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Waters Arc Premier 2475 Overview And Maintenance Manual

Multi-wavelength fluorescence detector
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Arc Premier 2475 Multi-Wavelength
Fluorescence Detector
Overview and Maintenance Guide
715007351
Copyright © Waters Corporation 2021
Version 00
All rights reserved

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  • Page 1 Arc Premier 2475 Multi-Wavelength Fluorescence Detector Overview and Maintenance Guide 715007351 Copyright © Waters Corporation 2021 Version 00 All rights reserved...

  • Page 2: General Information

    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. For the most recent revision of this document, consult the Waters website (www.waters.com).

  • Page 3: Contacting Waters

    We seriously consider every customer comment we receive. You can reach us at tech_comm@waters.com. Contacting Waters Contact Waters with enhancement requests or technical questions regarding the use, transportation, removal, or disposal of any Waters product. You can reach us via the Internet, telephone, fax, or conventional mail. Contact method Information www.waters.com...

  • Page 4: Safety Hazard Symbol Notice

    (GLP), and consult your organization’s standard operating procedures as well as your local requirements for safety. Safety hazard symbol notice symbol indicates a potential hazard. Consult the documentation for important information about the hazard and the appropriate measures to prevent and control the hazard. Considerations specific to the instrument Warning: To avoid electric shock, do not remove protective panels from the device.

  • Page 5: Applicable Symbols

    For compliance with the Waste Electrical and Electronic Equipment Directive (WEEE) 2012/19/EU, contact Waters Corporation for the correct disposal and recycling instructions For indoor use only August 19, 2021, 715007351 Ver. 00...

  • Page 6: Audience And Purpose

    (for example, 10kg) Serial number Part number, catalog number Audience and purpose This guide is intended for personnel who install, operate, and maintain the Arc Premier 2475 Multi-Wavelength Fluorescence Detector. Intended use of the Arc Premier 2475 Multi-Wavelength Fluorescence Detector Waters designed the Arc Premier 2475 Multi-Wavelength Fluorescence detector to analyze samples in high-performance liquid chromatography (HPLC) applications.

  • Page 7: Emc Considerations

    EMC considerations Canada spectrum management emissions notice This class A digital product apparatus complies with Canadian ICES-001. Cet appareil numérique de la classe A est conforme à la norme NMB-001. ISM classification: ISM group 1 class B This classification was assigned in accordance with CISPR 11 Industrial Scientific and Medical (ISM) instrument requirements.

  • Page 8: Table Of Contents

    Equipment misuse notice........................ iv Safety advisories..........................iv Operating the device..........................iv Applicable symbols.......................... v Audience and purpose........................vi Intended use of the Arc Premier 2475 Multi-Wavelength Fluorescence Detector......vi Calibrating............................vi Quality control..........................vi EMC considerations..........................vii Canada spectrum management emissions notice................vii ISM classification: ISM group 1 class B..................vii 1 Overview........................14...
  • Page 9 1.3.2 Emission wavelength selection..................... 17 1.3.3 Photomultiplier tube......................17 1.3.4 Scanning..........................17 1.3.5 Multichannel operation......................17 1.3.6 Fluorescence data........................ 17 1.3.7 References..........................18 1.4 Principles of operation........................19 1.4.1 Detector optics........................19 1.4.2 Optics assembly light path....................21 1.4.3 PMT calibration........................22 1.4.4 PMT sensitivity........................22 1.4.5 Filtering noise........................23 1.4.6 Electronics..........................
  • Page 10 2.6 Connecting other devices.......................50 2.6.1 Connecting cables........................ 50 2.6.2 Connecting a data system using an e-SAT/IN module............50 2.6.3 Connecting a Waters fraction collector................. 52 3 Using the detector..................... 55 3.1 Starting the detector........................55 3.1.1 Initializing the detector......................55 3.1.2 Startup failure........................56...
  • Page 11 3.4.3 Remote control operation for 474 emulation mode via RS-232..........77 3.4.4 Remote control operation via Ethernet connection using instrument control software..80 3.4.5 Verifying the detector......................80 3.4.6 Manual wavelength calibration....................81 3.4.7 Normalizing emission units....................82 3.4.8 Operating the detector in single-channel mode..............83 3.4.9 Operating the detector in multichannel mode...............
  • Page 12 5.3.9 Generating test peaks......................131 5.3.10 Overriding the optical filter setting..................131 5.3.11 Reducing PMT sensitivity....................132 5.4 Troubleshooting..........................133 5.4.1 Introduction......................... 133 5.4.2 Information needed when you contact Waters..............133 5.4.3 Power surges........................134 5.4.4 Hardware troubleshooting....................134 August 19, 2021, 715007351 Ver. 00 Page xii...
  • Page 13 A.1.1 Specific warnings........................137 A.2 Notices............................138 A.3 Bottles Prohibited symbol......................139 A.4 Required protection........................139 A.5 Warnings that apply to all Waters instruments and devices............139 A.6 Warnings that address the replacement of fuses.................143 A.7 Electrical symbols........................145 A.8 Handling symbols.........................146 B Specifications......................147 B.1 Physical specifications.........................

  • Page 14: Overview

    1 Overview The Arc Premier 2475 FLR detector is a multichannel, tunable, fluorescence detector designed for high-performance liquid chromatography (HPLC) applications. Figure 1–1: Front view, with door open The detector operates from 200 to 900 nm. It uses optics designed with an enhanced illumination system optimized for LC performance.

  • Page 15: Fluorescence Theory

    1.1 Fluorescence theory Fluorescence occurs when certain molecules absorb light at specific wavelengths, promoting the molecules to a higher energy state. As they return to their normal energy states, the “excited” molecules release their absorbed energy as photons. Many organic compounds absorb light, but few fluoresce. Liquid chromatography (LC) systems that incorporate fluorescence detection effectively identify polyaromatic hydrocarbons, aflatoxins, vitamins, amino acids, and so on.

  • Page 16: Excitation Sources

    1.2.2 Excitation sources A lamp that provides an intense, stable spectrum of light in the ultraviolet (UV) and visible ranges is the typical energy source used for fluorescence detection. The resulting fluorescence intensity is directly related to the intensity of the excitation spectrum. Thus, high-sensitivity detectors use the most intense excitation source available.

  • Page 17: Measuring Fluorescence

    1.3 Measuring fluorescence To measure fluorescence in the flow cell, the detector must balance the need for high selectivity (to distinguish very specific fluorescence wavelengths) with the need for high sensitivity (to measure low-fluorescence intensities). 1.3.1 Quantitation Fluorescence is linear at low concentrations, but can exhibit some non-linearity at high concentrations.

  • Page 18: Energy Units

    1.3.6.1 Emission units and normalization The 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 for changes that normally influence the signal strength of fluorescence measurements, such as lamp or optics degradations, by periodically renormalizing to the standard water reference.

  • Page 19: Principles Of Operation

    • Entrance slits • Exit slits • Blazed, plane, and concave holographic diffraction gratings • PMT • Waters axially illuminated flow cell The following diagrams show the optics assembly light paths and components. Figure 1–2: Excitation monochromator optics assembly Mercury-xenon lamp August 19, 2021, 715007351 Ver.
  • Page 20 Filter wheel Grating Parabolic mirror Exit slit Flow cell Entrance slit Ellipsoidal mirror Figure 1–3: Emissions monochromator optics assembly Ellipsoidal mirror Flow cell exit mask Flow cell Entrance slit Grating August 19, 2021, 715007351 Ver. 00 Page 20...

  • Page 21: Optics Assembly Light Path

    Exit slit 1.4.2 Optics assembly light path The detector demonstrates superior performance by employing several unique design elements. Its flow cell design minimizes stray background light and increases the detectability of low-level signals. Keeping the optics simple minimizes loss of signal and maximizes throughput. 1.4.2.1 Light source The detector uses a high-intensity 150-watt mercury-xenon arc lamp as its source.

  • Page 22: Pmt Calibration

    PMT to amplify and increase response. Controlling the high-voltage supply to the PMT achieves the gain. After the assembly and alignment of the detector, and whenever the PMT or any PC boards are replaced, Waters personnel calibrate PMTs using an onboard service diagnostic function.

  • Page 23: Filtering Noise

    mobile phase has a high background, is always a concern, even when the PMT gain setting is at the lowest level. For this reason, the detector has an Auto-Optimize Gain diagnostic function that lets you adjust the granularity of the gain. 1.4.5 Filtering noise The detector uses a digital filter to minimize noise.

  • Page 24: Wavelength Verification And Test

    This component ensures the best rejection of common mode noise in the two beams, leading to a quiet baseline. • Personality board – Receives inputs from the preamplifier board and external events. It also provides control of the optics positioning subsystems and the lamp power supply. •...

  • Page 25: Multichannel Mode

    In Single Channel mode, the detector automatically engages the second-order filter for excitation wavelengths of 400 nm and longer, and removes it for wavelengths shorter than 399 nm. The second-order filter is an optical filter that blocks unwanted UV light from reaching the diffraction grating, which can interfere with fluorescence detection of 400 nm and longer.

  • Page 26: Spectrum Scanning Mode

    1.5.2.2 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. See also: The Console online Help. 1.6 Spectrum Scanning mode You can use the detector as a fluorometer to acquire spectra and store them as a file.

  • Page 27: Auto-Optimize Gain

    Verifying reference energy alone to evaluate performance assumes that every lamp has the same longevity, degradation patterns, and spectral output characteristics. To reduce this uncertainty, Waters designed the detector to operate as independently of lamp output as possible. After the instrument verifies the calibration of the monochromator, it evaluates the energy levels in a number of characteristic regions across the spectrum.

  • Page 28: Method Optimization

    when you use the analog outputs during data collection. This value is also computed assuming a 2× margin for error. 1.8.1 Method optimization You can download a method that includes timed event changes. The timed event changes that alter gain, excitation wavelength, or emission wavelength are critical “light condition” changes. These are the points at which the signal peak maximum search gets renewed.

  • Page 29: Ensuring Gain Optimization For Each Peak Of Interest

    Table 1–1: Example of method development Time (min) Event Initial (0.0) Excitation = 375 nm Emission = 410 nm Gain = 100 Gain = 1 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.

  • Page 30: Startup Diagnostic Tests

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

  • Page 31: Wavelength Selection

    1.11 Wavelength selection Warning: To avoid severe damage to the instrument, injury to the operator, or both, do not use incompatible solvents. 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.

  • Page 32: Setting Up The Detector

    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 can report damage and discrepancies to Waters via the methods of contact listed in Contacting Waters (Page iii).
  • Page 33 Figure 2–1: Aligning pins with slots Alignment pin (2) Alignment slot (2) 2. Lower the front of the module that you are adding so that its front alignment pin rests in the front alignment slot on the previously added module. 3.

  • Page 34: Connecting To The Electricity Source

    Figure 2–2: Arc Premier System with 2475 FLR detector installed, example configuration Solvent tray Detector Column heater Sample manager Solvent manager (QSM-R or BSM-R) 2.3 Connecting to the electricity source The detector requires a separate, grounded electricity source. The ground connection in the electrical outlet must be common and connected near the system.

  • Page 35: Plumbing The Detector

    • Use SVT-type power cords in the United States and HAR-type power cords, or better, in Europe. For requirements elsewhere, contact your local Waters distributor. • Do not replace power cords with inadequately rated power cords.

  • Page 36: Connecting Columns In Arc Premier Systems

    Prohibited: To avoid equipment damage caused by spilled solvent, do not place reservoir bottles directly atop an instrument or device or on its front ledge. Instead, place the bottles in the bottle tray, which serves as secondary containment in the event of spills.

  • Page 37: Making Tubing Connections In Hplc Systems

    Figure 2–3: Ferrule and compression screw Tube Compression screw Ferrule Tubing end (cut straight and smooth to achieve maximum column efficiency) Distance (determined by each application, such as union or column fitting) 2.4.4 Making tubing connections in HPLC systems To make tubing connections in an HPLC system: 1.

  • Page 38: Making Signal Connections

    Waters recommends that you connect the Arc Premier 2475 FLR detector to the data system components via an Ethernet connection. The following table summarizes the signal connections used to connect the Arc Premier 2475 FLR detector to the HPLC or UHPLC system.

  • Page 39: Connecting The Ethernet Cable

    Connection hardware requires one standard Ethernet cable per Waters instrument and a standard Ethernet cable between the network switch and the acquisition computer. You must install the Waters instrument control software on the acquisition computer so that the computer can control the Waters instrument.
  • Page 40 2.5.2.3 Network installation guidelines Configurations for multiple Waters instruments use a dedicated local area network (LAN). See the following figure. The LAN requires a design based on the following guidelines: • Ethernet cable • A maximum distance of 100 meters (328 feet) You must use a network switch with multiple Ethernet instruments.

  • Page 41: Choosing Signal Connections

    2.5.2.4 Making inject-start signal connections In an Ethernet-controlled system, no inject-start is required. However, the Arc Premier 2475 FLR detector must receive an inject-start signal from the autosampler or manual injector to initiate the data collection and time-based programs if they are not Ethernet-controlled.

  • Page 42: Signal Connections

    – Switch 2 – Auto zero The following table describes each signal available on the I/O connectors. Table 2–3: Arc Premier 2475 FLR detector analog-out/event-in connections Signal Description Inject Start TTL contact closure. Configurable input to initiate sequencing of time- programmed events.
  • Page 43 Requirement: To meet the regulatory requirements of immunity from external electrical disturbances, you must install connection covers over the signal connectors. To make signal connections: 1. Attach the positive and negative leads of the signal cable to the connector. Figure 2–8: Attaching the signal cable to the connector Connector Signal cable 2.
  • Page 44 Shield Connection cover 4. 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. Figure 2–10: Tightening the clamp over the cable leads Screw Clamp Cable leads...

  • Page 45: Connecting An Alliance E2695 Separations Module

    Cover 2.5.6 Connecting an Alliance e2695 separations module Requirement: To connect an Alliance e2695 separations module, the Arc Premier 2475 FLR detector must be connected to the data system using an IEEE connection, instead of the recommended Ethernet connection. An Alliance e2695 separations module performs the following functions (when the detector is not under the control of Empower software): •...
  • Page 46 To generate a chart mark at the start of an injection from an Alliance e2695 separations module: 1. Make the following connections between connector II of the Alliance e2695 and connector I of the Arc Premier 2475 FLR detector. August 19, 2021, 715007351 Ver. 00 Page 46...

  • Page 47: Starting A Method

    To enable the detector to start a method when an injection from an Alliance e2695 separations module begins: Make the following connections between connector II of the Alliance e2695 and connector I of the Arc Premier 2475 FLR detector. Figure 2–14: Connections for starting a method injection Alliance...

  • Page 48: Making Rs-232 Signal Connections

    To turn the lamp on or off from an Alliance e2695 separations module: 1. Make the connections below between connector II of the Alliance e2695 and connector I of the Arc Premier 2475 FLR detector. Figure 2–15: Connections for turning the lamp on or off...
  • Page 49 IEEE system RS-232 communication does not support multichannel mode. When connecting the detector to a Waters data system, all detector parameters not configurable by the data system in use defer to local control. 2.5.7.1 Connecting an RS-232 device to the detector To connect an RS-232 device to the detector: 1.

  • Page 50: Connecting Other Devices

    To connect a system running under Empower software control to the detector: 1. Connect the e-SAT/IN module to the Ethernet card in a system running under Empower software control according to the instructions in the Waters e-SAT/IN Module Installation Guide (71500049404).
  • Page 51 Notice: • Do not turn on the e-SAT/IN module until you perform all procedures in the Waters e-SAT/IN Module Installation Guide (71500049404). Improper startup can damage the unit and void the warranty. • To prevent damage to the module, always disconnect the power cord at the wall outlet or the power supply before attaching or removing the power connection to the module.

  • Page 52: Connecting A Waters Fraction Collector

    • A chart mark signal generates at the chart mark inputs on the analog. 2.6.3 Connecting a Waters fraction collector Your Waters fraction collector communicates with your Alliance HPLC system through the I/O terminal. The I/O terminal is a two-part (male/female) connection. The female connector is permanently mounted on the rear panel of the fraction collector.
  • Page 53 Note: The signal cables should be as short as possible to minimize interference. 2. Using a wire insulation stripper, remove approximately 5 mm of insulation from the end of each signal cable wire. 3. Remove the male connector from the rear panel of the WFC III by pulling firmly on the orange body of the I/O terminal.
  • Page 54 Table 2–5: I/O terminal connector specifications Parameter Value Maximum terminal voltage 60 Vac Maximum terminal current 0.3 A Maximum switch wattage 10 W Minimum resistance capacity 100 mA 5. On the terminal block, locate the pin you want to connect. Make positive connections to even-numbered terminals and negative connections to odd- numbered terminals.

  • Page 55: Using The Detector

    Before you start the detector, ensure that the power cord is properly installed at the detector’s rear panel and plugged into the power source. Service keypad inputs are coded for use only by Waters service engineers for troubleshooting purposes. To power-on the detector: 1.

  • Page 56: Startup Failure

    The detector displays the following messages: • Initializing grating • Initializing system • Lighting lamp • Warmup time left (counts down from five minutes) • Homing optical filter • Searching for Zero Order Peaks • Finding erbium calibration peaks • Restoring last setup When initialization completes, the detector displays the Home screen.

  • Page 57: Using The Operator Interface

    Figure 3–2: Idle mode screen 3.2 Using the operator interface 3.2.1 Using the display The detector’s operator interface includes a 128 × 64 bitmap graphic display and a 24-key membrane keypad located behind the front door. The Home screen appears as follows: Figure 3–3: Home screen parameters Channel selector Next screen...

  • Page 58: Fluorescence And Message Icons

    Run time (minutes) You can display the Home screen anytime by pressing HOME. When you first use the detector, the Home screen shows factory-set defaults. Afterward, it shows the settings displayed before the detector was last shut down. The Home screen continues to change as the run continues. In real time, the detector monitors fluorescence in terms of emission or energy units of one or more wavelength pairs.
  • Page 59 Icon or field Description Function Numerical field (0.00) Fluorescence in Displays current normalized emission units or emission units or sample energy units for the selected channel sample energy units that are not normalized. The units displayed depend on the output units selected on the second screen of the operator interface.

  • Page 60: Using The Keypad

    Icon or field Description Function Sticky diagnostic on Indicates a sticky diagnostic setting is active. Local method number Indicates that the detector is not controlled by a data system. It displays either a cursive “m” and the current method number or an asterisk (*), which indicates current conditions are not stored as a method.
  • Page 61 • A/B – Toggles between channels A and B. • Direct access to specific screens – HOME, METHOD, CONFIGURE, DIAG (Diagnostics), TRACE, and SCAN. • Primary functions – Chart Mark, Auto Zero, and Run/Stop. Primary function keys take effect immediately, with no further entry required. •...
  • Page 62 Figure 3–4: Detector keypad The following table explains the functions of the primary and secondary keys. To initiate a secondary function, press Shift and then the key. Description Unshifted After pressing shift HOME – Displays the Home screen, ? – Displays context-sensitive Help, which displays icons, excitation and when available.
  • Page 63 Description Unshifted After pressing shift Auto Zero from the fourth Home screen. Run/Stop – Starts or stops (freezes) Reset – Resets the detector run clock the run clock and initiates scans. The to zero minutes and returns the detector elapsed time appears near the lower- to initial conditions for a current method.
  • Page 64 Description Unshifted After pressing shift 0-9 – Enters the corresponding 0-9 – See the descriptions that follow for number into the current field. specific, shifted, numeric keys. Also positions the cursor at the corresponding entry in a list. (0 = tenth item). Selects the corresponding number from a list.

  • Page 65: Navigating The User Interface

    Description Unshifted After pressing shift CE – Clears an editing change, and Clear Field – Blanks the current entry returns the contents of a field to its field before you specify a new value. previous value. Sets the value to a unique word for some fields.
  • Page 66 • Time constant • Data units selection • Voltage offset • Chart polarity • Enable/disable several inputs • Enable/disable external events The values and settings you specify in the secondary function fields become part of a current method’s conditions and are retained when you store the method. When you press Next, the detector displays three additional home screens, labeled 2 of 4, 3 of 4, and 4 of 4.

  • Page 67: Preparing To Start A Run

    3.3 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 68 By using emission units, you can eliminate optics deterioration as a variable in your measurements. When emission units are used, measurements taken on different Arc Premier 2475 FLR detectors are fully compatible with one another. Energy Energy units do not have the normalization advantage.
  • Page 69 Voltage offset Adjusts the charted analog output signal. Specified in millivolts, voltage offset adjusts the 1-V signal by the entered value. This is useful for making minor adjustments, and for nulling any offset between the detector and a connected external data system. Chart polarity Inverts the chromatogram on the analog output.
  • Page 70 Table 3–1: Primary and secondary function (method) parameters (continued) Function Screen Type Units Range Default Analog out 2 (of 4) Choice None • Emission A Emission A (single λ) • Reference energy A • Output off Analog out 2 (of 4) Choice None •...

  • Page 71: Operating The Trace And Scale Functions

    Table 3–1: Primary and secondary function (method) parameters (continued) Function Screen Type Units Range Default Auto Zero on λ 4 (of 4) Choice None • To baseline To baseline changes • To zero • Disable 3.3.3 Operating the trace and scale functions The trace function displays a fluorescence signal for the last n minutes (up to and including 60) of detector operation.
  • Page 72 Figure 3–6: Scaled trace of continuous injections with T1 changed to -60 The following figure shows a 4-minute scaled trace (or zoom) of the 60 minutes of continuous injections shown in the previous figure. T1 is changed to -4. T2 is changed to 0. EU1 and EU2 remain as auto.

  • Page 73: Configuring The Detector

    3.3.4 Configuring the detector You can configure the detector to emulate the Waters 474 detector communication protocol in the Configuration screens. Press CONFIGURE (Shift, DIAG). The first of three Configuration screens appears. Then, select 474 emulation. You can perform additional functions, such as specifying event inputs and enabling pulse periods, from the Configuration screens.

  • Page 74: Setting Pulse Periods

    The second Configuration screen includes four editable entry fields. Inject You can specify an inject-start input to signal the start of a run, an event that resets the run-time clock and applies initial method conditions immediately. • High – Starts run when the contact closure changes from Off (open) to On (closed). •...

  • Page 75: Setting The Display Contrast

    • Single pulse (in seconds) – If you program SW1 or SW2 to generate a pulse as a timed or threshold event, then this field specifies the period of the signal (single pulse width; range 0.1 to 60 seconds). • Rectangular wave (in seconds) – If you program SW1 or SW2 to initiate a rectangular wave as a timed or threshold event, then this field specifies the period of the signal (the width of one pulse period in a rectangular wave or pulse train;...

  • Page 76: Using Online Help

    3.3.9 Using online Help The detector has limited context-sensitive Help. When you press ? (Shift, HOME) from a point in the program associated with a Help screen, the screen appears. If Online Help is not available, pressing ? effects no response. Press Enter to return to the Home screen. Figure 3–15: Example of an online Help screen 3.4 Operating the detector If you are operating the detector under the control of an external data system, you can program...

  • Page 77: Remote Control Operation For 474 Emulation Mode Via Rs-232

    3.4.3.2 Method parameters The initial conditions for the method are specified on the General tab of the fluorescence method editor in chromatography data software. Note: The Arc Premier 2475 FLR detector and the 474 detector interpret some method parameters differently. Parameter Value Excitation λ...
  • Page 78 Setting the time constant of the filter (3, 5, 10, 20, or 40). The 474 detector interprets these numbers in terms of seconds. However, values of this magnitude are too high for chromatography, so the Arc Premier 2475 FLR detector interprets these inputs at one tenth the specified value.
  • Page 79 Software selection 2475 FLR detector 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. Typically, you can configure this function to a value that is comfortably longer than the runtime of any injection.

  • Page 80: Operation Details

    Separation methods for the Arc Premier 2475 FLR and 474 detectors are compatible except for their gain values. The higher sensitivity of the Arc Premier 2475 FLR detector’s flow cell produces significantly higher signals. Thus, gain values optimized for the 474 detector can yield saturated, highly distorted measurements when you apply them to the Arc Premier 2475 FLR detector.

  • Page 81: Manual Wavelength Calibration

    Before you pump solvent or mobile phase through the system, flush the lines with filtered, degassed, and sparged HPLC-grade water. Then pump the mobile phase, provided you encounter no miscibility problems, at 1 mL/min for 15 minutes, minimum. 3.4.6 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 occur during startup.

  • Page 82: Normalizing Emission Units

    You can select either emission or energy units in the Output field of page 2. 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 Arc Premier 2475 FLR detectors.

  • Page 83: Operating The Detector In Single-Channel Mode

    3.4.8 Operating the detector in single-channel mode The detector is optimized for single-channel (λ) operation, which is the default operating mode. To specify single-channel mode when the detector is in multichannel mode: 1. From the Home screen, press λ/λλ (Shift, Auto Zero). Result: The detector displays an appropriate message while switching to single-channel operation.

  • Page 84: Setting Gain And Eufs

    Result: This key toggles between single and multichannel mode, and the detector displays a message indicating it is setting up multichannel operation. 2. Specify the excitation wavelength to monitor in the xλ field, and then press Enter. 3. Specify the emission wavelength to monitor in the eλ field, and then press Enter. 4.
  • Page 85 Figure 3–19: Gain set too high (fluorescence pinned to –9999.9 EU) Figure 3–20: Gain set too high alarm message The value of -9999.9 EU in the Emission/Energy Units field and the alarm help differentiate between too high a gain setting and too low an EUFS setting. When the EUFS is set too low, flat- topped peaks appear, because the upper limit of the output range is exceeded.
  • Page 86 If you are using Empower software, you must enter the method into the method editor. The method is then downloaded to the Arc Premier 2475 FLR detector when you make an injection. After you program the method into the detector (or Empower editor), press DIAG, and then select 3 Auto-Optimize Gain.
  • Page 87 Figure 3–22: Selecting the auto-optimize gain diagnostic test Selecting Auto-Optimize Gain prepares the diagnostic to execute on the next injection. The (sticky diagnostic) icon appears on the Home screen, and appears in the emission field. 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 panel.

  • Page 88: Programming Methods And Events

    Figure 3–24: Results in the auto-optimize gain table After you clear the display by pressing HOME, the auto-optimize gain diagnostic test automatically disengages. With the proper gains and EUFS values specified in the method, the chromatogram is on scale. Figure 3–25: Gain and EUFS optimally set Minutes 3.5 Programming methods and events 3.5.1 Storing methods...

  • Page 89: Programming Timed Events

    appears on the Home screen. An asterisk in the method number icon indicates that conditions are not stored. If you edit a parameter such as wavelength or EUFS, you are editing the current conditions (Method *). You may store the method in one of the 10 available method storage slots, or you can replace the current method with one previously stored.
  • Page 90 Number Event Units Range or Specify channel default Chart mark (10% Does not apply Does not apply of full scale) Polarity 1. – 2. + Auto Zero Does not apply Does not apply Lamp 1. Off 2. On Switch 1 1.

  • Page 91: Programming Threshold Events

    Figure 3–27: Timed events screen 4. Press Enter. 5. To advance to the Set field (Events list), press ▼. 6. Press Enter again to display the list. Tip: If you know the event number, simply press it. 7. Input the appropriate selection in the To field, if the field appears. Tip: If you want the same event programmed on both channels, you must specify two events, one for channel A and one for channel B.

  • Page 92: Storing A Method

    Below the specified threshold, program the switch parameters as follows: Number Set to Below threshold switch state Pulse Rect wave (rectangular wave) To program a threshold event: 1. Press METHOD (Shift, A/B). Result: The Method list appears. 2. From the method list, select 2 Threshold events. Result: An active field (EU) for entering the threshold appears.

  • Page 93: Retrieving A Method

    To store a method: 1. Press METHOD (Shift, A/B). 2. From the method list, select 4 Store method *. Result: A method number field appears. Note: No warning message appears when the method number you specify in the method number box is already assigned a previously stored method. When you specify a number and press Enter, the current method conditions are stored, overwriting any previous method stored in the same slot.

  • Page 94: Resetting A Method

    If you change a timed or threshold event within a method, the asterisk appears (Method *), indicating that the method (*) is no longer the same as the stored method you retrieved in step 1. You can store the method containing the altered event or events in the same storage slot.

  • Page 95: Scanning Spectra

    Result: The method number icon has an asterisk. 3.6 Scanning spectra 3.6.1 Types of scanning The detector can collect sample scans for either excitation or emission fluorescence spectra. A zero-scan is desirable initially. • Zero-scan – A reference scan that characterizes the fluorescence spectrum of a solvent in the flow cell.
  • Page 96 Table 3–2: Pace and sampling resolution examples (continued) Pace (nm/min) Emission sampling Excitation sampling resolution (nm) resolution (nm) The following figures show two emission scans of anthracene. At a pace of 1000 nm/min, the second scan shows a reduced number of points scanned. The resolution is diminished relative to that in the original scan at a pace of 100 nm/min.
  • Page 97 Figure 3–32: Scan of water without tick marks Wavelength 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 98 Figure 3–34: Zero and sample scan screens Sample scan (screen 1 of 4 shown, screens 2, 3, and 4 not shown) Zero-scan (screen 2 of 4) Press Next Zero-scan (screen 3 of 4) Press Next 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 99: Scanning New Spectra

    Figure 3–35: Sample scan screens after a zero-scan Sample scan (screen 1 of 2) Press Next Sample scan (screen 2 of 4) 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. You must try to run sample scans within 15 minutes of the baseline zero scan.

  • Page 100: Parameters Used For Sample And Zero-Scans

    Figure 3–36: Scan list 2. From the scan list, select 1 New scan, or use ▲ and ▼ to scroll through the list. Result: The detector displays the first of four parameter screens. 3. Press Next to advance through the New scan parameter screens. 4.

  • Page 101: Programming A Zero-Scan

    Parameter Screen Units Range or default Gain Range: 1 to 1,000 λ other Default: 200 or 210 nm Monochromator scan Excitation or emission type 3.6.5 Programming a zero-scan To program a zero-scan: 1. Press SCAN (Shift, Chart Mark). 2. Select 1 New Scan, and then select2 Zero Scan. 3.

  • Page 102: Running A Sample Scan

    Figure 3–37: Zero-scan progress bar 3.6.6 Running a sample scan To run a sample scan: Note: Ensure that your sample and mobile phase are degassed. Tip: Run a zero-scan before running the sample scan. To ensure identical flow cell and solvent conditions, run the sample scan for the corresponding zero-scan within 15 minutes of running the zero-scan.
  • Page 103 Figure 3–39: Subtracting the Zero-scan progress bar After the scan is complete, the detector displays the sample scan graphically. Figure 3–40: Graph of sample anthracene scan 5. Press Next to display the parameters for up to three of the highest peaks scanned within the specified range.
  • Page 104 Figure 3–42: Anthracene scan with λ2 changed to 420 nm 9. If you change one or more scaling parameters, press Enter to reformat the graph. 10. Press Next to display the properties of the highest peaks of the scaled scan. Figure 3–43: Highest peaks from the scaled anthracene scan 11.

  • Page 105: Scanning Using A Static Flow Cell

    Figure 3–45: Zoom of sample emission scan 350 to 440 nm • Zoom of sample emission scan 350 to 440 nm • 20 to 35 EU • Excitation = 249 nm • Anthracene • λ2 changed to 440 nm Figure 3–46: Zoom of sample emission scan 360 to 420 nm •...

  • Page 106: Managing Results

    4. Run a sample scan, keeping the pressure below 145 psi to avoid over pressuring the flow cell. 5. Use the functions storage, review, subtract and review, and replay to compare the scanned data. 3.7 Managing results In stand-alone mode, after you run a spectrum, you can store it for later review, subtraction, or playback.

  • Page 107: Displaying Information About A Stored Spectrum

    3.7.2 Displaying information about a stored spectrum To display information about a stored spectrum: 1. Press SCAN (Shift, Chart Mark). 2. Select 3 Get scan info. Result: A slot number box appears. Its default is "Last" (for the most recently stored spectrum).

  • Page 108: Replaying A Spectrum

    2. Select 5 Subtract & Review. Note: To subtract one spectrum from another, the starting and ending wavelengths (λ1 and λ2) and the pace of both spectra must be identical. 3. 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 109: Manually Extinguishing The Lamp

    3.8.1 Manually extinguishing the lamp To manually extinguish the lamp: 1. Press Lamp (Shift, 1). Result: The lamp control screen appears. Figure 3–48: Lamp control screen 2. Press Lamp (Shift, 1) again to extinguish the lamp. Result: The Home screen appears with an "X" through the lamp indicator icon and the words Lamp off.

  • Page 110: Manually Lighting The Lamp

    Figure 3–49: Lamp off/on sequence Lamp off indicator Lamp on indicator 3.8.2 Manually lighting the lamp To manually light the lamp: 1. Press Lamp (Shift, 1). Result: The lamp control screen appears with 0 hours and 00 minutes in the "Lamp has been on"...

  • Page 111: Using A Timed Event Method To Program The Lamp

    3.8.3 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 112: Maintenance

    Off, and then unplug the power cord from the ac outlet. Afterward, wait 10 seconds before you disconnect an assembly. 4.1.2 Spare parts To ensure that your system operates as designed, use only Waters Quality Parts. Visit www.waters.com/wqp for information about Waters Quality Parts, including how to order them.

  • Page 113: Inspecting, Cleaning, And Replacing The Flow Cell

    erbium filter to verify the calibration for the diffraction gratings. The 365-nm mercury line serves as the second calibration point. • Flush buffered mobile phases from the detector with HPLC-grade water followed by a 90/10 methanol/water solution each time the detector shuts down. Flushing prevents these problems: •...

  • Page 114: Flushing And Passivating The Flow Cell

    4.3.1 Flushing and passivating the flow cell Flush and passivate the flow cell when you suspect that it is dirty. To flush and passivate the flow cell: 1. Discontinue the flow of mobile phase. 2. Remove the column. 3. Replace the column with a union or piece of tubing. 4.

  • Page 115: Replacing The Flow Cell

    7. If the flow cell will not be used for a period of time (such as a weekend), flush it with clean mobile phase, such as a water/acetonitrile mix. Recommendation: Waters recommends a mixture of 10% organic or more. 8. To store the flow cell, cap the flow ports. 4.3.3 Replacing the flow cell The detector is shipped with the analytical flow cell installed.

  • Page 116: Replacing The Lamp

    3. Insert the new flow cell assembly into the detector. 4. Tighten the captive screws. 5. Confirm that the flow cell seats properly. 6. Reconnect the inlet/outlet tubing. 7. Start the detector. 8. Calibrate and normalize the detector. 4.4 Replacing the lamp This section describes the procedure for removing and replacing the xenon lamp.

  • Page 117: Removing The Lamp

    Warning: Lamp gas is under positive pressure. To avoid shattering the glass, use care when disposing of the lamp. Waters suggests that you adequately cushion the old lamp by containing it in the packaging of its replacement before you dispose of it.

  • Page 118: Installing The New Lamp

    Figure 4–3: Removing the lamp assembly 4.4.3 Installing the new lamp Warning: To avoid eye injury from ultraviolet radiation exposure, • power-off the detector before changing the lamp; • wear eye protection that filters ultraviolet light; • keep the lamp in the housing during operation. Important: To avoid damaging the lamp, do not touch the glass bulb on the new lamp.

  • Page 119: Recording The New Lamp's Serial Number

    Warning: To avoid electric shock, power-off an instrument or device and disconnect its power cord before installing components or performing maintenance procedures. To install the new lamp: 1. Position the lamp cartridge, and insert it in the housing. Note: No additional alignment is required. 2.

  • Page 120: Replacing The Fuses

    Figure 4–4: Change lamp screen 5. Press Enter to store the serial number and move to the date installed field. 6. From the list, select the current month. 7. Press Enter twice to update the month and move to the day field. 8.

  • Page 121: To Replace The Fuses

    Suspect a fuse is open or otherwise defective when any of the following situations occurs: • The detector fails to power-on. • The display is blank. • The detector's fans do not operate. To replace the fuses: Requirement: Replace both fuses, even when only one is open or otherwise defective. 1.

  • Page 122: Error Messages, Diagnostic Tests, And Troubleshooting

    In most cases, you can cycle power (shut down, wait 10 seconds, and then restart) to correct the error. However, if the error persists, you must contact Waters Technical Service. Figure 5–1: <Error> display on the home screen August 19, 2021, 715007351 Ver.

  • Page 123: User-Selected Diagnostic Tests And Settings

    2. Ensure that the lamp access door is shut securely. 3. Cycle power to the detector. 4. If the error persists, contact Waters Technical Service. 5.3 User-selected diagnostic tests and settings The detector uses both user-selectable and service diagnostic tests and settings. Only qualified Waters Service personnel can access service diagnostic tests.
  • Page 124 Figure 5–3: Home screen showing active sticky diagnostic settings (wrench icon) • To disable a specific sticky diagnostic setting, reset it to the default settings. • To disable all active sticky diagnostic settings, press DIAG, and then select 4 Reset diagnostics.

  • Page 125: Sample And Reference Energy Diagnostic Tests

    2 Optical filter override 3 Previous choices << 9 Service Diagnostic tests used by Waters service personnel. 5.3.2 Sample and reference energy diagnostic tests The sample and reference energy tests plot the output of the analog channels, to examine noise fluctuations and to compare with the EU time trace.

  • Page 126: Raman Signal-To-Noise Diagnostic Test

    2. Specify a new wavelength number to change the wavelength, and then press Enter. Result: When the new wavelength shifts to the left, the corresponding sample and reference energies appear. 3. If you are operating the detector in multichannel mode, press A/B to view sample and reference energy on the other wavelength.
  • Page 127 • Fix (set) the voltage on the 1-V output. • Monitor contact closures and toggle event switches. • Generate test peaks. • Override the optical filter. To perform one of the input and output tests or change a setting, select 6 Input & output. A list of four diagnostic tests and settings appears.
  • Page 128 Figure 5–9: Fix EU screen 5.3.4.3 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, select 3 Fix voltage to select a voltage for the analog output. Note: You can select a voltage for both output channels (range -0.10 to +1.10-V).

  • Page 129: Change-Lamp Function

    Note: 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). 2.

  • Page 130: 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. 3. Select 4 to return to the diagnostics list.

  • Page 131: Generating Test Peaks

    Figure 5–14: Other diagnostic tests and settings 5.3.9 Generating test peaks The generate test peaks function changes the first entry on the list to Disable test peaks. To generate test peaks: 1. Press DIAG, and then select 8 Other diagnostics. 2.

  • Page 132: Reducing Pmt Sensitivity

    Figure 5–16: Optical filter setting screen 3. Press Enter. Note: The list of filters includes: • 1 Automatic • 2 Second Order • 3 None • 4 Erbium • 5 Shutter 4. In the list of filters, press a number to select the corresponding filter, or leave the default filter (Automatic) on.

  • Page 133: Troubleshooting

    Waters Technical Service. 5.4.2 Information needed when you contact Waters To expedite your request for service, keep the following information at hand when you call Waters Technical Service: • Detector serial number •...

  • Page 134: Power Surges

    Broken electrical connection Inspect electrical connections. illuminate Open (blown) fuse Inspect and, if necessary, replace fuses. Bad LCD or control board Contact Waters Technical Service. Front panel displays odd Faulty EPROMs or bad LCD Contact Waters Technical characters control board Service.
  • Page 135 Xenon lamp does not light Faulty lamp Replace the lamp. Lamp not plugged in Plug in the lamp connector. Bad lamp power supply Contact Waters Technical Service. Lamp switch off Inspect the rear panel connections. August 19, 2021, 715007351 Ver. 00...

  • Page 136: A Safety Advisories

    Heed all warnings when you install, repair, or operate any Waters instrument or device. Waters accepts no liability in cases of injury or property damage resulting from the failure of individuals to comply with any safety precaution when installing, repairing, or operating any of its instruments or devices.

  • Page 137: Specific Warnings

    (Risk of high-pressure gas release.) A.1.1 Specific warnings A.1.1.1 Burst warning This warning applies to Waters instruments and devices fitted with nonmetallic tubing. Warning: To avoid injury from bursting, nonmetallic tubing, heed these precautions when working in the vicinity of such tubing when it is pressurized: •...

  • Page 138: Biohazard Warning

    A.1.1.2 Biohazard warning The following warning applies to Waters instruments and devices that can process biologically hazardous materials. Biologically hazardous materials are substances that contain biological agents capable of producing harmful effects in humans. Warning: To avoid infection from blood-borne pathogens, inactivated microorganisms, and other biological materials, assume that all biological fluids that you handle are infectious.

  • Page 139: Bottles Prohibited Symbol

    Use eye protection when performing this procedure. Requirement: Wear clean, chemical-resistant, powder-free gloves when performing this procedure. A.5 Warnings that apply to all Waters instruments and devices When operating this device, follow standard quality-control procedures and the equipment guidelines in this section.
  • Page 140 Advertencia: cualquier cambio o modificación efectuado en esta unidad que no haya sido expresamente aprobado por la parte responsable del cumplimiento puede anular la autorización del usuario para utilizar el equipo. 警告： 未经有关法规认证部门明确允许对本设备进行的改变或改装，可能会使使用者 丧失操作该设备的合法性。 警告： 未經有關法規認證部門允許對本設備進行的改變或修改,可能會使使用者喪失操 作該設備的權利。 경고 규정 준수를 책임지는 당사자의 명백한 승인 없이 이 장치를 개조 또는 변경할 경 우, 이...
  • Page 141 Warnung: Bei der Arbeit mit Polymerschläuchen unter Druck ist besondere Vorsicht angebracht: • In der Nähe von unter Druck stehenden Polymerschläuchen stets Schutzbrille tragen. • Alle offenen Flammen in der Nähe löschen. • Keine Schläuche verwenden, die stark geknickt oder überbeansprucht sind. •...
  • Page 142 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。 This warning applies to Waters instruments fitted with nonmetallic tubing or operated with flammable solvents. August 19, 2021, 715007351 Ver. 00 Page 142...

  • Page 143: Warnings That Address The Replacement Of Fuses

    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. Avertissement : 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 144 Avertissement : pour éviter tout risque d'incendie, remplacez toujours les fusibles par d'autres du type et de la puissance indiqués sur le panneau à proximité du couvercle de la boite à fusible de l'instrument. Warnung: Zum Schutz gegen Feuer die Sicherungen nur mit Sicherungen ersetzen, deren Typ und Nennwert auf den Tafeln neben den Sicherungsabdeckungen des Geräts gedruckt sind.

  • Page 145: Electrical Symbols

    警告： 为了避免火灾，应更换“维护步骤”一章的“更换保险丝”一节中介绍的相同类型和 规格的保险丝。 警告： 為了避免火災，更換保險絲時，應使用「維護步驟」章節中「更換保險絲」所 指定之相同類型與規格的保險絲。 화재의 위험을 막으려면 유지관리 절차 단원의 “퓨즈 교체” 절에 설명된 것과 동일 경고 한 타입 및 정격의 제품으로 퓨즈를 교체하십시오. 警告 火災予防のために、ヒューズ交換ではメンテナンス項目の「ヒューズの交換」 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。 A.7 Electrical symbols The following electrical symbols and their associated statements can appear in instrument manuals and on an instrument’s front or rear panels.

  • Page 146: Handling Symbols

    A.8 Handling symbols The following handling symbols and their associated statements can appear on labels affixed to the packaging in which instruments, devices, and component parts are shipped. Symbol Description Keep upright! Keep dry! Fragile! Use no hooks! Upper limit of temperature Lower limit of temperature Temperature limitation August 19, 2021, 715007351 Ver.

  • Page 147: B Specifications

    B Specifications This appendix lists individual operating specifications for the Arc Premier 2475 FLR detector. Note: All performance specifications are measured following a warm-up period of one hour and ∆T ≤ ±2 °C/hr. B.1 Physical specifications Table B–1: Physical specifications...

  • Page 148: Electrical Specifications

    B.3 Electrical specifications Table B–3: Electrical specifications Attribute Specification Protection class Class I Overvoltage category Pollution degree Moisture protection Normal (IPXO) Line voltages Grounded AC, 100 to 240 VAC Maximum altitude 2000 m (6561.6 ft) Power requirements 100 to 240 VAC Line frequency 50 to 60 Hz Fuse ratings...

  • Page 149: Performance Specifications

    B.4 Performance specifications Table B–4: Performance specifications Attribute Specification Wavelength range • 200 to 890 nm (excitation) • 210 to 900 nm (emission) Bandwidth 20 nm Wavelength accuracy ±3 nm Wavelength repeatability ±0.25 nm Sensitivity Single λ mode: Excitation: 350 nm Emission: 397 nm (Signal-to-noise ratio of Raman peak of water ≥1000.

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