Waters 2489 Operator's Manual

Waters 2489

UV/Visible Detector

Operator’s Guide

71500142102 / Revision A

© 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/I N, 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 departme nt invites you to tell us of any errors you encounter in this document or to suggest ide as 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 tech_comm@waters.com.

Contacting Waters

Contact Waters® with enhancement requests or techn ical questions regard ing the use, transportation, removal, or disposal of any Waters prod uct. 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 worldwi de. Go to www.waters.com, and click About Waters >

Worldwide Offices. Telephone In the USA or Canada, phone 508 478-2000. Conventional mail Waters Corporation

34 Maple Street

Milford, MA 01757

USA

iii

Safety considerations

Some reagents and samples used with Waters® instruments ca n po se chemical, biological, and radiological hazard s. 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 “Pro tocol for the Ado ption of Analyt ical Methods in the Clinical Chemistry Laboratory,” Ame rican 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.

Symbols

Symbol Definition

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.

E C RE

Authorized representativ e of the European Community

Confirms that a manufactured product complies with all applicable European Community directives

For in vitro diagnostic use

IV D

The Waters 2489 UV/Visible Detector is CE-marked according to the European Union In Vitro Diagnostic Device Directive 98/79/EC.

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 quali ty-control samples, typical specimens, and atypical specimens.

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 col lected when qualit y control samples are ou t of range might not be valid. Do not r eport the se dat a until you ar e cert ain that the instrument performs satisfactorily.

When analyzing samples from a com plex matrix such as soil, tissue, serum/plasma, whole blood, and so on, note that the matrix co mponents can adversely affect results. To minimize these matrix effec ts, Waters recommends you adopt the following measures:

• Prior to the instrumental analysis, use appropria te 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.

IVD authorized representative

Waters Corp o ra tion (Microm a ss UK Ltd.)

Floats Road Wythenshawe Manchester M23 9LZ United Kingdom

Telephone: +44-161-946-2400 Fax: +44-161-946-2480 Contact: Quality manager

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.

vii

viii

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 assemb l y 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 wavelen g th mo de...... .. .. .............. .. .. ............. ... .......................... .. ... ..... 1-11

Primary para me t er s . ... ............. .. .. .............. .. .......................... .. .............. .. . 1-11

Secondary parameters .............................................................................. 1-12

Dual wavelength mode .................................................................................. 1-12

Chart Out selec t i on mo de s ............. ............. .. .......................... ... .. ............ 1-1 3

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 ix

Making fluid line connections ....................................................................... 2-6

Connecting columns .................................................................................... 2-7

Assembling the fittings ............................................................................... 2-8

Making connec ti o n s ............. ... .......................... .. ... ............. .. ....................... 2-9

Making electrical power connections .......................................................... 2-9

Detector rear panel ................................................................................... 2-10

Making signal connections ............................................................................ 2-11

Making I/O sign al co n n e ct i o n s ............................. .. .. ............. .. ................. 2-12

I/O signals .................................................................................................. 2-13

Making Ethernet connections ....................................................................... 2-14

Making Ethern et connectio n s w ith Wa ters data syste ms .............. ... .. ... 2-14

Starting a method ..................................................................................... 2-16

Turning the de t ec t or 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 mat eri a l s ....................... ... .......................... .. .. .............. .. ............ 2-21

Connecting the cables ............................................................................... 2-21

Connecting the de te ctor to Empower u si n g a n e-S A T /I N mo d ule ......... .. .. . 2-21

e-SAT/IN modu l e ............. .. ........................... .. .......................... ... ............. . 2-2 1

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 con n e ctions ............... ... ............. .. .. ............. ... ......................... 2-3 2

Pump and detect o r In je ct Start conne ct i o n s ............ .. .. .............. .. .. .......... 2-33

Connecting the detector to the Waters 717plu s Autos ampler..... ................ 2-33

x Table of Contents

Auto Zero connections ............................................................................... 2-34

Inject Start con n e ctions ............... ... ............. .. .. ............. ... ......................... 2-3 5

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 u se r i n te rface ................... .. ... ............. .. .. ............. ... .............. 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

Configuri ng ev e n t i n pu t s (contact closu res) .............. .. .............. .. ............ 3-2 3

Setting pulse pe riods ............................. .. .............. .. .. ............. .. ... ............. . 3-24

Setting displa y co n t ra st .. ........................... .. .. ............. .. ........................... . 3-2 5

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 contro l .......... ... ............. .. .. .............. .. ............. .. ........................... .. . 3-27

Verifying the detector.................................................................................... 3-27

Before you begin ........................................................................................ 3-27

Recording samp l e an d r ef e re n c e b ea m energies .. .. .......................... ... .. ... 3-28

Verifying peak response ............................................................................ 3-29

Wavelength calibration ................................................................................. 3-30

Operating the detector in single wavelength mode ..................................... 3-31

Table of Contents xi

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

xii Table of Contents

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 fr o nt le f t pa n e l co ve r............... .. .. ............. .. ........................... .. . 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

Disassemblin g a n d re a ssembling th e fl o w ce l l............. .. .. .............. .. .............. 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 th e fl o w ce l l . ............. .. .............. .. .. ............. .. .............. .. ............ 4-13

Replacing th e fl o w ce l l ............. .. ............. ... ............. .. ............. .. .............. .. . 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 la mp.......... ............. .. ............. ... ............. .. .. ............. ... ............. . 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 erro r me ssages ......... .............. .. ............. .. ............. ... ............. .. .. .......... 5-2

Table of Contents xiii

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 vol t age 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

xiv Table of Contents

Caution symbol .................................................................................................. A-5

Warnings that apply to all Waters instruments ......................................... A-5

Electrical and handling symbo l s ............. .. ... .. .. .. .. .............. .. .. .. .. ... .. .. .......... 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 misc ib ility ....... .. ... .. ............. .. ... .. .. ............. .. ... .. .. .. ............. ... .. .. .. ......... D-3

How to use miscibility numbers...................................................................... D-5

Buffered solvents ............................................................................................. D-6

Head height ....................................................................................................... D-6

Solvent visc os i t y ............. ... .. .. .. .. .............. .. .. .. ... .. ............. .. .. ... .. .. ............. ... .. .. .. D-6

Mobile phase solvent degassing ................................................................... D-6

Gas solubility ........................ .. .. .......................................................... .. .. ......... D-7

Effects of intermolecul ar fo rces ....................... .. .............. .. ............. .. .......... D-7

Effects of temp e ra t u re .... .. .............. .. .. ............. .. ........................... .. ............ D-7

Effects of par tia l pre ssure ........ ............. .. ........................... .. ............. ... .. ..... D-7

Table of Contents xv

Solvent degassing methods ............................................................................ D-8

Sparging ........................................................................................................... D-8

Vacuum degassi n g ..................................... .. ... ............. .. ........................... .. .. ... 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

xvi Table of Contents

1 Theory and Principles of

Operation

This chapter summarizes the Waters® 2489 UV/Visible Detector features and describes the theory and principles of operation.

Contents: Topic Page

Detector description 1-2 Principles of operation 1-4 Operational modes 1-11

See also: Appendix B for system specifications and Appendix D for

information on high-performance liquid chromatograp hy (HPLC) solvent considerations.

1-1

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

Inlet

The detector can oper ate ei ther as a s ta nd-alone unit (w ith a char t record er 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.

1-2 Theory and Principles of Operation

The detector has the following capabilities:

• Stand-alone programmability – Stores up to five user- def ined 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 addi tion 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 als o 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 me thod programming,

storage, and retrieval from the front panel.

• Full diagnostic capability – Supports buil t-in diagnostic t ools 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 acros s 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, t he 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.

Detector description 1-3

Principles of operation

To use the detector effectively, you should be famili ar wit h i ts 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 the following:

• High brightness deuterium (D

• 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

) lamp

1-4 Theory and Principles of Operation

Waters 2489 UV/Visible Detector optics assembly

D2 lamp

Filter

wheel

Slit

Grating

TaperSlit

Flow Cell

Beamsplitter

Reference

photodiode

Ellipsoidal

mirror

Sample

photodiode

Window

Optional

cuvette

Spherical

mirror

Optics assembly light path

The detector provides an extremely effic ient design for exceptionally high light throughput. It operates as follows:

1. The ellipsoidal mirror collects li ght from the lamp and focuses it through the filter wheel 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 det ecto r’s f ront pane l (o r through Empower™ or MassLynx™ softwar e ), the detector rotates the grating to the appropriate position.

Principles of operation 1-5

4. The preamplifier board integrates and digi tizes the currents from the photodiodes for processing by the signal processing electronics and output to a computer, chart recorder, or integrator.

Waters TaperSlit Flow Cell

The Waters TaperSlit Flow Cell 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 res ult from temperature or pump-induced pressure fluctuations.

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 spec tral 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 Taper Slit bore geometry prevents light from hitting the cell walls. Four beams go in, and four beams come out.

1-6 Theory and Principles of Operation

Comparison of flow cell characteristics

Conventional Cell

Light

Window

Waters TaperSlit™

Analytical Cell

Light

Lens

Window

TP0153

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 variabl e path length flow c ell (path length 0. 15 to 3 mm) is also available.

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 data rate. If you select Other, you can enter a value. However, the value you enter will be rounded up or down to a value based on the data rate.

Principles of operation 1-7

The filter time constant adjusts the f ilter response time to achieve an optimal signal-to-noise ratio. Select ing Other and entering a value of 0.0 disables all filtering.

Lower time constant settings produce these effects:

• Narrow peaks with minimal peak distortion a nd time delay

• Very small peaks become harder to discriminate from baseline noise

• Less baseline noise is removed

Higher time constant settings produce these effects:

• Greatly decrease baseline noise

• Shorten and broaden peaks

The software includes fast or normal filtering cons tants at each data 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.

1-8 Theory and Principles of Operation

Filter Time Constant comparison

Tip: Although the peak shape shows some distortion and the signal output is

delayed with different time constants, the peak area remains the same.

Wavelength verification and test

The detector deuterium arc lamp and the integral erbium filter exhibit peaks in the transmission spectrum at known wavelengths.

Upon startup, the detector verifies calibration by comparing the locations of these peaks with expected wavelengt hs based on cali bration data stored in the detector’s m e m o r y . If th e results of thi s v e ri fication dif fer from the stor ed calibration by more than 1.0 nm, the detector displays a Wavelength Verification Failure message. The detector verif ies rather than r ecalibrates on startup to avoid errors that may occur because of residual material s left i n the flow cell and /o r the cuvette.

TP02833

Principles of operation 1-9

Requirement: Always ensure that the cuvette is removed and the cuvette

holder and front left panel door are secured during startup verification. You can initiate a manual wavelength calibration at any time. A manual

calibration replaces the previous calibration data with new data. See

“Wavelength calibration” on pag e 3-30 for the manual wavelength calibration

procedure. The verification and calibration algorithms are virtually iden tical. However,

the verification algorithm may issue an error message indicatin g that actual data does not match stored data , wher e the calib ration algor ithm rep lace s the stored data with the new data.

The detector wavelength verification procedures establish an approximate Home position using a grating homing sensor. Once Home is established, the detector locates and references the 656. 1-nm peak in the deuterium lamp emission spectrum.

The integral erbium filter moves into the common light path ahead of the fl ow cell entrance slit, enabling the detector to locate three additional spectral features at these wavelengths:

• 256.7 nm (UV)

• 379.0 nm

• 521.5 nm

The verification tests for the det ect or require 5 minutes of lamp warmup time so the lamp can stabilize.

If you run the detector continuously, Waters recommend s that you perform wavelength verification weekly by turning off the detect or, then turning it on again.

See also: “Wavelength calibration” on page 3-30.

1-10 Theory and Principles of Operation

Operational modes

The detector operates in single or dual wavelength mode, allows spectrum scanning using a flow cell or a cuvett e, and provide s RatioPlot , differe nce plot, and MaxPlot functions.

Single wavelength mode

Single wavelength is the default mode of operation for the detector. The detector supports monitoring of a single wavelength from 190 nm to 700 nm, settable in 1-nm increments on channel A. You can configure the analog outputs for channel B while the detector is operating in single wavelength mode, so you can use channel B to obtain additional information about the wavelength selected on channel A.

In single wavelength mode, the detector automatically engages the second order filter for wavelengths 370 nm and above and removes it for wavelengths under 370 nm. The second or der filte r is an op tical filt er that bloc ks unwanted ultraviolet (UV) light from striking the diff raction grating and interfering with absorbance detection above 370 nm.

You can configure several additional parameters when using the det ector in single wavelength mode.

Primary parameters

The following are the major parameters you can use in single wavelength mode:

• Wavelength in nm – Specifies a wavelength for channel A from 190 to 700 nm settable in 1-nm increments.

• Sensitivity in AUFS – Specifies the scaling factor for the analog output channels and corresponds to the absorbance unit (AU) value where the analog outputs saturate at full-scale values. Absorbance units full-scale (AUFS) vary from 0.0001 to 4.000 AU.

Note: Changing the sensitivity (AUFS) setting affects the 2-V output.

• Chart polarity (+ or –) – Reverses the polarity of the charted chromatogram. Select + for a normal chromatogram, or – for an inverted chromatogram. This function changes t he direction o f the plot on the 2 -V output, similar to reversing the leads to an external chart recorder.

Operational modes 1-11

• Filter time constant – Programs a filter time in seconds. Options are Fast, Slow, Normal, or Other. If yo u sele ct Fast, S low, or Nor mal, you d o not need to enter a value. The filter constant is determined by the data rate. If you select Other, you can enter a value, but the value you enter will be rounded up or down to a value based on the data rate. Selecting Other and entering a value of 0.0 disables all filtering.

• Analog rate – Specifies a value up to 80 Hz.

Secondary parameters

When on the absorbance (or HOME) screen in single wavelength mode, pressing Next brings you to several pages of these secondary, or less frequently specified, parameters:

• Absorbance offset (in mV)

• Auto zero on inject

• Auto zero on λ changes

“Primary and secondary functions” on page 3-14 and the Table titled “Absorbance and message screen icons” on page 3-4 explain the functions,

ranges, and defaults of these parameter s.

Dual wavelength mode

In dual wavelength mode, the detector can monitor two wavelengths, one on channel A and one on channel B. The sampling frequency is reduced to 1 or 2 Hz, limiting use of this mode to more standard chromatography where peaks span at least 20 seconds to enable full characterization of a peak. You can use dual wavelength mode to obtain additional information about an analyte by running a RatioPlot or a MaxPlot.

The detector allows you to select any two wavelengths from 190 to 700 nm. In dual wavelength mode, the following conditions apply:

• If both selected wavelengths are greater than 370 nm, the detector applies the second order filter to block unwanted UV light.

• If both selected wavelengths are less than or equal to 370 nm, the detector removes the second order filter.

• If the selected wavelengths bracket the 370 nm threshold, the detector does not apply the second order filter and issues a warning message that any data collected for the wavelength above 370 nm may contain

1-12 Theory and Principles of Operation

inaccuracies because of possible UV light interference (second order effects).

Chart Out selection modes

When operating in dual wavelength mode, the d etector off ers these c hoices f or analog output in addition to the selections off ered in single wavelength mode and explained in “Single wavelength mode” on page 1-11. The default selection for dual wavelength mode is Absorbance.

• Absorbance (A and B) – This is the standard LC mode where the curr ent absorption is scaled and sent directly out the analog output. The scaling depends on the AUFS setting and the absor bance offset. The abs orbance value is scaled for the 2-V analog output. If a setting of 1 AU/V is desired, you can set an AUFS of 2.0000 for either the A or B output channels that can be controlled independently, even in single wavelength mode.

• MaxPlot – This mode results in the output of the larger of the two absorbance values, scaled to the selected AUFS sett ing. This mode is useful when looking at multiple compounds with absorbancies at two separate wavelengths with one data channel.

• RatioPlot (A/B) – This mode produces the ratio of absorbance from two wavelengths. Theoretically, the ratio is constant for a pure chromatographic peak and variable f or an impure peak. This re sults in a nonsquared response. Instead of a programmable AUFS, the detector provides minimum and maximum ratio values that scale the ratio plot proportionally. In addition, a configurable minimum absorbance threshold activates ratio output scaling only when it reaches the absorbance at both wavelengths.

• Difference Plot (A-B) – This mode plots the arithmetic difference in absorbance for the two monitored wavelengths.

Spectrum scanning

Note: When the detector is operating under the control of the Empower

software, the scanning function is disabled. You can use the detector as a spectrophot ometer to acquire spectr a from either

the flow cell or the cuvette. You can scan and store up to three sp ectra (three reference or zero scans, or three sample scans) in memory f or playback or to compare with other spectra.

Operational modes 1-13

The major difference between the detector and a double-beam spectrophotometer is that the detector employs only one flow cell or cuvette, rather than a simultaneous sample and a reference pair.

Recommendation: Use a matched pair of cuvettes for the zero and sample

scans. The detector obtains an absorbance spectrum by perfor ming two types of scan

on the flow cell or using the cuvette:

• Zero scan – Characterizes the baseline abs orbance spe ctrum of a solvent.

• Sample scan – Subtracts the zero scan, so the results displayed or charted are of the sample only.

To obtain a spectrum of a sample with the detector, you need to run a zero scan first, followed by a sample scan. Typ icall y, the zero scan i s run wi th pure solvent and the sample scan is of the analyte dissolved in that solvent.

Spectra can be simultaneously charted on the channel A output, or acquired and stored in memory for later playback.

See also: “Scanning using the cuvette” on page 3-59 and “Scanning using a

flow cell and a syringe” on page 3-62.

Cuvette operations

The detector cuvette option is used to measure the absorbance spectrum of a sample in a cuvette.

To generate and store a spectrum:

1. Acquire a zero scan, which measures the absorbance of the contents of the cuvette and flow cell over the desired wavelength range.

2. Acquire a sample (absorbance) scan, which measures the absorbance of the analyte dissolved in mobile phase.

The detector subtracts the zero scan from the sample scan to create a sample spectrum.

Since the cuvette scan is acquired by measuring the absorbance from a light path that includes both the flow cell and the cuvet te, the so lvent co ndit ions in the flow cell should be identical for both scans. For a detailed explanation of cuvette scanning, see “Scanning using the cuvette” on page 3-59.

1-14 Theory and Principles of Operation

RatioPlot

The detector allows ratio plotting: comparing the absorbances of a compound or analyte at two different wavelengths. The RatioPlot divides absorbanc es at two selected wa v e l en gths and plots th e resulting rati o on a ch a r t re c order or data system over one output channel (channel A). The RatioPlot is useful in detecting hidden components present within individual peaks.

The RatioPlot of a spectrally homogeneous peak appears as a rectangular wave. The RatioPlot of an impure peak appears as a distorted wave. When obtaining a RatioPlot, you m ust be operating the detector in dual wavelength mode; the RatioPlot is output on the selected channel.

For an explanation of the RatioPlot procedur e, see the discussio n “Obtaining a

RatioPlot” on page 3-34.

MaxPlot

The MaxPlot function monitors absorbance at two selected wavelengths and plots the maximum absorbance value for each sample component. To obtain a MaxPlot, you must operate the detector in dual wavelength mode. The MaxPlot outputs the greater of the two absorbance values on the selected channel.

For an explanation of the MaxPlot procedure, see the discussion “Obtaining a

MaxPlot” on page 3-35.

Thermal wander management

To mitigate thermal instability caus ed 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. You will hear the fan change speeds in response to the thermal changes. This is normal operating behavior.

Operational modes 1-15

1-16 Theory and Principles of Operation

2 Installing the Detector

The Waters® 2489 UV/Visible Detector require s connec tio ns to e lectric al power and to sample and waste lines to operate in any standard laboratory environment. This chapter describes how to install the detector and connect it to the electrical supplies and to other equipment in an HPLC system.

Contents: Topic Page

Preparing for installation 2-2 Site selection and power requirements 2-4 Making fluid line connections 2-6 Making electrical power connections 2-9 Connecting to other devices 2-20

2-1

Preparing for installation

Major steps in installing the detector

Start installation

procedure

Select appropriate

site

Unpack and

inspect

Install detector

Make fluid line

connections

Make power connections

Make signal connections

Make connections

to other devices

Installation

complete

After installing the detector, you should verify its functions and keep the verified chart output (if applicable) on file. Verification ensures proper operation of the detector optics and electronics. For proper verification procedures, see “Verifying the detector” on page 3-27.

2-2 Installing the Detector

Detector dimensions

28.4 cm (11.2 inches)

20.8 cm (8.2 inches)

50.3 cm (19.8 inches)

TP02804

Caution: Do not access the instrument through the top cover. Access

the instrument through the front left p anel where the la mp housi ng, flow cell assembly, and cuv ette holder are located.

Preparing for installati on 2-3

Site selection and power requirements

Site selection

Install the detector in an area that meets the requirements listed in the table below.

Installation site requirements

Parameter Requirement

Operating temperature range

Storage temperature range

Relative humidity 20% to <95%, noncondensing Storage humidity range 0 to <95%, noncondensing Bench space 12.7 cm (5 inches) clearance at rear Vibration Negligible Static electricity Negligible Power Grounded ac, 100/240 Vac, 50/60 Hz

Requirement: You must mount the detector on a level surface to allow proper

function of the drip management system (drain tube), which you can connect to a waste reservoir to divert solvent leaks from the flow cell.

4 to 40 °C (39 to 104 °F)

–30 to 60 °C

Power cord type required:

• SVT in the United States

• HAR type (or better) in Europe For information regarding the type of

cord to use in other countries, contact your local Waters distributor.

2-4 Installing the Detector

Power requirements

Warning: To avoid electric shock,

• use the SVT- ty pe power cord i n th e Un i ted States an d H AR -type (or better) co r d i n E u r ope.

• power-off and unplug the detector before performing any maintenance procedures on the instrument.

• connect all HPLC system components to a common ground.

The detector requires

• grounded alternating current (ac) power source.

• minimal power transients and fluctuations.

• a line voltage of 100 to 240 Vac. (Power consumption is 185 volt amps.)

• operation in nominal voltage range of 100 to 240 Vac.

• a 3.15-A 250-V fast-blo fuse

Warning: To avoid electric shock, power-off the detector and unplug

the power cord before you replace the fuse.

Warning: To reduce the risk of fire hazard, always replace the fuse

with the same type and rating.

Unpacking and inspecting

The detector is packed and shipped in one carton that contains the following items:

• Waters 2489 UV/Visible Detector Startup Kit, which includes this guide

• Power cord

•Release notes

Site selection and power requirements 2-5

Unpacking

To unpack the detector

1. Unpack the contents of the shipping carton. As you unpack the carton, check the contents to make sure you have received all items.

2. Check the contents of the startup kit.

3. Save the shipping carton for future transpo rt or shipment.

Inspecting

If you see any damage or discrepancy when you inspect the contents of the carton, immediately contact the shipping agent and Waters Technical Service at 1-800-252-4752, U.S. and Canadian customers only. Other customers, call your local Waters subsidiary or your local Waters Technical Service Representative, or call Waters corporat e headquarters for assistance at 1-508-478-2000 (U.S.).

Tip: Make sure the instrument serial number, found on the nameplate on the

rear panel or inside the front left pane l of the detector, corresponds to the number on the instrument integrity certificate.

For more information about the instrument warranty, see Waters Licenses, Warranties, and Support Services.

Making fluid line connections

Before initial startup of the detector

1. Complete the fluid line connections described in this section.

2. Complete the electrical connections described in “Making electrical

power connections” on page 2-9.

Caution: Observe Good Laboratory Practices when handli ng

solvents. Refer to the Materi al Safety Data Sheets for the solvents in use.

2-6 Installing the Detector

Requirement: You must make the following fluid line connections on your

detector unit:

• Column connections

• Drip management system connections

Recommendations:

• Before completing the column con n e ctions, perform the veri fi c a ti on procedures described i n “Verifying the detector” on page 3-27.

• Install a waste reservoir connected to the drain tube, located next to the rubber foot on the front lower-left section of the detector.

• Use Tygon

tubing to connect the drain tube to the waste reservoir.

Caution: The detector, as shipped, ha s a standard analytical fl ow cell

pressure-rated at 1000 psi. To prevent damage, do not connect any tubing or device that might cause backpressure to excee d the pressure rating of the tubing or flow cell.

Connecting columns

The fluid line connections to the detector are located in the front right-hand side of the flow cell assembly (see the figure below).

To make the inlet and outlet tubing connections

1. Attach the stainless s teel compression fitting and ferrule (supplied in the startup kit).

2. Connect the inlet tubing to the column outlet. Ensure the tubing is seated firmly, then tighten the compression screw.

3. Connect the Tygon

tubing to the flow cell outlet tubing and route to a

waste container.

Making fluid line connections 2-7

Detector fluid line connections

Inlet (labeled)

Outlet

Assembling the fittings

To assemble each fitting

1. Slide the compression screw over the tubing end, followed by the ferrule.

2. Mount the ferrule with its taper end facing the end of the tubing.

2-8 Installing the Detector

TP02810

Ferrule and compression screw assembly

Compression screw

Ferrule

Tubing end (straight and

Tube

TP01139

Distance (determined by each application, such as uni on or col umn fittin g)

smooth to achieve maximu column efficiency)

Making connections

To make connections at the column outlet and detector inlet, and at the detector outlet

1. Seat each tubing end in the appropriate fitting.

2. Seat each ferrule by tightening the compression screw 1/2-t urn past finger-tight.

Requirement: To ensure accu ra t e verificati o n , p o w e r-on the dete ct or before

pumping any mobile phase or solvent through the flow cell.

Recommendation: To prevent reabsor ption of dissolved oxygen (for systems

using vacuum degassers), Waters recommends that you run the solve nt degasser continuously when operating the detector at wavelengths less than 230 nm.

Making electrical power connections

To connect the detector to the ac power supply:

1. Plug the receptacle end of the power cord into the ac input connector on the rear panel of the detector (see the figure below).

2. Plug the other end of the power cord into a properly grounded ac power source.

Making electrical power connections 2-9

Detector rear panel

The detector connects to other Waters components through rear panel electrical connections.

Detector rear panel electrical connections

Inputs and

Outputs

Fan Vent

Power input

Fuse holder

TP02807

Rear panel connections enable the following signals:

• Analog outputs – There are two pairs of attenuated analog channel outputs with each pair supporting 2-V output to external devices or data systems. They are labeled I and II. For input/output voltage current specifications, see the Table titl ed “Operational specifications” on

page B-2.

– The 2-V output for I and II is scaled based on the AUFS (absorbance

units full scale) setting for each channel. The detector does not provide a traditional “unattenuated” dedicated 1-V/AU output

because of its enhanced working range above 2 AU. – The detector analog output range specifications are – 0.1 V to 2.1 V. – You can set the AUFS value individually for the output on each

channel. Volts per AU are calculated as follows:

Volts out = Absorbance × 2V/AUFS

2-10 Installing the Detector

Example: An AUFS setting of 2.0000 provides a traditional 1-V/AU

output. An AUFS setting of 4.0000 provides a 0.5-V/AU output,

which supports chromatography above 2 AU.

• Switched outputs – There are two switch contact closures you can program to turn on, off, toggle, pulse once for a defined duration, or pulse repetitively for a specified period of time.

• Event inputs – There are four general-purpose TTL contact closures on the detector A (inputs) terminal that support the following functions:

– Remote or inject start –Lamp on/off – Chart mark input –Auto zero

• Ethernet interface – The Ethernet connection on the rear panel of the detector allows remote control and direct data acquisition from Waters Empower and MassLynx workstations.

Making signal connections

The rear panel of the detector (see the fi gure on page 2-10) pro vides two analog connectors and an Ethernet communications port for operating the detector with e x te r n a l de vi ces.

Make the signal connections to your detector, considering the following conditions:

• Operating mode you select for the detector (stand-alone or remote control)

• Types of instruments that make up your HPLC system

This section describes the input/output (I/O) and digita l signal connections that you can make from the two rear panel connectors and the Ethernet connector.

The figure below shows an overview of the steps involved in making signal connections to the detector.

Making electrical power connections 2-11

Overview of making signal connections

Start signal

connection procedure

Connect to Ethernet bus?

Connect to other devices?

Signal connections

complete

Yes

Install Ethernet

and inject start cables

Install event and I/O

cable(s)

Making I/O signal connections

The rear panel includes two removable connectors that hold the pins for the I/O signals, as shown in the figure below. These connectors are keyed so that you can insert them one way only.

2-12 Installing the Detector

I/O signal inputs and outputs

TP01494

I/O signals

The table below describes each of the signals available on the I/O connectors. Refer to Appendix B for details on the electrical specifications for the signals.

I/O signals for the detector

Signal Description

Inject Start

Lamp On/Off

Chart Mark

Auto Zero

Analog 1

TTL contact closure. Configurable input to initiate sequencing of time-program med events. Defines the start of a run (typically an injection) and resets and starts the run-time clock to 0.00 minutes. Initial conditions apply immediately.

Configurable input to allow an external device to turn the deuterium lamp off and on.

Configurable input to add a chart mark (at 10% of full scale) to either or both analog output channels.

Configurable input to auto zero either or both channels.

2-V full-scale analog output signal of cha nnel A (sca led to the current AUFS setting).

Making electrical power connections 2-13

I/O signals for the detector

Signal Description

Analog 2

2-V full-scale analog output signal of cha nnel B (sca led

to the current AUFS setting). Switch 1 (2) Used to connect to a fraction collector. Can be Switch 2 (2)

a. Inject Start, Chart Mark, Auto Zero, and Lamp inputs are configurable from the detector first

Configuration screen by setting the appropriate parameter to High. See “Configuring event

inputs (contact closures)” on page 3-23 for more detail.

b. See the discussion of attenuation in the detector analog outputs in “Making electrical powe r

connections” on page 2-9.

controlled by threshold and timed events.

Making Ethernet connections

The detector rear panel also includes one Ethernet interface connector for digital signal communications. Use this connector for devices such a s thes e:

• Network ad a pter card in the E m power work st a ti o n

• Solvent manager

• MassLynx version 4.1 or higher

The Ethernet connector m ates with standard Ethernet cable.

Caution: To avoid possible damage to components, power-off all

instruments on the Ethernet connector before you connect an Ethernet cable to an instrument.

Making Ethernet connections with Waters data systems

When controlling the detector from a Waters data system or controller (Empower or MassLynx workstation), you can use the Ethernet interface to send and receive information from the data system.

When connecting via the Ethernet to these Waters data systems , you should be aware of the following:

• When in dual wavelength mode, you m ust select a data rate of 1 point per second in the data system method editor. In Empower software, the data rate para me te rs defaults to 1 point per secon d.

2-14 Installing the Detector

• The maximum range of the detector time constant setting depends on the wavelength mode and data rate selected. See the Table titled

“Primary and secondary function (method) parameters” on page 3-19.

• Empower allows the detector to operate in both single and dual wavelength modes with a wavelength range of 190 to 700 nm, and up to

4.0 AUFS.

To connect Ethernet cables from the detector to a Waters data system:

1. Connect the single receptacle end of the Ethernet cable to your data system by attaching the cable to the network adapter (laboratory acquisition and control environment, or LAC/E).

Note: The Ethernet cable is supplied with the Waters data system.

2. Connect the other end of the cable to the Ethernet connector on the detector rear panel.

Caution: The maximum total cable length between Ethernet devices

in a system is 20 meters (65 feet). The maximum recommended cable length between two Ethernet devices is 4 meters (13 feet). Longer total cable lengths can cause intermittent Ethernet co mmunication failures.

Ethernet connections to the detector in a Waters Empower System

busLAC/E or LAC/E32 card

Empower

workstation

Ethernet ca ble

Ethernet

connector

2489

Detector

Tip: When connecting the detector to a data system, all detec tor parameters

not configurable by the data system in use defer to local control.

Making electrical power connections 2-15

Starting a met hod

To start a method on the detector at the start of an injection from the separations module, make the c onnections summarized in the t able below and illustrated in the figure below.

2695 Separations Module (B Inputs and Outputs)

2489 Detector (II)

Pin 1 Inject Start Pin 1 Inject Start + Pin 2 Inject Start Pin 2 Inject Start –

2695 separations module connections to the detector for starting a method

Waters 2695

Waters 2489

Turning the detector lamp on or off

Before you ca n tu rn the detector la mp on or off from th e s ep a rations modu l e , you need to configure the lamp on/off signal at the front panel. You must change the default lamp configuration p arameter f rom Ignore to High or Low. See the discussion “Configuring event inputs (cont a ct closures)” on page 3-23 for more information.

2-16 Installing the Detector

TP01525

After configuring the detector lamp on/off signal, you can turn the lamp on or off from the separations module by making t he connecti ons shown in the t able and figure below.

Detector connections to separation module (lamp on or off)

2695 Separations Module (A Outputs)

2489 Detector (II)

Pin 1 Switch 1 Pin 4 Lamp On/Off + Pin 2 Switch 1 Pin 5 Lamp On/Off –

Separations module connections to the detector for turning the lamp on or off

Waters 2695

Waters 2489

TP01524

Making electrical power connections 2-17

Connecting the detector to a separations module

You can connect the detector to the separations module, when it is not under the control of the Empower software, to perform the following functions:

• Auto Zero

• Chart Mark on Inject

Generating Auto Zero

To generate the Auto Zero function on the detect or at t he start of an injectio n, make the connections summarized in the table and illustrated in the figure below.

Detector connections to a separ ation module to generate Auto Zero

2695 Separations Module (B Inputs and Outputs)

Pin 1 Inject Start Pin 9 Auto Zero + Pin 2 Inject Start Pin 10 Auto Zero –

Before you can generate an auto zero from the separations module, you need to configure the auto zero signal at the detector front panel. The de fault auto zero signal is Low. See the discussion “Configuring event inputs (cont act

closures)” on page 3-23 for more information.

2489 Detector (II)

2-18 Installing the Detector

Separations module connections to the det ector for Auto Zero on Inject

Waters 2695

Waters 2489

TP01527

Generating Chart Mark on Inject

To generate the Chart Mark function on the detector at the start of an injection, make the connections summari zed in the table and illustrated in the figure below.

2695 Separations Module (B Inputs and Outputs)

2489 Detector (II)

Pin 1 Inject Start Pin 6 Chart Mark + Pin 2 Inject Start Pin 7 Chart Mark –

Before you can generate a chart mark from the separations module, you need to configure the chart mark signal at the detector front panel. The default chart mark signal is Low. See the discussion “Configuring event inputs

(contact closures)” on page 3-23 for more information.

Making electrical power connections 2-19

Separations module connections to the detector for Chart Mark on Inject

Waters 2695 Separations Module

Connecting to other devices

Waters 2489

TP01526

You can connect the detector to a wide range of HPLC system devices. This section describes how to connect the det ector to the following devices:

• e-SAT/IN™ module (instead of Ethernet)

• Waters 745/745B/746 Data Module

• Chart recorder

• Waters 600 Series Pump

• Waters 717plus Autosampler

• Waters fraction collector

For details on connecting to other dat a modules , see the operator’s guide for the module you are using.

2-20 Installing the Detector

Required materials

When connecting cables to the terminals on the rear panel of the detector, you need the following tools:

• Small flat-b l a de sc re w d r iver

• Electrical insulation stripping tool

Connecting the cables

To connect cables from other HPLC system devices to the I and II terminals on the rear panel of the detector :

1. Remove terminal I or II (see Figure “I/O signal inputs and outputs” on

page 2-13).

2. Unscrew the connecting pin terminal.

3. Using the stripping tool, strip the wire down about 3 mm (1/ 8 inch ) from the end.

4. Insert the st ri pped wire into th e ap propriate con n e ct or.

5. Tighten the screw until the wire is held securely in place.

6. Reinsert the terminal.

7. Press firmly to ensure that it is inserted fully.

Connecting the detector to Empower using an e-SAT/IN module

Acquiring da ta and contro l l ing the detect o r wi th Empower so ftware, by usi n g the e-SAT/IN module instead of the Ethernet bus, requires connections between the following hardware:

• Laboratory acquisition and control environment (LAC/E) module (LAC/E

• Ethernet satellite interface (e-SAT/IN) module

e-SAT/IN module

The Waters e-SAT/IN module shown in the figure below translates analog signals from devices such as the detector into digital form. It then transmits

these digital signals to the busLAC/E or LAC/E Empower workstation.

Acquisition Server or busLAC/E card)

Connecting to other devices 2-21

card installed in the

e-SAT/IN module (front panel)

Power switch

LEDs

I/O connector

Analog inputs

To connect the detector to the Empower workstation:

1. Connect the detector to the e-SAT/IN module. (See “Connecting the

detector to th e e- SA T / IN module” on pag e 2-23.)

Caution:

• The e-SAT/IN module does not have a power switch. To prevent damage to the module, always disconnect the power cord at either the wall outlet or the power supply before attaching or removing the power connection to the e-SAT/IN module.

• To ensure proper startup of the e-SA T/IN module, do not turn on power to the module until you perform all procedures described in the Waters e-SAT/IN Module Installa ti on Guide. Improper startup can damage the unit and void the warranty.

TP02834

2-22 Installing the Detector

Connecting the detector to the e-SAT/IN module

The e-SAT/ IN module conn e ct s to the detecto r th rough the B (i np u ts and outputs) terminal on the rear panel of the detect or, as shown in the figure below.

To connect the detector to the e-SAT/IN module:

1. Using the electrical insulation stripping tool, strip off about 3 mm (1/8 inch) from one end of the e-SAT/IN 9-pin connector, exposing the white and black wires.

2. Connect the other end of the cable to either the Channel 1 or Channel 2 connectors on the front panel of the e-SAT/I N module.

3. For Channel 1: a. Connect the white wire to pin 1 on I (Analog 1 +). b. Connect the black wire to pin 3 on I (Ground).

e-SAT/IN module Channel 1 connection to detector

Waters 2489

(I)

4. For Channel 2: a. Connect the white wire to pin 4 on I (Analog 2 +). b. Connect the black wire to pin 8 on I (Ground).

Connecting to other devices 2-23

e-SAT/IN module Channel 2 connection to detector

Waters 2489

(I)

5. Configure the serial port for the e-SAT/IN module as described in the Empower 2 Installation and Configuration Guide.

The table below summarizes the detector connectio ns to the e-S AT/IN module .

Detector connections to a e-SAT/IN module

2489 Detector (I)

Pin 1 Analog 1 + (white) Pin 3 Ground (black) Pin 4 Analog 2 + (white) Pin 8 Ground (black)

2-24 Installing the Detector

e-SAT/IN Connector

Channel 1 or 2

Connecting the detector to a 745/745B/746 data module

You can connect the detector to a Waters 745/745B/ 746 data module using the analog output connector on the rear panel of the detector. The analog connector provides 2-V output that is scaled to the AUFS sensitivity setting and the voltage offset setting.

Caution: To prevent oversaturation of the signal from the detector to

the integrator, do not exceed the input voltage rating of the integrator.

To send the analog output signal from the detector to the dat a module, use the cable provided in the 2489 Detector Startup Kit to make the connections summarized in the table below and illustrated in the figure below.

Detector connections to a data module

2489 Detector (I)

Pin 1 Analog 1 + (red) + Pin 3 Ground (black) – Pin 4 Analog 2 + (red) + Pin 8 Ground (black) –

To minimize the chance of creating a ground loop that can adversely affect measurement, connect the shield of the cabl e to the chassi s ground at one end only.

745/745B/746 Terminal

Connecting to other devices 2-25

Data module connections to detector channels A and B

Waters 2489

TP01485

Waters 2489

2-26 Installing the Detector

TP01486

Connecting the detector to a chart recorder

Recorder signal

The A and B terminals on the rear panel of the detector provide 2-V analog output signals that you can use to connect the detector to a chart recorder.

To send 2-V signals from the detector to a chart recorder, use the cable provided in the 2489 Detector Startup Kit to make the connections summarized in the table and illustrated in the figure below.

Detector connections to a chart recorder

2489 Detector (B Inputs and Outputs)

Pin 1 Analog 1 + + Pin 3 Ground – Pin 4 Analog 2 + + Pin 8 Ground –

To minimize the chance of creating a ground loop that can adversely affect measurement, connect the shield of the cabl e to the chassi s ground at one end only. For connection to other data systems, a cable (part number 44000172) can help remove the incidence of ground loops.

Tip: The detector is optimized for use with the 2-V analog output.

Connect the detector to a chart recorder using the 2-V analog connection as shown in the following figures.

Chart Recorder Terminal

Connecting to other devices 2-27

Chart recorder 2-V output connections on the detector

Waters 2489

TP01487

Waters 2489

Chart marks

You can generate a chart mark from the front panel of the detector. A chart mark signal is generated whenever one of the following occurs:

• You press the Chart Mark key on the detector ke yp ad.

2-28 Installing the Detector

TP01488

• You program a timed event to generate a chart mark.

• A signal is received from one of the chart mark inputs on the analog connector.

Connecting the detector to the Waters 600 Series Pump

To connect the detector to the pump, locate the detector in a position that satisfies the sit e re q u irements in “Site selection and power requirements” on page 2-4.

Fluid line connections

Make the fluid line connections described in “Making fluid line connections” on page 2-6.

Lamp on/off connections

In addition to making the connections described below, you must configure the lamp on/off signal at the detector front panel. You must change the default from Ignore to High or Low. See the discussion “Configuring event inputs

(contact closures)” on page 3-23 for more information.

Using signal cable, make the connections between the pump controller and the detector that are summarized in the table and shown in the figure below.

Pump and detector lamp on/off connections

Waters 2489 Detector (II)

Pin 4 Lamp On/Off + S1, S2, or S4 Pin 5 Lamp On/Off – GND

Waters 600 Serie s Pump Terminal

Connecting to other devices 2-29

Pump lamp on/off connections

Waters 2489

CHART +

PRESSURE +

CHART

PRESSURE

INJECT

STOP

FLOW

_ _

GND

HOLD

GND

SWITCHES

GND

AUX.

+12V

Auto Zero connections

To make Auto Zero connections between the detector and the pump:

TP01489

1. Using a signal cable, make the connections between the detector and the pump that are summarized in the table and illustrated in the figur e below.

2. Program the pump to prov ide a pulse output on the applicable switch (S1, S2, or S4) at the beginning of each run. Refer to the Wate rs 600E Multisolvent Delivery System User’s Guide, Section 5.1.2, for more details.

Pump and detector Auto Zero connections

Waters 2489 Detector (II)

Pin 9 Auto Zero + S1, S2, or S4 Pin 10 Auto Zero – GND (one of four)

2-30 Installing the Detector

Waters 600 Series Pump Terminal

Pump Auto Zero connections

Waters 2489

CHART +

PRESSURE +

CHART

PRESSURE

SWITCHES

AUX.

+12V

INJECT

STOP

FLOW

HOLD

_ _

GND

Chart Mark connections

To make Chart Mark connections between the detector and pump:

TP01523

1. Using a signal cable, make the connections between the detector and the pump that are summarized in the table and illustrated in the figur e below.

2. Program the pump to provide a pulse output on the select ed switch at the beginning of each run. R efer to the Waters 600E Multisolvent Delivery System User’s Guid e for more details.

Pump and 2489 detector Chart Mark connections

Waters 2489 Detector (II)

Waters 600 Series Pump Terminal

Pin 6 Chart Mark + S1, S2, or S4 Pin 7 Chart Mark – GND (one of four)

Connecting to other devices 2-31

Pump Chart Mark connections

Waters 2489

CHART +

PRESSURE +

CHART

PRESSURE

SWITCHES

AUX.

+12V

INJECT

STOP

FLOW

HOLD

_ _

GND

Inject Start connections

Tip: If the detector is connected to an Empower data system, use the Inject

Start connections to initiate the start of data acquisition.

TP01491

T o make Inject St art connections between the pump and the detector to st art a method:

1. Using a signal cable, make the connections between the detector and the pump that are summarized in the table and illustrated in the figur e below.

2. Program the pump to provide a pulse output on the select ed switch at the beginning of each run. R efer to the Waters 600E Multisolvent Delivery System User’s Guid e for more details.

2-32 Installing the Detector

Pump and detector Inject Start connections

pump and detector Inject Start connections

Waters 2489 Detector (II)

Pin 1 Inject Start +

Waters 600 Series Pump Terminal

S1, S2, or S4

Pin 2 Inject Start – GND (one of four)

a. You can also connect the pump inject pin to the Pin 1

Inject Start + on the detector, and the inject ground pin to the Inject Start – on the detector.

Pump Inject Start connections

CHART +

PRESSURE +

INJECT

STOP

FLOW

GND

HOLD

GND

SWITCHES

GND

AUX.

+12V

Waters 2489

TP01493

Connecting the detector to the Waters 717plus Autosampler

The Waters 717plus Autosampler signals the start of an injection through a contact closure signal on its Inject Start terminals. You can use this contact closure signal to command the detector to perform an auto zero at the start of an injection.

Connecting to other devices 2-33

Auto Zero connections

To auto zero the detector at the start of an injection, make the connections described in the table and illustrated in the figur e below. Use any available pair of Inject Start te rminals on the autosampler.

Autosampler and detector Auto Zero connections

2489 Detector (A Inputs)

717plus Autosampler Terminal

Pin 9 Auto Zero + Inject Start + (any

one of three paired with –)

Pin 10 Auto Zero – Inject Start – (any

one of three paired with +)

Autosampler Auto Zero connections to the detector

Waters 717plus

Waters 2489

123456789101112131415

2-34 Installing the Detector

TP01522

Inject Start connections

You can also use the Inject Start connections on the autosampler to connect to the Inject Start signals on the detector to program the start of the active method.

To program an inject start, make the connections described in the table and illustrated in the figure below. Us e any available pair of Inject Start terminals on the autosampler.

Autosampler and detector Inject Start connections

2489 Detector (II)

717plus Autosampler Terminal

Pin 1 Inject Start + Inject Start + (any one of three

paired with + )

Pin 2 Inject Start – Inject Start – (any one of three

paired with – )

Detector Inject Start connections

Waters 717plus

Waters 2489

123456789101112131415

TP01522A

Connecting to other devices 2-35

Connecting the detector to a fraction collector

The detector can trigger a fraction collector based on

• Timed events (see “Timed events” on page 3-36).

• Threshold levels (see “Threshold events ” on page 3-39).

You can connect the fraction collector to one of the detector’s two programmable switches (SW1 or SW2) and program the timed event, threshold, or ratio at the detector front panel.

You can also connect the fraction collector to the detector to trigger a chart mark event input each time a tube is changed at the fraction collector.

The table below indicates the correct detector-to-fraction collector and autoinjector-to-fraction co llector connections.

Detector connections to the fraction collector

Waters 2489 Connection Waters Fraction Collector

I Pin 3 Ground Pin 1 Detector In – II Pin 6 Chart Mark + Pin 10 Event Marker + II Pin 7 Chart Mark – Pin 9 Event Marker – I Pin 6 SW1 Pin 7 External Count In + I Pin 8 Ground Pin 8 External Count In –

Waters 2695 Separations Module/717plus Autoinjector

Inject Start + External Start In + Inject Start – External Start In –

See also: Refer to the documentation provided with your fract ion coll ector for

complete informati on.

2-36 Installing the Detector

3 Preparing the Detector

Contents: Topic Page

Initializing the detector 3-2 Using the operator interface 3-3 Scanning spectra 3-44

After you install the detector, you are ready to set it up and operate it as a stand-alone instrument or as part of a data system.

• As a stand-alone instrument − You can use the detector as a stand-al one detector within a system, or with any fluid-handling unit, injector, integrator, or data sy stem. You can program the front panel of the detector for stand-alone operation, except when it is in remote mode.

See also: “Operating the detector” on page 3-26.

• As part of an Empower system − Use the detector configured with an Empower system to control and collect digital data. To configure the detector with this s yst em, foll ow the i nst ruct ions i n t he Empower online Help to specify pa r a m e ters and cont ro l th e de tector.

• As part of a MassLynx system − Use the detec tor configured with a MassLynx system to control and collect digital data. To configure the detector with this system, follow the instructions in the MassLynx online Help to specify parameters and cont rol the detector.

Requirement: To ensure accurate operation, and before pumping any mobile

phase or solvent through the flow cell, be sure to perform the procedures in

“Verifying th e detector” on page 3-27.

3-1

Initializing th e detector

Before you power-on the detec tor, be sure the connect or from the det ector re ar panel to the power source is properly installed.

To power-on th e de te ctor, press th e O n /Off switch lo c at e d o n th e front, lower-right corner of the unit.

At startup, the detector beeps thr ee times and runs a series of startup diagnostic tests. If it passes all startup diagnostic tests, the startup diagnostics OK messages appear.

Detector Startup Diagnostics display

STARTUP DIAGNOSTICS

TPU

SCI

OK OK

GPIB

QSPI

Following display of the Startup Diagnostics screen, the detector displays the following series of messages in sequence for about five minutes:

ROM RAM LCD CPU

OK OK OK OK

• Initializing grating

• Initializing system

• Lighting lamp

• Warmup time left: n minutes

• Homing optical filter

• Searching for 656 nm

• Optimizing system performance

• Finding calibration peaks

•Restoring last setup

• Completing initialization

When initialization is complete, the detector displays the absorbance screen shown in the figure below. The sections “Using the keypad” on page 3-6 and

“Navigating the user i nterface ” on page 3-12 provide more info rmation on t his

and subsequent screens.

3-2 Prepari ng the Detector

Tip: For normal use, allow the detector to warm up for at least 30 minutes

before operating.

Diagnostic test failure

If one or more of the internal startup diagnostic tests show a failin g result, the detector beeps and displays an error message. For serious errors, it displays the word “Error” in brackets (<Error>) in place of the run-time absorbance on the absorbance screen.

See also: “Error messages” on page 5-2, for a list of startup diagnostic test

failures, error message s, and recommended recovery actions. See “Hardware

troubleshooting” on page 5-20 for hardware-related causes of startup

diagnostic test failure and corrective actions.

Using the operator interface

Using the display

The detector employs a 128 × 64-bitmap graphic display and a 24-key membrane keypad for the operator interface. After the startup diagnostic tests are run successfully, the detector displays the absorbance (or HOME) screen.

Detector absorbance screen

Absorbance

Channel selector

Wavelength

Sensitivity

Lamp on/off

Shift on/off

Single/Dual wavelength

Keypad lock/unlock

Local (method #)/Remote control

Run time (minutes)

Sticky Diag nos tic test on/ of f

You can recall the absorbance screen at any time by pressing the HOME key. At the first use of the detector, the absorbance screen shows the factory-set defaults. After the firs t use, the absorbance screen shows the settings

Using the operator interface 3-3

displayed before the detector was last powered-off. The absorbance screen continues to change as the run continues.

The detector monitors absorbance of one or two wavelengths in real time, while allowing you to modify all the parameters discussed in the table below. You can use the A/B key to toggle between absorbance screens for channels A and B.

Absorbance and message icons

The absorbance screens and message screens in the detector program display the icons or fields shown in the figure on page 3-3 and described in the table below. For a list of ranges and defaults for the function icons and fields described in this table, see the Table titled “Primary and secondary function

(method) parameters” on page 3-19.

Caution: Changing the sensitivity (AUFS) setting affects the 2-V

output. For example, 1 AU gives 0.5 AU/V and 2 AU gives 1 AU/V.

Absorbance and message screen icons

Icon or Field Icon / Field Name Funct ion

Field requiring entry

3-4 Prepari ng the Detector

Sensitivity or AUFS

Wavelength Selects the wavelength monitored on the

Channel Selector Displays which channel is selected when

Selects the chart sensitivity in absorbance units full-scale (AUFS) for the selected channel (Ethernet signal is not affected).

selected channel. When in single wavelength mode there is no ind ependent control of wavelength on channel B.

you press the A/B key. The selected channel overlaps the other channel.

Displays the ON A or ON B icon for the channel on which a timed or threshold event is programmed.

When you press the TRACE key, displays only the channel being viewed.

Absorbance and message screen icons

Icon or Field Icon / Field Name Funct ion

Numerical field

Absorbance Displays current absorbance for the

selected channel.

Lamp On/Off On = Lamp icon

Off = Lamp icon with an X through it

Shift On/Off Blank = Shift off

▲ = Shift on

Single/Dual Wavelength

λ = Detector operating in single wavelength mode λλ = Detector operating in dual wavelength mode

Keypad Lock/Unlock

Sticky Diagnostic icon

Open lock = Unrestricted keypad entry Closed lock = Parameter changes not allowed

When the absorbance screen displays the wrench icon, a sticky diagnostic test is active. See “User-selected diagnostic

tests” on page 5-8 for a complete

explanation.

Using the operator interface 3-5

Absorbance and message screen icons

Icon or Field Icon / Field Name Funct ion

Numerical field

Local (Method #) /Remote Control

Run Time (Minutes)

Next Indicates that pressing Next brings you

Local control/Method number – If the detector is n ot controll ed by a data system or other control device over the Ethernet bus, it displays a cursive “m” and the current method number or an asterisk (*) that indicates current conditions are not stor ed as a method.

Remote control/Ethernet – If the detector is controlled by a data system or other control device over the Ethernet bus, it displays a remote control icon cont aining the letter E.

Displays the time elapsed since you pressed the Run key, or since an Inject Start signal was received.

to additional screens.

Message screen icons (from left): Error, Question, Warning, Information, and Standby.

Using the keypad

The detector keypad (shown below) consists of 24 keys providing

• full numeric entry (10 digits plus a decimal point).

• Enter, Shift, CE (Clear Entry), Next, and Help functions.

• ▲ and ▼ (used for navigation only; pressing ▲ may also move you to the left, ▼ to the right).

• A/B for channel selection.

• navigation to specific screens (HOME [absorbance], DIAGnostics, TRACE, CONFIGURE, METHOD, and SCAN).

3-6 Prepari ng the Detector

• primary function keys (Chart Mark, Auto Zero, and Run/Stop).

• secondary function keys (Scale, Single or Dual Wavelength, Res et Clock, Lamp, Lock, Calibrate, System Information, Contrast, Previous, Cancel, +/–, and Clear Field).

Detector keypad

HOME

METHOD Calibrate

A/B

CONFIGURE

DIAG

Scale

TRACE

Shift

SCAN

Chart Mark

Lamp Lock

System Info

Cancel

λ/λλ

Auto Zero Run/Stop

+/−

Reset

Contrast

Clear Field

Enter

Primary function keys take effect imm ediately, with no further entry required. Secondary function keys require you to enter information into parameter fields and to press Enter for the functions to take effect.

Keys that appear in all caps (HOME, DIAG, TRACE, METHOD, CONFIGURE, and SCAN) take you directly to a function from most screens.

For numerical entries from 1 to 9 on choice lists or menus, enter the number corresponding to the desired item, then press Enter. For the number 10, select 0, then pres s E n te r. To go to the en d of a choice list, select •. For entries

Using the operator interface 3-7

numbered 11 or 12, scroll to the desired item on the choice list, then press Enter.

The table below explains the functions of the primary and secondary keys on the detector k e y pa d.

Detector keypad description

Description

Key

Unshifted Shifted

HOME

SCAN

Chart Mark

λ/λλ

Auto Zero

Reset

Run/Stop

HOME – Displays the absorbance screen containing the icons, and the Wavelength and AUFS fields.

Chart Mark – Causes a momentary pulse to the analog output (A and/or B, depending upon the current settings). This key has no effect if chart mark is disabled on both channels.

Auto Zero – Sets the absorbance offset so that the analog output (A and/or B, depending on the current settings), reads 0 AU. You can enable or disable the auto zero function from the third absorbance screen (see the figure on page 3-18).

Run/Stop – Starts or stops (freezes) the run clock. (The elapsed time appears near the lower-right corner of the absorbance screen.) Initiates scans.

? – Displays context-sensitive help when available.

SCAN – Displays the list of options for generating and manipulating spectra.

λ/λλ – When you are on the absorbance screen, use this key to toggle between single and dual wavelength modes. The current mode is i ndica ted by an icon on the display.

Reset – Resets the detector run clock to 0 minutes. Returns the detector to initial conditions for the current method.

3-8 Prepari ng the Detector

Detector keypad description

Description

Key

Unshifted Shifted

Arrow keys − On screens with entry fields (edit, check box, or choice list), the active field has a thick border (highl ight). The arrow keys can b e used to make a differe nt fi e l d a ct i ve . (U p moves up or left; Down moves down or right.) On screens with a scrollable list, these keys move the highlight up (toward the beginning of the list) or down (toward the end). Other s creens may have special instructions for the use of the Up and Down arrow keys (for example, the Display Contrast screen).

METHOD

A/B

CONFIGURE

DIAG

Scale

TRACE

Next – Displays a screen with additional options related to the current screen. Repeated pressing of this key always brings you back to where you started. On most screens where this key is active, the NEXT arrow appears in the lower-right corner of the display.

A/B – On screens that have the A/B icon in the upper-left corner, this key toggles between channel A and channel B parameters.

DIAG – Displays the choice list of diagnostic tests.

TRACE – Displays the absorbance monitor trace for channel A or B.

Previous – When the Next key is available, Previous navigates through the screens in the reverse order.

METHOD – Displays the list of options for creating and clearing timed and threshold events, storing, retrieving, and resetting methods.

CONFIGURE – Displays the first Configuration screen.

Scale – When the wavelength trace or spectrum screen is visible, this function permits modification of the disp lay range in the X (time or wavelength) and Y (absorbance) dimensions.

Using the operator interface 3-9

Detector keypad description

Description

Key

Unshifted Shifted

Shift – Enables the shifted functions (identified by the text at

Shift

the top of most keys). The shifted state is temporary, and is reset after th e n ext ke y stroke.

0-9

Lamp

Lock

0-9 – Enters the corresponding number into

0-9 – See descriptions for

specific shifted numeric k eys. the current field. Also positions the cursor at the corresponding entry in a list (0 = tenth item). Selects the corresponding number from a choice list.

1 – See 0-9 above. Lamp – Displays the lamp use

statistics for the currently

installed lamp and allows you

to turn the lamp on or off. The

current state of the lamp is

indicated by an icon on the

absorbance screen. 2 – See 0-9 above. Lock – When you are on the

absorbance screen, enables or

disables the keypad lock

feature. Use the lock to

prevent inadvertent changes

to detector settings . The

current lock state is indicated

by an icon on the absorbance

screen.

Calibrate

3-10 Preparing the Detector

3 – See 0-9 above. Calibrate – Initiates the

wavelength calibration

routine.

Detector keypad description

Description

Key

Unshifted Shifted

System Info

Contrast

Cancel

+/–

•

Clear Field

Enter

4 – See 0-9 above. System Info – Displays

system information including

firmware version and

instrumen t se ri a l nu mber. 6 – See 0-9 above. Contrast – Permits

adjustment of contrast

(viewing angle) of the liquid

crystal display. 0 – See 0-9 above. Cancel – In some modes,

Cancel backs out of a prompt

without completing the task .

The word “Cancel” appears as

a cue in the lower-right border

of the message text.

• – Enters a decimal point. Also positions the cursor at the last entry in a list.

+/– – Some edit fields accept

negative number entry. Use

this function to invert the sign

of the number in the active

field. CE – Clears an editing

change, and returns the contents of a field to its

Clear Field – Clears the

current entry field before you

enter the desired values. previous value. Sets the value to a unique word for some fields. For example, in the voltage offset diagnostic display, you can enter e ither a numeric offset value or press CE to change it to OFF.

Enter – Completes the entry in an edit field. Also advances the active field as if the Down arrow had been pressed (except after editing the wavelength on the absorbance screen). Press Enter to acknowledge error messages and other prompts. In these cases, the word “Enter” appears as a cue in the lower-right border of the message text.

Using the operator interface 3-11

Navigating the user interface

To operate the detector:

1. Press the Enter key or the up and down arrow keys to navigate among editable fields on a display.

Tip: A thick border appears around the active field.

2. Press Enter to advance the active field once you have completed an entry.

3. When you make an error, press CE (Clear Entry) to undo any changes and return to the active entry field .

Tip: An active field containing a choice list is indicated by a number to

the right of th e fi e l d w i th i n th e th i ck border.

4. To display a choice list, press Enter. Then do one of the following:

• Press the corresponding num ber key to select an item immediately.

• Use the up and down arrow keys to scroll through the lis t, then

press Enter.

Tip: If you know the number corresponding to the desired choice, you

can press that number without pressing Enter first.

Rule: The up and down arrow keys do not increment or decrement numerical

field entries. Use the numerical keypad.

Navigating to and from the absorbance screen

Pressing HOME from most screens brings you to the absorbance screen. Fr om the absorbance screen, you can ac cess several secon dary functions . To move to the secondary function screens of the absorbance screen, press Next. The secondary functions include:

• Analog output specifications

• Time constant

• Absorbance offset

• Voltage offset

• Chart polarity

• Enable/disable inputs

• Enable/disable external events

3-12 Preparing the Detector

Tip: The parameters you enter into the secondary function fields become part

of the current method conditions and are stored when you store the method (see “Programming timed events, threshold events, and methods” on page 3-35).

When in single wavelength mode, the detector displays three additional screens labeled 2 of 4, 3 of 4, and so on. When in dual wavelength m ode, the detector displays four additional screens, labeled 2 of 5, 3 of 5, and so on (see

page 3-18).

Setting up a run

Once you press HOME to return to the absorbance screen and select a wavelength mode (λ or λλ), you are ready to set up the detector for a run. In addition to wavelength mode, you must program the following parameters before beginning a run:

• Operating wavelength

•Sensitivity

• Time constant

• Analog output sensitivity

Depending on other functions you may want to perform during a run, there are several other parameters you need to program. The section “Primary and

secondary functions” on page 3-14, and the Table titled “Primary and secondary function (method) para me ters” on page 3-19 contain the function

descriptions, fields, screen number, type of function, display units, allowable ranges, and the default settings for the absorban ce screen and the secondary function screens, for both single and dual wavelength opera tion.

Using the operator interface 3-13

Primary and secondary functions

You can access the following functions directly fr om the absorbance screen or by pressing the Next key on that screen.

Detector functions

Function Description

Wavelength Defines the operating wavelength for the channel. AUFS (absorbance

units full-scale)

Analog out (single λ)

Defines the relationship between the absorbance and the output voltage. The output voltage reaches full scale (2 V) when the current absorbance attains the AUFS value.

Caution: Changing the sensitivit y (AUFS) setting

affects the 2-V output. You can chart out the analog connections for the

following parameters:

• Absorbance - Charts the current a bsorbance of the selected channel analog output 2-V connector (scaled to the AUFS setting for that channel) and adjusted based on the voltage and absorbance offsets for that channel. For the 2-V output with 0 used for both the voltage and absorbance offsets:

Volts out = Absorbance × 2 V/AUFS

3-14 Preparing the Detector

Detector functions

Function Description

Analog out (dual λ)

In addition to the selections for single l above, you can chart the same parameters on the other channel at a different wavelength, and you can cha rt the foll owing parameters:

• MaxPlot – Charts the absorbance of multiple compounds with different absorbances at two different wavelengths on a single data channel. Scaling for MaxPlot is the same as for Absorbance above, except that the charted absorb ance is the larger of the two absorbances measured on the wavelengths on channel A and channel B. The detector uses the AUFS, absorbance offset, and voltage offset of the selected channel regardless of which absorbance (channel A or channel B) is larger.

Volts out = Larger absorbance (A or B) × 2 V/AUFS (of selected channel)

• RatioPlot (A/B) – Charts the ratio of absorbances at two wavelengths. Theoretically, the ratio is constant for a pure chromatographic peak and variable for an impure peak. This mode uses the three ratio parameters on screen 5 of 5 (see the figure below).

– Minimum AU: This setting defines the minimum AU value for either wavelength (A or B) before the actual ratio is calculated. Both absorbance values (A and B) must be above this value or 0 V is charted. If both absorbances are above this value, the absorbances are divided (A/B) and char ted. The output voltage is scaled proportionally to the ratio depending on the selected channel minimum ratio and maximum ratio settings.

– Minimum ratio: An actual ratio equal to the minimum ratio value results in 0 V being charted.

Using the operator interface 3-15

Detector functions

Function Description

– Maximum ratio: An actual ratio equal to the maximum ratio results in a full-scale output of 2 V. Absorbance offset is ignored with this selection.

For a RatioPlot, the actual voltage charted is Volts out = 0 V if Abs orbance A and B < minimum

AU Volts out = (Absorbance rat io – minimum ratio ) × 2

V/(maximum ratio – minimum ratio) To ensure proper operation of the RatioPlot

function, be su r e th e se l e cted time constants for both channels are set to the same value.

• Difference Plot (A-B) – Charts the difference in absorbances at two different wavelengths. The scaling for the difference plot is identical to the Absorbance selection above, except that the charted absorbance is the difference (subtracted) in value of the two abso rbances measured on t he A and B wavelengths. The detector uses the AUFS, absorbance offset, and volt age offset of the s elected channel for scaling.

Volts out = Absorbance difference (A – B) × 2 V/AUFS (of selected channel)

Filter time constant Adjusts the noise filter (time constant) to achieve the

Voltage offset Adjusts the charted analog output signal. Entered in

Chart polarity Inverts the charted chrom atogr am. Enter ing the plus

3-16 Preparing the Detector

optimum signal-to-noise ratio without changing the sensitivity settin g. S ee th e disc u ssion “Filtering

noise” on page 1-7 in for more information.

millivolts, this function adjusts the 2-V signal by the entered value. This function is useful for making minor adjustments and for nulling any offset between the detector and a connected external data sys tem.

sign (+) produces a normal chromatogram; entering the minus sign (–) produces an inverted chromatogram.

Detector functions

Function Description

Auto zero on inject Selected by default, this pa rameter res ults in an aut o

zero each time an Inject Start signal is received by the detector via contact closure, Ethernet, or through the front panel. You can disable this parameter by pressing any numerical key to clear t his box for either or both channels.

Auto zero on λ changes

This parameter results in an auto zero each time a wavelength change is requested. If you disable this function, significant chan ges in measured abs orbance can occur after each wavel ength change. S electing “ to zero” sets the signal level to zero. Selecting “t o baseline” maintains the previous baseline level when the wavelength change is made. The default is “to zero.”

Using the operator interface 3-17

Secondary functions of the absorbance screen

Absorbance screen (HOME)

Press Next.

Analog rate and filter

tie constant (available

for both λ and λλ)

Absorbance offset, and

auto zero on inject

(available for both λ and λλ)

Press Next.

Voltage offset and

chart polarity

(available for both λ and λλ)

Press Next.

Minimum AU, minimum ratio,

and maximum ratio

(available only for λλ)

Press Next.

3-18 Preparing the Detector

Primary and secondary function (method) parameters

Function Screen Type Units Range Default

(Wavelength)1 (Absorbance

Numeric nm I nteger 190 nm to

700 nm

254 nm

screen) AUFS 1 Numeric AUFS 0.0001 to 4.0000 2.0000 Analog rate 2 (of 4) or

2 (of 5) Filter time

constant

2 (of 4) or

2 (of 5)

Choice Hz (λ): 10, 20, 40, 80

(λλ): 1 or 2

Numeric Sec Slow

Normal

10 1

Normal

Fast Other: (λ): 0.0125 to 5.0

(for Other:)

1.000 (λλ): 0.5 to 5.0 0 to disable filtering

Data out (single λ)

Data out (dual λ)3 (of 5) Choice None Absorbance A

3 (of 4) Choice None Absorbance A Absorbance

A Absorbance

Absorbance B

A Maxplot A, B Difference A-B Ratio A/B

Auto zero on inject

Auto zero on λ changes

3 (of 4) or 3 (of 5)

3 (of 4) or 3(of 5)

Check box

None Selected/

Not selected

Choice None To baseline

To zero

Checked

To zero Disable

Voltage offset 4 (of 4) or

4 (of 5)

Chart polarity 4 (of 4) or

4 (of 5)

Numeric mV Integer –2000 to

2000

Choice None +

–

Minimum AU 5 (of 5) Numeric AU 0.0001 to 4.0000 0.1000

Using the operator interface 3-19

Primary and secondary function (method) parameters

Function Screen Type Units Range Default

Minimum ratio

Maximum ratio

5 (of 5) Numeric None 0.00 to 999.99 0.00

5 (of 5) Numeric None 0.00 to 999.99 2.00

Operating the Trace and Scale functions

The Trace function allows you to display an absorbance trace for the last n minutes (up to 60) of detector operation.

• When you press the TRACE key, the detector displays the absorbance acquired over the last 30 minutes by default. It updates the trace once every 20 seconds.

• When you press the Scale key (Shift TRACE), the detector displays the scaled trace with T1 (ending time) displayed (–30 for the last 30 minutes) by default.

You can change the ending time parameter to any number from 1 to 60. You can use the Scale function to “zoom” in on a particular secti on of the trace.

To display the Scale parameters after you press the Scale key:

1. Press Next to display T2 (starting time). The default is 0.

2. Press Next again to display AU1 (starting or low absorbance). The default is auto.

3. Press Next again to display AU2 (ending or high absorbance). The default is auto.

By entering appropriate times and absorbance numbers in the four scaling parameter boxes, you can zoom in on one section of the current absorbance trace.

• For AU1 and AU2, press CE to reset to auto.

• T1 represents the left-hand side of the trace, or ending time to be

displayed (default is –30).

• T2 represents the right-hand side of the trace, or starting time

(default is 0).

3-20 Preparing the Detector

The figure below shows a 60-minute trace of continuous injections of caffeine and propyl paraben in 50% methanol/50% water.

Scaled trace of continuous injections with T1 changed to –60

The figure below shows a 5-minute scaled trace (or zoom) of the 60 minutes of continuous injections shown in the previous figure. T1 is changed to –5. T2 is changed to 0. AU1 and AU2 remain as “auto.”

Scaled trace for 5 minutes of continuous injections with T1 changed to –5

The figure below shows a 60-minute scaled trace similar to that shown in the

preceding figure titled “Scaled trace of continuous injections with T1 changed to –60” with the starting absorbance or AU1 changed from auto to 1. T1

remains at 60. T2 remains at 0.

Scaled trace for 60 m inutes of continuous injections with AU1 changed to 1

Using the operator interface 3-21

The figure below shows a 60-minute trace on channel B scaled to the last 45 minutes. T1 is changed to –45.

Scaled trace changing T1 to –45

As you modify the output using the Scale function, the Trace function continues to display the detect or output in real time on either or both channels.

Operating other detector functions

Configuring the detector

To change default configurations, use the Configuration screens. Press the CONFIGURE key (Shift DIAG). The first of two Configuration

screens appears (see figure below).

Tip: Other functions, such as specifying event inputs and enabling pulse

periods, are stored within the Configuration screens.

Configuration screens

Configuration screen 1 of 2

Requirement: To prevent acquisition of incorrect data when operating the

detector in dual wavelength m ode under the control of the Empower software

3-22 Preparing the Detector

Configuration screen 2 of 2

or a MassLynx system, you must select a data sampling rate of 1 point per second.

Configuring event inputs (contact closures)

You also use the CONFIGURE key to edit event input settings and specify switched output settings.

Using the Enter key and the numeric keypad (or the ▲ and ▼ keys) to select the appropriate entry, you can edit four field s on the second Configuration screen shown above:

Tip: The default for Inject, Chart Mark, and Auto Zero is Low; the default for

Lamp is Ignore.

•Inject – You can specify the Inject input to signal the start of a run. This event resets the run-time clock and applies initial method conditions immediately:

– High – Start run when contact closure changes from off (open) to on

(closed).

–Low – Start run when contact closure changes fro m on (clos ed) to off

(open).

– Ignore – Do not respond to Inject start input.

• Chart mark – You can specify the chart mark input to create a chart mark on channel A and/or channel B. You determine the resp onse of the channel using the enable chart mark function explained in the table on

page 3-19 and shown in the figure on page 3-18.

– High – Create chart mark(s) when contact closure changes from off

(open) to on (closed).

– Low – Create chart mark(s) when contact closure changes from on

(closed) to off (open).

– Ignore – Do not respond to chart mark input.

• Auto zero – You can configure the auto zero input to auto zero absorbance readings on channel A and/or channel B. You determine the response of the channel using th e enable auto zero function explai ned in the table on page page 3-19 and shown in the figure on page 3-18.

–High – Auto zero the channel when cont act closure changes f rom off

(open) to on (closed).

Using the operator interface 3-23

– Low – Auto zero the channel when contact closure changes from on

(closed) to off (open).

– Ignore – Do not respond to auto zero input.

•Lamp – You can configure the lamp input level to turn the deuterium lamp on or off from an external device as follows:

– High – Turn lamp off when contact closure is on (closed). – Low – Turn lamp off when contact closure is off (open). – Ignore – Do not respond to Lamp input.

Setting pulse periods

You use the second Configuration screen (shown on page 3-22) to set pulse or signal width or to activate a pulse or rectangular wave on SW1 or SW2.

• Single pulse (in seconds ) – If SW1 or SW2 is programmed to generate a pulse as a timed or threshold event, then the period of the signal (single pulse width) is as specified in this field (range is 0.1 to 60 seconds).

• Rectangular wave (in seconds) – If SW1 or SW2 is programmed to initiate a rectangular w ave as a timed or threshold event, then the period of the signal (the widt h of one pulse period in a rectangular wave or pulse train) is as specified in this field (range is 0.2 to 60 seconds).

The figure below shows the difference between a single pulse and a rectangular wave.

Setting the pulse period or signal width using SW1 or SW2

n Seconds

Single pulse

n Seconds

Rectangular wave

3-24 Preparing the Detector

Setting display contrast

The Contrast function allows you to adjust the contrast of the detector dis play screen. When you press the Contrast key (Shift 6), the Display Contrast screen appears.

Display Contrast screen

Use the ▲ and ▼ keys to adjust the contrast of the display.

Displaying system info

The System Info key (Shift 4) displays information about the dete ctor, including the serial num ber and the firmware version number.

Tip: Use the scroll bar to see all of the message. The Firmware Rev and

ChkSm values shown here are examples only. They do not indicate the released version information.

System info screen example

Start of message

End of message

Using the operator interface 3-25

Using Help

The detector has limited context-sens itive help. When you press ? (Shift HOME) from a point in the program that has a Help screen associated with it, the appropriate screen appears.

Help screen

Enter the desired wavelength for detection on channel A. Range: between 190 and 700.

To exit the Help screen, press Enter. If help is not available for the function you are working on, pressing ? gets no response.

Operating the detector

Overview of detector operation

Tip: If you are operating the detector under the control of an external system,

you can program any parameters not controlled by the external data system at the front panel of the detector before the external system takes control.

Recommendation: To prevent reabsorption of dissolved oxygen, run the

solvent degasser continuously when operating the detector at wavelengths less than 230 nm.

Requirement: To maintain optimum system performance, be sure to replace

the front left panel cover before resuming nor mal operation of the detector.

Operating modes

You can use the detector in either single or dual wavelength mode ov er a range of 190 to 700 nm. The detector defaults to the mode of operation when the instrument was la st powered-off.

3-26 Preparing the Detector

Stand-alone operation

When using the detector as a stand-alone instrument, you can store up to five methods containing up to 50 t imed and 2 t hreshold events eac h. An aster isk in the method number field on the absorbance screen indi cates current conditions, not a stored method. See “Programming timed events, threshold

events, and methods” on pag e 3-35 for information on how to store a method.

Remote control

To operate under Empower or MassLynx control, the detector uses the Ethernet bus connector (see “Making Ethernet connections” on page 2-14).

To connect the detector to your HPLC system, see “Making fluid line

connections” on page 2-6.

When under th e control of an ex t ernal data sy st em, the detector is operati n g under remote control conditions. The Remote Control icon, containing the letter E, is present on the absorbance screen (see the table on page 3-4).

See “Making signal connections” on page 2-11, for more information on connecting the detector to an external system.

Verifying the detector

After you have installed the detector, verify that it is operating properly by performing the procedures in this section.

For complete validation procedures, you must obtain the Waters accuracy and linearity cuvette kits and system qualification tool for the detector. See

Appendix C for part numbers and ordering information.

Tip: Before you pump solvent or mobile phase through the system, flush the

lines with filtered, degassed, and sparged HPLC-grade methanol. Then pump your mobile phase, provided that there are no miscibility problems, at 1 mL/min for 15 minutes minimum.

Before you begin

Because the detector is shipped dry, you must pump solvent through the unit before initial use.

Rule: To ensure accurate verification, be sure to start up the detector and

follow steps 1 to 3 in this section and 1 to 4 in “Recording samp le and

Using the operator interface 3-27

reference beam energies” on page 3-28 before pumping any mobile phase or

solvent through the flow cell.

To pump solvent through the unit before initial use:

1. Connect the detector to a data system or chart recorder. Refer to

Chapter 2 for complete information on connecting the detector to

external devices.

2. Power-on the detector. The front panel displays a series of initialization messages for about 5 minutes. Refer to “Initializing the detector” on page 3-2.

3. When initialization is complete, the detector displays the absorbance screen. For an explanation of the detector keypad, see “Using the

keypad” on page 3-6.

4. Allow the detector to warm up for at least 30 minutes before operating.

Tip: I f th e sta r tup verificati o n di a g n o st i c te st s fa i l , n o te the error

message to determine the corrective action. Refer to Chapter 5.

Recording sample and reference beam energies

To determine baseline values on your detector for future reference and to monitor lamp aging (decreased lamp output energy), you need to record the baseline sample and reference beam energies to compare against future readings. Use these baseline values to troubles hoot the detector to determine whether

• the solvent is contaminated.

• the flow cell is contaminated.

• the lamp needs to be replaced.

• there is an air bubble in flow cell.

To record the sample and reference beam energies:

1. On the absorbance screen, use the arrow keys to highlight the λ field.

2. Enter 230 into the λ field, and t hen pr ess Enter.

3-28 Preparing the Detector

3. Press DIAG and then press 2, Sample & ref energy. The sample and reference energy diagnostic display appears.

Sample and reference energy diagnostic display

4. Record th e nu mbers for later comparison.

Rule: Run this procedure each time you change the detector lamp.

5. Flush the flow cell with approximately 30 to 60 mL of HPLC-grade methanol at 1 mL/min for 15 minutes minimum to clean the cell.

You are now ready to run the peak response verification test. If the following test fails, repeat it.

Make the fluid connections described in “Making fluid line connections” on page 2-6, before beginning the peak response test.

Ve rifying peak response

This test checks the detector peak res ponse.

To check peak response:

1. On the absorbance screen, use the arrow keys to highlight the λ field.

2. Enter 254 into the λ field, and then pres s Enter.

3. Press Enter again to activate the sensitivity field.

4. Set the sensitivity to 2.0 AUFS.

Using the operator interface 3-29

5. Set the pump flow rate in your HPLC system to 1.0 mL/min.

6. Inject 1 µL acetone.

When the test is successful, your chart recorder or data system displays a peak.

Wavelength calibration

You can calibrate the detector manually from the keypad. You can use the manual calibration key to recalibrate the detector at any time during operation, or if there are any calibration errors during startup. You do not have to reboot the detector after a successful wavelength calibration.

To calibrate the detector manually:

1. Press Calibrate (Shift 3) from the detector keypad. A message appears asking whether you re moved the cuvette and flushed

the flow cell with a transparent solvent (Waters recommends methanol or water).

Cuvette wavelength calibration message

2. Press Enter to continue the calibration cycle, or press Cancel to return to the absorbance screen without calibrating the detector.

After you press Enter, the detect or cycles through the calibration procedure and briefly displays a series o f i nitializatio n messages s imilar to those you saw at startup (see “Initializing the detector” on page 3-2).

If calibration is successful, the detector beeps three times and displays the maximum error in nanometers of the furthest calibration shift from the last cal i bration.

3-30 Preparing the Detector

Calibration successful message

3. Press Enter to complete calibration. A “Calibration complete” message is displayed momentari ly. Other

messages su ch a s “O ptimizing system performance” an d “R e storing last setup” may appear before the display returns to the absorbance screen.

Result: If calibr ation is succe ssful, the erro r message (<Err or>) that was

displayed on the absorbance screen before you recalibrated the detector disappears.

4. If calibration is not su ccessful, r etry, recycle power to the det ector, or se e

Chapter 5.

Operating the detector in single wavelength mode

The detector is optimized for singl e wavelength operation, whic h is the default operating mode.

To invoke single wavelength mode:

1. From the absorbance (or HOME ) screen, press the λ/λλ key (Shift Auto Zero) if the detector is in dual wavelength (the wavelength icon shows λλ) mode. The detector displays a message indicating it is switching to single wavelength operation.

Wavelength mode change message (for single wavelength)

Using the operator interface 3-31

2. Enter the wavelength and sensitivity on the absorbance screen, as well as any secondary parameters and timed or threshold eve nts.

Waters 2489 Operator's Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Table of Contents

Detector description

Principles of operation

Operational modes

2 Installing the Detector

Preparing for installation

Site selection and power requirements

Making fluid line connections

Making electrical power connections

Connecting to other devices

3 Preparing the Detector

Initializing th e detector

Using the operator interface