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Waters 2489 Operator's Manual

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

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Summary of Contents for Waters 2489

  • Page 1 Waters 2489 UV/Visible Detector Operator’s Guide 71500142102 / Revision A Copyright © Waters Corporation 2007. All rights reserved.
  • Page 2 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.
  • Page 3 Contacting Waters ® Contact Waters with enhancement requests or technical questions regarding the use, transportation, removal, or disposal of any Waters product. You can reach us via the Internet, telephone, or conventional mail. Waters contact information Contacting medium Information Internet The Waters Web site includes phone numbers for Waters locations worldwide.
  • Page 4 Safety considerations ® Some reagents and samples used with Waters instruments can pose chemical, biological, and radiological hazards. Be sure you are aware of the potentially hazardous effects of all substances you work with. Always observe Good Laboratory Practice (GLP) guidelines, published by the U.S. Food and Drug Administration, and consult your organization’s safety representative...
  • Page 5 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.
  • Page 6 When analyzing samples from a complex matrix such as soil, tissue, serum/plasma, whole blood, and so on, note that the matrix components can adversely affect results. To minimize these matrix effects, Waters recommends you adopt the following measures: •...
  • Page 7 Limited) is registered in the United Kingdom with the Medicines and Healthcare Products Regulatory Agency (MHRA) at Market Towers, 1 Nine Elms Lane, London, SW8 5NQ. The reference number is IVD000167. Waters Corporation (Micromass UK Ltd.) Floats Road Wythenshawe Manchester M23 9LZ United Kingdom Telephone:...
  • Page 8 viii...

  • Page 9: Table Of Contents

    Features ....................... 1-2 Principles of operation ..................1-4 Detector optics....................1-4 Optics assembly light path ................. 1-5 Waters TaperSlit Flow Cell ................ 1-6 Filtering noise ..................... 1-7 Wavelength verification and test ..............1-9 Operational modes ..................1-11 Single wavelength mode ................1-11 Primary parameters ..................
  • Page 10 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 ................
  • Page 11 Auto Zero connections ................2-34 Inject Start connections ................2-35 Connecting the detector to a fraction collector..........2-36 3 Preparing the Detector ................. 3-1 Initializing the detector .................. 3-2 Diagnostic test failure ................. 3-3 Using the operator interface ................3-3 Using the display .....................
  • Page 12 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 ..................
  • Page 13 4 Maintaining the Detector ..............4-1 Contacting Waters Technical Service ............4-2 Maintenance considerations ................4-2 Safety precautions ................... 4-2 Spare parts ....................... 4-2 Proper operating procedures ................. 4-3 Removing the front left panel cover..............4-3 Routine maintenance..................4-4 Maintaining the flow cell ................4-4 Flushing the flow cell..................
  • Page 14 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 ..............
  • Page 15 Caution symbol ....................A-5 Warnings that apply to all Waters instruments ......... A-5 Electrical and handling symbols ..............A-13 Electrical symbols ..................A-13 Handling symbols ..................A-14 B Detector Specifications ............... B-1 Operational specifications ................B-2 Optical specifications ..................B-4 Optional Waters TaperSlit Flow Cell specifications .......
  • Page 16 Solvent degassing methods ................D-8 Sparging ......................D-8 Vacuum degassing ................... D-8 Solvent degassing considerations ..............D-8 Sparging ....................... D-8 Vacuum Degassing ..................D-9 Wavelength selection ..................D-9 UV cutoffs for common solvents ..............D-9 Mixed mobile phases..................D-10 Wavelength selection for chromophore detection......... D-11 Table of Contents...

  • Page 17: Theory And Principles Of Operation

    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 Principles of operation Operational modes 1-11 Appendix B for system specifications and...

  • Page 18: Theory And Principles Of Operation

    Waters 2489 UV/Visible Detector Inlet The detector can operate either as a stand-alone unit (with a chart recorder or integrator) or as an integral part of a Waters chromatography system. The detector can be configured with Empower™ or MassLynx™ software systems.

  • Page 19: Detector Description

    Cuvette qualification – Facilitates qualification of the detector by insertion of a standard in a cuvette without breaking any fluid line connections. Waters qualification kits are available in cuvette form to support this feature. This feature also allows you to use the detector as a benchtop spectrophotometer.

  • Page 20: Principles Of Operation

    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 ) lamp • Two mirrors: one off-axis ellipsoidal mirror and one spherical mirror •...

  • Page 21: Optics Assembly Light Path

    Waters 2489 UV/Visible Detector optics assembly lamp Spherical mirror Filter Window wheel Slit Ellipsoidal mirror Grating Optional cuvette TaperSlit Flow Cell Sample photodiode Beamsplitter Reference photodiode Optics assembly light path The detector provides an extremely efficient design for exceptionally high light throughput.

  • Page 22: Waters Taperslit Flow Cell

    As shown in the figure below, in a conventional cell, light bends and hits the wall of the flow cell. Four beams go in, but only two come out. In the Waters TaperSlit Analytical Cell, the combination of the lens and TaperSlit bore geometry prevents light from hitting the cell walls.

  • Page 23: Filtering Noise

    Comparison of flow cell characteristics Conventional Cell Window Light Window Waters TaperSlit™ Analytical Cell Window Light Lens TP01530 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.
  • Page 24 The filter time constant adjusts the filter response time to achieve an optimal signal-to-noise ratio. Selecting Other and entering a value of 0.0 disables all filtering. Lower time constant settings produce these effects: • Narrow peaks with minimal peak distortion and time delay •...

  • Page 25: Wavelength Verification And Test

    Filter Time Constant comparison TP02833 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.
  • Page 26 The verification tests for the detector require 5 minutes of lamp warmup time so the lamp can stabilize. If you run the detector continuously, Waters recommends that you perform wavelength verification weekly by turning off the detector, then turning it on again.

  • Page 27: Operational Modes

    Operational modes The detector operates in single or dual wavelength mode, allows spectrum scanning using a flow cell or a cuvette, and provides RatioPlot, difference 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.

  • Page 28: Secondary Parameters

    • Filter time constant – Programs a filter time in seconds. Options are 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.

  • Page 29: Chart Out Selection Modes

    inaccuracies because of possible UV light interference (second order effects). Chart Out selection modes When operating in dual wavelength mode, the detector offers these choices for analog output in addition to the selections offered in single wavelength mode and explained in “Single wavelength mode”...

  • Page 30: Cuvette Operations

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

  • Page 31: Ratioplot

    RatioPlot The detector allows ratio plotting: comparing the absorbances of a compound or analyte at two different wavelengths. The RatioPlot divides absorbances at two selected wavelengths and plots the resulting ratio on a chart recorder or data system over one output channel (channel A). The RatioPlot is useful in detecting hidden components present within individual peaks.
  • Page 32 1-16 Theory and Principles of Operation...

  • Page 33: Installing The Detector

    Installing the Detector ® The Waters 2489 UV/Visible Detector requires connections to electrical 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.

  • Page 34: Preparing For Installation

    Preparing for installation Major steps in installing the detector Start installation procedure Make power connections Select appropriate site Make signal connections Unpack and inspect Make connections to other devices Install detector Installation complete Make fluid line connections After installing the detector, you should verify its functions and keep the verified chart output (if applicable) on file.
  • Page 35 Detector dimensions 20.8 cm (8.2 inches) 50.3 cm (19.8 inches) 28.4 cm (11.2 inches) TP02804 Caution: Do not access the instrument through the top cover. Access the instrument through the front left panel where the lamp housing, flow cell assembly, and cuvette holder are located. Preparing for installation...

  • Page 36: Site Selection And Power Requirements

    • HAR type (or better) in Europe For information regarding the type of cord to use in other countries, contact your local Waters distributor. 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.

  • Page 37: Power Requirements

    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...

  • Page 38: Unpacking

    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 corporate headquarters for assistance at 1-508-478-2000 (U.S.).

  • Page 39: Connecting Columns

    You must make the following fluid line connections on your Requirement: detector unit: • Column connections • Drip management system connections Recommendations: • Before completing the column connections, perform the verification procedures described in “Verifying the detector” on page 3-27. •...

  • Page 40: Assembling The Fittings

    Detector fluid line connections Inlet (labeled) Outlet TP02810 Assembling the fittings To assemble each fitting Slide the compression screw over the tubing end, followed by the ferrule. Mount the ferrule with its taper end facing the end of the tubing. Installing the Detector...

  • Page 41: Making Connections

    To prevent reabsorption of dissolved oxygen (for systems Recommendation: using vacuum degassers), Waters recommends that you run the solvent degasser continuously when operating the detector at wavelengths less than 230 nm.

  • Page 42: Detector Rear Panel

    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 Fuse holder Power input 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.

  • Page 43: Making Signal Connections

    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 figure on...

  • Page 44: Making I/O Signal Connections

    Overview of making signal connections Start signal connection procedure Install Ethernet Connect to Ethernet bus? and inject start cables Install event and I/O Connect to other devices? cable(s) Signal connections complete 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.

  • Page 45: I/O Signals

    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 TTL contact closure.

  • Page 46: Making Ethernet Connections

    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: •...
  • Page 47 190 to 700 nm, and up to 4.0 AUFS. To connect Ethernet cables from the detector to a Waters data system: 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).

  • Page 48: Starting A Method

    To start a method on the detector at the start of an injection from the separations module, make the connections summarized in the table below and illustrated in the figure below. 2695 Separations Module 2489 Detector (II) (B Inputs and Outputs) Pin 1 Inject Start + Pin 1 Inject Start Pin 2 Inject Start Pin 2 Inject Start –...
  • Page 49 Detector connections to separation module (lamp on or off) 2695 Separations Module 2489 Detector (II) (A Outputs) Pin 4 Lamp On/Off + Pin 1 Switch 1 Pin 2 Switch 1 Pin 5 Lamp On/Off –...

  • Page 50: Connecting The Detector To A Separations Module

    Detector connections to a separation module to generate Auto Zero 2695 Separations Module 2489 Detector (II) (B Inputs and Outputs) Pin 9 Auto Zero + Pin 1 Inject Start Pin 2 Inject Start Pin 10 Auto Zero –...

  • Page 51: Generating Chart Mark On Inject

    To generate the Chart Mark function on the detector at the start of an injection, make the connections summarized in the table and illustrated in the figure below. 2695 Separations Module 2489 Detector (II) (B Inputs and Outputs) Pin 6 Chart Mark + Pin 1 Inject Start Pin 2 Inject Start Pin 7 Chart Mark –...

  • Page 52: Connecting To Other Devices

    Separations module connections to the detector for Chart Mark on Inject Waters 2695 Separations Module Waters 2489 TP01526 Connecting to other devices You can connect the detector to a wide range of HPLC system devices. This section describes how to connect the detector to the following devices: •...

  • Page 53: Required Materials

    • 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 card installed in the Empower workstation.
  • Page 54 • To ensure proper startup of the e-SAT/IN module, do not turn on power to the module until you perform all procedures described in the Waters e-SAT/IN Module Installation Guide. Improper startup can damage the unit and void the warranty.

  • Page 55: Connecting The Detector To The E-Sat/In Module

    Connect the white wire to pin 1 on I (Analog 1 +). Connect the black wire to pin 3 on I (Ground). e-SAT/IN module Channel 1 connection to detector Waters 2489 For Channel 2: Connect the white wire to pin 4 on I (Analog 2 +).
  • Page 56 Empower 2 Installation and Configuration Guide. The table below summarizes the detector connections to the e-SAT/IN module. Detector connections to a e-SAT/IN module e-SAT/IN 2489 Detector (I) Connector Pin 1 Analog 1 + (white) Channel 1 or 2 Pin 3 Ground (black)

  • Page 57: Connecting The Detector To A 745/745B/746 Data Module

    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.
  • Page 58 Data module connections to detector channels A and B Waters 2489 TP01485 Waters 2489 TP01486 2-26 Installing the Detector...

  • Page 59: Connecting 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.

  • Page 60: Chart Marks

    Chart recorder 2-V output connections on the detector Waters 2489 TP01487 Waters 2489 TP01488 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: •...

  • Page 61: Connecting The Detector To The Waters 600 Series Pump

    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 site requirements in “Site selection and power requirements”...

  • Page 62: Auto Zero Connections

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

  • Page 63: Chart Mark Connections

    Program the pump to provide a pulse output on the selected switch at the beginning of each run. Refer to the Waters 600E Multisolvent Delivery System User’s Guide for more details. Pump and 2489 detector Chart Mark connections...

  • Page 64: Inject Start Connections

    Program the pump to provide a pulse output on the selected switch at the beginning of each run. Refer to the Waters 600E Multisolvent Delivery System User’s Guide for more details. 2-32...

  • Page 65: Pump And Detector Inject Start Connections

    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.

  • Page 66: 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 figure below. Use any available pair of Inject Start terminals on the autosampler. Autosampler and detector Auto Zero connections 717plus 2489 Detector Autosampler (A Inputs) Terminal Pin 9 Auto Zero +...

  • Page 67: Inject Start Connections

    To program an inject start, make the connections described in the table and illustrated in the figure below. Use any available pair of Inject Start terminals on the autosampler. Autosampler and detector Inject Start connections 2489 Detector 717plus Autosampler (II) Terminal...

  • Page 68: Connecting The Detector To A Fraction Collector

    Pin 9 Event Marker – Pin 7 External Count In + I Pin 6 SW1 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 –...

  • Page 69: Preparing The Detector

    Preparing the Detector Contents: Topic Page Initializing the detector Using the operator interface 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 −...

  • Page 70: Initializing The Detector

    the detector Initializing Before you power-on the detector, be sure the connector from the detector rear panel to the power source is properly installed. To power-on the detector, press the On/Off switch located on the front, lower-right corner of the unit. At startup, the detector beeps three times and runs a series of startup diagnostic tests.

  • Page 71: Diagnostic Test Failure

    For normal use, allow the detector to warm up for at least 30 minutes Tip: before operating. Diagnostic test failure If one or more of the internal startup diagnostic tests show a failing result, the detector beeps and displays an error message. For serious errors, it displays the word “Error”...

  • Page 72: Absorbance And Message Icons

    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.
  • Page 73 Absorbance and message screen icons Icon or Field Icon / Field Name Function Numerical Absorbance Displays current absorbance for the field selected channel. Lamp On/Off On = Lamp icon Off = Lamp icon with an X through it Shift On/Off Blank = Shift off = Shift on λ...

  • Page 74: Using The Keypad

    Absorbance and message screen icons Icon or Field Icon / Field Name Function Local (Method #) Local control/Method number – If the /Remote Control detector is not controlled 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 stored as a method.
  • Page 75 • primary function keys (Chart Mark, Auto Zero, and Run/Stop). • secondary function keys (Scale, Single or Dual Wavelength, Reset Clock, Lamp, Lock, Calibrate, System Information, Contrast, Previous, Cancel, +/–, and Clear Field). Detector keypad λ/λλ SCAN Reset HOME Chart Mark Auto Zero Run/Stop METHOD Lamp Lock...
  • Page 76 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 keypad. Detector keypad description Description Unshifted Shifted HOME – Displays the ? –...
  • Page 77 Detector keypad description Description Unshifted Shifted Arrow keys − On screens with entry fields (edit, check box, or choice list), the active field has a thick border (highlight). The arrow keys can be used to make a different field active. (Up moves up or left;...
  • Page 78 Detector keypad description Description Unshifted Shifted Shift – Enables the shifted functions (identified by the text at the top of most keys). The shifted state is temporary, and is Shift reset after the next keystroke. 0-9 – Enters the 0-9 – See descriptions for corresponding number into specific shifted numeric keys.
  • Page 79 Detector keypad description Description Unshifted Shifted 4 – See 0-9 above. System Info – Displays System Info system information including firmware version and instrument serial number. 6 – See 0-9 above. Contrast – Permits Contrast adjustment of contrast (viewing angle) of the liquid crystal display.

  • Page 80: Navigating The User Interface

    Navigating the user interface To operate the detector: Press the Enter key or the up and down arrow keys to navigate among editable fields on a display. A thick border appears around the active field. Tip: Press Enter to advance the active field once you have completed an entry.

  • Page 81: Setting Up A Run

    The parameters you enter into the secondary function fields become part Tip: of the current method conditions and are stored when you store the method (see “Programming timed events, threshold events, and methods” page 3-35). When in single wavelength mode, the detector displays three additional screens labeled 2 of 4, 3 of 4, and so on.

  • Page 82: Primary And Secondary Functions

    Primary and secondary functions You can access the following functions directly from 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 Defines the relationship between the absorbance and units full-scale) the output voltage.
  • Page 83 Detector functions Function Description Analog out In addition to the selections for single l above, you can (dual λ) chart the same parameters on the other channel at a different wavelength, and you can chart the following parameters: • MaxPlot – Charts the absorbance of multiple compounds with different absorbances at two different wavelengths on a single data channel.
  • Page 84 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 Absorbance A and B < minimum Volts out = (Absorbance ratio –...
  • Page 85 Detector functions Function Description Auto zero on inject Selected by default, this parameter results in an auto 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 this box for either or both channels.
  • Page 86 Secondary functions of the absorbance screen Absorbance screen (HOME) Voltage offset and Press Next. chart polarity (available for both λ and λλ) Press Next. Analog rate and filter Minimum AU, minimum ratio, tie constant (available and maximum ratio for both λ and λλ) (available only for λλ) Press Next.
  • Page 87 Primary and secondary function (method) parameters Function Screen Type Units Range Default λ Numeric Integer 190 nm to 254 nm (Wavelength) (Absorbance 700 nm screen) AUFS Numeric AUFS 0.0001 to 4.0000 2.0000 Analog rate 2 (of 4) or Choice (λ): 10, 20, 40, 80 2 (of 5) (λλ): 1 or 2 Filter time...

  • Page 88: Operating The Trace And Scale Functions

    Primary and secondary function (method) parameters Function Screen Type Units Range Default Minimum 5 (of 5) Numeric None 0.00 to 999.99 0.00 ratio Maximum 5 (of 5) Numeric None 0.00 to 999.99 2.00 ratio 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.
  • Page 89 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.

  • Page 90: Operating Other Detector Functions

    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 detector output in real time on either or both channels.

  • Page 91: Configuring Event Inputs (Contact Closures)

    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 keys) to select the appropriate entry, you can edit four fields on the second Configuration screen shown above:...

  • Page 92: Setting Pulse Periods

    – 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: –...

  • Page 93: Setting Display Contrast

    Setting display contrast The Contrast function allows you to adjust the contrast of the detector display screen. When you press the Contrast key (Shift 6), the Display Contrast screen appears. Display Contrast screen Use the keys to adjust the contrast of the display. Displaying system info The System Info key (Shift 4) displays information about the detector, including the serial number and the firmware version number.

  • Page 94: Using Help

    Using Help The detector has limited context-sensitive 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.

  • Page 95: Stand-Alone Operation

    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.

  • Page 96: Recording Sample And Reference Beam Energies

    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: Connect the detector to a data system or chart recorder. Refer to Chapter 2 for complete information on connecting the detector to external devices.

  • Page 97: Verifying Peak Response

    Press DIAG and then press 2, Sample & ref energy. The sample and reference energy diagnostic display appears. Sample and reference energy diagnostic display Record the numbers for later comparison. Run this procedure each time you change the detector lamp. Rule: 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.

  • Page 98: Wavelength Calibration

    To calibrate the detector manually: Press Calibrate (Shift 3) from the detector keypad. A message appears asking whether you removed the cuvette and flushed the flow cell with a transparent solvent (Waters recommends methanol or water). Cuvette wavelength calibration message Press Enter to continue the calibration cycle, or press Cancel to return to the absorbance screen without calibrating the detector.

  • Page 99: Operating The Detector In Single Wavelength Mode

    Calibration successful message Press Enter to complete calibration. A “Calibration complete” message is displayed momentarily. Other messages such as “Optimizing system performance” and “Restoring last setup” may appear before the display returns to the absorbance screen. Result: If calibration is successful, the error message (<Error>) that was displayed on the absorbance screen before you recalibrated the detector disappears.

  • Page 100: Operating The Detector In Dual Wavelength Mode

    Enter the wavelength and sensitivity on the absorbance screen, as well as any secondary parameters and timed or threshold events. See also: • Figure “Secondary functions of the absorbance screen” on page 3-18 • Table titled “Primary and secondary function (method) parameters” on page 3-19 through Table titled “Threshold events “To”...

  • Page 101: Changing From Single To Dual Wavelength Mode

    Changing from single to dual wavelength mode To change from single to dual wavelength mode: From the absorbance (or HOME) screen when in single wavelength mode (the wavelength icon shows λ), press the λ/λλ key (Shift Auto Zero). This key toggles between single and dual wavelength modes. A momentary message (“Setting up dual wavelength mode”) appears.

  • Page 102: Obtaining A Ratioplot

    contain inaccuracies because of possible UV light interference (second order effects). When in dual wavelength mode, select a wavelength pair Recommendation: either below or above 370 nm. If one or both of the selected wavelengths spans the 370 nm threshold, the detector beeps three times and issues the warning message shown in the figure below.

  • Page 103: Obtaining A Maxplot

    Enter the minimum AU, then press Enter. Tip: The minimum AU field contains a threshold value. The RatioPlot function does not plot if either wavelength does not exceed the minimum AU threshold. Enter the minimum ratio for the RatioPlot, then press Enter. Enter the maximum ratio for the RatioPlot, then press Enter.

  • Page 104: Timed Events

    The method number displayed on the absorbance screen is that of the retrieved method until you make a change. Any parameter change (for example, wavelength or AUFS) alters the current conditions so that the original recalled method is no longer in effect, causing the method number to change to an asterisk.
  • Page 105 Timed event parameters Specify Number Event Units Range or Default Channel Auto zero Does not Does not apply apply Lamp 1. Off 2. On Switch 1 1. High High 2. Low 3. Pulse 4. Rect wave Switch 2 1. High High 2.
  • Page 106 Timed events screen Press Enter to enter the time. To advance to the Set field (Events choice list), press the key. Press Enter again to display the choice list, or, if you know the event number, press the number for the event you are programming (Table titled “Timed event parameters”...

  • Page 107: Threshold Events

    Tip: • If the detector is under the control of the Waters 717plus Autosampler or another external device, the Inject Start programmed from that device runs the method. • If you are working in real time under current conditions (method *), at a power failure or shutdown you lose all timed or threshold events if you do not store them as a method.

  • Page 108: Storing A Method

    To program a threshold event: Press the METHOD (Shift A/B) key on the detector keypad. The Method choice list appears (Figure “Method choice list” on page 3-37). Press 2, Threshold events. An active field (AU) for entering the threshold appears. When you begin to enter a number in the AU field, additional fields appear.

  • Page 109: Retrieving A Method

    Storing a method, method number field There is no warning message if the method number you select Caution: is already being used by a previously stored method. When you enter a number and press Enter, the current method conditions are stored, overwriting any previous method stored in the same slot.

  • Page 110: Viewing Events Within A Method

    Viewing events within a method To view timed and threshold events that make up a stored method: Retrieve the method (see “Retrieving a method” on page 3-41). Once you have entered the method number to retrieve, the Method choice list appears with the method number displayed within the method number icon.

  • Page 111: Clearing Events

    If you press Enter: • All timed events are deleted. • All threshold events are disabled. • All other operating parameters of the method (λ, AUFS, etc.) are set to defaults. If you press Cancel (Shift 0), the display returns to the Method choice list.

  • Page 112: Scanning Spectra

    If you press Enter: • All timed and threshold events in the method are cleared. • All other operating parameters of the method (λ, AUFS, etc.) are unaffected. If you press Cancel (Shift 0), the display returns to the Method choice list.
  • Page 113 Pace and sampling resolution examples Pace (in nm/min) Sampling Resolution (in nm) 100 and less The figure below shows two scans of anthracene overlaid. At a pace of 1000 nm/min, the overlaid scan (with dotted line) shows a reduced number of points scanned, lowering the resolution in comparison with the original scan done at a pace of 100 nm/min.
  • Page 114 • Tickmarks – This value allows tickmarks to be generated at the specified wavelength increment to assist in the interpretation of charted data. The figures below show a 190 to 600-nm scan of erbium standard in a cuvette at a pace of 200 nm/min with tickmarks specified every 20 nm and the same scan without tickmarks.
  • Page 115 When you select a zero scan: • The detector displays three additional screens, labeled 2 of 4, 3 of 4, and 4 of 4. • You can change all parameters on these screens, including starting and ending wavelength, and the pace parameter. When you select a sample scan: •...

  • Page 116: Scanning New Spectra

    Scanning new spectra To specify a new spectrum: Press SCAN (Shift Chart Mark). A Scan choice list appears. Scan choice list Press 1, New scan, or use the keys to move through the Scan choice list. The detector displays the first of three parameter screens for a sample scan or four parameter screens for a zero scan (Figure “Zero and sample scan screen”...

  • Page 117: Zero Scan

    Sample and zero scan parameters Parameter Screen Scan Type Units Range or Default Type Sample and Sample scan: 1 zero Zero scan: 2 Default: 1 λ range Zero scan only Range: 190 to 700 nm Default: 190 to 700 nm Pace Zero scan only nm/min Range: 30 to 1000...
  • Page 118 To specify tickmarks, enter a number from 10 nm to 100 nm, then press Enter. Press CE to disable tickmarks (changes the value to OFF). Refer to the figures on page 3-46 for examples of scans made with tickmarks on or off.
  • Page 119 Zero and sample scan screen Zero scan (Screen 1 of 4) Sample scan (Screen 1 of 3) Zero scan (Screen 2 of 4) Sample scan (Screen 2 of 3) Sample scan (Screen 3 of 3) Zero scan (Screen 3 of 4) Zero scan (Screen 4 of 4) Scanning spectra 3-51...

  • Page 120: Running The Sample Scan

    Running the sample scan Run the zero scan before running the sample scan. To Recommendation: ensure identical flow cell and solvent conditions, you should run the sample scan for the corresponding zero scan within 15 minutes of running the zero scan.
  • Page 121 Sample erbium scan graphical display To return to the Scan choice list at the completion of the sample Tip: scan, press SCAN (Shift Chart Mark). Press Next to display up to four highest peaks scanned (if four are found) within the specified range. The figure below shows the display of the four highest peaks for the sample erbium scan shown in the graphical display (see above).
  • Page 122 Sample erbium scan with l1 changed to 225 nm and l2 changed to 600 nm Once you have changed one or more scaling parameters, press Enter to reformat the graphical display. 10. Once the scan reappears press Next to display the four highest peaks of the scaled scan.
  • Page 123 Series of scans of anthracene in acetonitrile Sample scan 200 nm to 400 nm –0.001 AU to 0.5 AU anthracene Zoom of sample scan 200 nm to 300 nm –0.001 AU to 0.5 AU anthracene, 230 to 270 nm λ2 changed to 300 nm Zoom of sample scan 230 nm to 270 nm –0.001 AU to 0.5 AU...

  • Page 124: Storing A Spectrum

    Storing a spectrum Once you have run a spectrum, you can store it for later review, subtraction, or playback. You may store up to three spectra. To store a spectrum: From the graphic display of a sample scan, return to the first Scan screen by pressing SCAN (Shift Chart Mark).

  • Page 125: Reviewing A Stored Spectrum

    Press Enter to exit the information screen and return to the Scan choice list. Reviewing a stored spectrum Once you have stored a spectrum, you can retrieve it for review from one of the five available storage slots by selecting the Review option from the Scan choice list.

  • Page 126: Replaying A Spectrum

    Enter the storage slot number (1 to 3) of the spectrum you want to subtract out from the current (or retrieved) spectrum. Press Enter. The message “Subtracting spectrum n” appears. The detector reviews and subtracts the spectrum specified from the current spectrum and, after a brief wait, displays the difference spectrum.

  • Page 127: Scanning Using The Cuvette

    Scanning using the cuvette The detector cuvette option allows the operator ease of use in: • Sample handling • Instrument verification and qualification The detector uses a standard 10-mm path length spectrophotometric cell (quartz cuvette). You insert the cuvette with a frosted side facing up in the cuvette holder and place it in the detector flow cell assembly.

  • Page 128: Before You Begin

    Before you begin To ensure accurate results, use a 10-mm path length Recommendation: quartz cuvette and matched pairs (from the same manufacturing lot) of quartz cuvettes for your zero and sample scans. Before beginning a scan using the cuvette: Flush the flow cell with the eluent you are going to scan. To ensure a clear and clean cuvette, wipe the clear portion of the cuvette with low-lint, nonabrasive tissue wipes.
  • Page 129 With the spring guide facing you, gently insert the cuvette (containing eluent) up under the guide, with the cap facing upward (into the holder) and a frosted side of the cuvette facing up. Refer to the figure on page 3-59. Recommendations: •...

  • Page 130: Scanning Using A Flow Cell And A Syringe

    Rules: • To prevent invalid subsequent chromatographic results, remove the cuvette from the detector and replace the empty holder after running your cuvette scans. • To maintain optimum system performance, replace the front left panel cover before resuming normal operation of the detector. Insert a reference cuvette containing the mobile phase standard and run a zero scan.
  • Page 131 Waters recommends that you program the lamp to shut off Recommendation: or turn the lamp off manually only if the “Lamp off” time is more than 4 hours. Without powering off the system, you can conserve lamp life by •...
  • Page 132 Press Lamp (Shift 1) again to turn the lamp off. The absorbance screen appears with an X through the lamp indicator icon, and the display shows the words “Lamp off.” Lamp off/on sequence Lamp off indicator Lamp on indicator To turn on the lamp manually: When there is an X through the lamp icon on the absorbance screen, press Lamp (Shift 1).

  • Page 133: Shutting Down The Detector

    Shutting down the detector If you need to power-off the detector for an extended length of time, you must remove any buffered mobile phase present in the fluid path. To avoid damage to the column, remove it before you Caution: perform the following procedure.
  • Page 134 3-66 Preparing the Detector...

  • Page 135: Maintaining The Detector

    Read the appropriate maintenance instructions before starting the procedures for any of the tasks in this chapter. If you are uncertain about how to perform the procedures, call the Waters Technical Service Department to have a trained service representative perform the procedure.

  • Page 136: Contacting Waters Technical Service

    Contacting Waters Technical Service For problems with HPLC equipment, computer software, or hardware other than for the Waters 2489 UV/Visible Detector, consult the documentation for the applicable instrument or program. If you encounter a problem with the detector that you cannot troubleshoot, contact Waters Technical Service at 1-800-252-4752, U.S.

  • Page 137: Proper Operating Procedures

    Proper operating procedures Removing the front left panel cover Caution: • There are no user-serviceable parts inside the detector. Do not remove the top cover. • To maintain optimum performance, replace the front left panel cover before resuming normal operation of the detector. To remove the front left panel cover: Holding on to the top of the cover, gently pull the bottom away from the unit.

  • Page 138: Routine Maintenance

    Routine maintenance The detector requires minimal routine maintenance. To achieve optimal performance, • replace solvent reservoir filters in your HPLC system regularly. • filter and degas solvents to prolong column life, reduce pressure fluctuations, and decrease baseline noise. • flush buffered mobile phases out of the detector with HPLC-grade water followed by a 5 to 10% methanol solution each time the detector is powered-off.

  • Page 139: Flushing The Flow Cell

    Flushing the flow cell Flush the flow cell when it becomes contaminated with the residues of previous runs and after each detector shutdown. A dirty flow cell can cause baseline noise, decreased energy levels, calibration failure, and other problems. Always flush and purge the flow cell as your initial attempt to correct these problems.

  • Page 140: Removing And Cleaning The Flow Cell

    If the mobile phase is not miscible in water, first use an Caution: intermediary solvent. Resume pumping mobile phase. Reattach the column. Waters recommends renormalizing in 100% water before resuming Tip: analyses. If the flow cell remains dirty or blocked, reverse flush it. Removing and cleaning the flow cell If flushing the flow cell is not effective, follow the procedures to remove the flow cell and inspect for dirty or broken windows or dirty gaskets.

  • Page 141: Tools Required

    • To prevent contamination, use powder-free finger cots or gloves when touching the flow cell lenses or window. • Take care to prevent scratching of the flow cell parts. • Use a clean nonparticulating cloth or a similar clean surface in the work area where you are removing, rebuilding, and replacing the flow cell.
  • Page 142 Unscrewing the flow cell assembly captive screws Captive screws (3) Gently pull the assembly toward you. Maintaining the Detector...
  • Page 143 Removing the flow cell assembly TP02813 Once you remove the flow cell assembly from the detector chassis, remove the cuvette holder from the flow cell assembly. Maintaining the flow cell...

  • Page 144: Disassembling The Flow Cell

    To prevent contamination, use powder-free finger cots or gloves Caution: when disassembling, inspecting, cleaning, or replacing parts within the Waters TaperSlit™ Flow Cell or when removing or replacing the flow cell within its assembly. The TaperSlit flow cell consists of these components: •...
  • Page 145 • Entrance lens • Entrance lens mount • Two gaskets Waters TaperSlit flow cell Cell exit Cell entrance For replacement parts for the TaperSlit flow cell, use the Flow Cell Rebuild Kit (part numberWAS081346). Tip: Use nitrogen to clean the flow cell. Use ethanol or methanol to clean the lenses and window.
  • Page 146 Using the plastic tweezers or the lint-free swab, tap and remove the clear plastic gasket. The figure below shows an exploded view of all parts of the flow cell within the flow cell assembly. Waters TaperSlit flow cell, exploded view Split ring Cuvette lens Cuvette lens screw...

  • Page 147: Inspecting, Cleaning, And Replacing Damaged Flow Cell Components

    Caution: gloves when disassembling, inspecting, cleaning, or replacing parts within the Waters TaperSlit Flow Cell or when removing or replacing the flow cell within its assembly. Work on a clean flat surface, such as a nonparticulating cloth or similar surface.
  • Page 148 With the tan-colored entrance lens holder curve facing down, use the torque wrench to tighten the entrance lens screw into the flow cell body to 0.904 Nm (128 in-oz or 8 in-lb). Flip the flow cell body over to the exit window side. Using plastic tweezers, inspect the second new gasket for cleanliness.

  • Page 149: Replacing The Flow Cell

    To prevent contamination, use powder-free finger cots or Caution: gloves when disassembling, inspecting, cleaning, or replacing parts within the Waters TaperSlit Flow Cell or when removing or replacing the flow cell within its assembly. The detector is shipped with a standard analytical flow cell installed. Replace the flow cell when •...

  • Page 150: Replacing The Lamp

    Replacing the lamp Recommendation: Waters recommends that you change the detector lamp when it repeatedly fails to ignite, or when the detector fails to calibrate. See also: The tables titled “Start up, calibration and operating error messages” (page 5-3), “Instruments error messages”...

  • Page 151: When To Replace The Lamp

    The Waters 2489 Detector uses a combination of front-end electronics and lamp optimization software that makes the best use of the energy present to optimize instrument performance. The design also compensates for the lamp energy changes that occur across the deuterium spectrum as well as lamp aging.

  • Page 152: Removing The Lamp

    If the lamp no longer provides an adequate signal-to-noise ratio for your specific application, replace it. The 2489 UV/Visible Detector source lamp is warranted to light and pass startup diagnostic tests for 2000 hours or one year from the date of purchase, whichever comes first.
  • Page 153 After the lamp is extinguished, allow the lamp to cool for a minimum of 30 minutes. Remove the front left panel cover, as described in “Proper operating procedures” on page 4-3. Disconnect power to the lamp and disconnect the cord. Lamp assembly and power connector Loosen the two captive screws in the lamp base.
  • Page 154 Loosening the captive screws at the lamp housing base Pull the lamp assembly out of the lamp housing. Gently pull the lamp out. Lamp gas is under slight negative pressure. To prevent Caution: shattering the glass, use care when disposing of the lamp. Cushion the old lamp by placing it in the packaging material of the new lamp before disposal.

  • Page 155: Installing The New Lamp

    Removing the lamp TP0281 Installing the new lamp Warning: To avoid the possibility of exposing your eyes to ultraviolet radiation, never ignite a lamp while it is outside the instrument or if it is not properly screwed in place. Do not touch the glass bulb on the new lamp. Dirt or Caution: fingerprints on the bulb affect detector operation.
  • Page 156 Record the serial number located on a label attached to the lamp connector wire using the procedure in “Recording the new lamp serial number” on page 4-23. Make sure the detector is powered-off and the power cord is Requirement: disconnected. To install the new lamp: When you change the lamp, always power-off the detector.

  • Page 157: Recording The New Lamp Serial Number

    Aligning the lamp Recording the new lamp serial number Caution: • Always run the Change Lamp diagnostic test after you install a new lamp (see “Using the lamp, display, and keypad diagnostic tests” on page 5-15). • If you do not record the new lamp serial number following the procedure in this section, the lamp warranty is voided.
  • Page 158 Be sure to enter the 9-digit lamp serial number and not the lamp Tip: part number when performing this procedure. Enter the 9-digit serial number of the new lamp in the active field. This field accepts numeric entries only. Change Lamp screen Press Enter to store the serial number and move to the Date installed field.

  • Page 159: Setting The Lamp Threshold

    Lamp serial number OK to store message 11. When the confirmation message appears, press Enter. Lamp serial number confirmation message 12. Perform a manual wavelength calibration (see “Wavelength calibration” on page 3-30). Requirement: To run the verification procedures with the new lamp installed, recalibrate the detector or recycle power to the detector after replacing the lamp.

  • Page 160: Replacing Fuses

    Lamp alarm threshold screen Replacing fuses The fuse holder is located on the rear panel of the detector (see figure below). Two fuses are shipped in place with the detector, rated as indicated in “Power requirements” on page 2-5. Detector rear panel fuse holder TP02807 Fuse holder 4-26...
  • Page 161 Warning: To avoid electric shock, power-off and unplug the instrument before checking the fuses. For continued protection against fire hazard, replace fuses Warning: only with those of the same type and rating. The detector requires two 100 to 240-VAC, 50 to 60-Hz, F 3.15-A, 250-V fast blo, 5 ×...
  • Page 162 Removing and replacing the rear panel fuses and fuse holder Remove and discard the fuses. Make sure that the new fuses are properly rated for your requirements, and then insert them into the holder and the holder into the power entry module, gently pushing until the assembly locks into position.
  • Page 163 Error Messages, Diagnostic Tests, and Troubleshooting The Waters 2489 UV/Visible Detector provides both user and service diagnostic tests to troubleshoot system problems. • Error messages – Describe powerup, calibration, and other error messages and recommended actions for correcting the errors.

  • Page 164: Error Messages, Diagnostic Tests, And Troubleshooting

    Messages requiring you to perform corrective action. These include messages encountered at startup, and during calibration or operation. • Messages requiring you to recycle power, then contact Waters Technical Service personnel if the error persists (see “Contacting Waters Technical Service”...
  • Page 165 Startup, calibration, and operating error messages Error Message Description Corrective Action Wavelengths span In dual wavelength Select wavelengths that are 370 nm: Order filter mode: both above or both below not in use • If both selected 370 nm. wavelengths > 370 nm, the detector applies the second order filter to block...
  • Page 166 Startup, calibration, and operating error messages Error Message Description Corrective Action Lamp lighting failure The lamp failed to 1. Recycle power to the ignite. detector. 2. Check lamp power connection. 3. Replace lamp. Lamp external input A timed event or 1.

  • Page 167: Error Messages Preventing Operation

    1. Recycle power to the performs a complete detector. verification, which 2. Perform manual includes calibration. remeasurement of all 3. Contact Waters calibration points. Technical Service. New calibration points are compared to stored information from the most recent manual calibration.
  • Page 168 • the front left panel door is shut securely. Recycle power to the detector. If the fatal error persists, contact Waters Technical Service. Instrument error messages Error Message Description Corrective Action Electronic A/D failure Lamp optimization is Recycle power. adjusted at the minimum level.
  • Page 169 Corrective Action System cannot respond Error occurs while unit 1. Recycle power to the is positioning next detector. wavelength or changing 2. Contact Waters modes. Occurs during Technical Service. initialization or calibration. Filter initialization Unit sensors cannot find 1. Recycle power to the...

  • Page 170: User-Selected Diagnostic Tests

    System not calibrated. The calibration read 1. Recycle power to the from nonvolatile detector. memory is not valid. 2. Perform manual calibration. 3. Contact Waters Technical Service. Communication failure: A/D communication test 1. Recycle power to the Reference A/D failed. detector.
  • Page 171 To perform user-selectable diagnostic tests: Press the DIAG key from the detector front panel. The detector displays the diagnostic tests choice list. Diagnostic tests choice list Reset diagnostics To access a specific diagnostic test, press the up or down arrow key to highlight the test you want to run and then press Enter, or press a number from 1 to 6 corresponding to the test number on the detector keypad.
  • Page 172 The user-selectable sticky diagnostic tests are: – Fix (Set) Voltage Output – Fix (Set) Absorbance Input – Generate Test Peaks – Optical Filter Override Caution: Application of sticky diagnostic tests affects results. To clear changes to the voltage output or absorbance input, or to make a manual optical filter change, select 1, “Reset instrument”...

  • Page 173: Using The Diagnostic Tests

    • Previous choices << Using the diagnostic tests The detector employs both user-selectable and service diagnostic tests. You access user diagnostic tests by pressing the DIAG key. Only qualified Waters service personnel can access service diagnostic tests. Contacting Waters Technical Service...

  • Page 174: Using The Input & Output Diagnostic Tests

    AU time trace. The current sample and reference energy readings are displayed in nanoamps (nA). Sample and reference energy diagnostic test To use the sample and reference energy diagnostic test: Press DIAG, and then press 2. To change the wavelength, enter a new wavelength number. Press Enter.

  • Page 175: Displaying Auto Zero Offsets

    Input & output diagnostic test choice list Auto zero offsets Displaying Auto Zero offsets From the Input & Output choice list, press 1 (Auto zero offsets). This diagnostic test also allows you to display and reset the offset to zero on both channels by pressing Cancel (Shift 0).

  • Page 176: Setting Fixed Voltage Output

    This diagnostic test sets the voltages on the analog output channels based on the current AUFS setting. This is a sticky diagnostic test. Setting fixed voltage output From the Input & Output choice list, press 3 (Fix voltage) to select a voltage for the analog output.

  • Page 177: Using The Lamp, Display, And Keypad Diagnostic Tests

    For the outputs (SW1 and SW2): Press Enter to display the active switch (surrounded by a dotted-line border). Press any numerical key to change the status of the switch (ON to OFF or vice versa). Press Enter to select the second switch. Using the lamp, display, and keypad diagnostic tests To access the lamp, display, and keypad diagnostic tests, press DIAG and then press 4.
  • Page 178 The display fills from top to bottom and from right to left, then returns to the Lamp, display & keypad choice list. If the display does not completely fill either horizontally or vertically, contact your Waters service representative. From the Lamp, display & keypad choice list, press 4 to return to the diagnostic tests choice list.

  • Page 179: Using The Other Detector Diagnostic Tests

    Using the other detector diagnostic tests The user diagnostic test display provides two additional features. You can: • Generate test peaks – Use this diagnostic test to specify the generation of test peaks to calibrate your chart recorder or data system. •...
  • Page 180 The amplitude of the test peaks is affected by your choice of filter time constant. This is a sticky diagnostic test. When this routine is selected, the choice list changes to read “Disable test peaks.” Press 1 (Disable test peaks) from the Other diagnostics choice list to turn off this diagnostic test.

  • Page 181: Service Diagnostic Tests

    Most detector problems are relatively easy to correct. If you are unable to correct a problem or a failed condition, contact Waters Technical Service at 1-800-252-4752, U.S. and Canadian customers only. Other customers, call...

  • Page 182: Diagnostic Tests

    Front panel display Broken electrical Check electrical connections. fails to illuminate connection Fuse blown Check and, if necessary, replace fuse(s). Bad LCD or control Contact Waters Technical board Service. Front panel Faulty EPROMs Contact Waters Technical displays odd Service. Bad LCD control...
  • Page 183 Keypad not Keypad defective 1. Recycle power to the detector functioning and run the keypad diagnostic test. 2. Contact Waters Technical Service. Deuterium lamp Faulty lamp Replace the lamp. does not light Lamp not plugged Plug in the lamp connector.
  • Page 184 250 nm. 3. Ensure there are no air bubbles in the flow cell. 4. Recalibrate the detector. 5. If problems persist, contact Waters Technical Service. Analog output AUFS setting Reset the AUFS setting. incorrect changed 5-22...
  • Page 185 Safety Advisories Waters instruments display hazard symbols designed to alert you to the hidden dangers of operating and maintaining the instruments. Their corresponding user guides also include the hazard symbols, with accompanying text statements describing the hazards and telling you how to avoid them.

  • Page 186: A Safety Advisories

    Heed all warnings when you install, repair, and operate Waters instruments. Waters assumes no liability for the failure of those who install, repair, or operate its instruments to comply with any safety precaution.

  • Page 187: Warnings That Apply To Particular Instruments, Instrument Components, And

    The following warnings can appear in the user manuals of particular instruments and on labels affixed to them or their component parts. Burst warning This warning applies to Waters instruments fitted with nonmetallic tubing. Pressurized nonmetallic, or polymer, tubing can burst. Warning: Observe these precautions when working around such tubing: •...

  • Page 188: Mass Spectrometer Shock Hazard

    Standby mode before touching areas marked with this high voltage warning symbol. Biohazard warning This warning applies to Waters instruments that can be used to process material that might contain biohazards: substances that contain biological agents capable of producing harmful effects in humans.

  • Page 189: Chemical Hazard Warning

    Chemical hazard warning This warning applies to Waters instruments that can process corrosive, toxic, flammable, or other types of hazardous material. Warning: Waters instruments can be used to analyze or process potentially hazardous substances. To avoid injury with any of these materials, familiarize yourself with the materials and their hazards, observe Good Laboratory Practices (GLP), and consult your organization’s safety...
  • Page 190 Attention: Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user’s authority to operate the equipment. Important: Toute modification sur cette unité n’ayant pas été expressément approuvée par l’autorité responsable de la conformité à la réglementation peut annuler le droit de l’utilisateur à...
  • Page 191 • Keine Schläuche verwenden, die stark geknickt oder überbeansprucht sind. • Nichtmetallische Schläuche nicht für Tetrahydrofuran (THF) oder konzentrierte Salpeter- oder Schwefelsäure verwenden. • Durch Methylenchlorid und Dimethylsulfoxid können nichtmetallische Schläuche quellen; dadurch wird der Berstdruck des Schlauches erheblich reduziert. Warnings that apply to all Waters instruments...
  • Page 192 Attenzione: prestare attenzione durante l’utilizzo dei tubi di polimero pressurizzati: • Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero pressurizzati. • Estinguere ogni fonte di ignizione circostante. • Non utilizzare tubi soggetti che hanno subito sollecitazioni eccessive o son stati incurvati.
  • Page 193 Warnings that apply to all Waters instruments...
  • Page 194 Warning: The user shall be made aware that if the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired. Attention: L’utilisateur doit être informé que si le matériel est utilisé d’une façon non spécifiée par le fabricant, la protection assurée par le matériel risque d’être défectueuses.
  • Page 195 Attenzione: per una buona protezione contro i rischi di incendio, sostituire i fusibili con altri dello stesso tipo e amperaggio. Advertencia: sustituya los fusibles por otros del mismo tipo y características para evitar el riesgo de incendio. Warnings that apply to all Waters instruments A-11...
  • Page 196 Warning: To avoid possible electrical shock, disconnect the power cord before servicing the instrument. Attention: Afin d’éviter toute possibilité de commotion électrique, débranchez le cordon d’alimentation de la prise avant d’effectuer la maintenance de l’instrument. Vorsicht: Zur Vermeidung von Stromschlägen sollte das Gerät vor der Wartung vom Netz getrennt werden.

  • Page 197: Electrical And Handling Symbols

    Electrical and handling symbols Electrical symbols – These can appear in instrument user manuals and on the instrument’s front or rear panels. Electrical power on Electrical power off Standby Direct current Alternating current Protective conductor terminal Frame, or chassis, terminal Fuse Recycle symbol: Do not dispose in municipal waste.

  • Page 198: Handling Symbols

    Handling symbols These handling symbols and their associated text can appear on labels affixed to the outer packaging of Waters instrument and component shipments. Keep upright! Keep dry! Fragile! Use no hooks! A-14 Safety Advisories...
  • Page 199 Detector Specifications This appendix lists individual operating specifications for the Waters 2489 UV/Visible Detector, as follows: • Operational specifications • Optical specifications • Specifications for the optional flow cells Contents: Topic Page Operational specifications Optical specifications Optional Waters TaperSlit Flow Cell specifications...

  • Page 200: B Detector Specifications

    Operational specifications Operational specifications Condition Specification Wavelength range 190 to 700 nm Bandwidth <5 nm Wavelength accuracy +1.0 nm Wavelength +0.1 nm repeatability Single λ dry noise <5 µAU (at 230 nm, 1-second digital filter, 30-second segments, 10-Hz data rate, analytical cell) <8 µAU Wet noise (at 230 nm, 1-second digital filter, 30-second...
  • Page 201 Operational specifications Condition Specification Analog output 10, 20, 40, 80 Hz (channel A) data rates 10 Hz only (channel B) (single λ mode) Optical Component Specifications Lamp source 30-W High Brightness Deuterium lamp, 0.5 nm aperture, pre-aligned 2000-hour warranty, front accessible Photodiodes 2 silicon photodiodes (matched pair) Second order filter...

  • Page 202: Optical Specifications

    Operational specifications Condition Specification Fuse ratings Two fuses: 100 to 240-Vac, 50 to 60-Hz F 3.15-A 250-V fast blo, 5 × 20 mm (IEC) Attenuated analog Attenuation range: 0.0001 to 4.000 AU 2-V output range: –0.1 to +2.1 V output channel: 2 VFS Two event outputs Type: Contact closure...

  • Page 203: Optional Waters Taperslit Flow Cell Specifications

    Optional Waters TaperSlit Flow Cell specifications Optional Waters TaperSlit Flow Cell specifications Sample Tube Path Volume Internal Pressure Length (µl) Diameter Rating (mm) (Inches) In/Out psi/bar Analytical Cell 0.009 0.009 1000/70 Semi-Prep Cell 0.040 0.040 1000/70 Microbore Cell 0.005 0.005...
  • Page 204 Detector Specifications...

  • Page 205: C Spare Parts

    Spare Parts The table below lists the spare parts for the Waters 2489 UV/Visible Detector. These spare parts are user-replaceable. Spare parts Item Part Number Fuses Fuse, 3.15 A, 5 × 20 mm WAS163-16 Fuse Holder WAT055426 Optical Parts/Assemblies Deuterium Lamp Assembly/Preventive...
  • Page 206 Spare parts Item Part Number Optional TaperSlit Flow Cells Analytical Cell WAS081140 Semi-Prep Cell WAT081158 Microbore Cell WAT081159 Inert (titanium) Cell WAT081157 High Pressure (3000 psi) Cell for LCMS WAT081321 Autopurification Cell 289000614 Optional Variable Prep Flow Cells Variable Prep Flow Cell 700000923 Variable Prep Flow Cell Quartz Rods and Seals 700000688...
  • Page 207 Solvent Considerations Contents: Topic Page Introduction Solvent miscibility Buffered solvents Head height Solvent viscosity Mobile phase solvent degassing Solvent degassing methods Wavelength selection To avoid chemical hazards, always observe Good Laboratory Warning: Practices when operating your system.

  • Page 208: D Solvent Considerations

    Introduction Clean solvents Clean solvents provide reproducible results and permit you to operate with minimal instrument maintenance. A dirty solvent can cause baseline noise and drift. It can also block the solvent filters with particulate matter. Solvent quality Use HPLC-grade solvents for the best possible results. Filter solvents through 0.45-µm filters before their use.

  • Page 209: Tetrahydrofuran

    Tetrahydrofuran When using unstabilized tetrahydrofuran, ensure that your solvent is fresh. Previously opened bottles of tetrahydrofuran contain peroxide contaminants, which cause baseline drift. Warning: Tetrahydrofuran contaminants (peroxides) are potentially explosive if concentrated or taken to dryness. Solvent miscibility Before you change solvents, refer to the table below to determine the miscibility of the solvents to be used.
  • Page 210 Solvent miscibility Boiling Miscibility λ Cutoff Polarity Viscosity Solvent Point °C Number Index CP, 20 °C (nm) (1 atm) Isopropyl ether 0.33 68.3 –– Toluene 0.59 100.6 P-xylene 0.70 138.0 Benzene 0.65 80.1 Benzyl ether 5.33 288.3 –– –– Methylene chloride 0.44 39.8 Ethylene chloride...

  • Page 211: How To Use Miscibility Numbers

    Solvent miscibility Boiling Miscibility λ Cutoff Polarity Viscosity Solvent Point °C Number Index CP, 20 °C (nm) (1 atm) Dimethylsulfoxide 2.24 189.0 –– Methanol 0.60 64.7 Formamide 3.76 210.5 –– Water 1.00 100.0 –– –– How to use miscibility numbers Use miscibility numbers (M-numbers) to predict the miscibility of a liquid with a standard solvent (see “Solvent miscibility”...

  • Page 212: Buffered Solvents

    Buffered solvents When using a buffer, use a good quality reagent and filter it through a 0.45-µm filter. Do not leave the buffer stored in the system after use. Flush all fluid line pathways with HPLC-quality water before shutting the system down and leave distilled water in the system (flush with 90% HPLC-quality water: 10% methanol for shutdowns scheduled to be more than one day).

  • Page 213: Gas Solubility

    • stable pump operation. Gas solubility Only a finite amount of gas can be dissolved in a given volume of liquid. This amount depends on • the chemical affinity of the gas for the liquid. • the temperature of the liquid. •...

  • Page 214: Solvent Degassing Methods

    Solvent degassing methods This section describes the solvent degassing techniques that will help you to attain a stable baseline. Degassing your solvent also improves reproducibility and pump performance. You can use either of the following methods to degas solvents: • Sparging with helium •...

  • Page 215: Vacuum Degassing

    To increase the length of membrane, you can connect two or more vacuum chambers in series. The in-line degasser is available as an option or factory-installed in the  Waters 2695 Separations Module, XE model. Wavelength selection This section includes UV cutoff ranges for •...
  • Page 216 UV cutoff wavelengths for common chromatographic solvents UV Cutoff UV Cutoff Solvent Solvent (nm) (nm) Acetone Isopropanol Acetonitrile Isopropyl chloride Amyl alcohol Isopropyl ether Amyl chloride Methanol Benzene Methyl acetate Carbon disulfide Methyl ethyl ketone Carbon tetrachloride 265 Methyl isobutyl ketone Chloroform Methylene chloride...

  • Page 217: Wavelength Selection For Chromophore Detection

    Triton-X™ 100, 0.1% Hydrochloric acid, 0.1% ® Waters PIC Reagent A, 1 vial/liter MES, 10 mM, pH 6.0 Waters PIC Reagent B-6, 1 vial/liter Potassium phosphate, Waters PIC Reagent B-6, monobasic, 10 mM low UV, 1 vial/liter dibasic, 10 mM...
  • Page 218 particular analysis. Because of the diversity possible within a given sample, scanning over a range of wavelengths may be necessary to determine the best wavelength for a particular analysis. Electronic absorption bands of representative chromophores* λ ∈ λ ∈ Chemical Chromophore Configuration (nm)
  • Page 219 Electronic absorption bands of representative chromophores* λ ∈ λ ∈ Chemical Chromophore Configuration (nm) (L/m/cm) (nm) (L/m/cm) Nitrate —ONO 2 (shoulde Allene —(C=C) 2 — 210-230 21,000 (acyclic) Allene —(C=C) 3 — 35,000 Allene —(C=C) 4 — 52,000 Allene —(C=C) 5 — 118,000 Allene —(C=C) 2 —...
  • Page 220 D-14 Solvent Considerations...
  • Page 221 1-10 starting a method from 2-16 analog outputs turning the lamp on or off from 2-16 channel outputs 2-10 600 Series Pump. See Waters 600 connections 2-21 Series Pump dual wavelength 3-15 dual wavelength signal 3-19 signal adjusting 3-14...
  • Page 222 3-23 generating 2-28 generating from the 2695 bandwidth specification separations module 2-19 beamsplitter timed event parameter 3-36 benefits of degassing Waters 600 Series Pump biohazard warning connections 2-31 buffered mobile phase Chart Mark key flushing chart polarity removing 3-65 function...
  • Page 223 2-36 qualification features other systems using Ethernet sample analysis 2-14 – 2-15 scanning 3-44 3-59 – 3-62 Waters 600 series pump 2-29 – 2-33 using Waters 717plus Autosampler 2-33 – 2-35 damage Waters 745/745B/746 Data Module to the detector 2-25 –...
  • Page 224 operating 3-26 – 3-65 setting a fixed voltage output 5-14 operating under remote control startup 3-36 sticky optics test display 5-16 powering-off 3-65 user-selected – 5-18 setting up to run 3-13 using – 5-21 site requirements for verification failure 3-28 specifications –...
  • Page 225 Empower, controlling the detector RatioPlot function 1-15 using 2-15 spectral 1-10 enabling filter time constant chart mark event inputs 3-23 changing 3-12 external events 3-12 function 3-16 inputs 3-12 parameter 3-19 ending wavelength 3-44 filters Enter key 3-11 filter setting specification entering negative numbers 3-11 noise...
  • Page 226 scanning 3-44 3-62 generating specifications 4-15 auto zero on inject from the 2695 static 3-62 2-18 tools required to remove, clean, or chart mark from the 2695 2-19 replace chart marks 2-28 window 4-13 spectra flow cell assembly, removing test peaks 5-17 flow cell rebuild kit 4-11...
  • Page 227 Inject Start HOME key λ/λλ key Waters 600 Series Pump 3-31 3-33 connections 2-32 Lamp key 3-10 Waters 717plus Autosampler lamp, display, and keypad connections 2-35 diagnostic tests 5-11 5-15 input and output diagnostic tests 5-10 Lock key 3-10 5-12...
  • Page 228 4-21 – 4-23 method – removing 4-18 4-20 active 3-41 – replacing 3-28 4-16 4-18 choice list serial number 4-23 – 4-25 current conditions 3-13 timed event parameter 3-37 initial conditions 3-23 3-41 turning off 3-62 – 3-64 Method * 3-35 3-41 turning on or off...
  • Page 229 filtering 3-16 overriding the optical filter 5-18 numerical keys 3-10 pace 3-44 obtaining parameters MaxPlot 3-35 absorbance threshold timed event RatioPlot 3-34 3-37 stored spectrum information 3-56 analog out (dual wavelength) 3-19 offset AUFS 3-19 absorbance 3-12 auto zero on inject 3-17 3-19 Auto Zero Offset diagnostic test...
  • Page 230 performing verification procedures reassembling the flow cell – 4-15 – 3-27 3-30 rebuild kit, flow cell 4-11 – photodiodes rebuilding the flow cell 4-13 4-14 Plus/minus key 3-11 recalling the absorbance screen using polarity timed event parameter 3-36 the HOME key polarity, chart 3-12 recording...
  • Page 231 resolution, reduction in 3-44 cuvette 3-44 3-59 – 3-62 retrieving a method 3-41 Erbium 3-46 returning to initial conditions flow cell 3-44 3-62 reviewing a scan 3-57 initiating routine maintenance new spectra 3-48 – 3-54 run clock, stopping pace 3-44 run time icon reference energy 3-47...
  • Page 232 Waters 5-11 viscosity considerations diagnostic tests 5-11 5-19 spare parts Set absorbance diagnostic test 5-10 sparging set voltage diagnostic test 5-10 specifications setting bandwidth detector up to run 3-13 dimensions fixed absorbance value 5-13 drift fixed voltage output 5-14...
  • Page 233 – 3-22 switched outputs 2-11 TRACE key 3-20 switches transient energy 5-20 programming 3-24 troubleshooting setting 5-14 contacting Waters 5-19 symbols diagnostic tests – 5-21 caution hardware 5-20 electrical A-13 turning lamp off to conserve lamp life – handling A-14...
  • Page 234 Waters data system control 2-14 scale function to zoom 3-20 Waters TaperSlit flow cell static flow cell for scanning 3-62 description – the 2489 as a spectrophotometer exploded view 4-12 1-13 frontal view 4-11 specifications 4-15 Waters Technical Service, contacting vacuum degassing.

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