Waters 2489 Manual

Waters 2489
UV/Visible Detector
Operator’s Guide
71500142102 / Revision A
Copyright © Waters Corporation 2007.
All rights reserved.

ii
Copyright notice
© 2007 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, its use.
Trademarks
Waters is a registered trademark of Waters Corporation. Empower, e-SAT/IN,
and MassLynx are trademarks of Waters Corporation.
Other trademarks or registered trademarks are the sole property of their
respective owners.
Customer comments
Waters’ Technical Communications department invites you to tell us of any
errors you encounter in this document or to suggest ideas for otherwise
improving it. Please help us better understand what you expect from our
documentation so that we can continuously improve its accuracy and
usability.
We seriously consider every customer comment we receive. You can reach us
at [e-mail].

iii
Contacting Waters
Contact Waters®with enhancement requests or technical questions regarding
the use, transportation, removal, or disposal of any Waters product. You can
reach us via the Internet, telephone, or conventional mail.
Waters contact information
Contacting medium Information
Internet The Waters Web site includes phone numbers
for Waters locations worldwide. Go to
www.waters.com, and click About Waters >
Worldwide Offices.
Telephone In the USA or Canada, phone [phone].
Conventional mail Waters Corporation
34 Maple Street
Milford, MA 01757
USA

iv
Safety considerations
Some reagents and samples used with Waters®instruments can pose
chemical, biological, and radiological hazards. Be sure you are aware of the
potentially hazardous effects of all substances you work with. Always observe
Good Laboratory Practice (GLP) guidelines, published by the U.S. Food and
Drug Administration, 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.
Operating this detector
When operating this detector, follow standard quality control procedures and
the guidelines presented in this section.

v
Symbols
Intended use
The Waters®2489 UV/Visible detector can be used for in vitro diagnostic
testing to analyze many compounds, including diagnostic indicators and
therapeutically monitored compounds. However, only professionally trained
and qualified laboratory personnel should use the instrument for those
purposes.
Calibrating the detector
To calibrate the detector, follow acceptable calibration methods using at least
five standards to generate a standard curve. The concentration range for
standards should cover the entire range of quality-control samples, typical
specimens, and atypical specimens.
Symbol Definition
Authorized representative of the
European Community
Confirms that a manufactured
product complies with all applicable
European Community directives
For in vitro diagnostic use
The Waters 2489 UV/Visible Detector is CE-marked according to
the European Union In Vitro Diagnostic Device Directive
98/79/EC.
EC RE
P
IVD

vi
Quality control
Routinely run three quality-control samples that represent subnormal,
normal, and above-normal levels of a compound. Ensure that quality-control
sample results fall 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 these data until you are certain that
the instrument performs satisfactorily.
When analyzing samples from a complex matrix such as soil, tissue,
serum/plasma, whole blood, and so on, note that the matrix components can
adversely affect results. To minimize these matrix effects, Waters
recommends you adopt the following measures:
• Prior to the instrumental analysis, use appropriate sample
pretreatment such as protein precipitation, liquid/liquid extraction
(LLE), or solid phase extraction (SPE) to remove matrix interferences.
• Whenever possible, verify method accuracy and precision using
matrix-matched calibrators and QC samples.
• Use one or more internal standard compounds, preferably isotopically
labeled analytes.

vii
IVD authorized representative
Waters Corporation (Micromass UK
Limited) is registered in the United
Kingdom with the Medicines and
Healthcare Products Regulatory Agency
(MHRA) at Market Towers, 1 Nine Elms
Lane, London, SW8 5NQ. The reference
number is IVD000167.
Waters Corporation (Micromass UK Ltd.)
Floats Road
Wythenshawe
Manchester M23 9LZ
United Kingdom
Telephone: [phone]
Fax: [phone]
Contact: Quality manager

viii

Table of Contents ix
1 Theory and Principles of Operation .................................................. 1-1
Detector description ........................................................................................ 1-2
Features ....................................................................................................... 1-2
Principles of operation .................................................................................... 1-4
Detector optics.................................................................................................. 1-4
Optics assembly light path ......................................................................... 1-5
Waters TaperSlit Flow Cell ........................................................................ 1-6
Filtering noise ............................................................................................. 1-7
Wavelength verification and test.................................................................... 1-9
Operational modes .......................................................................................... 1-11
Single wavelength mode................................................................................ 1-11
Primary parameters .................................................................................. 1-11
Secondary parameters .............................................................................. 1-12
Dual wavelength mode .................................................................................. 1-12
Chart Out selection modes ....................................................................... 1-13
Spectrum scanning ........................................................................................ 1-13
Cuvette operations......................................................................................... 1-14
RatioPlot......................................................................................................... 1-15
MaxPlot .......................................................................................................... 1-15
Thermal wander management...................................................................... 1-15
2 Installing the Detector .......................................................................... 2-1
Preparing for installation ............................................................................... 2-2
Site selection and power requirements ....................................................... 2-4
Site selection .................................................................................................... 2-4
Power requirements......................................................................................... 2-5
Unpacking and inspecting............................................................................... 2-5
Unpacking ................................................................................................... 2-6
Inspecting .................................................................................................... 2-6
Table of Contents

x Table of Contents
Making fluid line connections ....................................................................... 2-6
Connecting columns .................................................................................... 2-7
Assembling the fittings ............................................................................... 2-8
Making connections ..................................................................................... 2-9
Making electrical power connections .......................................................... 2-9
Detector rear panel ................................................................................... 2-10
Making signal connections............................................................................ 2-11
Making I/O signal connections ................................................................. 2-12
I/O signals .................................................................................................. 2-13
Making Ethernet connections ....................................................................... 2-14
Making Ethernet connections with Waters data systems ...................... 2-14
Starting a method ..................................................................................... 2-16
Turning the detector lamp on or off ......................................................... 2-16
Connecting the detector to a separations module ................................... 2-18
Generating Auto Zero ............................................................................... 2-18
Generating Chart Mark on Inject ............................................................ 2-19
Connecting to other devices ......................................................................... 2-20
Required materials .................................................................................... 2-21
Connecting the cables ............................................................................... 2-21
Connecting the detector to Empower using an e-SAT/IN module .............. 2-21
e-SAT/IN module ....................................................................................... 2-21
Connecting the detector to the e-SAT/IN module .................................... 2-23
Connecting the detector to a 745/745B/746 data module............................ 2-25
Connecting the detector to a chart recorder................................................. 2-27
Recorder signal .......................................................................................... 2-27
Chart marks .............................................................................................. 2-28
Connecting the detector to the Waters 600 Series Pump............................ 2-29
Fluid line connections ............................................................................... 2-29
Lamp on/off connections ........................................................................... 2-29
Auto Zero connections ............................................................................... 2-30
Chart Mark connections ........................................................................... 2-31
Inject Start connections ............................................................................ 2-32
Pump and detector Inject Start connections ............................................ 2-33
Connecting the detector to the Waters 717plus Autosampler..................... 2-33

Table of Contents xi
Auto Zero connections ............................................................................... 2-34
Inject Start connections ............................................................................ 2-35
Connecting the detector to a fraction collector............................................. 2-36
3 Preparing the Detector ......................................................................... 3-1
Initializing the detector .................................................................................. 3-2
Diagnostic test failure ................................................................................. 3-3
Using the operator interface .......................................................................... 3-3
Using the display............................................................................................. 3-3
Absorbance and message icons ................................................................... 3-4
Using the keypad............................................................................................. 3-6
Navigating the user interface ....................................................................... 3-12
Navigating to and from the absorbance screen ....................................... 3-12
Setting up a run ........................................................................................ 3-13
Primary and secondary functions................................................................. 3-14
Operating the Trace and Scale functions ................................................. 3-20
Operating other detector functions............................................................... 3-22
Configuring the detector ........................................................................... 3-22
Configuring event inputs (contact closures) ............................................ 3-23
Setting pulse periods ................................................................................. 3-24
Setting display contrast ............................................................................ 3-25
Displaying system info .............................................................................. 3-25
Using Help ................................................................................................. 3-26
Operating the detector................................................................................... 3-26
Overview of detector operation ................................................................. 3-26
Operating modes ....................................................................................... 3-26
Stand-alone operation ............................................................................... 3-27
Remote control ........................................................................................... 3-27
Verifying the detector.................................................................................... 3-27
Before you begin ........................................................................................ 3-27
Recording sample and reference beam energies ...................................... 3-28
Verifying peak response ............................................................................ 3-29
Wavelength calibration ................................................................................. 3-30
Operating the detector in single wavelength mode ..................................... 3-31

xii Table of Contents
Operating the detector in dual wavelength mode........................................ 3-32
Changing from single to dual wavelength mode ..................................... 3-33
Obtaining a RatioPlot ............................................................................... 3-34
Obtaining a MaxPlot ................................................................................. 3-35
Programming timed events, threshold events, and methods...................... 3-35
Timed events ............................................................................................. 3-36
Threshold events ....................................................................................... 3-39
Storing a method ....................................................................................... 3-40
Retrieving a method .................................................................................. 3-41
Viewing events within a method .............................................................. 3-42
Resetting a method ................................................................................... 3-42
Clearing events ......................................................................................... 3-43
Scanning spectra ............................................................................................. 3-44
Before you begin............................................................................................. 3-44
Scanning new spectra.................................................................................... 3-48
Zero scan .................................................................................................... 3-49
Running the sample scan .......................................................................... 3-52
Storing a spectrum......................................................................................... 3-56
Getting information about a stored spectrum.............................................. 3-56
Reviewing a stored spectrum ........................................................................ 3-57
Subtracting a spectrum................................................................................. 3-57
Replaying a spectrum.................................................................................... 3-58
Scanning using the cuvette........................................................................... 3-59
Before you begin ........................................................................................ 3-60
Cuvette scanning procedure ..................................................................... 3-60
Scanning using a flow cell and a syringe...................................................... 3-62
Conserving lamp life...................................................................................... 3-62
Shutting down the detector........................................................................... 3-65
Removing buffered mobile phase ............................................................. 3-65
Shutting down the detector ...................................................................... 3-65

Table of Contents xiii
4 Maintaining the Detector ..................................................................... 4-1
Contacting Waters Technical Service .......................................................... 4-2
Maintenance considerations .......................................................................... 4-2
Safety precautions ........................................................................................... 4-2
Spare parts....................................................................................................... 4-2
Proper operating procedures ......................................................................... 4-3
Removing the front left panel cover................................................................ 4-3
Routine maintenance....................................................................................... 4-4
Maintaining the flow cell ................................................................................ 4-4
Flushing the flow cell....................................................................................... 4-5
Removing and cleaning the flow cell............................................................... 4-6
Disassembling and reassembling the flow cell............................................... 4-6
Before you begin .......................................................................................... 4-6
Tools required .............................................................................................. 4-7
Removing the flow cell assembly ................................................................ 4-7
Disassembling the flow cell ...................................................................... 4-10
Inspecting, cleaning, and replacing damaged flow cell components ...... 4-13
Rebuilding the flow cell ............................................................................. 4-13
Replacing the flow cell .............................................................................. 4-15
Replacing the lamp ......................................................................................... 4-16
Lamp characteristics ................................................................................. 4-16
Lamp energy and performance ................................................................. 4-16
When to replace the lamp ......................................................................... 4-17
Removing the lamp........................................................................................ 4-18
Installing the new lamp................................................................................. 4-21
Recording the new lamp serial number........................................................ 4-23
Setting the lamp threshold............................................................................ 4-25
Replacing fuses ................................................................................................ 4-26
5 Error Messages, Diagnostic Tests, and Troubleshooting ............. 5-1
Error messages .................................................................................................. 5-2
Startup error messages ................................................................................... 5-2

xiv Table of Contents
Error messages preventing operation............................................................. 5-5
User-selected diagnostic tests ........................................................................ 5-8
Overview........................................................................................................... 5-8
Using the diagnostic tests ............................................................................. 5-11
Contacting Waters Technical Service ...................................................... 5-11
Using the sample and reference energy diagnostic tests ........................ 5-11
Using the Input & Output diagnostic tests ............................................. 5-12
Displaying Auto Zero offsets ..................................................................... 5-13
Setting fixed absorbance value ................................................................. 5-13
Setting fixed voltage output ..................................................................... 5-14
Using the lamp, display, and keypad diagnostic tests ............................ 5-15
Using the Change Lamp diagnostic test .................................................. 5-15
Using the other detector diagnostic tests ................................................ 5-17
Service diagnostic tests ................................................................................. 5-19
Troubleshooting .............................................................................................. 5-19
When you contact Waters.............................................................................. 5-19
Diagnostic tests.............................................................................................. 5-20
Power surges.................................................................................................. 5-20
Hardware troubleshooting ............................................................................ 5-20
A Safety Advisories .................................................................................. A-1
Warning symbols ............................................................................................... A-2
Task-specific hazard warnings........................................................................ A-2
Warnings that apply to particular instruments, instrument components, and
sample types A-3
Burst warning ............................................................................................. A-3
Mass spectrometer flammable solvents warning ...................................... A-3
Mass spectrometer shock hazard ............................................................... A-4
Biohazard warning ...................................................................................... A-4
Chemical hazard warning ........................................................................... A-5

Table of Contents xv
Caution symbol .................................................................................................. A-5
Warnings that apply to all Waters instruments ......................................... A-5
Electrical and handling symbols ................................................................. A-13
Electrical symbols.......................................................................................... A-13
Handling symbols .......................................................................................... A-14
B Detector Specifications ....................................................................... B-1
Operational specifications ............................................................................. B-2
Optical specifications ..................................................................................... B-4
Optional Waters TaperSlit Flow Cell specifications ............................... B-5
C Spare Parts ............................................................................................ C-1
D Solvent Considerations ....................................................................... D-1
Introduction ...................................................................................................... D-2
Clean solvents.................................................................................................. D-2
Solvent quality................................................................................................. D-2
Preparation checklist....................................................................................... D-2
Water................................................................................................................ D-2
Using buffers.................................................................................................... D-2
Tetrahydrofuran .............................................................................................. D-3
Solvent miscibility ........................................................................................... D-3
How to use miscibility numbers...................................................................... D-5
Buffered solvents ............................................................................................. D-6
Head height ....................................................................................................... D-6
Solvent viscosity ............................................................................................... D-6
Mobile phase solvent degassing ................................................................... D-6
Gas solubility ................................................................................................... D-7
Effects of intermolecular forces .................................................................. D-7
Effects of temperature ................................................................................ D-7
Effects of partial pressure ........................................................................... D-7

xvi Table of Contents
Solvent degassing methods ............................................................................ D-8
Sparging ........................................................................................................... D-8
Vacuum degassing........................................................................................... D-8
Solvent degassing considerations ................................................................... D-8
Sparging ....................................................................................................... D-8
Vacuum Degassing ...................................................................................... D-9
Wavelength selection ...................................................................................... D-9
UV cutoffs for common solvents...................................................................... D-9
Mixed mobile phases...................................................................................... D-10
Wavelength selection for chromophore detection......................................... D-11

1-1
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 D for
information on high-performance liquid chromatography (HPLC)
solvent considerations.
Contents:
Topic Page
Detector description 1-2
Principles of operation 1-4
Operational modes 1-11

1-2 Theory and Principles of Operation
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.
Waters 2489 UV/Visible Detector
The detector can operate either as a stand-alone unit (with a chart recorder or
integrator) or as an integral part of a Waters chromatography system.
The detector can be configured with Empower™ or MassLynx™ software
systems.
Features
The detector operates from 190 to 700 nm. The detector uses optics with an
enhanced illumination system for improved performance. These design
features increase the optical throughput and sensitivity and reduce
bandwidth for an overall increase in signal-to-noise ratio and improved
linearity.
Inlet

Detector description 1-3
The detector has the following capabilities:
• Stand-alone programmability – Stores up to five user-defined programs
(or methods) consisting of up to 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
370 nm and greater and removed for wavelengths 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 fluid line
connections. Waters qualification kits are available in cuvette form to
support this feature. This feature also allows you to use the detector as a
benchtop spectrophotometer.
• Cuvette sample analysis – Allows the spectrum of any sample placed in
the cuvette to be recorded.
• 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 carry, and 0.5-A current switching. The switches (SW1 and
SW2) can trigger fraction collectors and other external devices, as well
as activate based on time, absorbance threshold, or ratio criteria.
• Improved thermal wander management– To mitigate thermal
instability caused by ambient temperature changes, the detector has
improved insulation (for better air flow across the optics bench) and a
variable speed fan that will run at higher or lower speeds as needed.
• Median Baseline Filter (MBF) - A variation of the data mode, the MBF is
intended to decrease the effects of gradient separations on the
chromatographic baseline. The MBF enhances the UV detector's
baseline stability by decreasing its curvature, making the development
of integration methods easier.

1-4 Theory and Principles of Operation
Principles of operation
To use the detector effectively, you should be 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
Detector optics
The Waters 2489 UV/Visible Detector optics are based on a Fastie-Ebert
monochromator and include the following:
• High brightness deuterium (D2) lamp
• Two mirrors: one off-axis ellipsoidal mirror and one spherical mirror
• Filter wheel
• 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