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

+ 204 hidden pages

Waters 2489 Operator's Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Table of Contents

Detector description

Principles of operation

Operational modes