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Waters 2465 Operator's Manual

Electrochemical detector
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Waters 2465
Electrochemical Detector
Operator's Guide
34 Maple Street
Milford, MA 01757
71500246502, Revision B

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  • Page 1 Waters 2465 Electrochemical Detector Operator’s Guide 34 Maple Street Milford, MA 01757 71500246502, Revision B...
  • Page 2 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, the use of this document.
  • Page 3 Disconnect the instrument from all power sources before disassembly. Caution: To avoid electrical shock, power off the 2465 Detector and unplug the power cord before maintaining or servicing the instrument. The I/O connectors on the rear of the instrument have a risk of electrical shock.
  • Page 4 Note: When you use the instrument, follow generally accepted procedures for quality control and methods development. If you observe a change in the retention of a particular compound, in the resolution between two compounds, or in peak shape, immediately determine the reason for the changes.
  • Page 5 Caution: Use caution when working with any polymer tubing under pressure: • Always wear eye protection when near pressurized polymer tubing. • Extinguish all nearby flames. • Do not use Tefzel tubing that has been severely stressed or kinked. • Do not use Tefzel tubing with tetrahydrofuran (THF) or concentrated nitric or sulfuric acids.
  • Page 6 Precauzione: prestare attenzione durante le operazioni con i tubi di polimero sotto pressione: • Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero pressurizzati. • Estinguere ogni fonte di ignizione circostante. • Non utilizzare tubi Tefzel soggetti a sollecitazioni eccessive o incurvati. •...
  • Page 8 Caution: 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 9 Caution: To protect against fire hazard, replace fuses with those of the same type and rating. Attention : Remplacez toujours les fusibles par d’autres du même type et de la même puissance afin d’éviter tout risque d’incendie. Vorsicht: Zum Schutz gegen Feuergefahr die Sicherungen nur mit Sicherungen des gleichen Typs und Nennwertes ersetzen.
  • Page 10 Caution: To avoid possible electrical shock, disconnect the power cord before servicing the instrument. Attention : Afin d’éviter toute possibilité de commotion électrique, débranchez le cordon d’alimentation de la prise avant d’effectuer la maintenance de l’instrument. Vorsicht: Zur Vermeidung von Stromschlägen sollte das Gerät vor der Wartung vom Netz getrennt werden.
  • Page 11 Commonly Used Symbols Direct current Courant continu Gleichstrom Corrente continua Corriente continua Alternating current Courant alternatif Wechselstrom Corrente alternata Corriente alterna Protective conductor terminal Borne du conducteur de protection Schutzleiteranschluss Terminale di conduttore con protezione Borne del conductor de tierra...
  • Page 12 Commonly Used Symbols (Continued) Frame or chassis terminal Borne du cadre ou du châssis Rahmen- oder Chassisanschluss Terminale di struttura o telaio Borne de la estructura o del chasis Caution or refer to manual Attention ou reportez-vous au guide Vorsicht, oder lesen Sie das Handbuch Prestare attenzione o fare riferimento alla guida Actúe con precaución o consulte la guía Caution, hot surface or high temperature...
  • Page 13 Commonly Used Symbols (Continued) Caution, risk of electric shock (high voltage) Attention, risque de commotion électrique (haute tension) Vorsicht, Elektroschockgefahr (Hochspannung) Precauzione, rischio di scossa elettrica (alta tensione) Precaución, peligro de descarga eléctrica (alta tensión) Caution, risk of needle-stick puncture Attention, risques de perforation de la taille d’une aiguille Vorsicht, Gefahr einer Spritzenpunktierung Precauzione, rischio di puntura con ago...
  • Page 14 Commonly Used Symbols (Continued) Fuse Fusible Sicherung Fusibile Fusible Electrical power on Sous tension Netzschalter ein Alimentazione elettrica attivata Alimentación eléctrica conectada Electrical power off Hors tension Netzschalter aus Alimentazione elettrica disattivata Alimentación eléctrica desconectada...

  • Page 15: Intended Use

    2465 Electrochemical Detector Information Intended Use ® The Waters 2465 Electrochemical Detector is designed for HPLC applications. Biological Hazard When you analyze physiological fluids, take all necessary precautions and treat all specimens as potentially infectious. Precautions are outlined in “CDC Guidelines on Specimen Handling,”...

  • Page 17: Table Of Contents

    Table of Contents Preface ......................xxxiii Chapter 1 2465 Detector Theory of Operation ............... 1 Detector Introduction ..................1 1.1.1 Electrolysis Reactions................1 1.1.2 Current-Potential Curves ..............4 Detector Features....................5 Detector Design....................7 1.3.1 Electronics and Data Acquisition ............7 1.3.2 Electronics ..................
  • Page 18 Site Selection and Power Requirements............35 2.1.1 Site Selection ..................35 2.1.2 Power Requirements ................. 37 Unpacking and Inspecting the 2465 Detector ..........38 Making Electrical Power Connections ............39 Making Fluidic Connections ................40 2.4.1 Installing the 2465 Detector............... 43 2.4.2 Connecting a Column ...............
  • Page 19 3.1.6 Using the Keypad to Change Parameters ......... 67 3.1.7 Function Key Commands ..............67 3.1.8 Status and Control Parameters ............70 Overview of the 2465 Detector Modes ............74 3.2.1 DC Mode.................... 75 3.2.2 Pulse (PAD) Mode ................77 3.2.3 Scan Mode ..................
  • Page 20 Optimizing the Working Potential..............120 3.7.1 Constructing a Hydrodynamic Voltammogram ....... 120 3.7.2 Constructing a Scanning Voltammogram ........122 Shutting Down the 2465 Detector..............125 3.8.1 Turning Off the Flow Cell ............... 125 3.8.2 Shutting Down for a Short Time ............. 126 3.8.3 Shutting Down for a Long Time .............
  • Page 21 Troubleshooting Tables ................. 157 Physical Symptoms ..................161 Appendix A 2465 Detector Specifications ................ 163 Appendix B 2465 Detector Components and Spare Parts ........... 167 Flow Cells .................... 167 Startup Kit Components ................ 172 Spare Parts .................... 173 Table of Contents...
  • Page 22 Appendix C Sample ECD Methods ................... 175 Appendix D 2465 Detector Glossary ................. 179 Index ......................... 183 xxii Table of Contents...
  • Page 23 Installing the Flow Cell................49 2-11 Rear Panel Connections on 2465 Detector ..........52 2-12 I/O Signal Inputs and Outputs ..............53 2-13 2695 Separations Module Connections to the 2465 Detector....56 2-14 busSAT/IN Module (Front Panel)............57 List of Figures xxiii...
  • Page 24 2-15 Connecting a busSAT/IN Module Channel 1 to the 2465 Detector..58 2-16 Config Screen ..................59 2-17 IEEE-488 and RS-232 Connections in a Waters Empower System ..61 Calculating Checksum Screen ..............64 Checksum Screen..................64 Main Screen ..................... 64 2465 Detector Display and Keypad ............
  • Page 25 3-29 Overwrite Time Screen ................98 3-30 Prog Screen After Changing Line 1............99 3-31 Prog Screen After Changing Cell Potential ..........99 3-32 Prog Screen After Adding Line 2 ............99 3-33 Prog Screen After Adding Line 3 ............100 3-34 End Cycle Time Screen................
  • Page 26 3-61 Scan Setup Screen.................. 118 3-62 Switch Cell On Screen ................118 3-63 Switch Cell Off Screen ................118 3-64 Scan Setup Screen.................. 119 3-65 Scan Stat Screen..................119 3-66 Starting a Scan ..................120 3-67 Constructing a Hydrodynamic Voltammogram for Norepinephrine ..121 3-68 Scanning Voltammetry of Norepinephrine ..........
  • Page 27 Noise Test with Dummy Cell..............154 Noise Test Screen................... 155 Testing a Key on the Keypad ..............156 Display Test ................... 157 Configuration Screen ................157 List of Figures xxvii...
  • Page 28 xxviii List of Figures...
  • Page 29 List of Tables Ranges and Maximum Compensation ............ 11 Potential Limits and Applications for Working Electrodes ....16 Recommended Flow Cells with Different Columns....... 17 Flow Cell Volume with Spacers.............. 17 Potential of the Ag/AgCl Reference Electrode........19 Mass of Anhydrous Sodium and Potassium Chloride per Liter for Various Molar Concentrations ............
  • Page 30 Dummy Cell Test Settings ..............154 No Detector Response ................158 High Cell Current.................. 158 Noisy Baseline ..................159 Drifting Baseline................... 159 Decreased Sensitivity (Low S/N Ratio)..........160 Baseline Oscillations................160 Saturation of Output................160 5-10 Physical Symptoms................161 General Specifications ................163 Physical Specifications .................
  • Page 31 8-Hydroxy-2’-deoxyguanosine ............. 177 Lactose, Sucrose, and Maltose.............. 177 Performance Qualification ..............178 List of Tables xxxi...
  • Page 32 xxxii List of Tables...

  • Page 33: Preface

    Chapter 2 describes how to install the 2465 Detector, make power, fluidic, and signal connections, and connect the detector to other devices. Chapter 3 describes how to set up and operate the 2465 Detector in remote and stand-alone modes. Chapter 4 describes how to clean and replace various parts of the 2465 Detector.

  • Page 34: Documentation Conventions

    Empower software. Included as part of the Empower software. Printed Documentation Waters Bus SAT/IN Module Installation Guide: Describes installation of the Waters Bus SAT/IN™ Module. Millennium System Installation and Configuration Guide: Describes Millennium software installation.
  • Page 35 Note: Record your result before you proceed to the next step. Attentions Attentions provide information about preventing damage to the system or equipment. For example: Attention: Never lift the 2465 Detector by the door at the front, but only by its sides, STOP or you may damage the detector. Cautions Cautions provide information essential to the safety of the operator.
  • Page 36 xxxvi...

  • Page 37: 2465 Detector Theory Of Operation

    This chapter introduces the Waters 2465 Electrochemical Detector (2465 Detector). It summarizes the 2465 Detector’s features and major components, and describes the theory and principles of operation. To use the detector effectively, you should understand its principles of operation and design.

  • Page 38: Electrolysis At Working Electrode

    The rate of this step is determined primarily by the applied potential. Generally, the potential selected is high so that this step is very rapid relative to the rate of mass transport. The Nernst equation describes this behavior: E = E + (RT/nF) ln [Ox]/[Red] 2465 Detector Theory of Operation...
  • Page 39 where [Ox] and [Red] are the surface concentrations of the oxidized and reduced forms of the analyte, or: ∆ E = (E – E ) = RT/nF ln [Ox]/[Red] where: E = Potential = Standard potential of the analyte R = Gas constant T = Temperature n = Number of electrons [Ox] = Concentration of oxidized form...

  • Page 40: Current-Potential Curves

    In general, select the smallest potential at the plateau that can oxidize all peaks of interest. Operating at greater potential does not increase the signal and is likely to increase noise. 2465 Detector Theory of Operation...

  • Page 41: Detector Features

    Waters HPLC System and Empower software. The 2465 Detector is configurable with a variety of flow cells, variable volumes, reference electrodes (REFs), materials for working electrodes (WEs), and working electrode diameters.
  • Page 42 Open Detector Oven Figure 1-4 2465 Detector Oven The 2465 Detector has the following capabilities: • Stand-alone programmability – Stores up to nine user-defined programs (also called time files or methods), each consisting of up to 50 programmable timed events and two threshold events.

  • Page 43: Detector Design

    • Multiple reference electrodes (REF) for the standard flow cell – Offers three reference electrodes: salt-bridge silver/silver chloride (sb REF), in-situ silver/silver chloride (ISAAC), and hydrogen reference (Hy-REF). • Dummy cell – Facilitates troubleshooting by enabling you to isolate and test the electronics and control without the variability introduced by the presence of a real flow cell.

  • Page 44: Electronics

    The control board receives inputs from the keypad and displays information on the LCD display. The control board takes the signal from the sensor board’s processor and generates the visual information for the LCD display and 2465 Detector Theory of Operation...

  • Page 45: Rear Panel

    Interconnections The control board is central to all operations of the 2465 Detector. As such, most subassemblies connect directly to it or through intermediate cables. The power supply subassembly generates a filtered +24 V to the control board, which then generates operating voltages of +5 V and +13.5 V for use by other subassemblies.

  • Page 46: Time Constant (Filter Setting) Comparison

    PW = Peak width at half height of the narrowest peak Figure 1-6 shows the relationship between the time constant and response times. TC = 0 sec TC = 1 sec TC = 2 sec Time (Minutes) Figure 1-6 Time Constant (Filter Setting) Comparison 2465 Detector Theory of Operation...

  • Page 47: Autozero And Maximum Compensation

    Comp=on Maximum compensation (MaxComp) is the maximum cell current that can be compensated in a particular measurement range (Table 1-1). The 2465 Detector autozero range or maximum compensation depends on the range of the current setting. Table 1-1 Ranges and Maximum Compensation...

  • Page 48: Startup Diagnostics

    2465 Detector. 1.3.7 Temperature Control The detector oven, a heated flow cell and column compartment at the front of the 2465 Detector, is used to stabilize the detector’s performance. It can also be effective in aiding the chromatographic separation at the column.

  • Page 49: Flow Cell With A Salt-Bridge Reference Electrode

    Ag/AgCl. This guide refers to the GC ISAAC reference electrode design as the most likely configuration for your use. The 2465 Detector electrochemical flow cell has been developed for ultra-trace analysis in standard, microbore, and capillary LC-EC. The 2465 flow cell confined wall-jet configuration has proven to yield stable, reliable results.

  • Page 50: Three-Electrode Potentiostat

    At the working electrode, which is kept at virtual ground, the electrochemical reaction takes place as electrons are transferred. This results in an electrical current to the I/E converter. An integrator can monitor the output voltage. The oxidation or reduction 2465 Detector Theory of Operation...

  • Page 51: Electrodes

    The 2465 electrodes are manufactured and tested to ensure that the fabrication process attains this requirement. Finally, the analyte of interest must be oxidized (or reduced) with favorable I/E characteristics, and a high signal must be obtained at a low working potential.

  • Page 52: Working Electrode Diameter

    Several working electrode diameters (nominally 0.7, 2, and 3 mm) are available for the standard flow cell. In a standard LC system the signal and the noise increase linearly with the working electrode diameter, so the S/N ratio remains approximately the same. 2465 Detector Theory of Operation...

  • Page 53: Spacer Thickness

    In a micro-LC system, an increase in the working electrode diameter increases the noise more than the signal. Therefore, in micro-LC, a decrease of the working electrode diameter results in a better S/N ratio. The choice for a flow cell is primarily based on the HPLC column diameter to obtain the best possible detection limit for a standard, microbore, or capillary column.

  • Page 54: Isaac Reference Electrode

    Installation is simple and there are no air bubbles to check. Note: Waters recommends 2 mM chloride ions for the ISAAC reference electrode Either potassium or sodium chloride may be used. In many cases sodium chloride is preferred, to avoid solubility issues in the presence of other substances, such as perchlorate, which is often used in the sample preparation stage of some samples.

  • Page 55: Potential Of The Ag/Agcl Reference Electrode

    The standard electrode potential of the Ag/AgCl electrode (in a 1.0 M chloride ion solution) for the following half-reaction is defined as E – – AgCl(s) + e <=> Ag(s) + Cl where E = 0.222 V. The potential of the reference electrode is dependent on the chloride concentration, as described by the following equation: •...
  • Page 56 This destabilizes the operation of the 2465 Detector ISAAC flow cell and is often manifested as a decreased response of analytes and increased noise during the analysis.

  • Page 57: Hy-Ref Reference Electrode

    Table 1-6 shows the concentration of sodium chloride or potassium chloride needed to obtain various concentrations of chloride ions. Table 1-6 Mass of Anhydrous Sodium and Potassium Chloride per Liter for Various Molar Concentrations – – Concentration Concentration NaCl (g/L) KCl (g/L) (mMol/L) (Mol/L)

  • Page 58: Salt-Bridge Ag/Agcl Reference Electrode

    Figure 4-8 for a schematic representation of the Ag/AgCl reference electrode). Electrical contact with the other electrodes in the flow cell is made through a salt bridge consisting of a wetted cotton wool frit, which is electrically conducting and slows down leakage of 2465 Detector Theory of Operation...

  • Page 59: Principles Of Detector Operation

    1.6 Principles of Detector Operation To use the 2465 Detector effectively, you should be familiar with the operational modes, the fluidics including the configured flow cell and electrodes, the electronic design of the detector, and the theory and principles of operation.

  • Page 60: Pulse (Pad) Mode

    Elevation of the temperature above ambient can reduce the sensitivity of electrochemistry to environmental changes. • Methods Programmability – Ability to program the 2465 Detector (using timed events to change the parameters) and run using the Events feature. Method time files 1 to 5 are reserved for DC mode.
  • Page 61 Pulse mode differs from DC mode as follows: • The output signal is sampled during only a part of the total pulse cycle. During the sampling time (t ) the signal generated at the working electrode is collected, and this value is sent to the detector output.

  • Page 62: Potential Steps In Pulsed Amperometric Detection

    Optimization depends on the working electrode material, the sample constituents, and the required detection frequency. The impression may arise that the number of variables (three potential steps and four time settings) may lead to a time-consuming optimization procedure, but in practice, pulse mode is straightforward. 2465 Detector Theory of Operation...

  • Page 63: Selection Of Pulse Parameters

    The WE material determines the potential for the cleaning steps, E and E . At alkaline pH, gold oxide is already formed at E > +200 mV (versus Ag/AgCl). At a higher potential, the formation of a metal oxide layer is accelerated and you may choose a shorter time setting.

  • Page 64: Change In Cell Current During Pad

    For platinum and glassy carbon, the negative peaks run far off-scale. However, the profile is similar to the mirror image of the positive peaks. 2465 Detector Theory of Operation...

  • Page 65: Magnified View Of A Chromatogram Obtained With Pad

    Data Acquisition in Pulse Mode An important difference between DC mode and pulse mode is the frequency of the output signal. In DC mode the signal is produced continuously up to 10 Hz, but in pulse mode the sum of t , and t determines the observed data acquisition rate.

  • Page 66: Chromatograms Acquired At Different Data Rates

    – Time when the detection potential is applied. Detection and data collection occur during time interval or sampling total time. • t – Time when a monolayer of metal oxide forms at the working electrode due to the high positive potential. 2465 Detector Theory of Operation...
  • Page 67 • Methods Programmability – Allows you to program the 2465 Detector using timed events to change the parameters. Method time files 6 to 9 are reserved for pulse mode.

  • Page 68: Scan Mode

    1.6.3 Scan Mode Scanning Voltammograms Scan mode of the 2465 Detector optimizes working potential by performing a scanning voltammogram (Figure 1-14), which plots E (potential) in volts versus I (current) in nanoamps. A scanning voltammogram is a forward scan of a substance and an eluent under continuous flow with no column.

  • Page 69: References

    Scan Mode Primary Parameters • E Potential – Potential applied at the beginning of the scan cycle. • E Potential – Potential reached at the end of the scan cycle. • Scan Cycle – Length of the cycle, which can be half, full, or continuous. Half indicates a single ramp between E and E .
  • Page 70 2465 Detector Theory of Operation...

  • Page 71: Installing The 2465 Detector

    Figure 2-1 Major Steps for Installing the 2465 Detector 2.1 Site Selection and Power Requirements 2.1.1 Site Selection Figure 2-2 shows the dimensions of the 2465 Detector. Install the 2465 Detector in an area that meets the requirements in Table 2-1. Site Selection and Power Requirements...

  • Page 72: Dimensions Of The 2465 Detector

    (44 cm) 17.3 Inches (44 cm) 8.7 Inches (22 cm) Figure 2-2 Dimensions of the 2465 Detector Table 2-1 Installation Site Requirements Parameter Requirement Operating temperature range 39 to 104 °F (4 to 40 °C) –104 to 158 °F (–40 to 70 °C)

  • Page 73: Power Requirements

    Weight 30.9 lb (14 kg) without flow cell or column Attention: You must mount the 2465 Detector on a level surface to allow the drip STOP management system (drain tube) to work properly. You can connect a drain tube to a waste container to collect solvent leaks from the detector oven.

  • Page 74: Unpacking And Inspecting The 2465 Detector

    • Waters 2465 Electrochemical Detector Operator’s Guide • Startup Kit (Table B-8) • Flow cell (not shipped in the 2465 Detector carton; at least one flow cell must be purchased separately). Table B-1 lists the flow cells that are available. Table B-2 through Table B-7 list the components in each flow cell kit.

  • Page 75: Making Electrical Power Connections

    Waters Technical Service at 800 252-4752, for U.S. and Canadian customers. Other customers, call your local Waters subsidiary or your local Waters Technical Service Representative, or call Waters corporate headquarters in Milford, Massachusetts 01757 (U.S.A.).

  • Page 76: Making Fluidic Connections

    Figure 2-5 Venting the Detector 2.4 Making Fluidic Connections The controlled detector oven, which optimizes the detection stability in the 2465 Detector, contains the flow cell and the HPLC column. The detector oven has an integrated Faraday cage and is accurately thermostatted to ensure stable working conditions. Installing the flow cell and column within a controlled environment is the minimum requirement for high-quality LC-EC trace analyses.

  • Page 77: Required Materials

    • Flow rate for micro flow cell: 1 µL/min to 2.0 mL/min • Maximum pressure, standard flow rate: 40 psi (2.76 bar, 276 kPa) Attention: Always position the 2465 Detector as the last detector in the series (if STOP the system includes more than one detector).

  • Page 78: Optional Materials

    • HPLC column, preconditioned • Drip management system, tubing, and waste container Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents. Refer to the Material Safety Data Sheets for the solvents in use.

  • Page 79: Installing The 2465 Detector

    (2.76 bar, 276 kPa). To prevent damage, do not connect any tubing or device that might cause backpressure to exceed the pressure rating of the tubing or flow cell. Attention: Do not operate the 2465 Detector with the standard flow cell above a flow rate STOP of 2.0 mL/min or 40 psi (2.76 bar, 276 kPa) backpressure.

  • Page 80: Installing The External Pump And Pulse Dampener

    Waste Container Figure 2-8 Installing the External Pump and Pulse Dampener Note: Waters recommends that the mobile phase contain 2 mM chloride (KCl or NaCl) ions when using the ISAAC reference electrode. c. Fill a mobile phase reservoir with mobile phase. Place inlet filters on the tubing.
  • Page 81 5. Power on the pump and ensure that the system has no leaks, then stop the pump. Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents.

  • Page 82: Connecting A Column

    Place the reservoir higher than the flow cell, such as in the solvent tray on top of the detector. 2.4.2 Connecting a Column The column must be electrochemically clean for use in the 2465 Detector. A new column is not considered electrochemically clean. See the manufacturer’s instructions for more information.

  • Page 83: Installing The Flow Cell

    8. Once the column is clean, place an inline filter after the injector and before the column to protect both the column and the flow cell from particulates. 9. If the 2465 Detector is used for reductive ECD (at a negative working potential), take the following additional actions to remove oxygen from the mobile phase: •...

  • Page 84: Connecting Tubing To The Flow Cell

    1/4-turn more. Note: The ISAAC reference electrode requires chloride ions in the mobile phase. Waters recommends 2 mM chloride ions (KCl or NaCl). Add and equilibrate before installing the ISAAC reference electrode.

  • Page 85: Installing The Flow Cell

    through the upper-left hole in the chassis and out to the waste container (Figure 2-10). Outlet Tubing Out to Waste Flow Cell Outlet Flow Cell Cable (LC Out) Connector (9-Pin) Flow Cell Blue Cable to AUX (Auxiliary Electrode) Red Cable to WE (Working Electrode) Black Cable to REF (Reference Electrode)
  • Page 86 WE, AUX, and REF on the flow cell using the red, blue, and black cell cable (WE – red, AUX – blue, REF – black). Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents.

  • Page 87: Making I/O Signal Connections

    (including RS-232) longer than 9.8 feet (3 meters) when you make connections. 2.5.1 Rear Panel Connections The signal connections you need to make to your 2465 Detector (Figure 2-11) depend on: • The operating mode (stand-alone or remote control) • The types of instruments in your HPLC system...

  • Page 88: Rear Panel Connections On 2465 Detector

    Line Connector Figure 2-11 Rear Panel Connections on 2465 Detector The 2465 Detector has several types of event inputs and outputs, relays, and TTL event switches. The 2465 power supply drives the relays. The TTL switches require a minimum pulse of 100-ms duration, low (where low is < 0.8 V). If repetitive activation is required, the next pulse should occur after 100 ms, high (where high is >...

  • Page 89: I/O Signal Inputs And Outputs

    Table 2-2 and Table 2-3 describe the I/O signals available on I/O connectors A and B (see Appendix A, 2465 Detector Specifications, for the electrical specifications for the signals). The initial switch setting for relays 1 and 2 is open and the initial switch setting for the output and input events is high.
  • Page 90 Always accessible when Ic is on screen. Level triggered GND or Ground Common Table 2-3 Connector B Name Description GND or 1, 2 Ground Common 3, 4, 6 – Not connected 12, 14, 15 Installing the 2465 Detector...

  • Page 91: Connecting To A 2695 Separations Module (Stand-Alone)

    Negative 2.5.2 Connecting to a 2695 Separations Module (Stand-Alone) You can connect the 2465 Detector to the Waters 2695 Separations Module, when it is not under the control of the Empower software, to perform the following: • Autozero on inject start •...

  • Page 92: Connector B

    Pin 2 Inject Start Pin 1 or 2 Ground – Starting a Method on Inject Start To start a method on the 2465 Detector at the start of an injection from a 2695 Separations Module, make the following connections: 2695 Separations Module...

  • Page 93: Connecting To A Bussat/In Module

    TP01138 TP01138 Figure 2-14 busSAT/IN Module (Front Panel) The Waters busSAT/IN Module translates analog signals from devices such as the 2465 Detector into digital form, then transmits the digital signals to the busLAC/E or LAC/E card installed in the Millennium workstation.

  • Page 94: Connecting A Bussat/In Module Channel 1 To The 2465 Detector

    Module. c. To obtain an inject start signal, connect one external I/O cable from the 2465 Startup Kit, using pins 13 and 15 on the 2465 Connector A to the Inject In signal on the module.

  • Page 95: Connecting To A 746 Data Module

    3. Ensure that the Config Output of the 2465 Detector is set to 1 V as follows: a. From the 2465 Detector Main screen, select F1 CONFIG. The Config screen appears (Figure 2-16). Figure 2-16 Config Screen b. If the V setting is 10 VFS, use the value keys to change it to 1 VFS.

  • Page 96: Making Rs-232 Connections

    2. Ensure that the Config Output of the 2465 Detector is set to 1 V as follows: a. From the 2465 Detector Main screen, select F1 CONFIG. The Config screen appears (Figure 2-16). b. If the V setting is 10 VFS, use the value keys to change it to 1 VFS.

  • Page 97: Verifying Com Port Settings

    3. Expand Ports in the tree, right-click the appropriate COM port, then select Properties. The Communications Port (COM1 or COM2) Properties dialog box appears. 4. Click the Port Settings tab. The correct settings for the 2465 Detector are: • Bits per Second = 38,400 •...
  • Page 98 • Data Length = 8 • Flow Control = None 5. Click OK. 6. Close the dialog boxes (Device Manager, System Properties, and other dialog boxes). Installing the 2465 Detector...

  • Page 99: Operating The 2465 Detector

    Note: The 2465 Detector does not have any audible alarms, bells, or beeps. To start up the 2465 Detector: 1. Power on the 2465 Detector by pressing the power switch on the rear panel of the detector (see Figure 2-11). The detector runs several diagnostic tests and displays a message (Figure 3-1).

  • Page 100: Using The Display

    3.1.2 Using the Display This section describes how to use the display and the keypad on the front panel of the 2465 Detector. All messages and screens appear on a 4 × 40 liquid crystal display (LCD). Operating the 2465 Detector...

  • Page 101: Using The Keypad

    Cursor Keys Value Keys Enter Key Figure 3-4 2465 Detector Display and Keypad 3.1.3 Using the Keypad The keypad consists of 12 keys: • Function keys F1 through F5 (5) – Allow you to select the screens that are named in the bottom row of the display.

  • Page 102: Finding The Parameters

    • F3 – Pulse Setup1 screen (see Section 3.2.2, Pulse (PAD) Mode) • F4 – Scan Setup screen (see Section 3.2.3, Scan Mode) • F5 – Diag (Diagnostics) screen for the diagnostic tests (see Chapter 5, Diagnostics and Troubleshooting) Operating the 2465 Detector...

  • Page 103: Using The Keypad To Change Parameters

    To return to the Main screen, select F1 repeatedly. 3.1.7 Function Key Commands Table 3-1 explains the commands that can appear in the bottom row of the 2465 Detector display for the function keys F1 through F5. Command names are capitalized. Table 3-1 Function Key Commands...
  • Page 104 Accesses the Events Setup screen. PULSE SETUP2 HOLD/ F2 or F5 RUN, SCAN STAT Toggles between holding and RESUME resuming the execution of a time file or scan. KEYB DIAG Accesses the Keyb (Keyboard) screen (Keyboard) for testing the keypad. Operating the 2465 Detector...
  • Page 105 Table 3-1 Function Key Commands (Continued) Command Function Screens Description Name MARK DC STAT, PULSE Triggers a marker signal (chart mark) STAT on output (baseline spike of 10% FS, 100 ms duration). NEXT F1 or F5 Several screens Accesses a related screen in the current mode.

  • Page 106: Status And Control Parameters

    You can change active control parameters using the cursor and value keys (see Section 3.1.3, Using the Keypad). Table 3-2 explains the status and control parameters that appear on the 2465 Detector screens, primarily in the top three rows of the display. Table 3-2 Status and Control Parameters...
  • Page 107 Table 3-2 Status and Control Parameters (Continued) Parameter Screens Description Type Name COMP DC STAT, PULSE Toggles between on and off, and Control (Compen- STAT, REMOTE releases the Azero offset. Switches on sation) (DC, PULSE, if Azero is selected. Affects Azero SCAN) compensation only, not the % offset.
  • Page 108 Control • DC mode – File numbers 1 to 5 • Pulse mode – File numbers 6 to 9 The time files remain stored in RAM even after switching off the 2465 Detector. Filt (Filter) DC SETUP, DC Displays filter time constant settings.
  • Page 109 Table 3-2 Status and Control Parameters (Continued) Parameter Screens Description Type Name Offs DC SETUP, DC Controls or displays the percentage Control (Offset) STAT, PROG, offset. Allowed values: –50% to +50% PULSE SETUP1, in 5% steps. PULSE STAT, SCAN SETUP, SCAN STAT Displays the percentage offset during Status execution of a time file.

  • Page 110: Overview Of The 2465 Detector Modes

    None or Present. 3.2 Overview of the 2465 Detector Modes The 2465 Detector can operate as a stand-alone detector or controlled by a data system such as Empower software (remote mode). As a stand-alone detector, it can operate in three modes: DC mode (Section 3.2.1), pulse or PAD mode (Section 3.2.2), and scan mode (Section 3.2.3).

  • Page 111: Dc Mode

    DC STAT Change Polarity? F2 (CELL= YES or NO F3 (RUN) F5 (NEXT) ON/OFF) Switch Cell See A on See B on On/Off? Next Page Next Page YES or NO Figure 3-5 DC Mode Navigation Overview of the 2465 Detector Modes...
  • Page 112 AUTOZERO Delete Overwrite Time=nnn.nn? Time=nnn.nn? YES or NO YES or NO F1 (QUIT) F3 (STOP) (Running) F4 (AZERO) TIME FILE (HOLD) LINE nnn.nn F1 (PREV) F4 (SCROLL) AUTOZERO (RESUME) (Holding) Figure 3-5 DC Mode Navigation (Continued) Operating the 2465 Detector...

  • Page 113: Pulse (Pad) Mode

    EVENTS SETUP Change Polarity? + or – F2 (CELL= F3 (RUN) F5 (NEXT) ON/OFF) Switch Cell See C on See D on On/Off? Next Page Next Page YES or NO Figure 3-6 Pulse Mode Navigation Overview of the 2465 Detector Modes...
  • Page 114 AUTOZERO Delete Overwrite Time=nnn.nn? Time=nnn.nn? YES or NO YES or NO F1 (QUIT) F3 (STOP) (Running) F4 (AZERO) TIME FILE (HOLD) LINE nnn.nn F1 (PREV) F4 (SCROLL) AUTOZERO (RESUME) (Holding) Figure 3-6 Pulse Mode Navigation (Continued) Operating the 2465 Detector...

  • Page 115: Scan Mode

    Figure 3-7 Scan Mode Navigation 3.2.4 Remote Mode The 2465 Detector operates in remote mode when samples are run under the control of an external data system such as Empower software, and an Empower method is setting up and/or running. The 2465 Detector operates in remote mode from the instant that the setup method starts until the time a run ends or is aborted.

  • Page 116: Remote Dc Mode

    When an Empower run is in process, remote mode prevents any use of the front panel keypad of the 2465 Detector. The status parameters are updated during the run. The front panel keypad of the 2465 Detector is available whenever Empower is not setting up a method or performing a run.

  • Page 117: Introduction To Time Files

    A time file contains a series of lines (or rows) in which you can change the events (settings of the 2465 Detector) with a time resolution of 0.01 minute (0.6 second). Time files 1 through 5 are reserved for DC mode, and time files 6 through 9 are reserved for pulse mode.

  • Page 118: Default Time File In Dc Mode

    You can increase the end cycle time. Run Commands (Stand-Alone Detector) When the 2465 Detector is operated as a stand-alone detector (not remotely controlled by Empower), several commands are available in DC, pulse, and scan modes (see Table 3-1, 2465 Detector Commands).

  • Page 119: Run (Waiting) Screen Before Starting A Run

    F2 RESUME. When the run resumes, the cycle counter increments until it reaches the number selected in the timed events file. Once the run and all cycles are completed, the Events Setup screen appears. Overview of the 2465 Detector Modes...

  • Page 120: Programming Output Event Functions In Time Files

    Four output event functions can be programmed at each time. The notation of the output 0000 corresponds to the four output controls at the back panel of the controller. These are relay 1, relay 2, AUX1, and AUX2 (see Section 2.5, Making I/O Signal Connections). Operating the 2465 Detector...

  • Page 121: Preparing The 2465 Detector For Operation

    1000 3.3 Preparing the 2465 Detector for Operation Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents. Refer to the Material Safety Data Sheets for the solvents in use.
  • Page 122 • Passivation with nitric acid is required for the metal parts of the HPLC system (see Section 2.4.1, Installing the 2465 Detector). Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents.

  • Page 123: Preparing The Detector For Remote Control From Empower

    Setting Up the Detector To set up the detector: 1. Connect the 2465 Detector to any available COM port on the Empower PC using a standard RS-232 cable. The COM port configuration is set up automatically by the 2465 Empower Instrument Interface software (see Section 2.6, Verifying COM Port Settings).

  • Page 124: Acquisition Server Dialog Box

    2. Insert the Empower Instrument Control Options Pack CD into the CD-ROM drive. The installation starts automatically and the Welcome page appears. 3. Follow the instructions in the InstallShield Wizard to install the Waters 2465 Software. 4. When the installation is finished, remove the CD from the Empower PC and store it in a protected location.

  • Page 125: Changing From Remote Mode To Stand-Alone Mode

    Detector with Empower software, see Section 3.2.4, Remote Mode. 3.3.2 Changing from Remote Mode to Stand-Alone Mode If you want to use the 2465 Detector in stand-alone mode after control by Empower software (remote mode), Waters strongly recommends that you restart the 2465 Detector (turn the 2465 Detector off, wait 10 seconds, then turn it on).

  • Page 126: Derating Curve For Detector Oven Temperature

    Figure 3-16 Derating Curve for Detector Oven Temperature 3. Select F5 NEXT. The DC Stat screen appears (Figure 3-17). Temperature Setting Actual Temperature Figure 3-17 DC Stat Screen Note: Allow the detector to warm up for approximately 30 minutes. Operating the 2465 Detector...

  • Page 127: Using Dc Mode

    3.4 Using DC Mode You can use DC mode to perform direct current (DC) analyses. After you are comfortable with the 2465 Detector, use Table 3-6 for a quick reference list of tasks in DC mode. Table 3-6 DC Mode Quick Reference List...

  • Page 128: Setting Initial Conditions In Dc Mode

    3. To check the polarity: • If , the polarity is positive, and an oxidation current gives a positive F3 POLAR=+ signal. • If , the polarity is negative, and a reduction current gives a positive F3 POLAR=- signal. Operating the 2465 Detector...

  • Page 129: Change Polarity To Negative Screen

    4. If you want to change the polarity setting, select F3 POLAR. • If the polarity is positive, the Change Polarity to Negative screen appears (Figure 3-19). To change it to negative, select F2 YES. The DC Setup screen appears. Figure 3-19 Change Polarity to Negative Screen •...

  • Page 130: Turning The Flow Cell On And Off In Dc Mode

    6. Following all precautions, turn on the flow cell (see Section 3.4.2, Turning the Flow Cell On and Off in DC Mode), and allow the 2465 Detector to equilibrate. 3.4.2 Turning the Flow Cell On and Off in DC Mode...

  • Page 131: Dc Stat Screen With Cell Off

    Figure 3-22 DC Stat Screen with Cell Off 3. Select F2 CELL=OFF. The Switch Cell On screen appears (Figure 3-23). Figure 3-23 Switch Cell On Screen 4. Select F2 YES. The DC Stat screen appears and the value for F2 CELL is ON. To turn the flow cell off in DC mode: 1.

  • Page 132: Creating A Time File In Dc Mode

    To create timed events in DC mode: 1. From the Main screen, select F2 DC. (Alternatively, from the DC Stat screen, select F1 PREV). The DC Setup Screen appears (Figure 3-25). Figure 3-25 DC Setup Screen Operating the 2465 Detector...

  • Page 133: Prog Screen With Initial Conditions

    Note: Select a cycle value of 1 when an “inject start event in” is connected to pins 13 and 15 of the 2465 Detector (see Section 2.5, Making I/O Signal Connections). d. Select F5 NEXT. The Prog screen appears (Figure 3-27). The number under PROG shows that this time file currently has one line.

  • Page 134: Delete Timefile Screen

    5. Change line 1 in time file 2: a. Verify that Time = 000.00. b. Select AZero, then use either value key to select SET. c. Select F2 ADD. The Overwrite Time 000.00 screen appears (Figure 3-29). Figure 3-29 Overwrite Time Screen Operating the 2465 Detector...

  • Page 135: Prog Screen After Changing Cell Potential

    d. Select F2 YES. The Prog screen appears (Figure 3-30). Figure 3-30 Prog Screen After Changing Line 1 6. Create line 2: a. Select Time. b. Move the cursor to minutes, then change the value to 4. c. Move the cursor to hundredths of a minute, then change the value to 80. d.

  • Page 136: End Cycle Time Screen

    Select F5 ENDCYCLE. The End Cycle Time screen appears (Figure 3-34). Figure 3-34 End Cycle Time Screen b. Increase the end cycle time to 005.00 minutes. c. Select F1 PREV. The Events Setup screen appears (Figure 3-35). Operating the 2465 Detector...

  • Page 137: Running A Time File In Dc Mode

    Figure 3-35 Events Setup Screen with Time File 2 9. Review all four lines in time file 2: a. Select F5 NEXT. The Prog 3 screen appears, and Time = 000.00 minutes. b. Review the values for the first line of time file 2. c.

  • Page 138: Run (Waiting) Screen

    • If Cycles is set to a number from 1 to 999, the Events Setup screen appears at the end of the run. • When you want to return to the Main screen, select F1 QUIT > F1 PREV > F1 PREV. Operating the 2465 Detector...

  • Page 139: Using Pulse (Pad) Mode

    3.5 Using Pulse (PAD) Mode You can use pulse mode to perform pulsed amperometric detection (PAD). After you are comfortable with the 2465 Detector, use Table 3-8 as a quick reference list of tasks in pulse mode. Table 3-8 Pulse Mode Quick Reference List...

  • Page 140: Setting Initial Conditions In Pulse Mode

    Control Parameters): • E – Cell potential settings of the pulse. • t – Duration of potential steps E , and E Note: The value of t will always be at least 60 msec less than t Operating the 2465 Detector...

  • Page 141: Change Polarity To Negative Screen

    4. You can check and change polarity in pulse mode as follows: • If F3 indicates , the polarity is positive, and an oxidation current gives a POLAR=+ positive signal. If F3 indicates , the polarity is negative, and a reduction POLAR=- current gives a positive signal.

  • Page 142: Changing The Range In Pulse Mode

    2. If Range is not selected, move the cursor to select it. 3. Use the value keys to change the range. 4. Select F5 NEXT. The Pulse Setup2 screen appears (Figure 3-39). 5. Select F5 NEXT. The Pulse Stat screen appears (Figure 3-41). Operating the 2465 Detector...

  • Page 143: Making A Chart Mark In Pulse Mode

    2. Select F5 NEXT. The Pulse Setup2 screen appears (Figure 3-39). 3. Select F5 NEXT. The Pulse Stat screen appears (Figure 3-41). 4. Select F4 AZERO. The 2465 Detector sets the output voltage to 0 V or to the offset voltage, and control parameter...

  • Page 144: Pulse Stat Screen With Cell Off

    Figure 3-43 Pulse Stat Screen with Cell Off 4. Select F2 CELL=OFF. The Switch Cell On screen appears (Figure 3-44). Figure 3-44 Switch Cell On Screen 5. Select F2 YES. The Pulse Stat screen appears and the value for F2 CELL is ON. Operating the 2465 Detector...

  • Page 145: Creating A Time File In Pulse Mode

    To turn the flow cell off in pulse mode: 1. From the Main screen, select F3 PULSE. The Pulse Setup1 screen appears. 2. Select F5 NEXT. The Pulse Setup2 screen appears. 3. Select F5 NEXT. The Pulse Stat screen appears and the value for F2 CELL is ON. 4.

  • Page 146: Pulse Setup1 Screen

    4. Ensure that the initial conditions are acceptable for your purpose. At a minimum, select the following initial conditions: a. Time file 6 (File = 6) is the default time file. To program time file 7, select File, then use the value keys to select File = 7. Operating the 2465 Detector...

  • Page 147: Prog Screen With Initial Conditions

    Note: Select a cycle value of 1 when an “inject start event in” is connected to pins 13 and 15 of the 2465 Detector (see Section 2.5, Making I/O Signal Connections). d. Select F5 NEXT. The Prog screen appears (Figure 3-49). The number under PROG shows that this time file currently has one line.

  • Page 148: Overwrite Time Screen

    Select E1, then change the value to +0.65V. The suffix # appears when the cell potential changes. f. Press the Enter key to confirm the change to E . The suffix # disappears. g. Select AZero, then change the value to NOT. Operating the 2465 Detector...
  • Page 149 h. Select F2 ADD. The Prog screen appears (Figure 3-53). Figure 3-53 Prog Screen After Adding Line 2 8. Create line 3: a. For Time, select hundredths of a minute, then increase the value to 90. b. Select AZero, then change the value to SET. c.

  • Page 150: Events Setup Screen

    Select F1 PREV. (If you were not at the end cycle time, the End Cycle Time screen appears. Select F1 PREV.) The Events Setup screen appears. Time file 7 is ready to run (see Section 3.5.7, Running a Time File in Pulse Mode). Operating the 2465 Detector...

  • Page 151: Running A Time File In Pulse Mode

    3.5.7 Running a Time File in Pulse Mode To run a time file in pulse mode: 1. From the Events Setup screen, select F3 RUN. The Run (Waiting) screen appears (Figure 3-57), and the system waits for a start command. This can be a keypad command or an external trigger from connector A on the rear panel (see Section 2.5, Making I/O Signal Connections).

  • Page 152: Using Scan Mode

    3.6 Using Scan Mode You can use scan mode to perform simple scans. After you are comfortable with the 2465 Detector, use Table 3-10 as a quick reference list of tasks in scan mode. Table 3-10 Scan Mode Quick Reference List...

  • Page 153: Turning The Flow Cell On And Off In Scan Mode

    Figure 3-60 Scan Stat Screen 4. You can change the Range and Offs (offset), as needed (see Table 3-2, Status and Control Parameters). 3.6.2 Turning the Flow Cell On and Off in Scan Mode Attention: Before powering on the flow cell, ensure that: STOP •...

  • Page 154: Switch Cell On Screen

    F3 CELL is ON. 2. Select F3 CELL=ON. The Switch Cell Off screen appears (Figure 3-63). Figure 3-63 Switch Cell Off Screen 3. Select F2 YES. The Scan Setup screen appears and the value for F3 CELL is OFF. Operating the 2465 Detector...

  • Page 155: Performing A Scan In Scan Mode

    3.6.3 Performing a Scan in Scan Mode To perform a scan in scan mode: 1. From the Main screen, select F4 SCAN. The Scan Setup screen appears (Figure 3-64). Figure 3-64 Scan Setup Screen 2. You can change E and E (cell voltage), Range, Offs (offset), SPD (scan speed), Cyc (cycle length), and Temp (detector oven temperature), as needed.

  • Page 156: Optimizing The Working Potential

    2. Allow approximately 60 minutes to stabilize the electrochemical detector in DC mode at a high potential, at the selected temperature, with the flow cell off. 3. Read the background current from the display of the detector (I ) and measure the noise. Operating the 2465 Detector...

  • Page 157: Constructing A Hydrodynamic Voltammogram For Norepinephrine

    4. Start the run by injecting the compound, and observe the signal. • If no signal is obtained at a high working potential, the compound is not electrochemically active. Derivatization of the compound may be an option. • If a peak is measured, decrease the working potential by 50 or 100 mV, and repeat steps 2 to 4 until the lowest potential setting is obtained.

  • Page 158: Constructing A Scanning Voltammogram

    Depending on your data acquisition software and experimental setup, you can select a half, full, or continuous scan cycle, as follows: • Cyc=half – A half scan sweeps the potential from low (E = 0.20 V) to high = 1.20 V; see Figure 3-68 and Figure 3-70). Operating the 2465 Detector...

  • Page 159: Overlay Of Four "Half" Forward Scans

    • Cyc=full – A full scan includes the forward and reverse scans, i.e., from low (0.20 V) to high (1.20 V) and back to low. • Cyc=cont – In the continuous scan cycle, the voltage is swept up and down continuously between the low and high potentials, E and E Figure 3-70 shows the scanning voltammograms of 2,4-dimethylphenol (DMP), phenol...

  • Page 160: Chromatogram Of A Sample In Dc Mode

    Change the temperature if needed, and allow the detector to stabilize. c. Select a lower potential (E ) and an upper potential (E d. Set the range (Range) at 5 µA. e. Leave the offset (Offs) at +10%. Operating the 2465 Detector...

  • Page 161: Shutting Down The 2465 Detector

    3.8.1 Turning Off the Flow Cell The 2465 Detector has been developed for continuous operation. If preferred, the flow cell can be turned off at night. To turn off the flow cell, use one of the following options from the Main screen: •...

  • Page 162: Shutting Down For A Short Time

    STOP a saturated KCl solution. Note: If you plan to restart the 2465 Detector after a long shutdown period, and a water/methanol mixture has been in the flow cell, Waters recommends that you polish the electrode before restarting the detector (see Section 4.5, Maintaining the Salt-Bridge Reference Electrode).

  • Page 163: Maintaining The 2465 Detector

    This instrument has a lithium battery. Only qualified service personnel should replace the battery. Dispose of the battery properly as chemical waste. When you perform the maintenance procedures in this chapter on your 2465 Detector, follow these safety considerations. Caution: Untrained personnel should not open the instrument. Removing protective panels on the instrument can result in exposure to potentially dangerous voltages.
  • Page 164 Power off the detector before making any electrical connections. Power off the detector and disconnect the power cord before performing maintenance procedures. Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents.

  • Page 165: Frequency Of Electrode Maintenance

    2465 Detector, consult the documentation or the manufacturer for the applicable instrument or program. If you encounter a problem with the 2465 Detector that you cannot troubleshoot, contact Waters Technical Service at 800 252-4752, U.S. and Canadian customers only. Other...

  • Page 166: Disassembling The Flow Cell

    The flow cell is assembled properly when it arrives. The force on the bolts is preset to 30 Ncm. Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents.

  • Page 167: Disconnecting The Flow Cell

    Flow Cell Cable Spacer Outlet WE Block Inlet Tubing (Top) Block (Bottom) Inlet Tubing Working Electrode Reference Electrode AUX Electrode Figure 4-1 Disconnecting the Flow Cell 4. Slide the flow cell upward or downward out of the clamp. Attention: To avoid damaging the flow cell, do not remove it by pulling it STOP forcefully toward you.

  • Page 168: Cleaning The Working Electrode

    (reduction) reaction products that cause electrochemical changes to the electrode surface. Excessively high currents also may change the electrode surface. Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents.
  • Page 169 To clean the working electrode: 1. For the glassy carbon working electrode only, use electrochemical cleaning (pulsing). In pulse mode, let the potential jump between +1 and –1 V for 10 minutes. Use the following settings: • t = 1000 msec •...

  • Page 170: Maintaining The Isaac Reference Electrode

    When the flow cell is opened to service the working electrode, the reference electrode should also be serviced. Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents.

  • Page 171: Polishing The Isaac Reference Electrode

    Attention: To prevent damage to the electrodes, do not use the same STOP polishing disc for both the ISAAC reference electrode and the working electrode. Note: The polishing kit contains a bottle of diamond slurry and an ISAAC polishing disc specifically for the ISAAC electrode. 4.

  • Page 172: Storing The Isaac Reference Electrode

    3. Dry the flow cell with lint-free tissues and store it dry. 4.5 Maintaining the Salt-Bridge Reference Electrode Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents.

  • Page 173: Inspecting The Salt-Bridge Reference Electrode

    • The salt bridge must allow proper electrical contact with the mobile phase. The higher the leakage through the frit, the better the conductivity. • Because of their extreme compressibility, air bubbles inside or close to the salt bridge will lead to instability of the three-electrode configuration and to changes in conductivity and the ionic equilibrium of the reference electrode.

  • Page 174: Cleaning The Salt-Bridge Reference Electrode

    Salt-Bridge Reference Electrode. 4.5.2 Cleaning the Salt-Bridge Reference Electrode Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents. Refer to the Material Safety Data Sheets for the solvents in use.

  • Page 175: Replacing The Cotton Wool Frit

    Reference Electrode. 4.5.3 Replacing the Cotton Wool Frit Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents. Refer to the Material Safety Data Sheets for the solvents in use.

  • Page 176: Installing The Salt-Bridge Reference Electrode

    7. Continue with Section 4.5.4, Installing the Salt-Bridge Reference Electrode. 4.5.4 Installing the Salt-Bridge Reference Electrode Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents.
  • Page 177 2. Place the small Viton ring over the Ag/AgCl electrode and slowly insert it at an angle of 45° into the salt bridge. Make sure not to enclose an air bubble. 3. Tighten the black swivel of the reference electrode, but do not overtighten the swivel.

  • Page 178: Storing The Salt-Bridge Reference Electrode

    KCl solution to prevent drying out. 4.6 Reassembling the Flow Cell Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents. Refer to the Material Safety Data Sheets for the solvents in use.
  • Page 179 Note: If you use the ISAAC reference electrode, add and equilibrate with chloride ions in the mobile phase before installing the electrode. Waters recommends 2 mM chloride ions. Note: If you use the salt-bridge reference electrode, first connect the inlet and outlet, then fill the cell with mobile phase while keeping the REF hole on top (not the outlet hole).

  • Page 180: Assembling The Flow Cell

    • AUX – Blue wire to the auxiliary electrode 10. Inspect the flow cell for leaks. If you detect a leak, tighten the flow cell bolts by 1/4-turn only. Overtightening the bolts will deform the spacer and leakage will persist. Maintaining the 2465 Detector...

  • Page 181: Replacing The Micro Flow Cell

    You do not need to install the capillary on a new micro flow cell. Use this procedure if you need to replace the capillary in the micro flow cell. Caution: When you handle solvents, change tubing, or operate the 2465 Detector in general, always observe good laboratory practices. Know the physical and chemical properties of the solvents.

  • Page 182: Mounting The Fused Silica Connector

    5. Carefully push the block on the glass plate until the silica capillary is flush with the surface. 6. Fix the fused silica capillary firmly with the fingertight fitting while keeping a slight pressure of the block on the glass plate. Maintaining the 2465 Detector...

  • Page 183: Other Procedures

    • Section 4.8.4, Cleaning the Detector 4.8.1 Replacing Fuses Caution: To avoid electrical shock, power off the 2465 Detector and unplug the power cord before you replace the fuses. The I/O connectors on the rear of the instrument have a risk of electrical shock (Figure 4-13).

  • Page 184: I/O Connector Warning

    To replace the fuses: 1. Power off the 2465 Detector and remove the power cord. 2. Remove the fuse holder from the rear panel of the 2465 Detector (Figure 4-14). Power Switch AC Input Connector Figure 4-14 Removing the Fuse Holder 3.

  • Page 185: Changing A Spacer In The Flow Cell

    0.053 × a. Calculated as area above working electrode (WE area spacer thickness). The 2465 Detector has a post-detection volume of about 1.5 mL. b. The 3-mm WE has an actual diameter of 2.74 mm. c. The 2-mm WE has an actual diameter of 2.00 mm.

  • Page 186: Changing A Column

    Caution: Do not allow flammable and/or toxic solvents to accumulate. Follow a regulated, approved waste disposal program. Never dispose of such products through the municipal sewage system. Attention: To prevent permanent damage to the 2465 Detector, use only water to clean STOP the detector.

  • Page 187: Diagnostics And Troubleshooting

    – Baseline oscillations – Saturation of output • Other physical symptoms such as leaks – see Section 5.4 If there is a problem, contact Waters Technical Service (see Section 4.1.4). Note: The current firmware version appears on the Main screen.

  • Page 188: Error Messages

    Figure 5-1 Diagnostics Screen 5.2.1 Dummy Cell Test A successful dummy cell test confirms that the 2465 Detector functions properly. If the result of the noise measurement with the dummy cell is within specification, the 2465 Detector is excluded as a cause of the problem.

  • Page 189: Dummy Cell

    3. Attach cable ends from the flow cell cable to the dummy cell as in Figure 5-2 (red to red, blue to blue, and black to black). Keep the flow cell in the clamp and keep the large end of the flow cell cable attached to the 2465 Detector. Blue...

  • Page 190: Diagnostics Screen

    Table 5-2 Dummy Cell Test Settings Parameter Setting Cell potential 800 mV Cell current 2.67 +0.05 nA (read-out) Detector oven temperature 30 °C, stable Filter time constant 1.0 sec (or as specified) Range 100 pA/V a. Using these settings, the results of the output voltage (highest noise peak) should be no greater than 20 mV at Integrator Out.

  • Page 191: Stop Flow Test

    8. Measure the noise. The noise generated during the test should be less than 2 pA. 5.2.2 Stop Flow Test The 2465 Detector can detect noise but often is not the cause of the noise. Use the stop flow test to isolate the origin of excessive noise, which can be caused by: •...

  • Page 192: Keyboard Test

    4. Select each key until you have tested all 12 keys. 5. Select F1 PREV twice (rapidly) to return to the Diagnostics screen. 6. If there is a problem, contact Waters Technical Service (see Section 4.1.4). 5.2.4 Display Test Use the display test to verify that all pixels in the 4 × 40 LCD display work correctly.

  • Page 193: Troubleshooting Tables

    5. Use the cursor and value keys to change the contrast (allowed values: 1 to 20, where 20 is the darkest). 6. Select F1 PREV to return to the Main screen. 7. If there is a problem, contact Waters Technical Service (see Section 4.1.4). 5.3 Troubleshooting Tables Use the following tables to troubleshoot the following symptoms: •...

  • Page 194: No Detector Response

    Table 5-3 No Detector Response Possible Cause Remedy No power Check line voltage for local power outage. Ensure that power cord is plugged in correctly. Power switch off Turn the power switch on (at the rear panel). Faulty or blown detector Replace detector fuse.

  • Page 195: Noisy Baseline

    Table 5-5 Noisy Baseline Possible Cause Remedy Salt bridge in reference Refill salt bridge with saturated KCl; add wet KCl crystals. electrode not saturated Air bubble in reference Remove air bubble; continuously degas the mobile phase. electrode or in flow cell Slow temperature Isolate detector cell;...

  • Page 196: Decreased Sensitivity (Low S/N Ratio)

    Table 5-7 Decreased Sensitivity (Low S/N Ratio) Possible Cause Remedy Fouled working electrode Polish the working electrode. by dirty samples Dilute samples. Flow cell potential too low Optimize potential. Contaminated buffer Replace buffer; do not recycle the buffer. (high I Table 5-8 Baseline Oscillations Possible Cause Remedy...

  • Page 197: Physical Symptoms

    Table 5-9 Saturation of Output (Continued) Possible Cause Remedy Damaged working Replace flow cell block. electrode Flow cell incorrectly Check connections of flow cell cable (REF – black, WE – connected red, AUX – blue). Flow cell potential too high Optimize flow cell potential. 5.4 Physical Symptoms Table 5-10 lists other symptoms you may need to diagnose and repair.
  • Page 198 Table 5-10 Physical Symptoms (Continued) Symptom Possible Cause and Remedy No power evident Check cables and connections. (no movement, Check building or lab bench power. fans, or characters Check for blown fuse; replace. in the display) No communication Power off, wait 10 seconds, then power back on to reboot. with Empower Make sure Empower software is set up properly.

  • Page 199: Appendix A 2465 Detector Specifications

    Appendix A 2465 Detector Specifications ® Table A-1 lists general specifications for the Waters 2465 Electrochemical Detector. Table A-1 General Specifications Condition Specification Line frequency 50 to 60 Hz Line voltage 100 to 240 VAC (self-selecting) Operating modes DC, Pulse, and Scan Potential range Between +2.00 and –2.00 V in 10-mV increments...

  • Page 200: Physical Specifications

    Current ranges, recorder Filter (time constants) 0.1 to 5 sec in 1, 2, or 5 steps Noise < 2 pA with a dummy cell using 100 pA range (load of 300 MΩ and 0.5 µF) with 1.0 sec filter 2465 Detector Specifications...

  • Page 201: Pulse Mode

    Table A-8 Flow Cell Specifications Component Specification Design Confined wall-jet design; working volume determined by spacer thickness and WE diameter Spacers 25, 50, or 120 µm WE diameters 2.00 and 2.74 mm (0.75 mm with micro flow cell) 2465 Detector Specifications 165...
  • Page 202 PCTFE, FEP, 316-SS (stainless steel), Viton , silver, silver chloride, gold, and platinum Pressure Maximum 40 psi (2.76 bar, 276 kPa) Flow rate • Standard flow cell: 25 µL/min to 2.0 mL/min • Micro flow cell: 1 to 25 µL/min 2465 Detector Specifications...

  • Page 203: 2465 Detector Components And Spare Parts 167

    A flow cell must be ordered separately. B.1 Flow Cells You can order the flow cells listed in Table B-1 for the 2465 Detector. Note: A flow cell, such as the glassy carbon (GC) WE ISAAC flow cell, must be ordered separately (see Table B-2 through Table B-7).

  • Page 204: Flow Cell, 2-Mm Gc We, Salt-Bridge Reference Electrode

    700001952 Spacer, 25 µm 700001955 10-mL diamond slurry (1 µm) and Material Safety Data Sheet (MSDS) 700001959 KCl solution for salt-bridge REF (50 mL) and MSDS WAT057241 Connector for SAT/IN™ to 2465 analog cable 2465 Detector Components and Spare Parts...

  • Page 205: Flow Cell, 2-Mm Gc We, Isaac Reference Electrode

    PEEK tubing, 0.01 in. (0.3 mm) ID × 11.8 in. (30 cm) 700001950 Fingertight fitting 700001985 Hex key for flow cell assembly 700001954 Polishing disc for WE 700002069 Polishing disc for REF 700001951 Spacer, 120 µm 700001953 Spacer, 50 µm 700001952 Spacer, 25 µm 2465 Detector Components and Spare Parts 169...

  • Page 206: Flow Cell, 3-Mm Au We, Hy-Ref Electrode

    PEEK tubing, 0.02 in. (0.5 mm) ID × 3.28 ft (1 m) 700002104 PEEK tubing, 0.01 in. (0.3 mm) ID × 11.8 in. (30 cm) 700001950 Fingertight fitting 700001985 Hex key for flow cell assembly 2465 Detector Components and Spare Parts...

  • Page 207: Micro Flow Cell, 0.7-Mm Gc We, Salt-Bridge Ref Electrode

    10-mL diamond slurry (1 µm) and Material Safety Data Sheet (MSDS) 700001958 Salt-bridge Ag/AgCl reference electrode 700001959 KCl solution for salt-bridge REF (50 mL) and MSDS 700002103 Capillary flow cell and glass plate for mounting capillary tubing connection 2465 Detector Components and Spare Parts 171...

  • Page 208: Startup Kit Components

    B.2 Startup Kit Components Table B-8 lists the parts included in the Waters 2465 Electrochemical Detector Startup Kit (part number 200002465). Table B-8 Startup Kit Part Number Quantity Component Description 700001004 Fuse 5 × 20, 2.5 AT 250 V Detector fuses...

  • Page 209: Spare Parts

    Spacer, 120 µm 700001952 Spacer, 25 µm 700001953 Spacer, 50 µm 700001954 Polishing disc 700001955 10 mL diamond slurry (1 µm) and MSDS 700001956 Swivel for salt-bridge REF 700001957 Body for salt-bridge REF 2465 Detector Components and Spare Parts 173...
  • Page 210 700002103 Capillary Connection Kit for micro flow cell 700002105 PEEK tubing, 0.02 in. (0.5 mm) ID × 3.28 ft (1 m) 700002104 PEEK tubing, 0.01 in. (0.3 mm) ID × 11.8 in. (30 cm) 2465 Detector Components and Spare Parts...

  • Page 211: Appendix C Sample Ecd Methods

    Appendix C Sample ECD Methods You can adapt methods from extensive literature on electrochemical detection, and apply them to your own samples and mobile phases. Here are some electrochemical methods to get started (Table C-1 through Table C-6). Table C-1 Norepinephrine Parameter Conditions Column...
  • Page 212 Table C-2 Catecholamines (Continued) Parameter Conditions Injection volume 20 µL Temperature 35 ºC Flow cell 2-mm glassy carbon WE, ISAAC reference electrode Spacer 50 µm Mode Cell potential +700 mV Table C-3 Homocysteine Parameter Conditions Column , 3 µm, 4.6 × 100 mm Flow rate 1.0 mL/min Mobile phase...
  • Page 213 Table C-4 8-Hydroxy-2’-deoxyguanosine Parameter Conditions Column , 5 µm, 4.6 × 250 mm Flow rate 1.0 mL/min Mobile phase 50 mM citrate, pH = 3.5, 10% methanol Sample Hydrolyzed DNA Injection volume 50 µL Temperature Ambient Flow cell 3-mm glassy carbon WE, salt-bridge reference electrode Spacer 50 µm Mode...
  • Page 214 Table C-6 Performance Qualification Parameter Conditions Column Symmetry C Flow rate 0.5 mL/min Mobile phase 50 mM acetic acid, 50 mM sodium acetate, 5 mM potassium chloride Sample 0.1 mg/L acetaminophen Injection volume 20 µL Temperature 35 ºC Flow cell 2-mm glassy carbon WE, ISAAC reference electrode Spacer 50 µm...

  • Page 215: Appendix D 2465 Detector Glossary

    Electrolysis current that is determined by the rate of mass transfer current through a thin layer of solvent adding to the electrode surface by viscous drag. Flow cell potential, measured in volts (V). Electrochemical detection or detector. 2465 Detector Glossary 179...
  • Page 216 ® Empower software Waters database software for Windows XP or Windows 2000, which acquires, processes, reports, and manages chromatographic information. Potential of reference electrode. event A programmed change of a parameter in a time file; a timed event Faraday’s constant, 96,500 coulombs/mole of electrons Flow injection analysis mode, where the column is deleted from the flow path (not fluorescence immunoassay).

  • Page 217: Timed Events Mode

    Gas constant. Reference electrode. remote mode An external data system controls the 2465 Detector. Input on the front panel of the detector is not allowed during remote mode. reproducibility The degree of agreement between replicate measured values; usually expressed as a standard deviation (SD) or percent relative standard deviation (%RSD).
  • Page 218 An electronic feedback circuit that compensates for polarization effects at the electrodes. voltammogram Graph of the measurement of a current-voltage (I/E) relationship. Working electrode. working potential Potential of the working electrode relative to that of the reference electrode. 2465 Detector Glossary...
  • Page 219 Index Numerics Alliance Separations Module 56 Autozero command 67 2465 Bootloader program 12 control 70 2465 Detector defined 179 cleaning 150 maximum compensation 11 connections, power 39 damage 39 signal 54 AUX1 signal 54 dimensions 35 AUX2 signal 54 features 5...
  • Page 220 DB-25 8 generating from the 2695 56 output 52 Chassis ground connection 52 RS-232 52 Checksum 64 Contacting Waters Technical Service 39 Cleaning the detector 150 Contrast control 71 . See Concentration limit of detection Control board 8 Column clamps 44...
  • Page 221 description 40 software, remote mode 79 temperature control 74, 89 End cycle time 74, 84, 100 temperature derating curve 90 Endcycle command 68 Developing a method 79 Enter key 65, 67 Diag (Diagnostics) command 68 Environmental specifications 36 Diagnostics, startup 12 Equilibrating the column 47 Diffusion limited current, defined 179 Equinox card 60...
  • Page 222 Full scan 180 Inline degasser 42 Function keys 65, 66, 67 Inline filter 47 Fuses, replacing 148 Input signal 55 Inspecting the 2465 Detector 38 Installing detector, selecting a site 36 Generating waste tubing 46 autozero on inject from the 2695 55...
  • Page 223 Leaking flow cell 159 temperature range 36 Limiting current equation 3 Optimizing Line connector 52 wave forms 26 Linear flow velocity 17 working potential 120 Locating the detector 35 Other equipment, RS-232 connections 60 Out of Range event 54 Outp (Output) control 73 Output connector 52, 55 Main screen 64...
  • Page 224 switch 52 RELAY 1 signal 53 Pressure, maximum 41, 43 RELAY 2 signal 53 Prev (Previous) command 69 Relay output switches 53 Programming a time file Remote mode 60, 63, 80, 181 in DC mode 96 Replacing fuses 148 in pulse mode 109 Reproducibility 181 Protecting columns 47 Reset signal 53...
  • Page 225 Turning off the flow cell, 2695 Separations Module 57 Turning on flow cell 94, 107, 117 , control of potential step E , control of potential step E , control of potential step E Temperature control 12 Unpacking the 2465 Detector 38 Index 189...
  • Page 226 120 creating hydrodynamic 120 creating scanning 122 defined 182 Voltammogram, hydrodynamic 4 , status 74 Waste tubing, installing 46 Waters Technical Service, contacting 39 Working electrode 14 capacitance 28 diameter 16 material 28 size 16 Working potential 15, 182...

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