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: Customer Comments
800-252-4752 or fax 508-872-1990. For other locations worldwide, phone and fax numbers appear in the Waters Local Offices information. Conventional mail Waters Corporation Global Support Services 34 Maple Street Milford, MA 01757 December 16, 2021, 715007395 Ver. 00 Page iii...
Page 4: Intended Use
Intended use The 3465 Electrochemical Detector (ECD) is used in combination with Ultra Performance Liquid Chromatography for the electrochemical detection and quantification of suitable analytes in liquid samples. You can use the instrument for the chromatographic analysis of a wide range of electroactive analytes in the following fields: •...
Page 5: Ism Classification: Ism Group 1 Class B
Do not use the equipment in close proximity to sources of strong electromagnetic radiation (for example, unshielded intentional RF sources). The radiation can interfere with the equipment’s proper operation. Legal manufacturer Waters Corporation 34 Maple Street Milford, MA 01757 Safety considerations Some reagents and samples used with Waters instruments and devices can pose chemical, biological, or radiological hazards (or any combination thereof).
Page 6 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 December 16, 2021, 715007395 Ver. 00...
Page 7: Safety Hazard Symbol Notice
Symbol Definition No pushing Do not connect to an LC system Indicates the maximum load you can place on 10kg that item (for example, 10kg) Serial number Part number, catalog number 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.
Page 8 AC power source, line voltage 100 – 240 VAC. Connect the instrument to a protective earth via a ground socket. Only use the 3465 ECD with appliances and power sources with proper protective grounding to prevent damage through build-up of static electricity. The power source should exhibit minimal power transients and fluctuations.
Page 9 Replace blown fuses with fuses of the proper type and rating as indicated on the rear panel and as noted in the list of accessories and spares. The fuse holder is integrated in the mains connector. Ensure that the instrument is never put in operation with fuses of a different type. This could cause fire.
Page 10 When you analyze biological fluids, you must take all possible precautions and treat all specimens as potentially infectious. To avoid personal contamination with biologically hazardous, toxic, or corrosive materials, and to avoid spreading contamination to uncontaminated surfaces, wear clean, chemical-resistant, powder-free gloves when performing procedures with the 3465 ECD. Ventilation hazard...
Page 11: Safety Advisories
waste” laws also apply in other jurisdictions. In all cases, ensure that a certified electronics recycler processes end-of-life instruments. • Some Waters instruments use batteries, mercury-containing lamps, or other replaceable components during the life span of the instrument. Handle such materials in accordance with local laws governing their processing and safe disposal.
Page 12: Table Of Contents
Table of contents General information......................ii Audience and purpose..........................ii Copyright notice............................ii Trademarks list............................ii Customer comments..........................iii Contacting Waters..........................iii Intended use............................iv EMC considerations..........................iv FCC radiation emissions notice.......................iv Canada spectrum management emissions notice................iv ISM classification: ISM group 1 class B................... v EMC emissions..........................
Page 13 3.1.4 Cleaning..........................40 3.1.5 Replacement of fuses......................40 3.2 Shutting down the system ......................40 4 Detector controller.....................42 4.1 Introduction.............................42 4.2 Overview of 3465 Detector screens....................44 4.3 Parameters.............................46 5 Detection and parameters..................51 5.1 Introduction.............................51 5.2 Three-electrode configuration......................51 5.3 Internal organization........................52 5.4 Dual flow cell control (optional)......................
Page 14 5.5.1 Range........................... 54 5.5.2 Offset............................ 55 5.5.3 Polarity..........................56 5.6 Filter............................... 56 5.6.1 DC mode..........................56 5.6.2 Pulse mode........................... 57 5.6.3 Pulse 2 mode (available in remote control only) ..............57 5.7 Autozero............................57 6 Measurement modes....................59 6.1 DC mode............................59 6.2 Pulse mode............................
Page 15 9.3.1 Hydrodynamic voltammogram....................77 9.3.2 Scanning voltammogram...................... 77 9.4 Optimization using a voltammogram....................78 9.5 Construction of a hydrodynamic voltammogram................79 10 3465 Detector specifications.................. 81 10.1 Environmental, dimensional, weight, and power requirements............ 81 10.2 3465 General..........................82 10.3 3465 DC Mode..........................82 10.4 3465 PULSE mode........................83...
Page 16 12.1.1 Erratic cell current value or offset after current compensation..........93 12.2 Analytical troubleshooting......................93 12.3 Dummy cell test..........................93 12.3.1 External dummy cell......................93 12.3.2 Internal dummy cell......................95 12.4 Stop flow test..........................95 12.4.1 Possible solutions for analytical problems ................. 97 13 Detector accessories.....................100 13.1 Detector accessory kit........................
Page 17 15 SenCell........................123 15.1 The electrochemical flow cell..................... 123 15.1.1 Introduction........................123 15.1.2 Three-electrode configuration................... 123 15.1.3 Working electrode......................124 15.1.4 Detection limit........................125 15.1.5 Cell working volume adjustment..................127 15.2 Reference electrodes......................... 131 15.2.1 ISAAC reference electrode....................131 15.2.2 Salt bridge Ag/AgCl reference electrode................133 15.2.3 HyREF reference electrode....................
Page 18 16.2.3 HyREF reference electrode....................163 16.3 Installation..........................164 16.3.1 VT-03 flow cell with HyREF or ISAAC................164 16.3.2 VT-03 flow cell with salt bridge REF................. 165 16.3.3 VT-03 micro flow cell......................168 16.4 Maintenance ..........................175 16.4.1 HyREF..........................175 16.4.2 ISAAC..........................175 16.4.3 Ag/AgCl salt bridge......................176 16.4.4 Working electrode......................
Page 19: Introduction
1 Introduction Congratulations on your purchase of the 3465 Detector. This detector enables you to perform all LC/UPLC applications using electrochemical detection. The 3465 Detector includes a highly stable Faraday-shielded oven compartment accommodating column and flow cell. The flow cell provides an unsurpassed S/N ratio for extremely sensitive electrochemical analyses.
Page 20: Instrument Description
1.1 Instrument description Figure 1–1: 3465 Detector - Front side Instrument housing LC tubing inlet/outlet Instrument door panel 4 x 40 Ch LCD display Function keys <Enter> key December 16, 2021, 715007395 Ver. 00 Page 20...
Page 21 "+" and "-" value keys Cursor keys Door handle (for opening door) Figure 1–2: 3465 Detector - Back side Instrument rear panel Digital I/O connector (25-p sub-D fem) Analog data (9-pin sub-D fem) Valve connector (9-pin sub-D male) LAN connector (RJ45 jack) USB connector (USB B) Type label (PN, SN, etc.)
Page 22 Fuse and power rating Grounding stud Mains switch/inlet Ventilation holes Fuse compartment Figure 1–3: 3465 Detector - Oven compartment Door panel, rear Type label Door sensor Door lock Drain Mounting hole (column clamp) December 16, 2021, 715007395 Ver. 00 Page 22...
Page 23 Bottom fan heater (exhaust) Mounting hole (cell clamp) Flow cell clamp Column clamp Mounting plate Top fan heater (intake) Cell connector (9-pin sub-D) Cell cabinet December 16, 2021, 715007395 Ver. 00 Page 23...
Page 24: Installation
2 Installation 2.1 Storage requirements The 3465 Detector is shipped to your facility in one box with the following dimensions: Parameter Requirement Height 44.0 cm (17.3 inches) Width 22.0 cm (8.7 inches) Depth 43.0 cm (16.9 inches) Ensure that you have sufficient space to store the packed instrument under the following storage...
Page 25: Laboratory Requirements
• Exhaustive scanning by virus scanners. (In your antivirus software, turn off the option Check Files at Change for the relevant data storage directory.) The computer should be placed in the vicinity of the 3465 Detector, within a maximum distance of 2.5 meters.
Page 26: Electrical And Power Requirements
Contact your local distributor with any questions. 4. The maximum power consumption of the 3465 Detector on full power is < 200 W. The typical power consumption is < 50 W.
Page 27: Chemicals
To unpack the detector, lift it from its box by both hands. Never lift the detector by its front door; lift it by the sides. Flow cells are not part of the 3465 Detector and must be ordered separately. December 16, 2021, 715007395 Ver. 00...
Page 28 With both hands under the instrument, lift the detector and bring it to its operation location. Install the detector in an area that meets the environmental conditions. Figure 2–2: Location of ventilation holes in the 3465 Detector (rear) Remove the protective tape from the detector LCD screen. Leave the instrument to adopt ambient temperature for at least half an hour in the place of installation.
Page 29: Main Power Connection
The instrument is delivered with a special crossover LAN (UTP) cable (Waters PN: 700013076), which is part of the 3465 Detector Startup Kit, part number 200000485 for a single flow cell unit and 200000492 for a dual flow cell unit (Waters PN 200000492).
Page 30: Software
8. Connect the other end of the crossover LAN (UTP) cable to the LAN port on the rear panel of the 3465 Detector. 9. Power-on the 3465 Detector. Set the detector temperature to 35 °C if an Operational Qualification (OQ) will be executed, or set it to the temperature at which your ECD application is running.
Page 31: Dialogue Elite
Figure 2–4: Select devices menu • When a 3465 Detector is available, it is automatically detected, and the IP address appears in the port settings box. If it does not, press port scan or type in the default IP address 192.168.0.65.
Page 32: Fluidics Connections
With respect to third-party UPLC equipment, such as LC pumps, auto samplers, injection valves, and column heaters used in combination with the 3465 Detector, the equipment connected to the system should be designed specifically for use in Ultra High Performance Liquid Chromatography and capable of delivering flow rates in the range of 1 µL/min to 2 mL/min.
Page 33 Essentially, when you create a connection, the ferrule on the tubing is compressed into the valve to create a leak-tight seal. Take the following into account when creating the connection: If L is too long, the ferrule cannot form a seal in the connection. This may cause irreparable damage to the port of the valve, column, or other part: •...
Page 34: Mobile Phase
Every ferrule type needs an appropriate length of tubing for connecting it to the connection port, depending on the depth of the connection port. Refer to the information provided by the manufacturer. 2.7.2 Mobile phase Electrochemical detection is a sensitive detection technique characterized by extremely low detection limits.
Page 35: Installation And Startup
2.7.3 Installation and startup For a successful installation and startup, follow the next steps carefully: 1. See the Installation of flow cell and column in the 3465 Detector figure. Figure 2–7: Installation of flow cell and column in the 3465 Detector 2.
Page 36 Therefore, the use of an in-line degasser is strongly recommended. A one-time degassing step of the HPLC buffer is almost never sufficient. If the 3465 Detector is used for reductive ECD (at a negative working potential), additional steps should be taken to remove oxygen from the mobile phase.
Page 37 Figure 2–8: SenCell mounted at an angle of approximately 45° in the detector Cell clamp Cell outlet (tubing connection from cell to waste); ensure that the outlet is positioned on the top side to prevent entrapped air bubbles Cell Inlet (tubing connection from column to cell) WE contact (red) AUX contact (blue) REF contact (black)
Page 38 11. Connect the 3465 Detector to the PC control software (Empower 3). Your system is now ready for use. The 3465 Detector has been developed for continuous operation. For maximum stability, it is recommended that you leave the system ON continuously.
Page 39: Maintenance And Shutdown
3.1.2 Periodic check of the oven temperature The operator should perform regular checks to verify if the actual oven temperature is in accordance with the set temperature of the 3465 Detector. Warning: If the actual temperature exceeds 70 °C, switch off the detector immediately and contact Waters or the local representatives for service.
Page 40: Cleaning
3.1.4 Cleaning In general, the 3465 Detector needs very little maintenance. The outside of the detector may be cleaned with a non-aggressive cleaning liquid. Notice: Do not use organic solvents to clean the exterior of the detector, because this may damage the paint layer.
Page 41 4. Flush and store the system with 50:50 water/acetonitrile (or methanol). Switch the injector valve between load and inject a few times. Ensure that all tubing and filters are flushed so no traces of salt are left that could precipitate and clog the system. 5.
Page 42: Detector Controller
4 Detector controller 4.1 Introduction The 3465 Detector has been designed for maximum functionality and ease of use. The control of ECD parameters via the keyboard and LCD display (3465 Detector only) is such that, without reading this chapter, it should be possible to operate the detector. This chapter is intended as a reference guide in case questions arise during operation.
Page 43 Figure 4–1: 3465 Detector keyboard. The cursor is on Range” which allows changes using the value buttons + and -. The Enter button is used only to confirm changes in potential (Ec) and range. December 16, 2021, 715007395 Ver. 00...
Page 44: Overview Of 3465 Detector Screens
4.2 Overview of 3465 Detector screens Figure 4–2: DC mode Figure 4–3: Pulse mode December 16, 2021, 715007395 Ver. 00 Page 44...
Page 45 Figure 4–4: Scan mode Figure 4–5: CONFIG menu December 16, 2021, 715007395 Ver. 00 Page 45...
Page 46: Parameters
Figure 4–6: DIAG menu 4.3 Parameters Explanation: Type S is status, F is function, and C is control. Parameter Screen Description Type 28 > 30 ˚C DC STAT, PULSE STAT, Displays the actual (left-hand value) and SCAN STAT, RUN the preset oven temperature (right-hand value).
Page 47 Parameter Screen Description Type compensation will be applied, which may be higher than the 0 Volt level. Boot SYSTEM Displays boot firmware version. CELL=ON/ DC STAT, PULSE STAT, Toggles between cell ON and OFF. SCAN SETUP, SCAN STAT Confirmation is required—"switch cell on (off)?”...
Page 48 Parameter Screen Description Type Filt (DC mode) DC SETUP, DC STAT Filter settings: RAW (100 Hz), Off (10 Hz), and 1 Hz to 0.001 Hz cutoff frequency, in 1, 2, 5 steps. Filt (PULSE PULSE SETUP, PULSE Filter settings: Off and 0.5 Hz to 0.001 Hz mode) STAT cutoff frequency, in 1, 2, 5 steps.
Page 49 Parameter Screen Description Type Offs DCS SETUP, DC STAT, Percentage offset; can be set between -50 PROG, PULSE and +50%. SETUP1 ,PULSE STAT, SCAN SETUP, SCAN STAT POLAR DC SETUP, PULSE Inverts output polarity; toggle between + SETUP2 and -. Requires confirmation. PREV SEVERAL SCREENS Return to previous screen.
Page 50 Parameter Screen Description Type t1, t2, t3, t4, t5 PULSE SETUP2, PULSE Duration of potential step E1, E2, E3, E4, SETUP3 and E5. Time can be set between 0 (t2 – t5) or 100 (t1) and 2000 ms in 10-ms increments.
Page 51: Detection And Parameters
The 3465 Detector covers a wide range—from 200 µA down to 10 pA full scale—without being limited by electronic noise. For this reason, the 3465 Detector is equipped with a 24-bit ADC and a 16-bit DAC for analog data output.
Page 52: Internal Organization
CONFIG menu by setting the parameter Output = I/E. 5.4 Dual flow cell control (optional) The 3465 Detector electronics are located on two different PCBs (printed circuit boards)—the control board and the sensor board. The control board is dedicated to communication with the PC (LAN), keyboard, and display.
Page 53: Serial Mode Detection
Column 1 Cell 1 Cell 2 Empower instrument 1 Channel 2 Empower instrument 1 Channel 1 3465 Detector Empower Chromatography with Software Dual Cell Control 5.4.2 Navigation in dual cell menu All menus for a dual flow cell system are similar to those for a single cell system, with two exceptions.
Page 54: Parameters
This is an inevitable consequence of the tremendous dynamic range that is covered by electrochemical detection. The information listed in the following tables is valid for 3465 Detectors with FW version 1.09 or higher.
Page 55: Offset
Table 5–1: Table 2. DC ranges and maximum compensation Range Max comp Range Max comp 200 µA 2.5 mA 20 nA 2.5 µA 100 µA 2.5 mA 10 nA 2.5 µA 50 µA 2.5 mA 5 nA 250 nA 20 µA 250 µA 2 nA 250 nA...
Page 56: Polarity
Figure 5–6: Signal to noise ratio is improved using a filter (A vs. B) The 3465 Detector is equipped with ADF (Advanced Digital Filter) as a tool to filter the acquired signal and improve the sensitivity (signal-to-noise ratio) of the analysis. The next chapter explains the filter setting, including detailed background information about filtering.
Page 57: Pulse Mode
Table 5–3: Table 4. DC mode filter setting and corresponding data rate (continued) Filter setting DC mode (Hz) Data rate (Hz) 0.05 0.02 0.01, 0.005, 0.002, 0.001 Filter OFF is also a special case. The data rate is fixed at 10 Hz, and the data is not filtered. Setting OFF is therefore the same as RAW at 10 Hz.
Page 58 compensation circuitry makes it possible to measure small current signals (analyte peaks) in a sensitive range even if the background cell current is high. For example, with a background current of 20 nA, you can still do measurements in the 1 nA range, because the maximum compensation for that range is 25 nA.
Page 59: Measurement Modes
6 Measurement modes 6.1 DC mode In Direct Current (DC) mode, a static potential is constantly applied to the EC flow cell to establish an electrochemical oxidation or reduction reaction. The resulting current signal is continuously measured and sent to the detector output. Figure 6–1: Plot of cell potential versus time Figure 6–2: Empower Instrument Method window: DC mode settings DC mode can be used for detection using relatively inert working electrode (WE) materials such...
Page 60: Pulse Mode
6.2 Pulse mode The 3465 Detector can also be operated in Pulse mode. Pulse mode is different from DC mode. Instead of a constant potential, a series of potential steps is applied in a cyclic manner. The signal is sampled during a fraction of the total pulse cycle. During the sampling time (ts) the signal is collected, and this value is sent to detector output.
Page 61: Pulse Mode 2
72). 6.3 Pulse mode 2 The 3465 Detector (FW 1.09 and up) offers an extended pulse mode, pulse mode 2. In pulse mode 2 it is possible to program a multi-step waveform with up to 30 time-potential (t,E) coordinates and a maximum pulse duration of 4 seconds (see the following figure).
Page 62: Scan Mode
Figure 6–6: The new pulse mode 2 with freely programmable t,E table; in red, the sampling of data for acquisition The measurement time interval in which the current is measured, marked by Begin and End markers, is freely programmable in the pulse table. This new pulse mode extends the application areas of the detector to analysis using PAD detection with more sophisticated potential waveforms, as in amino acid analysis.
Page 63 Figure 6–7: Scan mode example: plot of cell potential versus time (two full scans) Note: Scan mode can be used in method development, but it is not a measurement mode used in HPLC-ECD analysis itself. It is available only in stand-alone operation. Scan mode is not available in any CDS.
Page 64: Noise Suppression - Adf
In addition to its tremendous linear dynamic range and selectivity, electrochemical detection is well-known for its very low limits of detection. To further improve these detection limits, the 3465 Detector is equipped with ADF (Advanced Digital Filter). The improvement factor in signal-to- noise (S/N) ratio depends on the frequency relation of signal and baseline noise.
Page 65: Frequency Of Signal And Noise
Figure 7–2: A full period is 0.41 min (25 s), which corresponds to a frequency of 1/25 = 0.04 7.2.1 Frequency of signal and noise A chromatographic peak can also be expressed in terms of frequencies. The way to determine this frequency is the same.
Page 66 Figure 7–4: Overlay of a chromatographic peak with 0.07 Hz sine Narrow chromatographic peaks are typically in front of a chromatogram, while peaks with longer retention times get wider. As a consequence, frequencies are not constant but vary between 0.1 and 0.01 Hz, which corresponds to 10 –...
Page 67: Low Pass Noise Filters
Figure 7–6: Typical random noise in chromatography (lower trace) Looking closely at the lower noise trace, you can recognize both frequencies (and others). This is typical of noise in chromatography: a collection of more or less random frequencies. 7.2.2 Low pass noise filters Noise filters work by suppressing certain frequencies in the acquired signal.
Page 68: Amplitude Response Plot
In contrast to the previous equation, each data point has a different weighting factor (a). The sum of these weighting factors (a0…n) will always be 1. Characteristic of noise filters is that processing the signal results in a delay. This is inevitable; the mathematics of digital signal processing requires a number of previous data points to process a new data point.
Page 69: Applying Adf In Chromatography
Figure 7–9: Analog 6-pole Bessel filter A digital filter does not have poles, but it is characterized by the number of input data points used to calculate a new output data point. For example, a 9-point digital filter (Savitzky-Golay) is given Note that the sum of coefficients is exactly 1.
Page 70 The 3465 Detector has a number of filter settings to optimize for best possible signal-to-noise ratio. The width of the peaks of interest is important because wider peaks allow stronger filter settings simply because of the lower frequency of such peaks.
Page 71 Because suppressing noise will always result in some suppression of signal, it is best to switch the 3465 Detector to the highest acceptable sensitivity. December 16, 2021, 715007395 Ver. 00...
Page 72: Pulsed Amperometric Detection (Pad)
8 Pulsed amperometric detection (PAD) 8.1 Introduction The 3465 Detector can operate in pulsed amperometric detection (PAD) mode, in which the working electrode (WE) is regenerated at a frequency of 0.5 to 3 Hz by the application of a series of potential changes.
Page 73: High Ph Of Mobile Phase
(marked by “Begin” and “End” markers) is freely programmable in a pulse table. This new pulse mode extends the application areas of the 3465 Detector to analysis using PAD detection with more sophisticated potential waveforms, such as that used in amino acid analysis.
Page 74: Optimization Of Wave Forms
Figure 8–2: Potential steps in pulsed amperometric detection The detection potential is applied during t1, and detection occurs during ts. Steps t2, t3, and t4 are for regenerating the electrode. This process repeats itself continuously when the cell is on. During the next time intervals (t2 through t4), the electrode is “cleaned”...
Page 75: Working Electrode Material
Figure 8–3: A detailed part of a chromatogram acquired at different data frequencies The data rate is (A) 5x, (B) 2.5x, (C) 1.2x, (D) 0.6x, and (E) 0.3x the frequency of the pulse. C is 1 Hz data rate. 8.1.6 Working electrode material Gold and platinum are used as working electrodes for PAD.
Page 76: Optimization Of The Working Potential
LC conditions). Furthermore, under real chromatographic conditions, reliable information about the S/N ratio is obtained. • A scanning voltammogram is obtained in the “scan” mode of the 3465 Detector: the voltage runs between two preset potential values (E1 and E2) and scan speed (in mV/s), and the current is measured.
Page 77: Hydrodynamic And Scanning Voltammograms
9.3 Hydrodynamic and scanning voltammograms 9.3.1 Hydrodynamic voltammogram A hydrodynamic voltammogram is constructed when the pure analyte is not available and separation over an analytical column is required. Under real chromatographic conditions, reliable information about the S/N ratio is obtained. The peak heights obtained from the sequence of chromatograms are plotted against the working potential used.
Page 78: Optimization Using A Voltammogram
The current is plotted against the working potential to give a voltammogram (I/E curve), as shown in the scanning voltammetry potential waveform figure. In scanning voltammetry, no HPLC separation is involved. The signal is the sum of all EC active substances.
Page 79: Construction Of A Hydrodynamic Voltammogram
In the example, the selectivity for compound X is improved considerably by decreasing the potential to E or E . If compound Y is the compound of interest, optimization of selectivity in this way is not possible and the chromatography must be optimized. Electrochemical detection differs from most other LC detection methods in that a reaction takes place in the detection cell.
Page 80 1. A solution of the analyte at a concentration between 1 and 100 µmol/L is prepared in mobile phase. 2. The electrochemical detector is stabilized in DC mode at a high potential. After stabilization, the background current is read from the display of the detector (I-cell), and the noise is measured.
Page 81: 3465 Detector Specifications
10 3465 Detector specifications The following pages outline the 3465 Detector specifications. 10.1 Environmental, dimensional, weight, and power requirements Working temperature 10 to 35 °C (indoor use only) Storage temperature -25 to +50 °C Humidity 20% to 80% RH Safety and EMC...
Page 82: 3465 General
Valve control VICI Valco 2-pos electrically actuated valve (E2CA, EHCA) via serial cable, manual valve, 1x inject marker output 10.3 3465 DC Mode Range 10 pA - 200 µA in 1, 2, 5 increments Filter (ADF) RAW (100 Hz), OFF (10 Hz), 10 - 0.001 Hz in...
Page 83: 3465 Pulse Mode
100 ms - 2000 ms; t2, t3, t4, t5: 0 - 2000 ms in 10-ms increments Sampling times (ts) 20 ms - [t1 - 60] ms 10.5 3465 PULSE mode 2 Range 10 nA - 200 µA in 1, 2, 5 increments Filter (ADF) 0.5 - 0.01 Hz in 1, 2, 5 increments...
Page 84 Potential (Ec) -2.50 V to +2.50 V in 10-mV increments Data rate 1 Hz Scan rate 1 - 100 mV/s in 1, 2, 5 increments Cycle Half, Full, Continuous December 16, 2021, 715007395 Ver. 00 Page 84...
Page 85: Rear Panel I/O
11 Rear panel I/O This chapter describes all rear panel functionality. Besides the main power inlet, the 3465 Detector has five connectors on the rear panel for communication, data output, and I/O. Figure 11–1: 3465 Detector rear panel 11.1 USB B connector USB type B connector for serial instrument control over USB;...
Page 86: Lan Connector
11.3.2 Relays The 3465 Detector has two freely programmable contact closure outputs: • Relay1: pin 1 normally closed, pin 2 normally open, pin 3 common • Relay2: pin 4 normally closed, pin 5 normally open, pin 6 common The maximum rating for these contact closure outputs is 24 VDC (switching voltage) and 0.25 A.
Page 87: Aux
1 is out of range does the status of the overload output change to “low” 0V. For all other cells present in the 3465 Detector, an “out of range” situation does not trigger a response on pin 11. ...
Page 88: Cell On Cell Off
(see the previous chapter about overload output). The configuration settings of this input are 1, 2, 3, 4, 5, ' ', and all. If "all" is selected, the cell current of all cells present in the 3465 Detector are zeroed when the input is triggered.
Page 89: Chassis Grounding Stud
Figure 11–4: Digital I/O board and cable 11.4 Chassis grounding stud A chassis grounding stud is available on the rear panel in the lower-right, next to the ventilation holes of the power supply compartment. This grounding stud is connected to the central grounding point of the instrument and can be used for shielding purposes;...
Page 90 Figure 11–5: Left: 3465 Detector rear panel grounding stud. Right: example of shielding the flow cell by grounding the solvent outlet tubing Note: Use the chassis grounding stud for shielding only, not for safety grounding. December 16, 2021, 715007395 Ver. 00...
Page 91: Troubleshooting
12.1 Instrument errors Incidental fault conditions may occur in any instrument. The 3465 Detector generates an error message containing an error number with a short description for several hardware fault conditions. The error messages appear in the instrument's LCD display.
Page 92 Table 12–2: No detector response Possible cause Remedy No power Check line voltage setting, plug in power cord Power switch off Turn this switch ON (at the rear panel) Faulty fuse Replace fuse Divergent mains voltage Verify line voltage Warning: To avoid electric shock, connect the instrument to a grounded power source with a line voltage within the specified ratings.
Page 93: Erratic Cell Current Value Or Offset After Current Compensation
The first step is to determine if the problem is caused by the 3465 Detector or something else in the UPLC system. For that purpose, two basic checks should be performed: •...
Page 94 standalone, via the LCD display in combination with an A/D converter, or Empower software. A successful dummy cell test confirms that the controller, including the cell cable, functions properly. If the result of the noise measurement with the dummy cell is within specs, the controller is excluded in a troubleshooting procedure.
Page 95: Internal Dummy Cell
12.3.2 Internal dummy cell The 3465 Detector also affords the option to run an "internal dummy cell" test. This checks the performance of the electronic circuit boards (amplifier circuitry) only, so it excludes the cell cables and the external dummy flow cell. From the MAIN screen, select DIAG to enter the DIAG screen, and then select NOISE.
Page 96 4. Connect the other end of the tubing (the tubing connected to the inlet of the flow cell) to the outlet of the flow cell. 5. The fluidics path of the flow cell is now completely isolated from the rest of the LC system. 6.
Page 97: Possible Solutions For Analytical Problems
Figure 12–7: Noise-Stop Flow Note: If no significant drop in noise or cell current is observed, service or replace the cell. If you still cannot solve the problem, contact your local representative. 12.4.1 Possible solutions for analytical problems Bear in mind that analytical problems may also be caused by external influences like temperature or unstable samples.
Page 98 Table 12–6: High cell current Possible cause Remedy Contaminated buffer Replace buffer, do not recycle the buffer High WE potential Optimize potential, if possible: use smaller WE diameter Salt bridge in REF not saturated Refill with wetted KCl crystals Retained peaks from previous runs Wait for elution of these (very) broad peaks Column is “bleeding”...
Page 99 Table 12–10: Base line oscillations Possible cause Remedy Malfunctioning pump (regular pattern) Check pump (seals, valves) Over-tightened cell bolts Adjust cell bolts, check pump pressure Air bubbles in cell or REF Maintenance REF Temperature oscillations Set oven temperature Contaminated buffer (high Icell) Replace buffer, do not recycle the buffer Fouled WE Clean WE...
Page 100: Detector Accessories
700013078 Cell cable a. For Dual Cell ECD the quantity of 700001943, 700013160, 700013078 is 2. For these and other 3465 Detector parts or flow cells, see the Graphical Part Locator at www.waters.com/wqp. 13.2 Optional AD converter cables For several types of ADC converters in third-party (U)HLPC systems, there are dedicated output cables available.
Page 101 Figure 13–1: Part 700013073 - 3465 Detector SCC output cable D9 December 16, 2021, 715007395 Ver. 00 Page 101...
Page 102: Flexcell
14 FlexCell 14.1 The FlexCell 14.1.1 Introduction The FlexCell has been developed for analysis in standard and microbore LC-EC with an effective volume of only 0.5 µL. A range of different working electrode materials are available for the FlexCell and they are easily exchangeable, offering maximum flexibility for running various applications.
Page 103: The Three-Electrode Configuration
14.1.2 The three-electrode configuration A three-electrode configuration is used in the FlexCell (Figure 14–2: Schematic representation of an electrochemical cell with a three-electrode configuration (Page 103)). The working potential is set between the working electrode (WE) and the auxiliary electrode (AUX). The auxiliary electrode is kept at the same precisely defined potential as the reference electrode (REF) by means of a “voltage clamp”, an electronic feedback circuit that compensates for polarization effects at the electrodes.
Page 104: Requirements And Limitations
14.1.3 Requirements and limitations 14.1.3.1 Ions in running solution For the three-electrode configuration to work, the solution inside the flow cell should have a low electrical resistance. This is obtained by running solution through the flow cell that contains at least 10 mM ions.
Page 105: Reference Electrode
and noise. At negative potentials, the use of platinum electrodes is severely limited by the specific ease of reducing hydrogen ions to hydrogen gas. For metal electrodes, the formation of metal oxides is a limiting factor for running oxidative measurements. Table 14–1: Some features of different working electrode (WE) materials Limits of working potential vs.
Page 106 Notice: It is important to recognize that if the pH of the mobile phase is changed, the optimum working potential also changes. In such cases it is best to construct a hydrodynamic voltammogram to find the new optimum. 14.1.5.1.1 pH-dependent reference potential When comparing the reference potential of a HyREF with that of a salt bridge (Ag/AgCl), a pH- dependent difference can be observed.
Page 107 Figure 14–4: Magnified schematic representation of the salt bridge Ag/AgCl reference electrode AgCl KCl saturated KCl crystals Cotton wool Three aspects determine the proper function of an Ag/AgCl reference electrode. December 16, 2021, 715007395 Ver. 00 Page 107...
Page 108 1. The chloride concentration must be kept at a strictly fixed level. This is best guaranteed by using a saturated chloride salt solution at a constant temperature. 2. Absence of air bubbles inside or close to the salt bridge gives the best stability of the three- electrode configuration.
Page 109: General Precautions
Table 14–2: Potential of the Ag/AgCl reference electrode (mmol/L) (mV) dE (mV) Ag/AgCl 3500 2500 1500 a. Rounded to the nearest whole number A salt bridge Ag/AgCl reference is loaded with saturated KCl and the ISAAC is in contact with the concentration of chloride added to the mobile phase.
Page 110: Installation
2. Always make sure that the surfaces of the spacer and working electrode are dry and free from particulate matter before assembling the cell. 3. Clean fingerprints from spacer and electrode surfaces with a soft tissue soaked in acetone or methanol. If the auxiliary electrode needs to be cleaned, do not apply force, which could damage the electrode surface.
Page 111: Connecting A Flexcell To An Lc System
14.3.1 Connecting a FlexCell to an LC system To connect a FlexCell to an LC system: 1. Install a suitable length of sharply cut 1/16-inch-OD PEEK tubing to the column outlet. Choose a tubing ID that matches the column ID: •...
Page 112: Maintenance
Figure 14–6: Flow cell with cell cable connected. WORK, AUX, and REF electrodes are connected using the red, blue, and black leads of the cell cable, respectively 14.4 Maintenance Maintenance of the working electrode is necessary if the electrode surface has been electrochemically changed.
Page 113 Figure 14–7: Electrode assembly with a BDD WE The working electrode is fitted on the electrode shaft with electrode retaining ring Held in place by a silicon electrode holder Figure 14–8: Exploded view of FlexCell Auxiliary electrode inlet block Retaining ring Working electrode disk Electrode swivel nut December 16, 2021, 715007395 Ver.
Page 114: Working Electrode Cleaning
Working electrode assembly 50-μm spacer Reference electrode 14.4.2 Working electrode cleaning A typical reason to perform maintenance on the working electrode is a drop in flow cell performance. In most cases, a simple cleaning is sufficient to remove the chemical deposition that is causing the problem.
Page 115: Hyref And Isaac Reference Electrodes
type, there is a dedicated flattening and polishing kit available (not provided with a FlexCell). Ordering information: Flattening/polishing kit for metal WE (250.1045). The flattening/polishing kit is a tailor-made kit for a three-stage flattening-polishing procedure: 1. Flattening step on coarse plate with 30-µm coarseness 2.
Page 116: Salt Bridge Ag/Agcl Reference Electrode Check
Figure 14–9: Salt bridge Ag/AgCl reference electrode, assembled (bottom) and disassembled (top) sb REF swivel (700001956) sb REF Ag/AgCl electrode (700002254) sb REF silicon O-ring small (700002175 sb REF body (700001957) sb REF silicon O-ring large (700002175 sb REF sealing cap a.
Page 117: Refilling A Salt Bridge Ag/Agcl Reference Electrode
1. Noise in the system will slowly but continuously increase. 2. Background current will increase. 3. Sensitivity to movements and pump noise will increase. Visually inspect for the absence of air bubbles inside the REF If an air bubble is trapped in the salt bridge or in the cotton plug that separates the reference electrode from the mobile phase, the flow cell becomes extremely sensitive to flow fluctuations and vibrations.
Page 118: Refritting A Salt Bridge Ag/Agcl Reference Electrode
10. Cap the salt bridge until use to prevent drying of the cotton frit. Note: If the cotton frit is dried out or discolored, it must be replaced. 14.4.9 Refritting a salt bridge Ag/AgCl reference electrode If the cotton frit of the salt bridge Ag/AgCl electrode needs replacement, the following materials are necessary.
Page 119: Assembly Of The Flow Cell
14.4.9.2 Inserting a new cotton wool frit To insert a new cotton wool frit: 1. Place the salt bridge with the frit hole downward on a glass plate and add a few drops of saturated KCl solution. 2. Pull a small plug of wetted cotton wool into a thin string (using two tweezers, for example). 3.
Page 120: Specifications
Figure 14–11: Assembling the flow cell Working electrode assembly Electrode swivel nut Retaining ring 50-μm spacer First fit the spacer and the retaining ring, and then insert the WE assembly and secure it with the electrode swivel nut. If the flow cell is not in use and uncoupled from the LC system, we recommend that you disassemble the cell and clean and dry all surfaces.
Page 121: Parts List
Max. back pressure in cell 40 psi (2.8 bar) Fluidics connections 1/16-inch o.d. PEEK tubing, with 10-32 PTCFE fingertight connectors Electric connections Cell cable for use with 3465 Detector 14.6 Parts list Table 14–4: Spare parts for the FlexCell Part number Description FlexCell parts...
Page 122 Table 14–4: Spare parts for the FlexCell (continued) Part number Description 700013121 WE disk BDD Parts for polishing/flattening 700001954 Polishing disk (for WE) 700001955 10 mL diamond slurry, 1 µm 700013150 Flattening/polishing kit for metal WE Parts for reference electrode maintenance 700013253 30 mL KCl solution saturated with AgCl 700013145...
Page 123: Sencell
15 SenCell 15.1 The electrochemical flow cell 15.1.1 Introduction The SenCell, a new electrochemical flow cell for (U)HPLC with ECD, has several unique features, such as a stepless adjustable working volume (spacerless concept) and toolless assembly. The SenCell is available with a glassy carbon working electrode (WE). The SenCell design eliminates the use of plastic or metal spacers.
Page 124: Working Electrode
reference electrode (REF) potential by means a “voltage clamp”, an electronic feedback circuit that compensates for polarization effects at the electrodes. At the WE, which is kept at virtual ground, the electrochemical reaction takes place (electrons are transferred at the WE). This results in an electrical current to the I/E converter, which is a special type of operational amplifier.
Page 125: Detection Limit
choice. The SenCell is currently available only with a glassy carbon electrode of 2-mm diameter. Under certain circumstances, other materials are favorable. For example, for the analysis of iodide, a silver WE can be used. At the silver WE, the following oxidation reaction occurs for iodide: Ag + I →...
Page 126 Table 15–2: LC-EC conditions for analysis of norepinephrine (continued) Parameter Description Mobile phase 50 mM, citric acid 50 mM, 20 mg/L EDTA, 100 mg/L octane sulphonic acid (OSA), pH=3.1 with KOH, 5% methanol Sample 1.0 µmol/L norepinephrine, 20 µL injection Temperature 30 °C Cell...
Page 127: Cell Working Volume Adjustment
Figure 15–3: Example S/N ratio for norepinephrine (peak height: 80 nA, peak-to-peak noise: 1.5 pA). The amount injected is 20 pmol (1.0 µmol/L). The concentration detection limit based on three times sigma-noise in this case is 11 pmol/L. 15.1.5 Cell working volume adjustment In a traditional electrochemical flow cell that uses metal/plastic gaskets (spacers), the thickness of the gasket affects the linear flow velocity in the cell.
Page 128 Figure 15–4: Example chromatograms of 100 nM standard of catecholamines in 10 mM HAc recorded with the SenCell spacing adjustment set to position 3 and 0.5, corresponding to an approximate spacing setting of 100 µm and 12 µm, respectively The preceding chromatogram images show an example to demonstrate the effect of cell working volume on signal.
Page 129 Figure 15–5: Normalized peak height of dopamine as a function of spacing setting (red curve) based on chromatograms recorded with a 100 nM standard of catecholamines in 10 mM HAc with a SenCell The dotted curve is a simulated curve based on the Cotrell equation (F.G. Cottrell, Z. Phys. Chem 42 (1903) 385).
Page 130 Figure 15–6: ASTM noise values as a function of cell spacing Figure 15–7: Noise traces as a function of cell spacing Pump pulsations and pressure rise SenCell spacing position 3 corresponds to approximately 100 ± 10 µm. The spacings used with the SenCell under test in this experiment were determined using a stylus profilometer.
Page 131: Reference Electrodes
15.2 Reference electrodes The SenCell is available with an ISAAC (in situ Ag/AgCl) reference electrode, a salt bridge Ag/ AgCl reference electrode, or a HyREF reference electrode. 15.2.1 ISAAC reference electrode The ISAAC reference electrode is in direct contact with the mobile phase, which contains chloride ions.
Page 132 Table 15–3: Potential of the Ag/AgCl reference electrode (mmol/l) E Ag/AgCl (mV) dE (mV) 3500 2500 1500 a. Rounded to the nearest whole number The addition of chloride to the mobile phase has a few restrictions. For example, the ISAAC is not recommended at a high working potential (>...
Page 133: Salt Bridge Ag/Agcl Reference Electrode
Figure 15–9: Schematic representation of the Ag/AgCl reference electrode AgCI coating KCI saturated KCI crystals Cotton wool 15.2.2 Salt bridge Ag/AgCl reference electrode The reference electrode of the Ag/AgCl type with salt bridge consists of a silver rod, coated with solid AgCl and immersed in a solution of saturated KCl containing KCl crystals.
Page 134: Hyref Reference Electrode
15.2.3 HyREF reference electrode The HyREF is a hydrogen reference electrode. Its potential depends on the pH of the mobile phase. The HyREF is fully comparable to the standard Ag/AgCl REF with respect to baseline stability and S/N ratio. The HyREF is more user-friendly, and in principle this REF is completely free of maintenance requirements.
Page 135: Adjusting The Sencell Working Volume
Figure 15–10: Left: Photo of assembled SenCell inlet block (green) with in situ Ag/AgCl (ISAAC) reference electrode (REF). Right: Bottom side of the SenCell with the cell working volume adjustment system, auxiliary (AUX) electrode contact (opening on left-hand side, next to inscription '3'), and working electrode (WE) contact (opening in the center). The working volume is preset at the factory to position 2 (refer the preceding right-hand side image), corresponding to a spacing of approximately 50 ±...
Page 136: Installation In Lc System
1. If applicable, install the SenCell clamp (700013160) from the startup kit in the center position of the 3465 Detector with a Phillips screwdriver. 2. Connect the column to the flow cell inlet with 1/16-inch OD small-bore PEEK tubing (0.3- mm ID or smaller depending on the column bore size) using the PCTFE 10-32 finger-tight fitting (700013151).
Page 137 3. Connect 0.5 mm ID PEEK tubing to the outlet of the flow cell. Use only factory-supplied finger-tight fittings in the flow cell; others may cause serious damage. Do not over-tighten the fitting. 4. Turn on the HPLC pump. Keep some tissues at hand because you will probably spill some mobile phase during this mounting procedure.
Page 138 Figure 15–12: SenCell mounted at an angle of approximately 45° in the 3465 Detector Cell clamp Cell outlet (tubing connection from cell to waste); make sure that the outlet is positioned on the top side to prevent entrapped air bubbles...
Page 139 Figure 15–13: Installation of SenCell in 3465 Detector: oven compartment with column and SenCell installed Make the electrical connections as depicted in the following figure. Figure 15–14: Installation of flow cell WORK, AUX, and REF are connected using the red, blue, and black cell cables.
Page 140: Maintenance
15.4 Maintenance 15.4.1 Assembling/disassembling the cell Figure 15–15: Exploded view of SenCell WE block O-ring Inlet block Inlet block O-ring Closing ring The arrows indicate how to assemble the cell. December 16, 2021, 715007395 Ver. 00 Page 140...
Page 141 Figure 15–16: Left: unscrewing the salt bridge electrode. Right: WE block with O-ring placed in O-ring groove. 15.4.1.1 Disassembling the cell To disassemble the cell: 1. Hold the cell in the upward position (with the metal closing ring on the top side). 2.
Page 142: Hyref
4. Confirm that the black inlet block O-ring is undamaged and properly mounted on the inlet block. 5. Place the inlet block on top of the WE block. 6. Place the metal closing ring over the inlet block and close the cell by turning the closing ring clockwise.
Page 143 Figure 15–17: Apply a few drops on the polishing disc (left), and polish the electrode (right) 15.4.3.2 Coating with ISAAC solution Warning: Take the necessary precautions (gloves, lab coat, and glasses) because the ISAAC solution is corrosive. 1. After polishing, coat the ISAAC REF with the factory-supplied ISAAC solution (700001949). 2.
Page 144: Ag/Agcl Salt Bridge
15.4.4 Ag/AgCl salt bridge Three aspects determine the proper function of an Ag/AgCl reference electrode. The chloride concentration must be kept at a strictly fixed level. This is best guaranteed by using a saturated chloride salt solution at a constant temperature. •...
Page 145 Maintenance on Salt Bridge Reference: 1. Turn the cell OFF on the controller. 2. Stop the HPLC pump. 3. Disconnect the cell from the controller. 4. Remove the REF from the inlet block. 5. Disassemble the REF by unscrewing the black fitting. 6.
Page 146: Working Electrode
Figure 15–20: Pushing the cotton wool frit out with the SB ref replacement toot Follow the procedure for the maintenance of the cotton wool frit: 1. Clean the salt bridge thoroughly with tap water then demi-water. 2. Saturate a small piece of cotton wool in KCl to prevent trapping of air within the wool. 3.
Page 147 15.4.5.1 Decreased flow cell performance Warning: Use proper eye and skin protection when working with solvents. Several actions can be taken to address decreased flow cell performance. Avoid unnecessary polishing; take the next step only if the previous was not successful. Steps to address decreased flow cell performance: 1.
Page 148: Specifications
PEEK, silicone, REF material (palladium or Ag and AgCl) Max. pressure 5 bar (73 psi) Fluidics connections 1/16-inch o.d. PEEK tubing with 10-32 PCTFE finger-tight connections Electric connections Cell cable for use with 3465 Detector December 16, 2021, 715007395 Ver. 00 Page 148...
Page 149: Parts List
15.6 Parts list Table 15–6: SenCell parts Part number Description 700013136 SenCell O-ring Silicone, 4 pcs 700013137 SenCell sb REF O-ring Silicone, 4 pcs 700013138 SenCell inlet block O-ring FKM, 1 pc 700013139 SenCell sb REF 700013141 SenCell sb REF body 700013142 SenCell sb REF cap 700013143...
Page 150: Flow Cell
16 VT-03 flow cell 16.1 The FlexCell 16.1.1 Introduction The VT-03 flow cell is available with a glassy carbon, platinum, gold, silver, or copper working electrode. In combination with the spacer set (25, 50 and 120 μm) a variety of detection volumes (down to 5 nL) can be attained.
Page 151 Figure 16–1: The VT-03 electrochemical flow cell Inlet Outlet The upper part, the inlet block, is separated from the working electrode block by means of a gasket (spacer, not shown). The VT-03 electrochemical flow cell was developed for ultra-trace analysis in standard, microbore, and capillary LC-EC.
Page 152: Three-Electrode Configuration
configuration gives the best results. In addition, it was found that the quality and finishing of the electrode materials in the flow cell are decisive factors in the performance of an EC detector. While competitive designs usually deteriorate when in use, this flow cell, by design, improves in performance.
Page 153 Figure 16–2: Schematic of an electrochemical cell with a three-electrode configuration Essentially, for the oxidation or reduction reaction, it would be sufficient to use only two electrodes. However, the three-electrode configuration has several advantages over a two- electrode configuration. If the working potential were applied only over an AUX versus the WE (without REF), the working potential would continuously change due to polarization effects at the electrodes, resulting in highly unstable working conditions.
Page 154: Working Electrode
16.1.3 Working electrode Electrochemical detection puts high demands on the WE material. The WE should be made of an electrochemically inert material. Furthermore, to avoid an irregular flow profile over the electrode, it should have a very well defined surface. Finally, it is important that the analyte of interest can be oxidized (or reduced) with favorable I/E characteristics.
Page 155 Table 16–2: LC-EC conditions for analysis of norepinephrine Column ODS-2, 3 µm, 100 x 4.6 mm Flow rate 1.0 mL/min Mobile phase 50 mM, citric acid 50 mM, 20 mg/L EDTA, 100 mg/L octane sulphonic acid (OSA), pH=3.1 with KOH, 5% methanol Sample 1.0 µmol/L norepinephrine, 20-µL injection Temperature...
Page 156: Working Electrode Diameter
Figure 16–3: Typical S/N ratio for norepinephrine measured with a VT-03 glassy carbon flow cell (peak height: 80 nA, peak-to-peak noise: 1.5 pA) The amount injected is 20 pmol (1.0 µmol/L). The concentration detection limit based on three times the sigma-noise is 11 pmol/L. 16.1.5 Working electrode diameter The size of the WE is an important factor in LC-EC;...
Page 157: Spacer Thickness
Table 16–3: Flow cell recommendations Column diameter (mm) Recommended flow cell 3 and higher 3 mm GC 3 - 1 2 mm GC 1 and below 0.7 mm µGC The choice of flow cell is based primarily on the HPLC column diameter. This way the best possible detection limit for a standard, microbore, or capillary column is warranted.
Page 158 Figure 16–4: Signal to Noise for 1.0 μmol/L norepinephrine The signal and noise for 1.0 µmol/L norepinephrine measured at variable spacer thickness (given in µm). See Table 16–2: LC-EC conditions for analysis of norepinephrine (Page 155) for other conditions. December 16, 2021, 715007395 Ver. 00 Page 158...
Page 159: Reference Electrodes
Figure 16–5: Peak height versus spacer thickness to the power -2/3 Decreasing the spacer thickness is limited by an increased pressure drop over the flow cell that eventually leads to an obstruction of the flow. The minimum spacer thickness available is 25 µm. Applying these small spacers should be done with care.
Page 160 The potential of the reference electrode (REF) depends on the chloride concentration as described by the following equation: where R is the gas constant (8.314 Jmol-1K ), T is the absolute temperature (293 K), and F is the Faraday constant (96485 Cmol ).
Page 161 Table 16–5: Potential of the Ag/AgCl reference electrode; dE is the potential difference with EAg/AgCl in saturated KCl (continued) (mmol/L) (mV) dE (mV) Ag/AgCl a. Rounded to the nearest whole number The addition of chloride to the mobile phase has a few restrictions. For example, the ISAAC is not recommended at a high working potential (>...
Page 162 Figure 16–7: Schematic representation of the Ag/AgCl reference electrode AgCl coating KCl saturated KCl crystals Cotton wool December 16, 2021, 715007395 Ver. 00 Page 162...
Page 163: Salt Bridge Ag/Agcl Reference Electrode
16.2.2 Salt bridge Ag/AgCl reference electrode The Ag/AgCl reference electrode with salt bridge consists of a silver rod, coated with solid AgCl, immersed in a solution of saturated KCl containing KCl crystals. 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 KCl.
Page 164: Installation
16.3 Installation 16.3.1 VT-03 flow cell with HyREF or ISAAC The flow cell is assembled when it arrives. The force on the bolts is preset to 13 Ncm (a little bit beyond finger-tight). Familiarize yourself with this force, because over-tightening of the bolts strongly deteriorates the S/N ratio and eventually the cell itself.
Page 165: Flow Cell With Salt Bridge Ref
2. Do not over-tighten the finger-tight. Over-tightening affects the flow pattern through the tubing (turbulence) and may strongly decrease flow cell performance. 3. Connect 0.5 mm ID PEEK tubing to the outlet of the flow cell. Use only the factory-supplied finger-tights in the flow cell.
Page 166 Figure 16–9: Installation of flow cell WORK, AUX, and REF are connected using the red, blue, and black cell cables. Note: The LC outlet is placed on top, to prevent entrapment of bubbles. Warning: Use proper eye and skin protection when working with solvents. To install the flow cell with salt bridge REF: 1.
Page 167 4. Connect 0.5-mm ID PEEK tubing to the outlet of the flow cell. Use only the factory-supplied finger-tights in the flow cell; others may cause serious damage. Again, do not over-tighten the finger-tight. 5. Fill the flow cell, keeping it in an angle of 45° with the REF fitting on top. Filling is best done by blocking the outlet with a finger and letting the air escape via the REF fitting.
Page 168: Micro Flow Cell
Figure 16–10: Always install flow cell with outlet on top 16.3.3 VT-03 micro flow cell The micro flow cell is assembled when it arrives. The force on the bolts is preset to 13 Ncm (“a little bit beyond finger-tight”). Familiarize yourself with this force, because over-tightening of the bolts strongly deteriorates the S/N ratio and eventually the cell itself.
Page 169 Figure 16–11: Disassembled VT-03 micro flow cell with spacer Note: VT- 03 micro cells purchased after 2009 are delivered without the metal centering ring and have no groove in the WE and inlet block. To ensure proper installation and optimal performance, follow the instructions below for assembling a VT-03 micro flow cell.
Page 170 Figure 16–12: WE block with inserted bolds on the glass mounting plate 2. Place the metal ring on the WE block. Make sure that you hold the metal ring horizontally when pushing it onto the block. Subsequently, gently place the 25-µm spacer on top of the WE block using a pair of tweezers.
Page 171 the chance of scratching the inlet block surface). Then, turn the inlet block downward in horizontal position. The inlet block now rests on top of the WE block with the bolts positioned on the mounting holes. The black positioning markers on the sides of the blocks should now be aligned in vertical position.
Page 172 crosswise pattern with the hex key (tightening force approximately 13 Ncm). The cell is again ready for use. Figure 16–16: Fixing the two cell blocks with the hex key (tighten the bolts in a crosswise pattern) 16.3.3.2 Capillary connections The micro flow cell is supplied with a low dead-volume fused silica capillary connection for coupling with capillary columns (<...
Page 173 Figure 16–17: Mounting of the fused silica connector in the VT-03 micro flow cell The capillary connector is not necessary with a 1-mm (or larger ID) column. With larger columns, use narrow-bore PEEK tubing (100-µm ID), and install it as described in VT-03 flow cell with HyREF or ISAAC (Page 164) (ISAAC REF) or in...
Page 174 5. Mount the combination carefully in the injection block (Figure 16–17: Mounting of the fused silica connector in the VT-03 micro flow cell (Page 173)). 6. Let the fused silica protrude slightly (< 0.5 mm) through the injection hole. 7. Clean the factory-supplied glass mounting plate from particles. 8.
Page 175: Maintenance
Read the column instructions first. The flow cell is then filled at a higher flow rate. After filling, reconnect the column. Be careful not to include air bubbles. Warning: Never switch ON the flow cell when: • The cell cable is not correctly connected. •...
Page 176: Ag/Agcl Salt Bridge
16.4.2.1 Polishing Polishing the reference electrode is done using the factory-supplied polishing kit, which contains diamond slurry and a polishing disc. To initiate polishing: 1. Shake the diamond slurry thoroughly before use. 2. Rinse the polishing disc with demi-water before applying the diamond slurry. 3.
Page 177 16.4.3.2 Material • An over-saturated and thoroughly degassed KCl solution • Salt bridge REF tool (700013145), or a stainless steel rod of about 5 cm in length and 1 mm in diameter • Ordinary cotton wool 16.4.3.3 Procedure Note: Use proper eye and skin protection when working with solvents. 1.
Page 178 8. Clean all parts with demi-water. 9. The Ag/AgCl electrode must be cleaned if the silver on the tip (Figure 16–21: Exploded view of the reference electrode (Page 177) arrow) has a non-metallic appearance by gently grinding it with sanding paper; also, the AgCl can be gently resurfaced in this way. 10.
Page 179: Working Electrode
9. Tighten the black swivel so that a small droplet appears at the end of the salt bridge, but do not over-tighten the swivel. 10. Flush the complete, mounted REF with demi-water and dry it with a tissue, but keep the cotton wool frit soaked.
Page 180 3. Apply a few drops of slurry to the wetted polishing disc, and polish the electrode with a figure-8 motion for about one minute. Apply only gentle pressure. 4. Clean the electrode with an ethanol-wetted tissue and inspect the surface visually. Repeat the procedure if necessary, until the shining metal REF surface appears.
Page 181: A Safety Advisories
A Safety advisories Waters products display safety symbols that identify hazards associated with the product’s operation and maintenance. The symbols also appear in product manuals with statements that describe the hazards and advise how to avoid them. This appendix presents all safety symbols and statements that apply to Waters’...
Page 182: Specific Warnings
Warning: (Risk of exposure to ultraviolet radiation.) Warning: (Risk of contacting corrosive substances.) Warning: (Risk of exposure to a toxic substance.) Warning: (Risk of personal exposure to laser radiation.) Warning: (Risk of exposure to biological agents that can pose a serious health threat.) Warning: (Risk of tipping.)
Page 183: Notices
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 184: Bottles Prohibited Symbol
A.3 Bottles Prohibited symbol The Bottles Prohibited symbol alerts you to the risk of equipment damage caused by solvent spills. 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 185 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 186 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 187 警告：当有压力的情况下使用管线时，小心注意以下几点： • 当接近有压力的聚合物管线时一定要戴防护眼镜。 • 熄灭附近所有的火焰。 • 不要使用已经被压瘪或严重弯曲的管线。 • 不要在非金属管线中使用四氢呋喃或浓硝酸或浓硫酸。 • 要了解使用二氯甲烷及二甲基亚枫会导致非金属管线膨胀，大大降低管线的耐压能力。警告：當在有壓力的情況下使用聚合物管線時，小心注意以下幾點。 • 當接近有壓力的聚合物管線時一定要戴防護眼鏡。 • 熄滅附近所有的火焰。 • 不要使用已經被壓癟或嚴重彎曲管線。 • 不要在非金屬管線中使用四氫呋喃或濃硝酸或濃硫酸。 • 要了解使用二氯甲烷及二甲基亞楓會導致非金屬管線膨脹，大大降低管線的耐壓能力。 경고 가압 폴리머 튜브로 작업할 경우에는 주의하십시오. • 가압 폴리머 튜브 근처에서는 항상 보호 안경을 착용하십시오. •...
Page 188: 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 189 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 190: 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 191: 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 December 16, 2021, 715007395 Ver.

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