as a stand-alone unit (with an integrator) or as an
integral part of a Waters chromatography system.
The detector can be configured with either an Empower™ or a MassLynx™
software system.
The detector has the following capabilities:
• Stand-alone programmability – Stores as many as five user-defined
programs (or methods) consisting of as many as 50 programmable, timed
events and two threshold events each.
• Single or dual-wavelength – Monitors absorbance at one or two discrete
wavelengths.
• Wavelength verification reference filter – Ensures wavelength accuracy.
• Automatic, second-order filter – Automatically engaged for wavelengths
of 370 nm and greater and removed for wavelengths of 369 nm or less.
• Spectrum scan and storage – Supports spectrum scan, display,
subtraction, storage, and playback, in addition to standard absorbance
and UV/Vis functionality.
• Cuvette qualification – Facilitates qualification of the detector by
insertion of a standard in a cuvette without breaking any plumbing
connections. Waters qualification kits, available in cuvette form, support

Principles of operation
November 9, 2012, 715003800 Rev. A 19
this feature, which allows the detector to serve as a benchtop
spectrophotometer.
• Cuvette sample analysis – Allows recording of the spectrum of any
sample placed in the cuvette.
• Method editing and storage – Supports basic method programming,
storage, and retrieval from the front panel.
• Full diagnostic capability – Supports built-in diagnostic tools to optimize
functionality and performance.
• Two contact closure outputs – The detector has two configurable
switches, each of which can accommodate a maximum of +30 VDC, 1.2-A
current carrying capacity, and 0.5-A current switching. The switches
(SW1 and SW2) can trigger fraction collectors and other external
devices, as well as activate according to time, absorbance threshold, or
ratio criteria.
• Thermal wander management – The detector's insulation, fans and
baffling are designed to mitigate thermal instability caused by ambient
temperature changes.
• Median Baseline Filter (MBF) – A variation of the data mode, the MBF
decreases the effects of gradient separations on the chromatographic
baseline. It enhances the UV detector's baseline stability by decreasing
its curvature, making the development of integration methods easier.
Principles of operation
To use the detector effectively, become familiar with its optical and electronic
design and the theory and principles of its operation.
This section describes the following parts and functions of the detector:
•Optics
• Wavelength verification and test
• Flow cell
• Electronics
Detector optics
The Waters 2489 UV/Visible detector optics are based on a Fastie-Ebert
monochromator and include these components:

1 Theory and Principles of Operation
20 November 9, 2012, 715003800 Rev. A
• High brightness deuterium (D2) lamp
• Two mirrors: one off-axis, ellipsoidal mirror and one spherical mirror
•Filterwheel
• Shutter, wavelength calibration filter, and second-order filter
• Entrance slit
• Blazed, plane-holographic, diffraction grating
•Beamsplitter
• Sample and reference photodiodes
• Waters TaperSlit™ Flow Cell (its entrance is the exit slit of the
monochromator)
• Cuvette holder
Waters 2489 UV/Visible detector optics assembly:
Spherical
mirror
Slit
Window Filter
wheel
Grating
Reference
photodiode
Beamsplitter
TaperSlit
flow cell
Optional
cuvette
Sample
photodiode
Ellipsoidal
mirror
D2lamp