Waters 2489 Operating manual

2489 UV/Visible
Detector
Overview and Maintenance Guide
715004752/Revision A
Copyright © Waters Corporation 2015
All rights reserved

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General Information
Copyright notice
© 2015 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. For the most recent revision of this document, consult
the Waters Web site (waters.com).
Trademarks
ACQUITY, Empower, MassLynx, PIC, “THE SCIENCE OF WHAT’S POSSIBLE.”, and
Waters are registered trademarks of Waters Corporation, and ACQUITY Arc, e-SAT/IN,
FractionLynx, and TaperSlit are trademarks of Waters Corporation.
Tygon is a registered trademark of Saint-Gobain Performance Plastics Corporation.
Other registered trademarks or trademarks are the sole property of their owners.
Customer comments
Waters’ Technical Communications organization invites you to report any errors that
you encounter in this document or to suggest ideas for otherwise improving it. Help
us better understand what you expect from our documentation so that we can
continuously improve its accuracy and usability.
We seriously consider every customer comment we receive. You can reach us at
[email protected].

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Contacting Waters
Contact Waters with enhancement requests or technical questions regarding the use,
transportation, removal, or disposal of any Waters product. You can reach us via the
Internet, telephone, or conventional mail.
Safety considerations
Some reagents and samples used with Waters instruments and devices can pose
chemical, biological, or radiological hazards (or any combination thereof). You must
know the potentially hazardous effects of all substances you work with. Always follow
Good Laboratory Practice, and consult your organization’s standard operating
procedures.
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.
Considerations specific to the 2489 UV/Visible detector
Power cord replacement hazard
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 [phone], or fax 508
872 1990.
For other locations worldwide, phone and fax numbers
appear in the Waters Web site.
Conventional mail Waters Corporation
Global Support Services
34 Maple Street
Milford, MA 01757
USA
Warning: To avoid electric shock, use the SVT-type power cord in the United
States and HAR-type (or better) cord in Europe. The main power cord must be
replaced only with one of adequate rating. For information regarding what cord
to use in other countries, contact your local Waters distributor.

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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.
Electrical power safety notice
Do not position the instrument so that it is difficult to disconnect the power cord.
Equipment misuse notice
If equipment is used in a manner not specified by its manufacturer, protections
against personal injury inherent in the equipment’s design can be rendered
ineffective.
Safety advisories
Consult Appendix A for a comprehensive list of warning advisories and notices.
Operating this instrument
When operating this instrument, follow standard quality-control (QC) procedures and
the guidelines presented in this section.
Applicable symbols
Symbol Definition
Manufacturer
Date of manufacture
Authorized representative of the European Community
Confirms that a manufactured product complies with all
applicable European Community directives
or
Australia EMC compliant

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Audience and purpose
This guide is intended for personnel who install, operate, and maintain 2489
UV/Visible (UV/Vis) detectors.
Intended use of the 2489 UV/Visible detector
Waters designed the 2489 UV/Visible detector to analyze and monitor many
compounds.
Calibrating
To calibrate LC systems, follow acceptable calibration methods using at least five
standards to generate a standard curve. The concentration range for standards must
include the entire range of QC samples, typical specimens, and atypical specimens.
Quality control
Routinely run three QC samples that represent subnormal, normal, and above-normal
levels of a compound. If sample trays are the same or very similar, vary the location
of the QC samples in the trays. 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.
Confirms that a manufactured product complies with all
applicable United States and Canadian safety
requirements
Consult instructions for use
Alternating current
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.
Serial number
Part number catalog number
Symbol Definition
5()

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EMC considerations
Canada spectrum management emissions notice
This class A digital product apparatus complies with Canadian ICES-001.
Cet appareil numérique de la classe A est conforme à la norme NMB-001.
ISM Classification: ISM Group 1 Class B
This classification has been assigned in accordance with IEC CISPR 11 Industrial
Scientific and Medical (ISM) instruments requirements.
Group 1 products apply to intentionally generated and/or used conductively coupled
radio-frequency energy that is necessary for the internal functioning of the
equipment.
Class B products are suitable for use in both commercial and residential locations and
can be directly connected to a low voltage, power-supply network.
EC authorized representative
Waters Corporation
Stamford Avenue
Altrincham Road
Wilmslow SK9 4AX UK
Telephone: [phone]
Fax: [phone]
Contact: Quality manager

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General Information .................................................................. iii
Copyright notice ......................................................................................... iii
Trademarks ................................................................................................ iii
Customer comments ................................................................................... iii
Contacting Waters ...................................................................................... iv
Safety considerations .................................................................................. iv
Safety hazard symbol notice.............................................................. iv
Considerations specific to the 2489 UV/Visible detector ......................... iv
FCC radiation emissions notice........................................................... v
Electrical power safety notice............................................................. v
Equipment misuse notice .................................................................. v
Safety advisories ............................................................................. v
Operating this instrument ............................................................................ v
Applicable symbols........................................................................... v
Audience and purpose ...................................................................... vi
Intended use of the 2489 UV/Visible detector ...................................... vi
Calibrating ...................................................................................... vi
Quality control................................................................................. vi
EMC considerations .................................................................................... vii
Canada spectrum management emissions notice................................. vii
ISM Classification: ISM Group 1 Class B ............................................. vii
EC authorized representative ....................................................................... vii
1 Theory and Principles of Operation .......................................... 15
1.1 Detector description .................................................................................. 15
1.2 Principles of operation ............................................................................... 17
1.2.1 Detector optics................................................................................ 17
1.2.2 Wavelength verification and test........................................................ 21
1.3 Operational modes .................................................................................... 22
1.3.1 Single-wavelength mode .................................................................. 22
1.3.2 Dual-wavelength mode..................................................................... 24
1.3.3 Spectrum scanning .......................................................................... 25
1.3.4 Cuvette operations .......................................................................... 26
1.3.5 RatioPlot ........................................................................................ 26
Table of Contents

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1.3.6 MaxPlot.......................................................................................... 26
1.3.7 Thermal-wander management........................................................... 26
2 Installing the Detector ............................................................. 27
2.1 Before you begin ...................................................................................... 27
2.2 Unpacking and inspecting .......................................................................... 27
2.2.1 Unpacking the detector .................................................................... 27
2.2.2 Inspecting the detector .................................................................... 28
2.3 Selecting a laboratory site ......................................................................... 28
2.4 Stacking system modules .......................................................................... 29
2.5 Connecting to the electricity source ............................................................. 30
2.6 Connecting signal cables ............................................................................ 31
2.6.1 Connecting I/O cables ...................................................................... 32
2.6.2 Connecting signal cables from the rear panel and Ethernet connectors to the
detector......................................................................................... 33
2.6.3 Making Ethernet connections............................................................. 33
2.6.4 Starting a method ........................................................................... 35
2.6.5 Turning the detector lamp on or off .................................................... 36
2.6.6 Connecting the detector to the 2695 separations module ...................... 36
2.6.7 Connecting the detector to a chromatography data workstation using an
e-SAT/IN module ............................................................................ 38
2.6.8 Connecting the detector to a 745/745B/746 data module...................... 41
2.6.9 Connecting the detector to a fraction collector ..................................... 42
2.7 Plumbing the detector ............................................................................... 43
2.7.1 Connecting columns in HPLC systems................................................. 44
2.7.2 Connecting columns in ACQUITY Arc systems ...................................... 44
2.7.3 Assembling the fittings (HPLC only).................................................... 45
2.7.4 Making tubing connections in HPLC systems........................................ 45
2.7.5 Making tubing connections in ACQUITY Arc systems ............................. 45
3 Prepare the Detector ............................................................... 47
3.1 Initializing the detector .............................................................................. 47
3.1.1 Diagnostic test failure ...................................................................... 48
3.1.2 Idle mode....................................................................................... 48
3.2 Using the operator interface ....................................................................... 49
3.2.1 Using the display............................................................................. 49
3.2.2 Using the keypad............................................................................. 51
3.2.3 Navigating the user interface ............................................................ 55

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3.2.4 Primary and secondary functions ....................................................... 57
3.2.5 Operating other detector functions..................................................... 63
3.2.6 Operating the detector ..................................................................... 66
3.2.7 Verifying that the detector operates properly....................................... 67
3.2.8 Wavelength calibration ..................................................................... 69
3.2.9 Operating the detector in single-wavelength mode ............................... 69
3.2.10 Operating the detector in dual-wavelength mode ............................... 70
3.3 Scanning spectra ...................................................................................... 78
3.3.1 Before you begin ............................................................................. 78
3.3.2 Scanning new spectra ...................................................................... 80
3.3.3 Storing a spectrum .......................................................................... 86
3.3.4 Reviewing a stored spectrum ............................................................ 87
3.3.5 Subtracting a spectrum .................................................................... 87
3.3.6 Replaying a spectrum....................................................................... 88
3.3.7 Scanning using the cuvette ............................................................... 88
3.3.8 Scanning using a flow cell and a syringe ............................................. 90
3.3.9 Conserving lamp life ........................................................................ 90
3.3.10 Shutting down the detector ............................................................. 92
4 Maintaining the Detector ......................................................... 93
4.1 Contacting Waters technical service ............................................................. 93
4.2 Maintenance considerations ........................................................................ 93
4.2.1 Safety and handling......................................................................... 94
4.2.2 Spare parts .................................................................................... 94
4.3 Proper operating procedures ...................................................................... 94
4.3.1 Routine maintenance ....................................................................... 94
4.4 Maintaining the flow cell ............................................................................ 95
4.4.1 Flushing the flow cell ....................................................................... 95
4.4.2 Removing and cleaning the flow cell................................................... 97
4.4.3 Disassembling and reassembling the flow cell...................................... 98
4.5 Replacing the lamp ................................................................................. 103
4.5.1 Lamp characteristics ...................................................................... 104
4.5.2 Lamp energy and performance ........................................................ 104
4.5.3 Removing the lamp........................................................................ 105
4.5.4 Installing the new lamp .................................................................. 107

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4.5.5 Recording the new lamp serial number ............................................. 109
4.5.6 Setting the lamp threshold.............................................................. 110
4.6 Replacing fuses ...................................................................................... 111
5 Error Messages, Diagnostic Tests, and Troubleshooting ......... 113
5.1 Error messages ...................................................................................... 113
5.1.1 Startup error messages.................................................................. 113
5.2 User-selected diagnostic tests .................................................................. 113
5.2.1 Overview...................................................................................... 113
5.2.2 Using the diagnostic tests............................................................... 115
5.2.3 Service diagnostic tests.................................................................. 120
5.3 Troubleshooting ...................................................................................... 121
5.3.1 Diagnostic tests............................................................................. 121
5.3.2 Power surges ................................................................................ 121
5.3.3 Hardware troubleshooting............................................................... 121
5.3.4 Lamp troubleshooting .................................................................... 123
A Safety Advisories ................................................................... 125
A.1 Warning symbols .................................................................................... 125
A.1.1 Specific warnings .......................................................................... 126
A.2 Notices ................................................................................................. 127
A.3 Bottles Prohibited symbol ........................................................................ 128
A.4 Required protection ................................................................................ 128
A.5 Warnings that apply to all Waters instruments and devices ........................... 128
A.6 Warnings that address the replacing of fuses .............................................. 131
A.7 Electrical and handling symbols ................................................................ 133
A.7.1 Electrical symbols.......................................................................... 133
A.7.2 Handling symbols .......................................................................... 134

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B Specifications ........................................................................ 137
B.1 Physical specifications ............................................................................. 137
B.2 Environmental specifications .................................................................... 137
B.3 Electrical specifications ............................................................................ 138
B.4 Performance specifications ....................................................................... 139
B.5 Optical component specifications .............................................................. 139
B.6 Flow cell specifications ............................................................................ 140
C Solvent Considerations .......................................................... 141
C.1 Introduction ........................................................................................... 141
C.1.1 Preventing contamination ............................................................... 141
C.1.2 Clean solvents .............................................................................. 141
C.1.3 Solvent quality.............................................................................. 141
C.1.4 Preparation checklist...................................................................... 141
C.1.5 Water .......................................................................................... 142
C.1.6 Using buffers ................................................................................ 142
C.1.7 Tetrahydrofuran ............................................................................ 142
C.2 Solvent miscibility .................................................................................. 142
C.2.1 How to use miscibility numbers ....................................................... 143
C.3 Buffered solvents ................................................................................... 144
C.4 Head height ........................................................................................... 144
C.5 Solvent viscosity .................................................................................... 144
C.6 Mobile phase solvent degassing ................................................................ 145
C.6.1 Gas solubility ................................................................................ 145
C.7 Solvent degassing methods ..................................................................... 146
C.7.1 Sparging ...................................................................................... 146
C.7.2 Vacuum degassing......................................................................... 146
C.7.3 Solvent degassing considerations .................................................... 146
C.8 Wavelength selection .............................................................................. 147
C.8.1 UV cutoffs for common solvents ...................................................... 147
C.8.2 Mixed mobile phases...................................................................... 148
C.8.3 Wavelength selection for chromophore detection ............................... 149

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1Theory and Principles of
Operation
This chapter summarizes the 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.
1.1 Detector description
The 2489 UV/Vis detector is a two-channel ultraviolet/visible (UV/Vis) detector
designed for high-performance liquid chromatography (HPLC) applications.
Figure 1–1: 2489 UV/Vis detector
The detector can operate as a stand-alone unit (with an integrator) or as an integral
part of a Waters chromatography system.

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The detector can be configured with either Empower®or MassLynx®chromatography
data software.
Table 1–1: Detector capabilities
Capability Description
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.
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.
They can also 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.

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1.2 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
•Flowcell
• Electronics
1.2.1 Detector optics
The 2489 UV/Vis detector optics are based on a Fastie-Ebert monochromator and
include these components:
• 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 for flow cells requiring a cuvette.
If your flow cell requires a cuvette:
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 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.
Table 1–1: Detector capabilities
Capability Description

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Figure 1–2: 2489 UV/Vis detector optics assembly
1.2.1.1 Optics assembly light path
The detector provides an extremely efficient design for exceptionally high light
throughput. It operates as follows:
1. The ellipsoidal mirror collects light from the lamp and focuses it through the
filter wheel and onto the entrance slit. The spherical mirror directs light toward
the grating. A different portion of the spherical mirror focuses dispersed light of
a particular wavelength band, determined by the grating angle, onto the
entrance of the flow cell. Light exiting the flow cell passes through the cuvette
location to the sample photodiode.
2. The beamsplitter, located just ahead of the flow cell, diverts a portion of the
light to a reference photodiode.
3. When you enter a new wavelength through the detector’s front panel (or
through Empower or MassLynx software), the detector rotates the grating to the
appropriate position.
4. The preamplifier board integrates and digitizes the currents from the
photodiodes for processing by the signal processing electronics and output to a
computer, or integrator.
1.2.1.2 Waters TaperSlit flow cell
The Waters TaperSlit flow sell used in this detector renders the detector baseline less
sensitive to changes in mobile phase refractive index (RI). RI changes occur during
gradient separations or result from temperature or pump-induced pressure
fluctuations.
Spherical
mirror
Slit
Window Filter
wheel
Grating
Reference
photodiode
Beamsplitter
TaperSlit
flow cell
Optional
cuvette
Sample
photodiode
Ellipsoidal
mirror
D2lamp

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To achieve RI immunity, a combination of a spherical mirror, a lens at the entrance of
the flow cell, and a taper to the internal bore of the flow cell prevents light rays from
striking the internal walls of the flow cell. An additional feature of the TaperSlit flow
cell and the reason for its name is the shape of the flow cell entrance, which matches
the shape of the entrance slit. The detector achieves higher light throughput for a
given spectral resolution via the TaperSlit cell design, compared to a conventional flow
cell with a circular entrance.
As shown in the figure below, in a conventional cell, light bends and hits the wall of
the flow cell. Four beams go in, but only two come out. In the Waters TaperSlit
analytical cell, the combination of the lens and TaperSlit bore geometry prevents light
from hitting the cell walls. Four beams go in, and four beams come out.
Figure 1–3: Comparison of flow cell characteristics
The standard analytical, inert, and LC/MS cells have a path length of 10 mm. The
semi-prep and microbore cell path length is 3 mm. The autopurification cell path
length is 1.0 mm. A variable path length flow cell (path length 0.15 to 3 mm) is also
available.
1.2.1.3 Selecting the appropriate sampling rate
A sufficient number of points must fall across a peak to define its shape. Thus, at very
low sampling rates, the definition between peaks is lost. The chromatography data
software uses the index of the data point closest to the end time, minus the index of
the data point closest to the start time, to calculate a value for each integrated peak
in the chromatogram.
Tip: In Empower software, this value is called the Points Across Peak value, and it
appears in the Peaks table, at the bottom of the Review Main window. If the Points
Across Peak field is not visible, right-click anywhere in the table, and then click Table
Properties. Click the Columns tab, and then scroll down to find the Points Across Peak
field. Clear the check box, and then click OK.
UV light
UV light
Conventional cell
TaperSlit analytical cell
Window
Window
Window
Window

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If the value for the narrowest peak of interest is less than 15, you must specify a
higher sampling rate in the instrument method. If the value is greater than 30,
specify a lower sampling rate in the instrument method.
Set the sampling rate to the lowest value required to achieve 15 or more points
across the narrowest peak. Excessively high sampling rates can slow the system with
more data than you need for your analysis.
1.2.1.4 Filtering noise
The detector provides a Hamming filter to minimize noise. The Hamming filter is a
digital finite impulse response filter, which creates peak height degradation and
enhances the filtering of high frequency noise.
The behavior of the filter depends on the filter time constant you select. You can
program a filter time to be Fast, Slow, Normal, or Other. If you select Fast, Slow, or
Normal, you do not need to enter a value. The filter constant is determined by the
sampling rate. If you select Other, you can enter a value. However, the value you
enter is rounded up or down to a value based on the sampling rate.
The filter time constant adjusts the time window over which the data is filtered, thus
controlling the degree of baseline smoothing and the impact on peak height
degradation. Optimizing this parameter in the method ensures the highest
signal-to-noise ratios are achieved for a particular application.
Decreasing the time-constant settings produces these effects:
• Narrow peaks with minimal peak distortion and time delay.
• Very small peaks become harder to discriminate from baseline noise.
• Less baseline noise is removed.
Increasing the time-constant settings produces these effects:
• Greatly decrease baseline noise.
• Shorten and broaden peaks.
The software includes fast or normal filtering constants at each sampling rate that are
appropriate for high speed or high sensitivity applications respectively.
The following figure shows the relationship between increased filter time constant and
absorbance.