Waters 2475 Operator's Manual
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Waters 2475 Operator's Manual

Multi fluorescence detector
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2475 Multi λ
Fluorescence Detector
Operator's Guide
71500247502/Revision F
Copyright © Waters Corporation 2010
All rights reserved

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  • Page 1 2475 Multi λ Fluorescence Detector Operator’s Guide 71500247502/Revision F Copyright © Waters Corporation 2010 All rights reserved...

  • Page 2: Copyright Notice

    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: Customer Comments

    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...

  • Page 4: Safety Advisories

    Australia C-Tick EMC Compliant Confirms that a manufactured product complies with all applicable United States and Canadian safety requirements Consult instructions for use Audience and purpose This guide is intended for personnel who install, operate, and maintain the 2475 Multi λ Fluorescence detector.

  • Page 5: Intended Use Of The Instrument

    Intended use of the instrument Waters designed the 2475 Multi λ Fluorescence Detector to analyze samples in high-performance liquid chromatography (HPLC) applications. The 2475 Multi λ Fluorescence detector is for research use only Calibrating To calibrate LC systems, follow acceptable calibration methods using at least five standards to generate a standard curve.

  • Page 6: Ec Authorized Representative

    EC authorized representative Waters Corporation (Micromass UK Ltd.) Floats Road Wythenshawe Manchester M23 9LZ United Kingdom Telephone: +44-161-946-2400 Fax: +44-161-946-2480 Contact: Quality manager...

  • Page 7: Table Of Contents

    Table of Contents Copyright notice ....................ii Trademarks ......................ii Customer comments .................... iii Contacting Waters ....................iii Safety considerations ..................iii Safety advisories ....................iv Operating this instrument ................. iv Applicable symbols ..................... iv Audience and purpose..................iv Intended use of the instrument................v Calibrating ......................
  • Page 8 Photomultiplier tube..................1-4 Scanning ......................1-5 Multichannel operation ................... 1-5 Fluorescence data .................... 1-5 References ......................1-6 Detector description ..................1-7 Features......................1-7 Principles of operation ..................1-9 Detector optics....................1-9 Optics assembly light path ................1-10 Photomultiplier (PMT) calibration ............... 1-12 PMT sensitivity....................
  • Page 9 Plumbing the detector ..................2-3 Connecting columns..................2-3 Assembling fittings ..................2-4 Making tubing connections ................2-5 Making signal connections ................2-5 Component connection overview..............2-6 Connecting the Ethernet cable................ 2-7 Choosing signal connections................2-10 Making I/O signal connections ..............2-11 Signal connections ..................
  • Page 10 Two operating modes ..................3-29 Standalone operation..................3-29 Remote control operation for 474 emulation mode via RS-232 ....3-29 Remote control operation via Ethernet connection using 2475 instrument control software..................3-34 Verifying the detector ..................3-34 Manual wavelength calibration ..............3-35 Normalizing emission units ................
  • Page 11 Using a timed event method to program the lamp ........3-70 Shutting down the detector ................3-70 4 Maintenance Procedures ..............4-1 Contacting Waters technical service ............4-2 Maintenance considerations ................4-2 Safety and handling..................4-2 Spare parts ....................... 4-3 Routine maintenance ..................
  • Page 12 Hardware troubleshooting ................5-21 A Safety Advisories .................. A-1 Warning symbols ....................A-2 Task-specific hazard warnings................ A-2 Specific warnings ..................... A-3 Caution symbol ....................A-5 Warnings that apply to all Waters instruments ......... A-6 Electrical and handling symbols ..............A-12 Table of Contents...
  • Page 13 Electrical symbols ..................A-12 Handling symbols ..................A-13 B Specifications ..................B-1 C Solvent Considerations ............... C-1 Introduction ...................... C-2 Clean solvents ....................C-2 Solvent quality ....................C-2 Preparation checklist..................C-2 Water ........................ C-2 Buffers ......................C-3 Tetrahydrofuran (THF) ................... C-3 Solvent miscibility ...................
  • Page 14 Table of Contents...
  • Page 15 Theory of Operation This chapter explains the theory and technology supporting the ® 2475 Multi λ Fluorescence Detector and operation of the Waters describes the instrument’s features. Contents: Topic Page Fluorescence theory Fluorescence detection Measuring fluorescence Detector description Principles of operation...

  • Page 16: Theory Of Operation

    This type of detection can be accommodated by disabling the light source or (as in the case of the 2475 detector) enabling a shutter to stop any excitation light from reaching the flow cell.

  • Page 17: Fluorescence Detection

    • Measuring the emitted fluorescence • Amplifying the emitted signal Fluorescence detection Overview The scanning fluorescence detector illuminates a sample with a narrow band of high-intensity light. The detector then measures the low levels of fluorescence emitted by the sample. The emitted light is filtered, amplified, and converted to electrical signals that can be recorded and analyzed.

  • Page 18: Exciting The Sample

    also be selected. Detectors with excitation and emission monochromators can scan holding one monochromator at a constant setting while varying the setting on the other. This type of operation is necessary when you are evaluating mixtures or analyzing chemical structures. Exciting the sample The broad band of high-intensity light from the lamp passes through a filter or monochromator, which selects a narrow band of wavelengths.

  • Page 19: Scanning

    Emission units and normalization The 2475 detector offers two types of output units: emission and energy. Emission units are normalized to a standard water reference, and their magnitude is as independent of the PMT gain as possible. You can compensate...

  • Page 20: References

    Gain and Counts = values from the most recent execution of Raman Raman the normalize units function PMTCounts and Gain = values at the time of data collection Normalizing the emission units results in a water/Raman signal strength, at 350 nm/E 397 nm, of 100 emission units.

  • Page 21: Detector Description

    J. D. Winefordner, S. G. Schulman, and T. C. O’Haver, Luminescence Spectroscopy in Analytical Chemistry, Wiley-Interscience: New York, 1972. Detector description The 2475 Multi λ Fluorescence Detector is a multichannel, tunable, fluorescence detector designed for high-performance liquid chromatography (HPLC) applications.
  • Page 22 Fast Scan mode – Dynamically scans either the emission or excitation grating through a selectable wavelength range to monitor the fluorescence of a series of wavelengths. (This feature is available only in Empower systems through remote Ethernet interface with 2475 ICS software control.) Theory of Operation...

  • Page 23: Principles Of Operation

    Two ellipsoidal mirrors and one parabolic mirror • Shutter, wavelength calibration filter, and second-order filter • Entrance slits • Exit slits • Blazed, plane, and concave holographic diffraction gratings • Photomultiplier tube (PMT) • Waters axially illuminated flow cell Principles of operation...

  • Page 24: Optics Assembly Light Path

    The following diagrams show the optics assembly light paths and components. Excitation monochromator optics assembly Xenon lamp Grating Parabolic Filter mirror wheel Entrance slit Ellipsoidal Flow cell Exit slit, excitation mirror monochromator Emission monochromator optics assembly Photomultiplier tube Ellipsoidal mirror Exit slit Entrance slit Grating...
  • Page 25 and increases the detectability of low-level signals. Keeping the optics simple tends to minimize loss of signal and maximize throughput. Light source The detector uses a high-intensity 150-watt xenon arc lamp as its source. The emitted light passes through the excitation monochromator to flood the aperture of the flow cell.

  • Page 26: Photomultiplier (Pmt) Calibration

    PMT gain setting is at the lowest level. For this reason, the 2475 detector’s Auto Optimize Gain diagnostic function lets you adjust the granularity of the gain.

  • Page 27: Electronics

    The following figure shows the relationship between increased time-constant and response times. Effect of filter time constant 0 sec. 1 sec. 2 sec. TP02824 Time (minutes) Electronics The electronics consist of the following components: • Preamplifier board – Collects and processes the analog input signals from the PMT and photodiode to the microprocessor for further signal conditioning.

  • Page 28: Wavelength Verification And Test

    • Lamp power supply – Provides stable xenon lamp operation. • DC power supply – Provides voltage for the analog and digital circuitry. It is the DC power source for the detector. Wavelength verification and test The xenon arc lamp and the integral erbium filter exhibit peaks in the transmission spectrum at known wavelengths.

  • Page 29: Multichannel Mode

    that blocks unwanted ultraviolet (UV) light from reaching the diffraction grating, which can interfere with fluorescence detection of 400 nm and longer. Selecting the appropriate sampling rate A sufficient number of points must fall across a peak to define its shape. For this reason, the definition between peaks is lost at very low sampling rates.

  • Page 30: Spectrum Scanning

    wavelength pairs and plots the maximum fluorescence signal value for each sample component. The MaxPlot outputs the greater of the fluorescence values on the selected channels. Difference plot The detector allows you to obtain a difference plot in multichannel mode. The difference plot function monitors fluorescence at user-selected excitation/emission wavelength pairs and plots the difference in signal value between them.

  • Page 31: Auto-Optimize Gain

    The 2475 detector source lamp is warranted to light and pass startup diagnostic tests for 2000 hours or 1 year from the date of purchase, whichever comes first. The detector’s on-board diagnostics allow you to record lamp usage and report the lamp serial number.

  • Page 32: Method Optimization

    electronics. Too low a gain reduces sensitivity to emission signals, degrading signal-to-noise ratios. The detector therefore requires you to specify a gain setting for the PMT before you inject a sample. However, before the injection, you cannot know the magnitude of your fluorescence signal. Users traditionally resolve this difficulty by running several injections to determine a suitable gain setting, a tedious process, especially when they run timed event changes in gain and/or wavelength.

  • Page 33: Example Of Recommended Method Development Approach

    setting for all peaks in the chromatogram and no segregated peak region optimization. Example of recommended method development approach A method with two timed event changes optimizes the chromatogram shown below. Gain optimized chromatogram Emission Region 1 Region 2 Region 3 Time Gain: 10 Gain: 1000...

  • Page 34: Ensuring Gain Optimization For Each Peak Of Interest

    Example of method development (Continued) Time (min) Event Excitation = 375 nm, Emission = 410 nm (no need to change gain here) After you run the Auto-Optimize Gain diagnostic function, the detector displays recommended gain values. Recommended gain values EUFS: 2000 Best gain event time (min.) 0.0 (Initial)

  • Page 35: Startup Diagnostic Tests

    The gain for Region 2 is determined by the maximum signal level in Region 1. Therefore, a gain of only 10 would be used from time 0.0 to time 2.0, but the small peak may not be adequately resolved at this setting. If the detector did find it, the peak area integration would be far less accurate because of higher baseline noise.

  • Page 36: Wavelength Selection

    Wavelength selection In fluorescence, if the excitation monochromator is set below the UV cutoff of a mobile-phase component, the solvent absorbs some of the available excitation light intensity, which in turn reduces the fluorescence emission response for the sample. For a complete list of UV cutoff ranges for common solvents and common mixed mobile phases, refer to Appendix Using incompatible solvents can cause severe damage to...

  • Page 37: Setting Up The Detector

    Setting Up the Detector Contents Topic Page Before you begin Installing the detector Plumbing the detector Making signal connections Connecting other devices 2-23 Connecting to the electricity source 2-36...

  • Page 38: Before You Begin

    0 to 90%, noncondensing If you discover any damage or discrepancy when you inspect the contents of the cartons, immediately contact the shipping agent and your local Waters representative. Customers in the USA and Canada should report damage and discrepancies to Waters Technical Service (800 252-4752).

  • Page 39: Installing The Detector

    15.24 cm (6 inches) of clearance at the rear of the detector. To install the 2475 detector, place it on a level surface to allow proper function of the drip management system (drain tube), to which you can connect to a waste reservoir that diverts solvent leaks from the flow cell.

  • Page 40: Assembling Fittings

    ® Connect the Teflon tubing to the flow cell outlet tubing, and route it to a waste container. Plumbing connections Assembling fittings Slide the compression screw over the tubing end. Follow it with the ferrule mounted so that its taper faces the end of the tubing. Compression screw Tubing end (cut...

  • Page 41: Making Tubing Connections

    Making tubing connections To make tubing connections: Bottom each tubing end in the column outlet, detector inlet, or detector outlet fitting. Seat each ferrule by tightening the compression screw 1/2-turn past finger-tight. To ensure accurate verification during installation, be sure to pump fresh, Tip: degassed, and filtered 100% water through the flow cell before powering it.

  • Page 42: Component Connection Overview

    Fuse holder Power input Component connection overview Waters recommends you connect the 2475 detector to other system Tip: components via an Ethernet connection. The following table summarizes the signal connections needed to connect the 2475 detector to other HPLC system components.

  • Page 43: Connecting The Ethernet Cable

    (Consult the software installation instructions that accompany the instrument control software for details.) Single Waters instrument connection In a single Waters instrument system configuration, the connection hardware requires only one CAT 5, shielded, Ethernet cross-over cable (startup kit). Making signal connections...
  • Page 44 Ethernet switch communicates between Waters instruments and the acquisition computer. Connection hardware requires one standard, CAT 5, shielded, Ethernet cable per Waters instrument and a standard, CAT 5, shielded, Ethernet cable between the network switch and the acquisition computer. See the figure “Multiple Waters ethernet instrument connections”...
  • Page 45 Analog input 2 Analog input 1 Making inject-start signal connections The Ethernet data system or controller used with the 2475 detector must receive an inject-start signal from the autosampler or manual injector to initiate the data collection and time-based programs.

  • Page 46: Choosing Signal Connections

    Tip: inputs and outputs” on page 2-11. Choosing signal connections Connect the 2475 detector to other HPLC system components through an Tip: Ethernet connection. The rear panel provides two analog connectors and an RS-232 communications port for operating the detector with external devices. You can...

  • Page 47: Making I/O Signal Connections

    • RS-232 Interface – The RS-232 connection allows remote control and direct data acquisition from an Empower system or a Millennium workstation (version 3.2 and later) in 474 emulation mode. Making I/O signal connections The rear panel includes two removable connectors that hold the pins for the I/O signals (see the following figure).

  • Page 48: Signal Connections

    I/O signals for the detector (Continued) Signal Description Configurable input to allow an external device to turn Lamp On/Off the xenon lamp off and on. Configurable input to add a chart mark (at 10% of full Chart Mark scale) to either or both analog output channels (Detector Out 1 and Detector Out 2).
  • Page 49 To make signal connections: Attach the positive and negative leads of the signal cable to the connector. Connector Signal cable Slide the clamp (with the bend facing down) into the protective shield. Insert the clamp and shield (with the bend facing down) into the connection cover and loosely tighten with one self-tapping screw.

  • Page 50: Connecting An Alliance Separations Module

    Insert the connector with the signal cable into the connection cover, position the clamp over the cable leads, and then tighten the clamp into place with the second self-tapping screw. Cable leads Clamp Place the second connection cover over the first cover and snap it into place.
  • Page 51 Make the connections shown in the following table and figure. Connections for generating an auto zero on inject Alliance separations module 2475 detector (A inputs) (B inputs and outputs) Pin 9 Auto Zero + Pin 1 Inject Start Pin 2 Inject Start Pin 10 Auto Zero –...
  • Page 52 Make the connections shown in the following table and figure. Connections for generating a chart mark on inject Alliance separations module 2475 detector (A inputs) (B inputs and outputs) Pin 1 Inject Start Pin 6 Chart Mark + Pin 2 Inject Start Pin 7 Chart Mark –...
  • Page 53 Connections for starting a method Alliance separations module 2475 detector (A inputs) (B inputs and outputs) Pin 1 Inject Start + Pin 1 Inject Start Pin 2 Inject Start Pin 2 Inject Start –...

  • Page 54: Connecting Rs-232 Devices

    Make the connections shown in the following table and figure. Connections for turning the detector lamp on or off Alliance separations module 2475 detector (A Inputs) (A Outputs) Pin 1 Switch 1 Pin 4 Lamp On/Off + Pin 2 Switch 1 Pin 5 Lamp On/Off –...
  • Page 55 RS-232 communication does not support multichannel mode. Tip: When connecting the detector to a Waters data system, all detector Tip: parameters not configurable by the data system in use defer to local control. Making signal connections 2-19...

  • Page 56: Connecting Ethernet Devices

    To connect an RS-232 device such as a Waters data system to the detector: Connect the single receptacle end of the RS-232 cable (supplied with the detector) to an RS-232 device. Such a device may be an RS-232 communications port or Equinox card in an Empower system or a Millennium chromatography workstation.
  • Page 57 “Multiple Waters ethernet instrument connections” on page 2-22. You must install the Waters instrument control software in the acquisition computer before the computer can control the Waters instrument. See the software installation instructions that accompany the software instrument driver disk.
  • Page 58 Analog input 1 Making inject-start signal connections When you are using an Ethernet data system with the 2475 detector, the data system or controller must receive an inject-start signal from the autosampler or manual injector to initiate the data collection and time-based programs.

  • Page 59: Connecting Other Devices

    Chart recorder • Waters 600-series pump • Waters 717plus Autosampler • Waters Fraction Collector II or III Required materials To connect cables to the terminals on the detector’s rear panel, you need the following tools: • Small flat-blade screwdriver (startup kit) •...

  • Page 60: Connecting A Data System Using A Bus Sat/In Module

    Empower system or Millennium computer according to the instructions in the Waters Bus SAT/IN Module Installation Guide. Caution: • Do not turn start the Bus SAT/IN module until you perform all procedures in the Waters Bus SAT/IN Module Installation Guide.
  • Page 61 Using the electrical insulation stripping tool, strip about 1/8 inch from one end of the Bus SAT/IN connector, exposing the white and black wires. For channel A (see the figure “Connecting the Bus SAT/IN module channel 1 to the detector” on page 2-26 and the figure “I/O signal inputs and outputs”...
  • Page 62 Connecting the Bus SAT/IN module channel 1 to the detector Bus SAT/IN module 2475 detector B (inputs and outputs) Detector out 1 − Detector out 1 Ground Detector out 2 − Detector out 2 Switch 1 − Switch 1 Ground Switch 2 −...

  • Page 63: Connecting A 746 Data Module

    Pin 5 Detector Out 2 – (black) Connecting a 746 data module You can connect a Waters 746 data module to the detector using the analog output connector on the detector’s rear panel. The analog connector provides 1 V output, which is scaled to the EUFS sensitivity setting and the voltage offset setting.

  • Page 64: Connecting A Chart Recorder

    Detector inputs and chart recorder terminals Chart recorder 2475 detector (B inputs and outputs) terminals Pin 1 Detector Out 1 + (1 V) –...

  • Page 65: Connecting A 600-Series Pump

    Connecting a chart recorder to the detector 2475 detector B (inputs and outputs) Detector out 1 − Detector out 1 Ground Detector out 2 − Detector out 2 Switch 1 − Switch 1 Black Ground Switch 2 − Switch 2 –...
  • Page 66 Detector inputs and 600-series pump terminal connections 600-series pump terminal 2475 detector (A inputs) S1, S2, S3, or S4 Pin 4 Lamp On/Off + GND (any one of four) Pin 5 Lamp On/Off –...
  • Page 67 Make the connections shown in the following table and figure with a signal cable. Program the pump to provide a pulse output on the applicable switch (S1, S2, or S4) at the beginning of each run (see the Waters 600E Multisolvent Delivery System User’s Guide). Auto-zero connections for the 600-series pump...
  • Page 68 Program the pump to provide a pulse output on the selected switch at the beginning of each run. See the Waters 600E Multisolvent Delivery System User’s Guide. Chart-mark connections for the 600-series pump 600-series pump terminal 2475 detector (A inputs)

  • Page 69: Connecting A 717Plus Autosampler

    Auto zero Connecting a 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 auto zero at the start of an injection.
  • Page 70 To auto zero the detector at the start of an injection, make the connections in shown in the following table and figure. Auto-zero connections for the 717plus autosampler 717plus autosampler terminal 2475 detector (A inputs) Inject Start + (any one of many paired Pin 9 Auto Zero + with –) Inject Start –...
  • Page 71 (see the following table and figure). Inject-start connections for the 717plus Autosampler 717plus Autosampler terminal 2475 detector (A inputs) Inject Start + (any one of many paired with Pin 1 Inject Start + Inject Start – (any one of many paired with Pin 2 Inject Start –...

  • Page 72: Connecting To The Electricity Source

    Connecting to the electricity source The 2475 detector requires a separate, grounded electricity source. The ground connection in the electrical outlet must be common and connected near the system. To avoid electrical shock: Warning: • Use power cord SVT-type in the United States and HAR-type or better in Europe.

  • Page 73: Using The Detector

    • As a standalone instrument – You can use the detector as a standalone detector with a system such as the Waters Alliance system or with any pump, injector, chart recorder, or integrator. You can program the detector’s front panel unless it is controlled by a data system such as Waters Empower or Millennium software.

  • Page 74: Starting The Detector

    The detector beeps three times, displays the message “Booting System... Please Wait (Service Keypad Inputs Accessible for 6 sec.),” and runs a series of startup diagnostic tests. Service keypad inputs are coded for use only by Waters service engineers Tip: for troubleshooting purposes.

  • Page 75: Startup Failure

    2475 detector startup screen Homing optical filter Searching for Zero Order Peaks Finding erbium calibration peaks Restoring last setup When initialization is complete, the 2475 detector displays the Fluorescence home screen. See page 3-9 page 3-16 for more information. Fluorescence home screen...

  • Page 76: Idle Mode

    Idle mode When the detector is successfully started, it defaults to idle mode (see the figure “2475 detector idle mode screen” on page 3-4). When it is not performing any function requiring the shutter to be open (local methods, scans, noise test, and so on), the shutter is closed, and the detector remains in idle mode with the lamp lit.

  • Page 77: Fluorescence And Message Icons

    In real time, the detector monitors fluorescence in terms of emission or energy units of one or more wavelength pairs. Simultaneously, you can modify all parameters in the table titled “2475 detector home and message screen icons” on page 3-5. Press the A/B key to toggle between home screens for channels A and B.
  • Page 78 2475 detector home and message screen icons (Continued) Icon or field Description Function Gain Selects the PMT gain setting. Sensitivity (EUFS) Selects the chart sensitivity in emission or sample energy units full scale (EUFS) for the selected channel (digital data is not affected).
  • Page 79 2475 detector home and message screen icons (Continued) Icon or field Description Function Lamp off Indicates the lamp is off. Shift off Blank = Shift off Shift on Indicates the shift is on for one key press. Single wavelength Indicates the detector is operating in single-channel mode.
  • Page 80 2475 detector home and message screen icons (Continued) Icon or field Description Function Keypad unlock Indicates unrestricted keypad entry. Keypad lock Indicates parameter changes are not allowed; instrument is under control of an external data system (remote mode only). Sticky diagnostic Indicates a sticky diagnostic setting is active.

  • Page 81: Using The Keypad

    2475 detector home and message screen icons (Continued) Icon or field Description Function Message screen Indicates a question. icon. Message screen Indicates a warning message. icon. Message screen Indicates information is being icon. displayed. Message screen Indicates that you should standby.
  • Page 82 enter information into parameter fields, and then press Enter to actuate the specified functions. Keys that appear in all-uppercase letters (HOME, METHOD, CONFIGURE, DIAG, TRACE, and SCAN) display a function, directly, from most screens. Select a numerical entry on a list or menu as follows: •...
  • Page 83 Clear Field +/− Shift The following table explains the functions of the primary and secondary keys. To initiate a secondary function, press Shift and then the key. 2475 detector keypad description Description Unshifted After pressing shift HOME – Displays the home ? –...
  • Page 84 2475 detector keypad description (Continued) Description Unshifted After pressing shift Chart Mark – Causes a SCAN – Displays the list of momentary pulse to the analog options for generating and SCAN output (A and B, depending on manipulating spectra. Chart Mark specified settings).
  • Page 85 2475 detector keypad description (Continued) Description Unshifted After pressing shift Next – Displays a screen with Previous – When the Next additional options related to key is available, Previous Previous the current screen. Repeatedly navigates through the Next pressing this key always screens in the reverse order.
  • Page 86 2475 detector keypad description (Continued) Description Unshifted After pressing shift 0-9 – Enters the corresponding 0-9 – See the descriptions number into the current field. that follow for specific, Also positions the cursor at the shifted, numeric keys. 0 - 9 corresponding entry in a list (0 = tenth item).
  • Page 87 2475 detector keypad description (Continued) Description Unshifted After pressing shift 6 – See 0-9 above. Contrast – Use to adjust contrast (viewing angle) in Contrast the liquid crystal display. 0 – See 0-9 above. Cancel – In some modes, Cancel backs out of a...

  • Page 88: Navigating The User Interface

    Navigating the user interface Press Enter or to navigate among editable fields. A thick border appears around the active field. When you complete an entry, press Enter to advance to the next active field. If you make an error, press CE (Clear Entry) to undo any changes and return to the active entry field.
  • Page 89 When you press Next, the detector displays three additional home screens, labeled “2 of 4”, “3 of 4”, and “4 of 4” (see the figure “Secondary functions of the home screen” on page 3-17). Secondary functions of the home screen Press Next Fluorescence home screen Press Next...

  • Page 90: Preparing To Start A Run

    Preparing to start a run You must set up a run before you make fluorescence measurements. To start a run, you can press Run/Stop or trigger the detector’s operation via the inject- start terminals on the rear panel. When you start a run, the shutter opens automatically and the detector performs an auto-zero function (when the function is enabled).

  • Page 91: Accessing Primary And Secondary Functions

    When emission units are used, measurements taken on different 2475 detectors are fully compatible with one another. • Energy – Energy units do not have the normalization advantage. Those traditional units of measurement conform to currently established test methods.
  • Page 92 • Analog out (single λ pair) • Emission – Fluorescence output corresponding to the data units selected. • Reference Energy – Charts the lamp energy from the reference photodiode located in the excitation optic bench. Reference scaling is fixed at 10,000 units per volt. •...
  • Page 93 the minus symbol (–) produces an inverted chromatogram at the analog output channel. • Auto Zero-On-Inject – Selected by default, this parameter specifies the auto-zero behavior each time the detector receives an inject-start signal. You can disable this parameter by pressing any numerical key to clear this field for either or both channels.
  • Page 94 Primary and secondary function (method) parameters (Continued) Function Screen Type Units Range Default Analog out 2 (of 4) Choice None • Emission Emission A (single λ) • Reference energy A • Output off Analog out 2 (of 4) Choice None •...

  • Page 95: Operating The Trace And Scale Functions

    Primary and secondary function (method) parameters (Continued) Function Screen Type Units Range Default Auto Zero on 4 (of 4) Check None Checked Checked inject not checked Auto Zero on 4 (of 4) Choice None • To To baseline λ changes baseline •...
  • Page 96 • T1 represents the left-hand side of the trace, or ending time (default is -30). • T2 represents the right-hand side of the trace, or starting time (default is 0). The following figure shows a 60-minute trace of continuous injections of salicylic acid and naproxin with the excitation wavelength set to 240 nm and emission to 355 nm.

  • Page 97: Configuring The Detector

    As you modify the output using the scale function, the trace function continues to display the 2475 detector output in real time on either or both channels. Configuring the detector You can configure the detector to emulate the Waters 474 detector communication protocol in the Configuration screens.

  • Page 98: Setting Pulse Periods

    – High – Starts run when contact closure changes from off (open) to on (closed). – Low – (Default) starts run when contact closure changes from on (closed) to off (open). – Ignore – No response to inject-start input. • Chart mark –...

  • Page 99: Setting The Display Contrast

    SW2. The figure “Setting the pulse period or signal width on SW1 or SW2” on page 3-27 shows a single pulse and a rectangular wave. • Single pulse (in seconds) – If SW1 or SW2 is programmed to generate a pulse as a timed or threshold event, then this field specifies the period of the signal (single pulse width;...

  • Page 100: Displaying System Information

    (where applicable) the serial number, software version number with checksum, and version date. Press Enter to return to the home screen. Example of a System Info screen The 2475 detector release notes also reflect the checksum and version. Tip: Using Online Help The detector has limited context-sensitive Help.

  • Page 101: Two Operating Modes

    3-21 includes more information on operating parameters adapted in single or multichannel mode. When an Empower or Millennium data system controls the detector, it functions as a Waters 474 detector (see page 3-29). Standalone operation When using the detector as a standalone instrument, you can store up to 10...
  • Page 102 Method parameters The initial conditions for the method are specified on the General tab of the fluorescence method editor in Empower or Millennium software. The 2475 detector and the 474 detector interpret some method Tip: parameters differently. Example of method parameters...
  • Page 103 Filter Response – Time-constant setting of the filter (3, 5, 10, 20, and 40). The 474 detector interprets these numbers in terms of seconds. However, values of this magnitude are too high for chromatography, so the 2475 detector interprets these inputs at one tenth the specified value. Operating the detector...
  • Page 104 Digital time constant response settings Empower or Millennium selection 2475 time constant (sec.) • Lamp Off Time (hrs) – Determines how long after the start of an injection the lamp turns off. The timer resets at the beginning of every injection.
  • Page 105 Auto Zero – Corresponds directly with enabling (Auto) and disabling (Manual) the automatic Auto Zero on injections, gain, or wavelength changes. You change these settings via the boxes on page 4 of the 2475 operator interface. If you select manual, the detector performs an Auto Zero only when directly commanded to do so, either as a timed event, front panel button, or rear panel (terminal block) contact closure.

  • Page 106: Remote Control Operation Via Ethernet Connection Using 2475 Instrument Control Software

    “Fluorescence home screen” on page 3-3) with an “E” at it’s center (see the table titled “2475 detector home and message screen icons” on page 3-5). For this mode of operation, you must disable the Emulate 474 option from the Empower Configuration window.

  • Page 107: Manual Wavelength Calibration

    Manual wavelength calibration You can calibrate the detector manually from the keypad by pressing the manual calibration key at any time during detector operation or when calibration errors arise during startup. You need not restart the detector after a successful wavelength calibration. Before you pump solvent or mobile phase through the system, flush the Tip: lines with filtered, degassed, and sparged HPLC-grade water and continue to...

  • Page 108: Normalizing Emission Units

    3-19). If you select emission units, normalize to a standard water reference on a monthly basis to ensure that measured signal strengths are as consistent as possible with those measured by other 2475 detectors. Before you pump solvent or mobile phase through the system, flush the Tip: lines with filtered, degassed, and sparged HPLC-grade water.

  • Page 109: Operating The Detector In Single-Channel Mode

    Normalization values at completion The values are used in the emission units formula described on page 1-14. the lamp and optics age, the Raman Gain will gradually increase to a maximum of 1000, and the Raman Counts value can decrease. If the Raman signal for water occurs within 3 nm of 397 nm, the normalized units are embedded in the detector’s memory.

  • Page 110: Operating The Detector In Multichannel Mode

    can also use the Energy setting on channel B while making the primary fluorescence measurement specified by a EUFS on channel A. For example, while operating in single-channel mode, you can set an EUFS of 500 on the second channel, providing a different scaling factor on the channel B 1-V output.
  • Page 111 Specify the other operating parameters and any timed or threshold events, if desired. Specify the desired gain. Press A/B to switch channels. The home screen for the other channel appears. Specify the operating parameters for the second excitation and emission wavelength pair monitor, the gain, and any timed and threshold events, if desired.

  • Page 112: Setting Gain And Eufs

    Setting gain and EUFS Choosing a gain setting for the PMT before injecting sample is a necessary part of fluorescence measurement with detectors that use PMTs. For the best signal-to-noise ratio, set a gain that maximizes the dynamic range of the electronics (see page 1-17).
  • Page 113 474 Fluorescence Detector Instrument method editor. The method is then downloaded to the 2475 detector when you make an injection. After you program the method into the 2475 detector (or Empower or Millennium editor), press DIAG, and then press 3 Auto-Optimize Gain.
  • Page 114 Selecting Auto-Optimize Gain prepares the diagnostic to be executed on the next injection. The sticky diagnostic (wrench) icon appears on the home screen, and <Auto Gain> appears in the emission field. Auto-optimize gain diagnostic test is implemented Auto Gain diagnostic Sticky diagnostic tool You can start the injection after you arm the diagnostic with a start pulse trigger from an injector input to the inject event terminal on the detector rear...

  • Page 115: Programming Methods And Events

    (see the figure “Finding parameters in the fluorescence home screen” on page 3-4). An asterisk in the method number icon (see the table titled “2475 detector home Programming methods and events 3-43...

  • Page 116: Programming Timed Events

    When the detector is operating under remote control by Empower or Millennium software, the remote icon appears (see the table titled “2475 detector home and message screen icons” on page 3-5). Programming timed events You can program up to 48 timed events to the nearest 0.01 minute.
  • Page 117 Timed event parameters (Continued) Range or Specify Number Event Units default channel Time Seconds 0: Disable constant filter Hamming: (λ) 0.1 to 5.0, (λλ) 1 to 50 RC: (λ) 0.1 to 99 sec RC: (λλ) 1 to 99 sec Gain 0 to 1000 Sensitivity EUFS...
  • Page 118 Timed event parameters (Continued) Range or Specify Number Event Units default channel Threshold –100.0 to 1100.0 EUs or variable, depending on output selection To program a new timed event: Select METHOD (Shift, A/B). Method list From the method list, select 1 Timed events. An active field for specifying the time of the event appears.

  • Page 119: Programming Threshold Events

    Press Enter again to display the list. If you know the event number, simply press it (see the table titled “Timed event parameters” on page 3-44). Enter the appropriate selection in the To field if the field appears. If you want the same event programmed on both channels, you Tip: must enter two events, one for channel A and one for channel B.
  • Page 120 Threshold events “Set” parameters Number Event Set switch 1 Set switch 2 Below the specified threshold, program the switch parameters as in the table below. Threshold events “To” parameters Below threshold Number Set to switch state Pulse Rect wave (rectangular wave) To define the pulse period, or frequency, of a wave, see page...

  • Page 121: Storing A Method

    Threshold events screen Press Enter to advance to the Set field, or press to move among the three fields. When the Set field is active, press Enter to display the threshold events list, or press the number corresponding to the event you are programming (see the table titled “Threshold events “Set”...

  • Page 122: Retrieving A Method

    Specify the number of the method you wish to retrieve and press Enter. A brief message (“Retrieving method n”) appears, and then the method number you specified appears within the method number icon (see the table titled “2475 detector home and message screen icons” on page 3-5). Viewing events within a method...

  • Page 123: Resetting A Method

    Resetting a method Resetting a stored method is a two-step process. First you reset the current conditions to the defaults; then you save the defaults in a storage location. To reset a method: Select METHOD (Shift, A/B). From the method list, select 5 Reset method *. A message screen asks if you want to set the current conditions to the factory defaults.

  • Page 124: Scanning Spectra

    If you select Cancel (Shift, 0), the Method list appears. When you press HOME, the method number icon has an asterisk. Scanning spectra Types of scanning The detector can collect sample scans for either excitation and emission fluorescence spectra. A zero-scan is desirable initially. •...
  • Page 125 possible resolution for the specified pace. As shown by the following table, the higher the pace value, the poorer the resolution. Pace and sampling resolution examples Emission sampling Excitation sampling Pace (nm/min) resolution (nm) resolution (nm) 100 and less The following figure shows two emission scans of anthracene. At a pace of 1000 nm/min, the second scan (right) shows a reduced number of points scanned.
  • Page 126 Scan of water without tick marks Wavelength (nm) Scan of water with tick marks Wavelength (nm) Enter scanning parameters when you select the type of scan, zero or sample. Use the detector’s scan function to run a new zero or sample scan, store, review, subtract, obtain scan information, and replay.
  • Page 127 Zero and sample scan screens Zero-scan (screen 1 of 4) Sample scan (screen 1 of 4) (screens 2, 3, and 4 not shown) Zero-scan (screen 2 of 4) (screen 3 of 4) Zero-scan Zero-scan (screen 4 of 4) When you select a sample scan after performing a zero-scan, the detector displays one additional screen, labeled 2 of 2.
  • Page 128 Sample scan screens after a zero-scan (screen 1 of 2) Sample scan Sample scan (screen 2 of 2) When you run the zero-scan, you specify the starting and ending wavelengths, other wavelength, pace, tick marks, and sensitivity for the zero-scan and subsequent sample scans.

  • Page 129: Scanning New Spectra

    Scanning new spectra To scan new spectra: Select SCAN (Shift, Chart Mark). Scan list From the scan list, select 1 New scan, or use to scroll through the list. The detector displays the first of four parameter screens. Press Next to advance through the New scan parameter screens. On the first New scan screen, specify the type of scan: •...

  • Page 130: Parameters Used For Sample And Zero-Scans

    Parameters used for sample and zero-scans The following table provides the defaults and ranges for all parameters for the sample and zero-scans. Sample and zero-scan parameters Range or Parameter Screen Units default Type Sample scan: 1 Zero-scan: 2 Default: 1 λ...

  • Page 131: Programming A Zero-Scan

    Programming a zero-scan To program a zero-scan: Select SCAN (Shift, Chart Mark). Press 1 New Scan, then press 2 Zero Scan. Press Next. The second zero-scan parameter screen appears. Specify the zero-scan parameters: Select the type of zero-scan. Specify the starting wavelength for the zero-scan, and then press Enter.

  • Page 132: Running A Sample Scan

    10. Press Run/Stop to start the zero-scan, or press Next, to return to the first zero-scan parameter screen to review your parameter value, and then press Run/Stop. The Scanning screen displays a progress bar and instantaneous energy (EU). Zero-scan progress bar When the detector completes the zero-scan, the software returns to displaying the scan list.
  • Page 133 A progress bar reports the degree of fluorescence, in emission or energy units (EU). Sample scan progress bar When you perform a zero-scan before a sample scan, the detector Tip: indicates that the zero-scan is being subtracted from the sample scan in progress.
  • Page 134 Highest peaks of sample anthracene scan Press Next to return to the graph. Select Scale (Shift, TRACE) to change the scale and zoom in on one section (artifact) of the spectrum. The scale of the spectrum is affected by the EUFS setting. You can alter the following four scaling parameters: •...
  • Page 135 Highest peaks from the scaled anthracene scan 11. Press Next to return to the sample scan display. To display use of the software’s scaling function, the figure “Three scans of anthracene in acetonitrile” on page 3-64 shows a series of scans of anthracene dissolved in acetonitrile.

  • Page 136: Scanning Using A Static Flow Cell

    Three scans of anthracene in acetonitrile Sample emission scan 350 to 460 nm Excitation=249 nm Anthracene Zoom of sample emission scan 350 to 440 nm 20 to 35 EU Excitation=249 nm Anthracene λ2 changed to 440 nm Zoom of sample emission scan 360 to 420 nm Excitation=249 nm Anthracene...

  • Page 137: Managing Results

    Managing results In standalone mode, having run a spectrum, you can store it for later review, subtraction, or playback. You can store up to five spectra (see page 3-65). You can the retrieve the spectrum for purposes of reviewing it from one of the five storage slots by selecting the review function from the scan list (see page 3-66).

  • Page 138: Getting Information About A Stored Spectrum

    Getting information about a stored spectrum To get information about a stored spectrum Select SCAN (Shift, Chart Mark). Press 3 Get scan info. A slot number box appears, its default, Last (for the most recently stored spectrum). Press Enter for information about the last stored spectrum. As an alternative, press the number (1 to 5) of the stored spectrum about which you want information, and then press Enter.

  • Page 139: Creating A Difference Spectrum (Subtracting A Spectrum)

    Creating a difference spectrum (subtracting a spectrum) To create a difference spectrum: Select SCAN (Shift, Chart Mark). Press 5 Subtract & Review. To subtract one spectrum from another, the starting and ending Tip: wavelengths (λ1 and λ2) and the pace of both spectra must be identical. Specify the storage slot number (1 through 5) of the spectrum you want to subtract from the current (or retrieved) spectrum, and then press Enter.

  • Page 140: Manually Extinguishing The Lamp

    If the detector is operating under remote control, you can program the controller to extinguish the lamp without using the detector’s front panel. You should extinguish the lamp only if the lamp will remain unlit more Tip: than 4 hours. Use the Lamp key to light and extinguish the lamp manually.

  • Page 141: Manually Lighting The Lamp

    Lamp off/on sequence Lamp off indicator Lamp on indicator Manually lighting the lamp To manually light the lamp: Select Lamp (Shift, 1). The lamp control screen appears with 0 hours and 00 minutes in the “Lamp has been on” field. Select Lamp (Shift, 1) again to light the lamp.

  • Page 142: Using A Timed Event Method To Program The Lamp

    Using a timed event method to program the lamp You can conserve lamp life by programming it to light and extinguish (for example, overnight) using a timed event method. To program the lamp, select Timed events in the Method list, or program it through one of the external contact closures.
  • Page 143 Maintenance Procedures Contents: Topic Page Contacting Waters technical service Maintenance considerations Routine maintenance Inspecting, cleaning, and replacing the flow cell Replacing the lamp Replacing the fuses 4-14 Cleaning the instruments exterior 4-15...

  • Page 144: Maintenance Procedures

    Waters Technical Service (800 252-4752). Otherwise, phone the Waters corporate headquarters in Milford, Massachusetts (USA), or contact your local Waters subsidiary. Waters’ site includes phone numbers and e-mail addresses for Waters locations worldwide. Visit www.waters.com, and click About Waters > Worldwide Offices.

  • Page 145: Spare Parts

    Appendix C more information. Spare parts Replace only the component parts mentioned in this document. See the Waters Quality Parts Locator on the Waters Web site’s Service/Support page. Routine maintenance For sustained optimal performance, the 2475 detector requires minimal routine maintenance: Replace the HPLC system’s solvent reservoir filters regularly.

  • Page 146: Removing The Front-Left-Hand Panel Cover

    Pull the bottom of the cover away from the detector gently, all the while holding on to the cover’s top. Withdraw the top of the cover gently, and store the cover nearby. 2475 detector with the front-left-hand panel cover removed Maintenance Procedures...

  • Page 147: Inspecting, Cleaning, And Replacing The Flow Cell

    Inspecting, cleaning, and replacing the flow cell A dirty flow cell can cause baseline noise, decreased sample energy levels, calibration failure, and other problems. This section provides information about the following procedures: • Flushing the flow cell • Removing and cleaning the flow cell •...

  • Page 148: Removing The Flow Cell Assembly

    Removing the flow cell assembly To remove the flow cell assembly: Shut down the detector. Flush and dry the flow cell (see page 4-5), and then disconnect and cap the inlet and outlet LC tubing. Remove the front-left-hand panel cover. Use a 1/4-inch, flat-blade screwdriver to loosen the three captive screws on the flow cell assembly front plate.

  • Page 149: Replacing The Flow Cell

    Pull the assembly gently toward you, tilting the bottom of the cell upward to avoid disturbing the cell mask. 2475 detector flow cell assembly Place the flow cell assembly on a flat clean surface. Replacing the flow cell The detector is shipped with a standard analytical flow cell installed. Replace the flow cell when it becomes damaged.

  • Page 150: Replacing The Lamp

    This section describes the procedure for removing and replacing the xenon lamp. The 2475 detector source lamp is warranted to light and pass startup diagnostic tests for 2000 hours or 1 year from the date of purchase, whichever comes first.

  • Page 151: Removing The Lamp

    Removing the lamp The lamp housing becomes extremely hot during lamp Warning: operation. To prevent burn injuries, • allow the lamp to cool for 60 minutes before removing it. • keep the lamp in the housing when handling the lamp. To avoid eye injury from ultraviolet radiation exposure, Warning: •...
  • Page 152 Pinch the locking mechanism on the bottom connector before pulling it out. Unplugging the lamp assembly 4-10 Maintenance Procedures...
  • Page 153 • Lamp gas is under positive pressure. To prevent shattering the glass, use care when disposing of the lamp. Waters suggests that you adequately cushion an old lamp by containing it in the packaging of its replacement before you dispose of it.

  • Page 154: Installing The New Lamp

    Installing the new lamp To avoid exposing your eyes to harmful ultraviolet Warning: radiation, wear eye protection that filters ultraviolet light, and keep the lamp in the housing during operation. Do not touch the glass bulb on the new lamp. Dirt or skin oils Caution: on the bulb affect detector operation.

  • Page 155: Recording The New Lamp's Serial Number

    Recording the new lamp’s serial number If you do not record a new lamp’s serial number, the date of the previous Tip: lamp installation remains in the detector’s memory, voiding the new lamp’s warranty. Using the detector software, you can record and store the serial number and date of installation of a new lamp so that you can monitor the age of the lamp and its number of ignitions.

  • Page 156: Replacing The Fuses

    14. Perform a normalization procedure (see page 3-36). Replacing the fuses To avoid electric shock, power-off and unplug the 2475 Warning: detector before examining the fuses. For continued protection against fire, replace fuses with those of the same type and rating only.

  • Page 157: Cleaning The Instruments Exterior

    Removing and replacing the rear panel fuses Flat-blade Fuse holder screwdriver Remove and discard the old fuses. Make sure that the ratings of the new fuses suit your requirements. Insert the new fuses into the fuse holder. Insert the fuse holder into the receptacle, and gently push until it locks into position on the rear panel.
  • Page 158 4-16 Maintenance Procedures...

  • Page 159: Error Messages, Diagnostic Tests, And Troubleshooting

    Error Messages, Diagnostic Tests, and Troubleshooting The detector provides both user and service diagnostics to troubleshoot system problems. Only qualified Waters Service personnel can access the service diagnostics. • Error messages – Startup, calibration, and other error messages and recommended actions for correcting the errors.

  • Page 160: Startup Error Messages

    New • Perform manual calibration points are calibration. compared to stored • Contact Waters points from the most Technical Service. recent manual calibration. This message appears when any point differs by more than 2.0 nm.

  • Page 161: Operational Error Messages

    In most cases, you can cycle power (shut down, wait 10 seconds, then restart) to correct the error. However, if the error persists, contact Waters Technical Service (see page 4-2).
  • Page 162 Ensure that the flow cell is clean. Ensure that the lamp access door is shut securely. Recycle power to the detector. If the catastrophic error persists, contact Waters Technical Service (see page 4-2). The following table has error messages that prevent operation, listed in alphabetical order.
  • Page 163 Data acquisition via 1. Power-off and A/D converters is power-on again. interrupt-driven. If 2. If the problem interrupt is too long, persists, call Waters problem with data Technical Service. acquisition is indicated. Filter initialization Unit sensors cannot 1. Ensure solvent is...
  • Page 164 2. If the problem optical filter. persists, power-off and power-on again. 3. If the problem persists, call Waters Technical Service. Filter initialization Unit sensors cannot 1. Ensure solvent is failure: No response identify any dark flowing.
  • Page 165 Search for the home 1. Power-off and failure: No home sensor sensor failed. power-on again. 2. If the problem persists, call Waters Technical Service. Hardware failure: lamp The lamp failed to Close the lamp door, relay cannot open! extinguish when the...

  • Page 166: User-Selected Diagnostic Tests And Settings

    User-selected diagnostic tests and settings The detector uses both user-selectable and service diagnostic tests and Tip: settings. Only qualified Waters Service personnel can access service diagnostic tests. Overview of diagnostic tests and settings You may operate several diagnostic tests and settings for use in troubleshooting the detector and verifying proper functioning of the detector’s...
  • Page 167 Enter, or press a number from 1 through 8. Choices that display other choices are indicated by >> (see the table titled “2475 detector diagnostic tests and settings” on page 5-10).
  • Page 168 The following table lists the diagnostic tests and settings by number with a brief description. 2475 detector diagnostic tests and settings Diagnostic Description 1 Normalize Units Normalizes the emission units of the detector to 100 EU using a standard clean water reference.

  • Page 169: Sample And Reference Energy Diagnostic Tests

    2475 detector diagnostic tests and settings (Continued) Diagnostic Description 8 Other diagnostics >> Tests that allow you to generate test peaks to determine wavelength accuracy or override the default filter setting: 1 Generate test peaks 2 Optical filter override 3 Previous choices <<...

  • Page 170: Raman Signal-To-Noise Test Diagnostic Test

    Specify a new wavelength number to change the wavelength, and then press Enter. When the new wavelength shifts to the left, the corresponding sample and reference energies appear. If you are operating the detector in multichannel mode, press A/B to view sample and reference energy on the other wavelength.
  • Page 171 • Override the optical filter. To perform one of the input and output tests or change a setting, press 6 Input & output. A list of four diagnostic tests and settings appears. Input and output diagnostic tests and settings Displaying auto-zero offsets To display auto-zero offsets: From the Input &...
  • Page 172 Fix EU screen Setting fixed voltage output This function drives the voltage on the selected analog channel (A or B). To set the fixed voltage output: From the Input & output list, press 3 Fix voltage to select a voltage for the analog output.

  • Page 173: Change-Lamp Function

    Contact closures & events screen You can monitor the state of the contact closure inputs in real time. A solid (filled in) circle indicates the contact closure is closed (ON = High). An open (empty) circle indicates the contact closure is open (OFF = Low). For the outputs (SW1 and SW2) Press Enter to display the active switch (surrounded by a dotted-line border).

  • Page 174: Testing The Keypad

    If the keypad is operating properly, each key location is filled in and Tip: then cleared with a second press of the key. If any key does not respond when pressed, contact your Waters service representative. Press Enter twice to exit the keypad test. 5-16...

  • Page 175: Testing The Display

    The display fills from top to bottom and right to left, and then returns to the Lamp, display & keypad list. If the display does not completely fill horizontally or vertically, contact your Waters service representative. Press 4 to return to the diagnostics list.

  • Page 176: Overriding The Optical Filter Setting

    To generate test peaks: Press DIAG, then press 8 Other diagnostics. Press 1 Generate test peaks to generate test peak. Every 100 seconds, the detector generates a 100-EU peak with a Tip: standard deviation of 10 seconds on the trace, chart, or data system display.

  • Page 177: Reducing Pmt Sensitivity

    Press Enter. Automatic Second Order None Erbium Shutter In the list of filters, press a number to the corresponding filter, or leave the default filter (Automatic) on. Press DIAG, and then press 1, or select Automatic for the default filter. Reducing PMT sensitivity The detector’s design increases the limit of detection, but in some circumstances the photomultiplier tube can be overloaded by large...

  • Page 178: Troubleshooting

    Most detector problems are relatively easy to correct. If you are unable to correct a problem or failed condition after running the user diagnostics applicable to the problem, contact Waters Technical Service (see page 4-2).

  • Page 179: Power Surges

    “Overview of diagnostic tests and settings” on page 5-8 has diagnostic descriptions and instructions on how to use them. The table titled “2475 detector diagnostic tests and settings” on page 5-10 and the table titled “General hardware troubleshooting” on page 5-21 describe error messages that can appear onscreen as you start up or operate the detector, and it suggests corrective actions.
  • Page 180 Check electrical fails to illuminate connection connections. Open (blown) fuse Check and, if necessary, replace fuse(s). Bad LCD or control Call Waters Technical board Service. Front panel displays Faulty EPROMs or bad Call Waters Technical odd characters LCD control board Service.
  • Page 181 RS-232 cable. Xenon lamp does not Faulty lamp Replace the lamp. light Lamp not plugged in Plug in the lamp connector. Bad lamp power supply Contact Waters Technical Service. Lamp switch off Check the rear panel connections. Troubleshooting 5-23...
  • Page 182 5-24 Error Messages, Diagnostic Tests, and Troubleshooting...
  • Page 183 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 184: 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 185: Specific Warnings

    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 186 Also ensure a gas-fail connection is connected to the LC system so that the LC solvent flow stops if the nitrogen supply fails. Mass spectrometer shock hazard This warning applies to all Waters mass spectrometers. To avoid electric shock, do not remove the mass spectrometer’s Warning: protective panels.

  • Page 187: Caution 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. Waters instruments and software can be used to analyze or...

  • Page 188: Warnings That Apply To All Waters Instruments

    Warnings that apply to all Waters instruments When operating this device, follow standard quality control procedures and the equipment guidelines in this section. 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.
  • Page 189 • 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 190 Attenzione: fare attenzione quando si utilizzano tubi in materiale polimerico sotto pressione: • Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero pressurizzati. • Spegnere tutte le fiamme vive nell'ambiente circostante. • Non utilizzare tubi eccessivamente logorati o piegati. •...
  • Page 191 농축 질산 또는 황산과 함께 사용하지 마십시오. • 염화 메틸렌(Methylene chloride) 및 디메틸술폭시드(Dimethyl sulfoxide)는 비금속 튜브를 부풀려 튜브의 파열 압력을 크게 감소시킬 수 있으므로 유의하십시오. 警告：圧力のかかったポリマーチューブを扱うときは、注意してください。 • 加圧されたポリマーチューブの付近では、必ず保護メガネを着用してください。 • 近くにある火を消してください。 • 著しく変形した、または折れ曲がったチューブは使用しないでください。 • 非金属チューブには、テトラヒドロフラン(THF)や高濃度の硝酸または硫酸などを流 さないでください。 • 塩化メチレンやジメチルスルホキシドは、非金属チューブの膨張を引き起こす場合が あり、その場合、チューブは極めて低い圧力で破裂します。 Warnings that apply to all Waters instruments...
  • Page 192 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 193 警告 : 為了避免火災， 更換保險絲時， 請使用與儀器保險絲蓋旁面板上所印刷之相同類 型與規格的保險絲。 警告 : 为了避免火灾，应更换与仪器保险丝盖旁边面板上印刷的类型和规格相同的 保险丝。 경고: 화재의 위험을 막으려면 기기 퓨즈 커버에 가까운 패널에 인쇄된 것과 동일한 타입 및 정격의 제품으로 퓨즈를 교체하십시오. 警告 : 火災予防のために、ヒューズ交換では機器ヒューズカバー脇のパネルに記 載されているタイプおよび定格のヒューズをご使用ください。 Warnings that apply to all Waters instruments A-11...

  • Page 194: 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 195: 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! Electrical and handling symbols A-13...
  • Page 196 A-14 Safety Advisories...

  • Page 197: B Specifications

    Specifications Physical specifications Attribute Specification Height 20.8 cm (8.2 inches) Depth 50.3 cm (19.8 inches) Width 28.4 cm (11.2 inches) Weight 13.61 kg (30 pounds) Environmental specifications Attribute Specification Operating temperature 4 to 40 °C (39.2 to 104 °F) Operating humidity 20 to 80%, noncondensing Shipping and storage temperature –40 to 70 °C (–40 to 158 °F)
  • Page 198 Electrical specifications (Continued) Attribute Specification Altitude 2000 m (6561.6 feet) Line frequency 50/60 Hz Fuse ratings Two fuses: 100 to 240 VAC, 50 to 60 F 3.15 A, 250 V FAST BLO, 5 × 20 mm (IEC) Power consumption 280 VA (nominal) Two attenuated analog output Attenuation range: 1 to 100,000 channels: 1 VFS...
  • Page 199 Performance specifications (Continued) Attribute Specification Wavelength +0.25 nm repeatability Sensitivity, single Ex: 350 nm channel Em: 397 nm (Signal-to-noise ratio of water Raman peak ≥1000. Hamming filter TC = 1.5 sec) Sensitivity setting 1 to 100,000 EUFS range Filter setting range Single-channel: 0.1 to 5.0 seconds, Hamming (default) 0.1 to 99 seconds, RC...
  • Page 200 Specifications...
  • Page 201 Solvent Considerations Contents: Topic Page Introduction Solvent miscibility Buffered solvents Head height Solvent viscosity Mobile phase solvent degassing Wavelength selection...

  • Page 202: C Solvent Considerations

    Introduction To avoid chemical hazards, always observe safe laboratory Warning: practices when operating your system. Clean solvents Clean solvents provide • reproducible results • operation with minimal instrument maintenance A dirty solvent can cause • baseline noise and drift • blockage of the solvent filters with particulate matter Solvent quality Use HPLC-grade solvents to ensure the best possible results.

  • Page 203: Buffers

    Buffers When you use buffers, dissolve salts first, adjust the pH, then filter to remove insoluble material. Tetrahydrofuran (THF) When using unstabilized THF, ensure that your solvent is fresh. Previously opened bottles of THF contain peroxide contaminants, which cause baseline drift.
  • Page 204 Solvent miscibility (Continued) Boiling Miscibility λ Cutoff Polarity Viscosity Solvent point °C number index CP, 20 °C (nm) (1 atm) N-hexane 0.313 68.7 –– Cyclohexane 0.98 80.7 Butyl ether 0.70 142.2 –– Triethylamine 0.38 89.5 –– Isopropyl ether 0.33 68.3 ––...

  • Page 205: How To Use Miscibility Numbers

    Solvent miscibility (Continued) Boiling Miscibility λ Cutoff Polarity Viscosity Solvent point °C number index CP, 20 °C (nm) (1 atm) Methoxyethanol 1.72 124.6 –– Acetonitrile 0.37 81.6 11, 17 Acetic acid 1.26 117.9 –– Dimethylformamide 0.90 153.0 –– Dimethylsulfoxide 2.24 189.0 ––...

  • Page 206: Buffered Solvents

    Position the solvent reservoirs at a level above the HPLC equipment or on top of the pump or detector (with adequate spill protection). Since the 2475 detector contains a high voltage power Warning: source, all solvents should be isolated from the detector.

  • Page 207: Mobile Phase Solvent Degassing

    If the extent to which the pressure changes will affect the analysis is not known, monitor the pressure during the run using the Chart Out terminal. Mobile phase solvent degassing Mobile phase difficulties account for 70% or more of all liquid chromatographic problems.

  • Page 208: Solvent Degassing Methods

    temperature, but the solubility of He in benzene increases with an increase in temperature. Effects of partial pressure The mass of gas dissolved in a given volume of solvent is proportional to the partial pressure of the gas in the vapor phase of the solvent. If you decrease the partial pressure of the gas, the amount of that gas in solution also decreases.

  • Page 209: Solvent Degassing Considerations

    To increase the length of membrane, you can connect two or more vacuum chambers in series. The inline degasser is available as an option or factory-installed in the Waters Alliance System. Wavelength selection...

  • Page 210: Uv Cutoffs For Common Solvents

    UV cutoffs for common solvents The following table shows the UV cutoff (the wavelength at which the absorbance of the solvent is equal to 1 AU for some common chromatographic solvents). Operating at an excitation wavelength near or below the cutoff increases baseline noise due to the solvent’s ability to absorb excitation light energy.
  • Page 211 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 monobasic, 10 mM B-6, low UV, 1 vial/liter dibasic, 10 mM...
  • Page 212 Wavelength selection for chromophore detection Certain functional groups found in most compounds absorb light selectively. These groups, known as chromophores, and their behavior can be used to categorize the detection of sample molecules. The following table lists some common chromophores and their detection wavelengths ( max ), as well as the λ...
  • Page 213 Wavelength selection for chromophore detection (Continued) λ max ∈ max λ max ∈ max Chemical Chromophore Configuration (nm) (L/m/cm) (nm) (L/m/cm) Aldehyde —CHO strong 280–30 11–18 Carboxyl —COOH 200– 50–70 Sulfoxide >S→O 1500 Nitro —NO 2 strong Nitrite —ONO 220– 1000–2000 300–40 —N=N—...
  • Page 214 C-14 Solvent Considerations...

  • Page 215: Index

    Auto Zero on inject from biohazard warning 2-15 buffered solvents starting a method from 2-17 burst warning turning the 2475 lamp on or off Bus SAT/IN module, connecting 2-24 from 2-17 analog outputs Calibrate key 3-14...
  • Page 216 2-22 generating 2-29 3-12 Millennium chromatography generating from the Alliance workstation 2-24 Separations Module 2-16 multiple Waters instruments Waters 600 Series Pump 2-21 connections 2-31 other equipment 2-23 – 2-35 Chart Mark key 3-12 signal cables chart polarity single Waters instrument...
  • Page 217 5-10 setup configuring event inputs 3-25 DIAG key 3-13 monitoring 5-14 diagnostic settings contacting Waters Technical Service auto-zero offset 5-10 5-13 5-20 contact closures and events 5-10 context-sensitive Help 3-28 fix (set) EU 5-10 contrast...
  • Page 218 external events 3-16 filter inputs 3-16 scan 3-53 disassembling the flow cell error messages – 5-23 display errors diagnostic test 5-10 5-17 calibration 3-35 fluorescence trace 3-13 startup 3-35 lamp use statistics 3-14 Ethernet options 3-13 communications interface 1-13 system information 3-28 Ethernet cable, connecting 2-20...
  • Page 219 parameter 3-21 functions filters analog outputs, single-channel 3-20 changing the filter type 3-16 auto-zero on wavelength changes erbium 3-21 filter setting specification change lamp 5-16 optical override 5-11 5-18 generating test peaks 5-17 second-order MaxPlot 3-20 3-39 time constant 3-20 optical filter override 5-18 types of...
  • Page 220 DIAG key 3-13 inject-start Enter key 3-15 connection 2-22 functions 3-11 signal 2-22 Help key 3-11 3-28 Waters 600 Series Pump HOME key 3-11 connections 2-32 λ/λλ key 3-12 3-37 3-38 Waters 717plus Autosampler Lamp key 3-14 connections 2-35 lamp, display, and keypad...
  • Page 221 Run/Stop key 3-12 lamp on/off connections to 600 Series Scale key 3-13 3-23 Pump 2-30 SCAN key 3-12 3-54 light filters Shift key 3-13 light sources, excitation System Info key 3-14 line spikes 5-21 test 5-16 local/remote control icon test diagnostic test 5-10 lock icon TRACE key...
  • Page 222 3-38 operation in single-channel mode 3-37 multichannel mode, changing to single modes 3-29 3-38 under remote control 3-44 multiple Waters instruments, operation, theory and principles of connecting 2-21 optical multiwavelength mode 1-15 and electronic design description 3-12 component specifications...
  • Page 223 sensitivity 1-12 physical specifications pace 3-52 plus/minus key 3-15 parameters analog out (multiwavelength) 3-22 calibration 1-12 analog out (single wavelength) 3-22 gain setting 1-17 auto-zero on inject 3-21 3-23 sensitivity 1-12 auto-zero on wavelength changes sensitivity, reducing 5-19 3-23 polarity timed event parameter 3-45 auto-zero timed event 3-45...
  • Page 224 EUFS parameter 3-21 sample and reference energy diagnostic scanning 3-53 tests 5-10 5-11 setting specification sample energy 3-56 timed event parameter 3-45 sample scan serial number, lamp 4-13 procedure 3-55 – 3-56 service screens 3-55 contacting Waters 5-20 Index-10...
  • Page 225 2-22 generating 3-12 making 2-13 3-57 3-63 – single pulse signal 3-26 obtaining information about 3-66 single Waters instrument, connecting replaying 3-67 2-20 reviewing 3-66 single wavelength mode scanning 3-52 – 3-64 3-12 storing 3-65 operating in...
  • Page 226 A-13 TRACE key 3-13 3-23 warning transient energy 5-21 system troubleshooting displaying information 3-14 contacting Waters 5-20 information 3-28 diagnostic tests 5-23 – System Info key 3-14 hardware 5-21 turning lamp on or off from an external device...
  • Page 227 2475 detector with older external inject- start connections 2-35 data systems 3-29 Waters 746 data module, connecting A/B key 2-27 2-28 – diagnostic tests – 5-23 Waters Technical Service, contacting input and output diagnostic 5-20 settings 5-12 wavelength input and output diagnostic tests...
  • Page 228 Index-14...

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