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Page 3: Trademarks
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 Web site (www.waters.com).
Page 4: Customer Comments
We seriously consider every customer comment we receive. You can reach us at tech_comm@waters.com. Contacting Waters Contact Waters with enhancement requests or technical questions regarding the use, transportation, removal, or disposal of any Waters product. You can reach us via the Internet, telephone, or conventional mail. Waters contact information Contacting medium...
Page 5: Considerations Specific To The Waters Alliance E2695 Separations Module
For information regarding what cord to use in other countries, contact your local Waters distributor. Solvent leakage hazard The source exhaust system is designed to be robust and leak-tight. Waters recommends you perform a hazard analysis, assuming a maximum leak into the laboratory atmosphere of 10% LC eluate.
Page 6: Fcc Radiation Emissions Notice
High voltage hazard Warning: To avoid electric shock, observe these precautions: • Do not remove the mass spectrometer’s protective panels. The components they cover are not user-serviceable. • When the instrument is in Operate mode, avoid touching the areas marked with the high voltage warning symbol.
Page 7: Operating This Instrument
Electrical and electronic equipment with this symbol may contain hazardous substances and should not be disposed of as general waste. For compliance with the Waste Electrical and Electronic Equipment Directive (WEEE) 2012/19/EU, contact Waters Corporation for the correct disposal and recycling instructions. Serial number Part number catalog number February 27, 2018, 715003794 Rev.
Page 8: Audience And Purpose
Waters Alliance e2695 Separations Module. This guide sets forth procedures for unpacking, installing, using, maintaining, and troubleshooting the Waters Alliance e2695 Separations Module. It also includes appendixes for specifications, spare parts, and solvent considerations. Intended use of the Waters Alliance e2695 Separations Module Waters designed the Alliance e2695 Separations Module, an integrated solvent and sample management platform, to facilitate all critical separations functions.
Page 9: Ec Authorized Representative
There may be potential difficulties in ensuring electromagnetic compatibility in other environments due to conducted as well as radiated disturbances. EC authorized representative Waters Corporation Stamford Avenue Altrincham Road Wilmslow SK9 4AX UK Telephone:...
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Page 11: Table Of Contents
Contacting Waters ........................iv Safety considerations ........................ iv Safety hazard symbol notice..................iv Considerations specific to the Waters Alliance e2695 Separations Module ....v FCC radiation emissions notice ................... vi Electrical power safety notice ..................vi Equipment misuse notice..................... vi Safety advisories ......................
Page 12 1.3 Sample management system overview ..................26 1.3.1 Flow path ........................26 1.3.2 Normal flow........................27 1.3.3 Injection ......................... 27 1.3.4 Carousel configuration....................27 1.3.5 Vial presence ......................... 28 1.3.6 Syringe assembly ......................28 1.4 Operating configurations ......................29 1.4.1 System Controller mode ....................29 1.4.2 No Interaction mode ......................
Page 13 3.3 Configure the separations module ..................... 47 3.3.1 Set configuration parameters..................47 3.3.2 Configure the operating mode ..................52 3.4 Prepare the solvent management system ................. 56 3.4.1 Prepare solvent reservoirs..................... 57 3.4.2 Degassing solvents......................57 3.4.3 Prime the plunger seal wash pump ................58 3.4.4 Prime the solvent management system.................
Page 14 5 Automatic Runs ....................81 5.0.1 System Controller or No Interaction mode..............81 5.0.2 Controlled by Empower software ................... 81 5.0.3 Controlled by MassLynx software .................. 82 5.1 Perform automatic runs in a standalone mode ................82 5.1.1 Run a sample set......................82 5.1.2 View sample sets ......................
Page 15 7 Maintenance Procedures ..................119 7.1 Maintenance considerations ....................119 7.1.1 Proper operating procedures ..................119 7.1.2 Reinitialize the system ....................119 7.2 Spare parts ..........................119 7.3 Troubleshooting with Connections INSIGHT ................119 7.4 Safety and handling ......................... 120 7.5 Maintain the solvent management system ................
Page 16 A.3 Bottles Prohibited symbol ....................... 183 A.4 Required protection ......................... 184 A.5 Warnings that apply to all Waters instruments and devices ............ 184 A.6 Warnings that address the replacing of fuses ................. 187 A.7 Electrical and handling symbols ....................189 A.7.1 Electrical symbols......................
Page 17 B.2.7 Connect the column selection valve................203 B.2.8 Connect the detector ....................206 B.3 Signal connections ........................207 B.3.1 I/O signal connections ....................208 B.3.2 Digital signal connections .................... 211 B.4 Add hardware options ......................213 B.4.1 Install an optional sample loop ..................214 B.4.2 Install the column heater and column heater/cooler............
Page 18 D.7 Mobile phase solvent degassing ..................... 230 D.7.1 Gas solubility....................... 231 D.7.2 Vacuum degassing ...................... 231 D.8 Wavelength selection ......................232 D.8.1 UV cut-offs for common solvents ................232 D.8.2 Mixed mobile phases ....................233 D.8.3 Refractive indices of common solvents ............... 234 February 27, 2018, 715003794 Rev.
Page 19: Getting Started With The Alliance E2695 Separations Module
Getting started with the Alliance e2695 Separations Module Separations module overview The Alliance e2695 Separations Module is an integrated solvent and sample management platform. Integrating the two, traditional high performance liquid chromatography (HPLC) components—a solvent management system and a sample management system—facilitates all critical separations functions.
Page 20: Hplc System Configurations
• As a component of an HPLC system controlled by Empower software and that uses Ethernet communications • As a component of an HPLC system controlled by a non-Waters data system that uses RS-232 communications 1.1.2 Solvent bottle tray kits There are several solvent bottle tray kits available for use with Alliance HPLC system configurations.
Page 21 Figure 1–2: A-series solvent bottle organizer configuration Solvent bottle organizer Drip tray Detector e2695 separations module Figure 1–3: B-series solvent bottle tray configuration (only single detector shown) Detector Solvent bottle tray Drip tray and base Column heater or e2695 separations column heater/cooler module February 27, 2018, 715003794 Rev.
Page 22: Control Of Chromatographic Functions
Figure 1–4: C-series solvent bottle bucket configuration: Detector Solvent bottle bucket e2695 separations Column heater or module column heater/cooler 1.1.3 Control of chromatographic functions Once you program a method, the separations module controls these functions: • Solvent composition • Flow rate •...
Page 23: Record-Keeping Functions
1.1.5 Record-keeping functions The separations module automatically records the following information: • System errors • Programmed operating conditions for each run (local methods only) System errors may be viewed from the front panel by pressing the Log soft key. Local methods are viewable on the front panel, and they can be printed if a printer is attached or transferred to an external device using the RS-232 port.
Page 24: Solvent Blending
Just before the accumulator chamber empties, the solvent in the primary piston chamber is precompressed to a pressure slightly less than that indicated by the system pressure transducer. When the accumulator piston chamber is empty, the primary piston delivers solvent, under pressure, through the primary pressure transducer.
Page 25: Preferred Plunger Stroke Volume
1.2.5 Preferred plunger stroke volume The separations module’s discrete, programmable stroke volumes provide optimal flow delivery and solvent blending. Nevertheless, you can improve performance for specific applications by overriding the default (preprogrammed) stroke volume of 130 µL. To do so, you select a preferred stroke volume of 25 µL, 50 µL, or 100 µL, as shown in the following table.
Page 26: Sample Management System Overview
Table 1–3: Absolute pressure transducer attributes (continued) Item Absolute pressure transducer Theoretical maximum vacuum 0 psia (based on 1 atm at sea level) Operating range 22.1 to 0 kPa (0.2 to 0 bar, 3.2 to 0 psia) (using the default vacuum pressure threshold) (If pressure is out of range, an asterisk [*] appears in the pressure field.) “Typical”...
Page 27: Normal Flow
The flow path through the sample management system changes according to the function being performed. 1.3.2 Normal flow During normal flow, V1 is open, allowing solvent to flow along two paths: • 95 percent of the solvent flows through the body of V2, the sample loop pressure transducer, the sample loop, and needle.
Page 28: Vial Presence
1.3.5 Vial presence A light-emitting diode assembly searches for the presence of a vial before the needle moves to the Draw position. This searching prevents air from being accidentally injected into the solvent stream when a programmed vial position is accidentally left empty. See the Verify vial presence parameter “Set configuration parameters”...
Page 29: Operating Configurations
IEEE-488 (digital) for Waters detectors • I/O (analog, hard wire cables) • RS-232 (digital) for non-Waters data systems, printers, or integrators Follow the procedures in Chapter 4 to control the HPLC system in the System Controller mode. In System Controller mode, the separations module cannot control any other instruments/detectors via Ethernet communications.
Page 30: No Interaction Mode
In the System Controller mode, the separations module can control as many as three detector channels on the IEEE-488 bus (two UV detector channels and one RI detector channel). These Waters detectors can be used: • 2414 RI, 2410 RI, or 410 dRI •...
Page 31: Remote Control Mode
Waters MassLynx Mass Spectrometry Software for mass spectrometers • RS-232 communications by non-Waters data systems • An external autosampler (the Waters 2700 Sample Manager, for example) with the separations module in the Operate Gradient by Event In mode Use the procedures in Chapter 5 to set up the separations module for remote control.
Page 32 Operate Gradient by Event In control In Operate Gradient by Event In mode, the separations module is connected to an external autosampler (a Waters 2700 Sample Manager, for example). In this mode, the separations module provides gradient functionality, and the external autosampler provides the sampling/injection functionality.
Page 33: Options And Accessories
4 °C to 65 °C, with a set point temperature range that is ambient minus 15 °C or 4 °C (whichever is greater) to 65 °C, in 1 °C increments. The Waters Alliance Series Column Heater and Column Heater/Cooler Operator’s Guide presents the procedures for installing, operating, maintaining, and basic troubleshooting of the Alliance column heaters and column heater/coolers.
Page 34: Sample Loops
1.5.6 Sample loops You can install an additional 100-µL sample loop or you can install a larger one to inject sample volumes greater than 100 µL. (See “Install an optional sample loop” on page 214 for the procedure to install an optional sample loop.) 1.5.7 Column selection valve The motorized column selection valve enables the system to switch solvent flow to one of several...
Page 35 Figure 1–11: e2695 column heater Location of optional column selection valve 1.5.7.1 Two-column selection valve The firmware does not recognize the 2-column selection valve (6 port, 2 position) option. Instead, the option is controlled via one of the four I/O event switches on the separations module’s rear panel.
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Page 37: Install The E2695 Separations Module
Install the e2695 Separations Module Installation overview This chapter explains how to connect electrical cables, signal cables, tubing and fittings, and hardware options to the separations module. Figure 2–1: Major steps to installing the separations module Connect tubing Obtain required materials Review site Connect signal cables...
Page 38: Site Requirements
Site requirements The installation site must meet the specifications in the following table. Table 2–1: Installation site requirements Factor Requirement Temperature 4 to 40 °C (39 to 104 °F) Relative humidity 20 to 80%, noncondensing Bench space Width: 45.7 cm (18 inches). With optional column heater or column heater/cooler, 58.4 cm (23 inches).
Page 39: Firmware And Software Requirements
The Waters detectors that connect to the separations module must meet minimum firmware requirements to successfully communicate with the software installed in the separations module.
Page 40: External Connections
External connections Appendix B (“External Connections” on page 191) for instructions on making all external connections to the Alliance e2695 Separations Module. February 27, 2018, 715003794 Rev. C Page 40...
Page 41: Prepare The E2695 Separations Module For Operation
Prepare the e2695 Separations Module for Operation Power-on the separations module Power-on the separations module by moving the power switch (located at the top of the left-hand side panel) to I. The start-up diagnostic tests begins. 3.1.1 Start-up diagnostic tests On start-up, the separations module performs the following functions and tests: •...
Page 42: Operating Overview
Figure 3–1: Main screen Notes: • Develop Methods and Run Samples options are dimmed when locked on the Service Diagnostics page. • The option to disable Run Samples from the front panel ensures that injections can only be controlled through the software. Analysts cannot use the front panel. Banner area Data area Screen keys...
Page 43 • Remote control – Indicates Empower software or another remote control mode • Current separation method, sample set, or template 3.2.1.2 Data area The Data area of the screen’s two modes of display show operating information: • Menu – Shows all control features with which you perform tasks; configuring the separations module, and creating or editing separation methods, sample sets, or sample templates •...
Page 44: Use The Keypad
3.2.1.4 More screen key The More screen key appears on various screens. Pressing it displays an additional set of screen keys. 3.2.2 Use the keypad The following table describes the functions of the keypad keys. Table 3–2: Keypad functions Function Moves the cursor one field to the left.
Page 45: Enter Values In Parameter Fields
Table 3–2: Keypad functions (continued) Function Immediately stops the flow of solvent and terminates a current Stop Stop function (sample processing, priming, purging, and so on). Pressing Flow Flow this key also activates the Run Stopped output switch. Enters the corresponding numeral into a current field, selects the corresponding gradient curve (0 = curve 10), positions the cursor to the corresponding entry in a list (0 = tenth item).
Page 46 Figure 3–2: Steps for entering alphabetic data Select text field Move the cursor Press the screen key to the position for the appropriate immediately after letter group incorrect letter Press the Backspace Press the screen Screen key key for the letter Press the screen key for the appropriate Enter another...
Page 47: Configure The Separations Module
• Printer – Selects the type of printer and the baud (data transfer) rate. Tip: The baud rate is automatically set to 1200 when you set the Waters 746 Integrator as the printer type. • Date format – Selects between “Mmm DD YY” (for example, Aug 28 17) and “DD Mmm YY”...
Page 48 Table 3–3: Screen keys in the Configuration screen (continued) Screen key Description Options Displays a list of the hardware options installed on the separations module. Set Clock Displays the Set Clock dialog box, in which you establish the current time and date. Events In Specifies external signal conditions for Stop Flow and Inject Hold (see...
Page 49 To enable the auto shutdown function: Press the Config screen key in the Main screen, and then press the More screen key once. Press the Auto Shutdown screen key. Select a separation method to use while the separations module is shut down. Tip: Only the initial conditions in the method you select are used.
Page 50 3.3.1.3 Set report options Use the Report Options dialog box to define information sent to the printer or integrator. (Before you print a report, select the printer in the Printer section of the Configuration screen. You select the destination of the report in the print dialog box that appears when you press the Print screen key.) To define the information sent to a printer or integrator: Press the Reports screen key to display the Report Options dialog box.
Page 51 The following figure is an example of a report made with all options selected. Figure 3–3: Sample report REPORT: Alliance e2695 Separations Module SERIAL NUMBER: F01SM7885M PRINTED: 03/17/08 08:26:40am USER: User CONFIGURATION: Syringe (250 uL), Loop (100 uL) SEPARATION METHOD: Default...
Page 52: Configure The Operating Mode
Enter an appropriate value in each field, and then press OK. Tip: The following table lists the ranges and typical values for each parameter. Waters recommends you use the typical values for each parameter to best characterize the system. Table 3–4: Compression check parameters...
Page 53 In this standalone mode, the separations module controls up to three detector channels on the IEEE-488 bus. These can include two UV detector channels (Waters 2487 dual wavelength and/or 486 tunable absorbance) and one RI detector channel (Waters 2414 RI, 2410 RI, or 410 dRI). See “Digital signal connections” on page 211 for information on IEEE-488 connections.
Page 54 3.3.2.3 Controlled by Empower mode In this remote control mode, Empower software controls the operation of your HPLC system using the Ethernet interface or the IEEE-488 interface bus (but not both simultaneously). Connect the Ethernet or IEEE-488 cable as described in “Digital signal connections”...
Page 55 3.3.2.5 Controlled by Operate Gradient by Event In mode In this mode, an external autosampler (a Waters 2700 Sample Manager, for example) initiates the start of a chromatographic run and performs the inject function (instead of the separations module). The separations module has no control of IEEE-488 devices, but can control non-IEEE devices using the I/O connections on its rear panel.
Page 56: Prepare The Solvent Management System
To set the separations module to the Controlled via RS-232 (ASCII) or (Binary) mode: Select the System field in the Configuration screen, and then press Enter to display a list of operating modes. Select Controlled via RS232 (ASCII) or Controlled via RS232 (Binary), and then press Enter and then Exit.
Page 57: Prepare Solvent Reservoirs
Library under Waters Division/Services & Support. Choose solvent reservoirs that provide a snug fit for the reservoir caps supplied in the start-up kit. Waters recommends 1-L reservoirs. Place the reservoirs in the solvent bottle tray, and set the tray above the solvent management system components.
Page 58: Prime The Plunger Seal Wash Pump
3.4.3 Prime the plunger seal wash pump The seal wash solvent lubricates each plunger and flushes away any solvent or dried salts forced past the plunger seal from the high-pressure side of the piston chamber. This wash cycle extends the life of the seals. Position the plunger seal wash reservoir in a visible location above the solvent management system, and refill the reservoir as necessary.
Page 59: Prepare The Sample Management System For Operation
Prepare the sample management system for operation Prepare the sample management system for operation after you prepared the solvent management system (see “Prepare the solvent management system” on page 56). The following figure shows the steps involved in preparing the sample management system for operation. Figure 3–5: Preparing the sample management system Start Purge the sample...
Page 60: Prime The Needle Wash Pump
To prevent a compression test from running, leave the Compression Check check box blank the first time you use the system. Note: Calibrate the seal pack before running a compression test or it will likely fail. Press OK. 3.5.2 Prime the needle wash pump The needle wash pump flushes the needle in the sample management system, preventing carryover of sample between injections.
Page 61: Adjust The Seal Pack
Table 3–5: Suggested needle wash solvent Chromatographic condition Needle wash solvent Buffered aqueous, reversed-phase 80% organic/20% aqueous Nonaqueous, reversed-phase 100% methanol Normal phase Mobile phase Mobile phase Ion exchange Water 1. If the aqueous portion of the analytical solution is pH-adjusted to ensure sample solubility, match the pH of the aqueous portion of the needle wash solution to it.
Page 62: Load Carousels
3.5.4 Load carousels The following table lists the carousels and their color codes. Table 3–6: Carousel codes and colors Code Color Vial numbers Blue 1 to 24 Yellow 25 to 48 49 to 72 Green 73 to 96 White 97 to 120 To load the carousels: Open the carousel door.
Page 63: Prepare A New Or Dry Separations Module For Operation
3.6.1 Prepare a new or dry separations module for operation To change from a buffered solvent to a totally organic solvent: Prime the plunger seal wash pump (see “Prime the plunger seal wash pump” on page 58). Prime the needle wash pump (see “Prime the needle wash pump”...
Page 64: Change From A Buffered Solvent To An Organic Solvent
3.6.3 Change from a buffered solvent to an organic solvent Notice: To avoid salts from precipitating in the separations module, use an intermediate solvent such as water when you change from buffers to high-organic-content solvents (see “Solvent miscibility” on page 228).
Page 65: Power-Off The Separations Module
Replace the needle wash solvent with a solution of 90% methanol/10% water or a miscible solvent, and perform a needle wash prime (see “Prime the needle wash pump” on page 60). 3.7.2 Power-off the separations module To power-off the separations module: Ensure that the system is flushed properly (see “Flush the flow path”...
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Page 67: Front Panel Control
Front Panel Control This chapter explains how to manually control the components of your HPLC system through the front panel of the separations module. When the separations module is configured in the No Interaction or Operate Gradient by Event In mode, you control each HPLC system component individually via its front panel.
Page 68: Remove The Carousel
4.2.1 Remove the carousel To remove a carousel from the sample compartment: Open the carousel door. Press the Next screen key, or select the desired carousel screen key (A to E), to move the desired carousel to the front loading position. Remove the carousel from the sample chamber.
Page 69: Monitor Your Hplc System
Monitor your HPLC system You can use the Status screen to monitor the current status of your HPLC system during a run. Press Menu/Status to display the first page of the Status screen. The content and layout of the Status screen vary with the options installed in the separations module and with the mode of operation.
Page 70 Figure 4–3: Sample set queue The following table describes the functions of the parameters in the Status screen fields. You can make changes only in the fields outlined with a solid border. Table 4–1: Status screen parameters Parameter Description Method Indicates the current separation method.
Page 71 Table 4–1: Status screen parameters (continued) Parameter Description Pressure Ripple: Indicates pressure readings in the solvent management system for the last minute. • Max psi Maximum pressure. • Min psi Minimum pressure. • Delta psi Peak-to-peak pressure difference. Vial Indicates the number of the vial currently under the injector.
Page 72: Perform Direct Functions
• External Temp 1 • External Temp 2 1. Enabled if the optional column heater or column heater/cooler module is configured with the Waters 2414, 2410, or 410 detector. Perform direct functions The following figure outlines the procedure for using the front panel to set up the separations module to perform a direct function.
Page 73 77 mobile phase. As an option, performs a compression test. Purge 2410/410 Purges the Waters 2414, 2410, or 410 “Purge the 2410 and Reference detector reference cell. 410 reference cell” on (if applicable)
Page 74: Dry Prime The System
4.4.1 Dry prime the system Use the dry prime option to prime the system when the flow path in the solvent management system is dry. See “Prepare the separations module for operation” on page To dry prime the system: Set up the reservoirs as described in “Install needle wash and plunger seal wash reservoirs”...
Page 75: Run System Prep
11. Press a screen key corresponding to the solvent line with which you want to prime the system. Tip: To facilitate purging air from the lines, start the wet prime using the solvent with the lowest viscosity. 12. Specify a duration of 5 minutes and press Continue. Result: The solvent management system begins to operate.
Page 76: Wet Prime The System
Notice: • To avoid damaging the plunger seals, run System Prep only when solvent is in the solvent management system’s flow path. If necessary, fill all solvent reservoirs and dry-prime the system before you run System Prep. • To avoid precipitating salts in the separations module, use an intermediate solvent such as water when you change from buffers to solvents of high organic content.
Page 77 On the Status screen, press Direct Function. Select Wet Prime, and then press Enter. Gently shake the filters in the reservoirs to remove any bubbles. In the Wet Prime dialog box, specify the flow rate and duration of the priming (using the composition entered in the Status screen), and then press the OK screen key.
Page 78: Purge The 2410 And 410 Reference Cell
4.4.4 Purge the 2410 and 410 reference cell You can purge the reference and sample sides of the Waters 2414 RI, 2410 RI, and 410 dRI detectors using the Purge 410 Reference direct function. Purge the flow path whenever you change solvents or you experience an unexpected loss in sensitivity due to excess noise or drift.
Page 79: Inject Samples
To condition the column: Press Menu/Status to display the Status screen. In the Status screen, select the Method field, and then select the separation method with which you want to condition the column. Press the Direct Function screen key. Select Condition Column, and then press Enter. In the Condition Column dialog box, specify a length of time for conditioning the column that equals or exceeds the duration of the gradient, as defined in the specified separation method (including reequilibration time).
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Page 81: Automatic Runs
Automatic Runs You can set up the separations module to make automatic runs when it operates as follows: • In System Controller or No Interaction mode • Controlled by Empower software • Controlled by MassLynx software “Operating configurations” on page 29 for details about separations module control modes.
Page 82: Controlled By Masslynx Software
5.0.3 Controlled by MassLynx software If your HPLC system is controlled by MassLynx software, you must select an LC method that defines the separations module’s operating parameters and those of the system’s detector (but not the mass spectrometer, if included). For details on performing a run using MassLynx software, see “Perform automatic runs under MassLynx control”...
Page 83: View Sample Sets
The following table describes the icons used in the Run Samples screen. Table 5–1: Icons in the Run Sample screen Icon Description Separation method Sample set Sample template Select the sample set you want to run, and then press one of the following screen keys to perform the indicated tasks: •...
Page 84: Perform A Run From A Sample Template
• Loading View – Displays the sample set with one vial per line (see figure “Sample Set screen – Loading View” on page 84). This view shows you how to load samples in the carousel. Figure 5–3: Sample Set screen – Injection View Figure 5–4: Sample Set screen –...
Page 85: Modify A Sample Set During A Run
Repeat step 3 step 4 when the sample template includes multiple separation methods. Enter the number of standards in the run, and then press the Continue screen key. Enter the location of the first standard vial, and then press the Continue screen key. Requirement: You must use consecutive standard vials.
Page 86: Stop A Run
5.1.5 Stop a run To stop a run in progress, press one of the following keys from the Status screen: • Stop Inject (screen key) – Stops the current injection, and displays a dialog box requesting you to abort or resume the injection. •...
Page 87: Considerations When Performing Runs Controlled By Empower Software
If you need to modify these values, verify that the term “Idle” appears in the banner area before you edit the default separation method. You can edit the default separation method in the separations module to set parameter values appropriate for your applications. See “Edit a separation method”...
Page 88: Perform An Automatic Run Using Masslynx Software
5.3.1 Perform an automatic run using MassLynx software Tip: You can operate the separations module from its front panel whenever it is not being run by the MassLynx software. To perform an automatic run at the MassLynx workstation: Tip: See the MassLynx online Help for an explanation of how to perform the tasks in these steps. Configure an LC/MS system that includes the separations module.
Page 89: Create Methods, Sample Sets, And Sample Templates
Create Methods, Sample Sets, and Sample Templates Chapter 5 explains how to perform an automatic run in a standalone or remote control mode. You can create and store the separation methods, sample sets, and sample templates with which you make automatic runs when the separations module is in the standalone System Controller mode.
Page 90: Sample Templates
6.0.3 Sample templates Using sample templates, you pre-program a sample set so that the operator needs only to enter the number and location of samples and standards. The sample template then prompts you to load and prime the correct solvents for the sample set. As with any sample set, the sample template can use multiple separation methods.
Page 91: Create A Separation Method
Figure 6–1: Create or edit a separation method To create a new To edit an existing separation method: separation method: Enter the autosampler parameters in the Autosampler screen Press the Develop Press the Develop Methods screen key Methods, New, then the Separation Method screen keys Select the separation...
Page 92: Edit A Separation Method
Figure 6–2: Methods screen Press the New screen key. Press the Separation Method screen key. Enter a name for the method, and then press Enter. On the Mobile Phase screen, specify values for all applicable parameters and also in the 5 associated parameter screens.
Page 93: Copy And Edit A Separation Method
6.1.3 Copy and edit a separation method To copy and edit an existing separation method, leaving the original method unchanged: On the Main screen, press the Develop Methods screen key. In the Methods screen, select the separation method you want to copy. Press the Copy screen key.
Page 94: Set Separation Method Parameters
Set separation method parameters You specify separation method parameters in six different screens, which are arranged in the order shown in the following table. Each screen is described in the sections of this manual cited in the Reference column of this table. Table 6–1: Separation method parameter screens Screen name Reference...
Page 95 The following table describes the fields and screen keys in the Mobile Phase screen. Table 6–2: Mobile Phase screen parameters Parameter Function Value range Initial Flow Specifies the initial flow rate 0.000 and 0.010 to 10.000 of the method. For isocratic in 0.001 mL/min increments operation, this is the flow rate for the entire...
Page 96 The in-line degasser is disabled on any fault regardless of the alarm setting you select. Waters recommends that you enable either the “Stop Function” or “Stop Flow” alarm setting. Labels...
Page 97 Table 6–2: Mobile Phase screen parameters (continued) Parameter Function Value range Strk Vol The Preferred Stroke 130 µL (default) Seal Wsh Volume field sets the volume 100 µL (screen key) of solvent delivered with 50 µL each piston stroke. 25 µL You can override the default stroke volumes, but do not Off, 0.50 to 10.00...
Page 98 6.2.1.2 Set Gradient table parameter values The Gradient table allows you to make time-based changes to the composition of the mobile phase during a run. You can program up to 25 lines in the Gradient table. To set the parameters in the Gradient table: On the Mobile Phase screen, press the Gradient screen key to display the Gradient screen.
Page 99 Table 6–4: Gradient table parameters (continued) Parameter Function Value range Curve Sets the rate at which the Select the desired gradient solvent is to change to the new curve from the list of profiles, proportions and/or flow rates or select the curve number (1 (see “Gradient curves”...
Page 100: Set Sample Parameter Values
6.2.2 Set sample parameter values To enter parameters in the Sample screen: Press the Next or Prev screen key to display the Sample screen. Figure 6–6: Sample screen In the Sample screen, enter or select values in the Sample screen parameter fields. The following table describes the parameters in the Sample screen.
Page 101: Set Autosampler Parameter Values
Table 6–5: Sample parameters (continued) Parameter Function Value range Sample Draw Depth Adjusts the depth of the 0 to 20 needle tip to accommodate for in 1 mm increments sedimented samples or nonstandard vials. A value of 0 corresponds to the bottom of the vial.
Page 102 The following table describes the parameters in the Autosampler screen. Table 6–6: Autosampler parameters Parameter Function Value range Pre-column volume The sample management 0.0 to 10000.0 system starts the gradient and in 0.1 µL increments delivers this volume before making an injection. Use this parameter when transferring a method from a system that has a delay volume smaller than...
Page 103: Set Column Parameter Values
6.2.4 Set column parameter values To enter parameters in the Column screen: To display the Column screen, press Next or Prev. Figure 6–8: Column screen Enter values in the Column screen, as appropriate. The following table describes the parameters in the Column screen. Table 6–7: Column parameters Parameter Function...
Page 104: Set I/O Parameter Values
Table 6–7: Column parameters (continued) Parameter Function Value range Column Selection Selects a column position if a Position 1 through Position 3, 3-Column Valve column selection valve is or No Change installed. Tip: Select No Change when you link methods in a sample set and do not want to change the column from the previous method.
Page 105 To enter parameters in the I/O screen: Press the Next or Prev screen key to display the I/O screen. Figure 6–9: I/O screen Enter values in the I/O screen, as appropriate. The following table describes the parameters in the I/O screen. Table 6–8: I/O parameters Parameter Function...
Page 106 6.2.5.1 Edit the I/O Events table In the I/O Events table, you set the timing for the following events to occur during a run: • Changing the state of event switches • Setting the column temperature • Setting the sample compartment temperature •...
Page 107: Set Detector Parameters
In a standalone mode, System Controller or No Interaction, the separations module can control one Waters 2414 RI, 2410 RI, or 410 dRI detector and as many as two UV/Vis detector channels (Waters 2487 dual wavelength or 486 tunable absorbance detectors). Use the IEEE-488 interface bus to connect the separations module to the detector(s).
Page 108 The following table describes the selections in the Detectors screen. Table 6–11: Detector selections Parameter Function Selections Absorbance Detector (1) Enables a Waters 2487 or 2487, 486, or Not used 486 UV/Vis detector channel Absorbance Detector (2) Enables a second Waters 2487, 486, or Not used...
Page 109 111). parameters” on page 112. To enter control parameters for Waters detectors, refer to the “Setting absorbance parameters” and “Setting RI parameters” procedures that follow. 6.2.6.1 Set absorbance parameters To enter control parameters for a 2487 or 486 UV/Vis detector: Press the Abs (1) or Abs (2) screen key to display the appropriate absorbance detector screen.
Page 110 Enter detector parameters as required and click OK. See also: “Edit the detector table” on page 112 for steps on programming detector events. The following tables list the parameters for Waters 2487 and 486 detectors. Table 6–12: 2487 UV/Vis parameters Parameter Function Value range ...
Page 111 1. The Waters 2414 RI, 2410 RI, and 410 dRI detectors can control the temperature of one or two Waters Column Heater Modules (CHM) or column heater/cooler modules. These devices are separate from the column heater or column heater/cooler attached to the separations module.
Page 112 To enter events in the detector table: Press the Table screen key in the Detectors screen. The following figure shows the screen for a Waters 2487 detector using channels 1 and 2. Figure 6–15: Detector Events screen Enter parameters in the Detector Events screen and click Exit.
Page 113: Create And Edit A Sample Set
The following table describes the actions that you can specify with Waters 2487 and 486 tunable absorbance detectors. Table 6–16: 2487 and 486 detector actions Parameter Function Value range Set wavelength Sets the wavelength of the 2487: 190 to 700 in 1 nm detector.
Page 114 The following table describes the parameters in the Sample Set table. Table 6–17: Sample Set table parameters Parameter Function Value range Vials Selects the vial(s) from which 1 to 120 the injection is to be made. Use the “.” key to specify a vial range.
Page 115 Table 6–18: Sample Set functions (continued) Function name Description System Prep Performs the System Prep function using the parameters from the separation method. For more information on System Prep, see “Run System Prep” on page Purge Purges the sample management system. Press the Row Details screen key to enter additional purge parameters.
Page 116: Link Rows In A Sample Set
6.3.0.2 Auto Add Use the Auto Add function to perform an injection composed of aliquots from as many as 10 vials, one of which is the sample vial. The total volume of this injection must be less than the volume of the sample loop.
Page 117: Create A Sample Template
Figure 6–17: Linked rows in a sample set To link rows: Select the row you want to link. Press the More screen key, and then press the Make Link screen key. Result: The current row is linked with the previous row. To unlink lines: Select the row you want to unlink.
Page 118 February 27, 2018, 715003794 Rev. C Page 118...
Page 119: Maintenance Procedures
This procedure helps ensure that the mechanical and electrical components, as well as internal firmware, synchronize for proper operation. Spare parts To ensure that your system operates as designed, use only Waters Quality Parts. Visit www.waters.com/wqp for information about Waters Quality Parts, including how to order them.
Page 120: Safety And Handling
If you encounter a performance issue when using the Instrument Console, you can manually submit a Connections INSIGHT request to Waters customer support. Alternatively, you can use Remote Desktop, a real-time collaboration option that controls the two-way connection with the ACQUITY UPLC system by enabling the Connections INSIGHT iAssist service level.
Page 121: Maintain The Solvent Management System
Warning: The probe and source can be hot. To avoid burn injuries, take great care while working with these components. Notice: When performing maintenance inside the source enclosure, ensure that the following criteria are met: • Instrument is in Standby mode. •...
Page 122 Figure 7–1: Solvent management system components System Accumulator In-line transducer piston chamber filter Primary piston chamber TP01366A Prime/vent Inlet check Inlet check Primary seal wash valve valve valve transducer pump Table 7–1: e2695 solvent management system components and functions Component Function Primary piston chamber Draws in/expels solvent as part of serial flow...
Page 123: Remove The Head, Seal Wash Assembly, And Plunger
Table 7–1: e2695 solvent management system components and functions (continued) Component Function In-line filter Provides solvent filtering between the solvent management system and the sample management system. 7.5.2 Remove the head, seal wash assembly, and plunger For information about isolating problems in the solvent management system, see Chapter figure “Solvent management system piston chamber components (exploded view)”...
Page 124: Notes On Replacing The Plunger Seals
HPLC separations methods. If you find these seals perform unsatisfactorily for your applications, contact Waters for alternative plunger seals manufactured with graphite-filled PTFE material (WAT271066), or Alliance ClearSeal (700001326).
Page 125 7.5.3.1 Plunger seal replacement Notice: To avoid damaging the sealing surfaces, use the seal removal tool. Do not use a sharp tool to remove or install seals. To replace the plunger seal: Remove the head (see “Remove the head, seal wash assembly, and plunger” on page 123).
Page 126: Replace The Seal Wash Assembly Seals
After you replace the seals, observe the Head Removal and Replacement screen. Follow the on-screen instructions to reinsert the head, seal wash housing, washer, and plunger assemblies. Requirement: To avoid damaging the tube seals and to properly align the seal wash tubes with the seal wash housing, refer to step a through...
Page 127 Figure 7–4: Seal wash assembly seals seal wash face seal Tube seal (spring side faces Plunger wash Tube seal (spring seal (spring side side faces in) faces in) Wet the seal insertion tool, each new seal, and seal opening with 100% alcohol. Place the new plunger wash seal on the seal insertion tool with the spring side facing away from the seal insertion tool, and insert the tip of the tool into the seal opening, pushing firmly to install the seal.
Page 128: Clean And Replace The Plunger
7.5.5 Clean and replace the plunger Tip: For information about isolating problems in the solvent management system, see Chapter Required materials • Pumice, fine • Cotton swab • Water, HPLC-grade To clean the plunger: Remove the head, seal wash assembly, and plunger (see “Remove the head, seal wash assembly, and plunger”...
Page 129: Replace An Inlet Check Valve Cartridge
Inspect the ends of the seal wash tubes for damage. If necessary, use a razor blade to trim the ends. Reinsert the seal wash tubes into the manifold, and then fully tighten the head nut. Return the release ring to the run position. If the separations module’s solvent lines are empty, dry prime the solvent management system before you wet prime it or start delivering solvents (see “Dry prime the system”...
Page 130 Figure 7–6: Inlet check valve Check valve cartridge Check valve housing TP01367 Tip the inlet check valve housing upside down to dislodge the old check valve cartridge. Inspect the inlet check valve housing, clean it, if necessary, and wet it with 100% alcohol. Wet the new check valve cartridge with 100% alcohol.
Page 131: Replace The In-Line Filter
7.5.7 Replace the in-line filter The in-line filter provides solvent filtering between the solvent management system and sample management system. Clean and replace the in-line filter element when it is the source of elevated backpressure. Notice: It is important to maintain proper functioning of the in-line filter. Damage to downstream components can occur if you replace the in-line filter with a union, or remove the filter element.
Page 132 Figure 7–7: Replacing the in-line filter Inlet housing Outlet housing In-line filter element Reconnect the in-line filter inlet and outlet housings. Re-tighten the compression screw into the inlet housing. Flush the solvent management system with mobile phase at 1 mL/min for 10 minutes (see “Power-off the separations module”...
Page 133: Maintain The Sample Management System
Maintain the sample management system Perform the procedures in this section when you determine there is a problem with a sample management system component. Tip: For information about isolating problems in the sample management system, see Chapter Warning: To prevent injury, always observe good laboratory practices when you handle solvents, change tubing, or operate the separations module.
Page 134 Figure 7–9: Sample management system components (right-side view) Sample loop transducer Injector Sample loop assembly Table 7–2: e2695 sample management system assemblies/components Assembly/component Function Syringe Draws sample from the sample vial into the sample loop. Sample carousels and carousel carrier Five, color-coded carousels.
Page 135: Remove Side Panel Of Separations Module
7.6.1 Remove side panel of separations module Some maintenance procedures, such as sample loop and needle and seal-pack replacement, require that you remove the side panel of the separations module to access internal components. Note: This section only applies if you are using the column heater or column heater/cooler option and need to remove that unit to access the side panel on the separations module.
Page 136: Replace The Lower Needle Wash Frit (If So Equipped)
Open the column heater or column heater/cooler door and route the column tubing through the clip on the front of the unit. Slide the drip tray back into the bottom of the column heater or column heater/cooler compartment and close the door on the unit. Align the left side of the detector base with the two feet on the right side of the solvent bottle tray and lower the right side of the base into position.
Page 137 The plunger tip becomes worn • You want to change to the optional 25-µL or 2500-µL syringe size Tip: To ensure accurate results, use only syringes approved by Waters for use in the sample management system. Required material Replacement syringe February 27, 2018, 715003794 Rev.
Page 138 To remove the syringe: Set the flow rate to 0.000 mL/min. Open the syringe compartment door. Remove the knurled nut that holds the syringe barrel to the syringe mounting bracket. Figure 7–11: Syringe assembly components Syringe mounting bracket Syringe Knurled nut On the Main screen, press the Diag screen key.
Page 139 7.6.2.2 Install a new syringe If you need to install a syringe different in size from the original syringe, refer to the following table for available syringe sizes. Be sure to enter the new syringe size in the Configuration screen. (See page 140.) Notice:...
Page 140 Push the syringe plunger down so that the threaded end slides through the syringe guide mounting bracket. Hand tighten the syringe. On the Diagnostics screen, press Other Tests. 10. In the Other Tests screen, select the Syringe Position field, and then press Enter to display the drop-down list.
Page 141: Replace The Injector Needle And Seal Pack
7.6.3 Replace the injector needle and seal pack Replace the injector needle and seal pack when these conditions apply: • The needle is bent. • The tip of the needle is damaged. • Contaminant peaks appearing in the chromatogram cannot be corrected by changing the wash solvent in the needle wash system.
Page 142 Figure 7–13: Injector assembly components Injector motor cable Sensor cable Needle tee Compression screw Needle Upper frit retainer Captive mounting screws Seal pack assembly (exploded view) To access the injector assembly: Note: This procedure applies only to a separations module with no column heater or column heater/cooler option installed.
Page 143 Figure 7–14: Remove the injector assembly Injector motor cable and interconnect Optical switch cable Sample loop fitting Needle wash lines Restrictor loop Column fluidic Captive mounting line screws (2) To remove the injector: Cut and remove the tie wraps securing the needle wash lines and restrictor loop. Disconnect the column fluid line between the injector assembly and the column.
Page 144 To remove the seal pack with needle from the injector assembly: With the injector assembly removed from the separations module, use the two 5/16-inch open-end wrenches to disconnect the injector needle from the needle tee. Tip: Use one 5/16-inch wrench to hold the tee, while you use the second one to loosen the compression screw that secures the needle to the tee (see figure “Injector assembly components”...
Page 145 Figure 7–15: Align the needle port Injector base M3 seal pack (bottom view) screws (4) Seal pack Scallop marking assembly needle port alignment Restrictor loop connection Column fluidic line Needle wash connection lines To install the seal pack with needle: With the seal pack with needle removed from the injector, rotate the injector’s motor shaft until the needle tee is furthest from the injector base (see figure “Injector assembly...
Page 146 To reinstall the injector assembly: Notice: To avoid damaging the injector and/or needle wash tubing, ensure that the tubing is not pinched between the injector and the compartment floor when you reinstall the injector assembly. Lower the injector so that its base seats in the compartment floor, and then secure it with the two captive screws (see figure “Remove the injector assembly”...
Page 147: Clean The Sample Compartment
7.6.4 Clean the sample compartment Clean the sample compartment when these conditions apply: • The sample carousels do not turn properly. • The carousel carrier does not engage. Required materials • Wrench, hex, 3-mm • Nonabrasive cleaner To access the sample compartment: Open the sample compartment and syringe cover doors (see figure “Sample management system components (front view)”...
Page 148 To clean the sample compartment: Push down about 1/4-inch on the spring-retaining pin in the center of the carousel carrier to loosen the carousel carrier (see figure “Sample compartment components” on page 148). Figure 7–17: Sample compartment components Sample carousel motor Carrier carousel motor...
Page 149: Diagnostic Testing And Troubleshooting
Chapter 8.0.1.1 Spare parts To ensure that your instrument operates as intended, use only Waters Quality Parts (WQP). Visit www.waters.com/wqp for more information about WQP, including how to order them. Safety and handling When you troubleshoot the separations module, keep the following safety considerations in mind:...
Page 150: Use The Error Log
Diagnostics screen – Accesses certain system status information and diagnostic and performance tests for the solvent management system and the sample management system; also accesses utilities for Waters Technical Service representatives. • Other Diagnostics screen – Accesses diagnostic tests and functions that allow the user to perform maintenance procedures and control optional devices (such as the column heater, column heater/cooler, and sample heater/cooler).
Page 151: Exit The Diagnostics Screens
Figure 8–1: Diagnostics screen: 8.3.1 Exit the Diagnostics screens To avoid unexpected behavior, ensure that you exit from all diagnostic screens before you allow external control of the separations module via Empower, MassLynx, or other third-party system control. 8.3.1.1 Diagnostics screen fields The fields in the Diagnostics screen include: •...
Page 152: Prime The Plunger Seal Wash Pump
Table 8–1: Diagnostics screen keys (continued) Screen key Function Reference Service Utilities Displays a password-protected “Service utilities diagnostic list of service diagnostics tests” on page 156 Other Tests Displays the Other Diagnostics “Perform other diagnostic tests screen, which displays more and functions”...
Page 153 Perform a compression check if you suspect seal pack leakage. Symptoms of seal pack leakage include: • Dripping in the sample chamber • Abnormally low system pressure To perform the Compression Check diagnostic test: Install on the red outlet line a restrictor that generates at least 3447.4 kPa (34.5 bar, 500 psi) of backpressure when the solvent flow is 3.0 mL/min or less.
Page 154: Prime The Needle Wash Pump
8.3.4 Prime the needle wash pump The prime needle wash pump test procedure automatically primes the needle wash system and homes the needle into the stream position. Perform this procedure if these conditions apply: • A lack of flow in the needle wash system •...
Page 155 Ramp system pressure up to 31026.4 kPa (310.3 bar, 4500 psi); hold this pressure for 2 minutes. Repeat the adjust seals test. If the test fails again: Stop solvent flow. Monitor the displayed pressure value (which must be stable and approaching zero). Contact Waters Technical Service. February 27, 2018, 715003794 Rev. C Page 155...
Page 156: Service Utilities Diagnostic Tests
8.3.6 Service utilities diagnostic tests Service utilities diagnostic tests are for use by Waters Technical Service representatives only. These utilities are password-protected. Local injections can be disabled on the Service Utilities page by selecting Lock Run Samples, which will require a service call.
Page 157: Perform The Motors And Valves Diagnostic Test
“Column heater or column heater/cooler column heater/cooler module. heater/cooler test” on page 167 Defrost column cooler Defrosts the column Waters Alliance Series heater/cooler module. Column Heater and Column Heater/Cooler Operator’s Guide Rebuild injector valves Tests valves V1 and V2. “Rebuild injector valves function”...
Page 158 Figure 8–2: Motors and Valves diagnostic screen To control the needle or syringe drive: Select the motor position parameter (needle position or syringe position) that you want to change in the Motors and Valves screen. Press Enter to display a drop-down list of parameter values. Select the value you want, and then press Enter to save your selection.
Page 159: Sensors Diagnostic Function
8.4.1.1 Control the GPV valve The following table describes the valve positions available in the GPV drop-down list. Table 8–6: GPV position parameters GPV off Solvent A GPV selecting solvent from reservoir A Solvent B GPV selecting solvent from reservoir B Solvent C GPV selecting solvent from reservoir C Solvent D...
Page 160 The following table describes the function of each sensor when it is selected (enabled). Table 8–7: Sensors diagnostic parameters Component Sensor Description Carousel Home The carousel that is under the injector is in the home position. The carousel that is under the injector is identified during the injection sequence.
Page 161: Static Leak Test
8.4.3 Static leak test The static leak test performs a solvent pressure ramp up, and monitors the pressure decay in the solvent delivery system to determine whether the inlet check valves, tubing, fittings, or plunger seals are faulty. Perform the Static Leak test when these conditions apply: •...
Page 162: Valve Leak Test
• Compression fittings and head nuts are tight. • The appropriate solvent (methanol or water is recommended) was used. If necessary, ensure a good seal around the plunger and head surface by flowing at high pressure before rerunning the test. For example, install a restrictor sufficient to achieve 27579 to 31026 kPa (4000 to 4500 psi) backpressure at a flow rate of 0.5mL/min.
Page 163: Head Removal And Replacement Function
8.4.4.1 Required materials • Pin plug • Wrenches, 5/16-inch, open-end, two • Waste container To perform the valve leak test: In the Other Diagnostic screen, select Valve leak test, and then press the OK screen key. Tip: You can send the system pressure ramp and decay to an external device using the System Pressure Chart Output parameter.
Page 164: Inputs And Outputs Test
8.4.6 Inputs and outputs test The inputs and outputs test allows you to view the status of the input and output connections on the I/O connectors of the separations module. To perform the inputs and outputs test: In the Other diagnostics screen, select Inputs and outputs, and then press the OK screen key.
Page 165: Keypad Test
Confirm that the input to which the jumper wires are connected changes state (circles change from empty to full) as the switch is toggled. 8.4.7 Keypad test The keypad test tests the keypad keys. You press the key on the keypad and observe the key as it changes appearance in the Keypad diagnostic screen.
Page 166: Sample Heater/Cooler Test
8.4.10 Sample heater/cooler test The sample heater/cooler test tests the sample heater/cooler for proper functioning. It ramps down the temperature in the sample heater/cooler and then measures the rate at which the temperature rises. You can monitor the sample heater/cooler temperature by connecting a chart recorder to the Chart Out terminals on the rear panel of the separations module.
Page 167: Column Heater Or Column Heater/Cooler Test
To run the defrost sample heater/cooler function: In the Other Diagnostics screen, select Defrost, and then press OK. Press Start. Open the sample compartment door when you are prompted. Requirement: To complete this procedure, the sample compartment door must remain open during the entire procedure.
Page 168: Rebuild Injector Valves Function
8.4.13 Rebuild injector valves function Run the rebuild injector valves function to rebuild and test the sample loop and syringe valves, V1 and V2, respectively. The function causes the valve motor to move forward and reverse so that you can remove and replace valve components.
Page 169: Troubleshoot
Troubleshoot This section provides information for troubleshooting the separations module. It addresses these topics: • General troubleshooting hints • Chromatography troubleshooting • Hardware troubleshooting for the: – System – Solvent management system – Sample management system Tip: “Safety and handling” on page 149, for information on safety and handling considerations while you troubleshoot the separations module.
Page 170: When To Call Waters Technical Service
8.5.2 When to call Waters Technical Service You can correct many problems with a Alliance e2695 Separations Module relatively easy. However, if you cannot correct a condition, contact Waters Technical Service. When you do, have the following information available: •...
Page 171: Troubleshoot Chromatography Problems
8.5.3 Troubleshoot chromatography problems Warning: Always observe good laboratory practices when you handle solvents, change tubing, or operate the separations module. Know the physical and chemical properties of the solvents you use. Refer to the Material Safety Data Sheets for the solvents in use. The following table lists chromatographic symptoms, possible causes, and suggested corrective actions for the separations module.
Page 172 Table 8–9: Chromatography troubleshooting (continued) Symptom Possible cause Corrective action Decreased retention times Incorrect flow rate Change flow rate. Incorrect solvent composition Change composition, evaluate GPV performance. High column temperature Reduce column temperature. Incorrect mobile phase Replace with correct mobile phase.
Page 173 Table 8–9: Chromatography troubleshooting (continued) Symptom Possible cause Corrective action Baseline noise cycling, short Flow fluctuating Stabilize flow by checking for term leaks and for proper (30 to 60 sec) degassing (see “Erratic retention times” symptom). Solvent not well mixed (short- Stir solvent.
Page 174 Table 8–9: Chromatography troubleshooting (continued) Symptom Possible cause Corrective action Carryover is seen from Previous injection is Calculate mass injected. Try previous injection exceedingly high in re-injecting a larger volume of concentration less concentrated solution while keeping injected mass constant. Choice of needle wash solvent Choose a needle wash solvent is insufficient to solubilize all...
Page 175: Troubleshooting Hardware Problems
Table 8–9: Chromatography troubleshooting (continued) Symptom Possible cause Corrective action Sensitivity loss Leak in sample management Troubleshoot sample system management system. Degraded, contaminated, or Use fresh sample. improperly prepared sample Column contaminated Clean/replace column. Loss of column efficiency Clean/replace column. Peaks wider than expected Troubleshoot sample management system.
Page 176 Power cord not connected Establish line voltage is present. No power at outlet No response from keypad. Keypad broken Call Waters Technical Service. Screen is blank Unit not powered-on Power-on unit. Start-up diagnostic test failure Power-off and power-on again. If the failure is not corrected, call Waters Technical Service.
Page 177 8.5.4.2 Solvent management system troubleshooting The following table provides troubleshooting suggestions for solvent management system problems in the separations module. Table 8–11: Solvent management system troubleshooting Symptom Possible cause Corrective action Plunger head leaks solvent Worn plunger seals Replace plunger seals (see “Notes on replacing the plunger seals”...
Page 178 Diagnostics screen. Valve V3 (waste valve) faulty Call Waters Technical Service. Leak at seal pack Damaged needle, seal pack Replace needle and seal pack (see “Replace the injector needle and seal pack”...
Page 179 “Clean the sample compartment” on page 147). Carousel malfunction Carousels do not turn properly Clean/replace the carousel. Defective carousel sensor Call Waters Technical Service. Carousel carrier obstructed or Check for obstructions. Clean contaminated sample compartment (see “Clean the sample compartment” on page 147).
Page 180 Table 8–12: Sample management system troubleshooting (continued) Symptom Possible cause Corrective action Needle bottom sensor alarm Bent needle. Needle hitting Replace needle (see “Replace bottom of vial the injector needle and seal pack” on page 141). Adjust the syringe draw depth (see “Set sample parameter values”...
Page 181: A Safety Advisories
Heed all warnings when you install, repair, or operate any Waters instrument or device. Waters accepts no liability in cases of injury or property damage resulting from the failure of individuals to comply with any safety precaution when installing, repairing, or operating any of its instruments or devices.
Page 182: Specific Warnings
A.1.1.2 Biohazard warning The following warning applies to Waters instruments and devices that can process material containing biohazards, which are substances that contain biological agents capable of producing harmful effects in humans. Warning:...
Page 183: Notices
A.1.1.3 Biohazard and chemical hazard warning These warnings apply to Waters instruments and devices that can process biohazards, corrosive materials, or toxic materials. Warning: To avoid personal contamination with biohazards, toxic materials, or corrosive materials, you must understand the hazards associated with their handling.
Page 184: Required Protection
Use eye protection when refilling or replacing solvent bottles. Requirement: Wear clean, chemical-resistant, powder-free gloves when handling samples. Warnings that apply to all Waters instruments and devices When operating this device, follow standard quality-control procedures and the equipment guidelines in this section.
Page 185 Warning: 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 tubing that has been severely stressed or kinked. • Do not use nonmetallic tubing with tetrahydrofuran (THF) or concentrated nitric or sulfuric acids.
Page 186 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。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。 경고: 가압 폴리머 튜브로 작업할 경우에는 주의하십시오. • 가압 폴리머 튜브 근처에서는 항상 보호 안경을 착용하십시오. • 근처의 화기를 모두 끄십시오. •...
Page 187: Warnings That Address The Replacing Of Fuses
警告：使用者必須非常清楚如果設備不是按照製造廠商指定的方式使用，那麼該設備所提供的保護將被消弱。警告：使用者必须非常清楚如果设备不是按照制造厂商指定的方式使用，那么该设备所提供的保护将被削弱。 경고 : 제조업체가 명시하지 않은 방식으로 장비를 사용할 경우 장비가 제공하는 보호 수단 이 제대로 작동하지 않을 수 있다는 점을 사용자에게 반드시 인식시켜야 합니다 . 警告：ユーザーは、製造元により指定されていない⽅法で機器を使⽤すると、機器が提供し...
Page 188 警告：⽕災予防のために、ヒューズ交換では機器ヒューズカバー脇のパネルに記載されているタイプおよび定格のヒューズをご使⽤ください。 Finding fuse types and ratings when that information does not appear on the instrument or device Warning:...
Page 189: Electrical And Handling Symbols
Electrical and handling symbols A.7.1 Electrical symbols The following electrical symbols and their associated statements can appear in instrument manuals and on an instrument’s front or rear panels. Symbol Description Electrical power on Electrical power off Standby Direct current Alternating current Alternating current (3 phase) Safety ground Frame, or chassis, terminal...
Page 190: Handling Symbols
A.7.2 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 February 27, 2018, 715003794 Rev.
Page 191: B External Connections
External Connections This section provides instructions for making all external connections to the Alliance e2695 Separations Module. Connect to the electrical power supply Proper operation of the separations module requires that the AC power supply be grounded and that has no abrupt voltage fluctuations.
Page 192 Figure B–1: Rear panel of the separations module I/O connectors RS-232 connector Ethernet connector Column heater or IEEE-488 column heater/cooler Power connector connector TP03529 February 27, 2018, 715003794 Rev. C Page 192...
Page 193: Connect Tubing And Attachments
Connect tubing and attachments START Install solvent Install detector and supply tubing drip tray Connect column Connect waste line and detector tubing and reservoir Prime needle wash, Install needle wash seal wash, and the and seal wash system reservoirs Notice: To ensure proper drip protection, be sure that the retaining screws in the solvent delivery and solvent conditioning trays are tightened before you operate the system.
Page 194 Required materials • Diagonal cutters • Screwdriver, T-20 TORX To replace the feet and install the bottle tray/detector base: Disconnect all cabling and other connections from the detector. Tip the detector so that it rests on its side. Using a T-20 TORX screwdriver, remove the securing screws and rubber feet from the chassis.
Page 195: Install A- And C-Series Drip Tray And Support Bracket
Seat the detector on the detector base. Make sure the end of the detector drip tray tubing protrudes from the detector drain fitting into the black-colored drip tray, as shown in the figure below. In detectors with longer drip tray tubing, you may need to bend the tubing slightly to the right or left to ensure that the end of the tubing lays inside the drip tray.
Page 196 If the detector is a 2424, remove the barbed fitting from the underside of the detector, as this interferes with the mounting of the drip tray. Remove the T-10 screw located on the bottom right corner of the detector front panel and set it aside.
Page 197 To install the detector support bracket and detector: Power-off the separations module and disconnect the column module cable from the rear panel of the separations module. Refer to the figure below for locations referenced in the rest of this procedure. Figure B–5: Detector support bracket installed on rear of separations module Foot of detector in bracket...
Page 198 Figure B–6: A- and C-series detector drain routing without drip tray Drain fitting Tube routing location End of tube Syringe drip tray February 27, 2018, 715003794 Rev. C Page 198...
Page 199: Install Solvent Supply Tubing
Figure B–7: A- and C-series detector drain routing with drip tray Drain fitting location on drip tray Tube routing End of tube Syringe drip tray B.2.3 Install solvent supply tubing The tubing from the separations module to the solvent reservoirs is coiled and stored in front of the separations module, near the syringe compartment.
Page 200: Make Waste Line Connections
To install the solvent lines: Uncoil the solvent lines stored in front of the separations module, near the syringe compartment. Remove the filter from each solvent line and insert the free end of each solvent line through a reservoir cap (start-up kit). Reinstall a filter on the end of each solvent line.
Page 201: Install Needle Wash And Plunger Seal Wash Reservoirs
B.2.4.2 Condensation waste line The condensation waste outlet from the sample compartment is also located under the front panel of the separations module, to the right of the spill tray waste outlet. In addition to condensate from the sample heater/cooler, the condensation waste can contain spills from leaking sample vials. To install the condensation waste line: Cut a length of 1/4-inch ID tubing (start-up kit) long enough to reach from the condensation waste outlet to the front of the bench.
Page 202: Connect The Column
Remove the diffuser from the plunger seal wash inlet line (clear, labeled “Pump Wash In”), insert the line through a reservoir cap, reinstall the diffuser on the end of the line, and install the cap on the plunger seal wash reservoir. Place the plunger seal wash waste line (clear) in a suitable waste container.
Page 203: Connect The Column Selection Valve
Figure B–8: Side view of frame openings for tubing connections (side panel removed) Hole for detector drip tray waste line Syringe access door B.2.7 Connect the column selection valve These optional column selection valves switch the flow path among multiple columns: •...
Page 204 Restrictor 3 2 1 Waste In from Out to Alliance e2695 detector Install the 3-column selection valve after installing the column heater or column heater/cooler. (See “Install the column heater and column heater/cooler” on page 79.) Required material Open-end wrench, 5/16-inch...
Page 205 63.) Figure B–10: Six-column selection valve connections (example) Column 1 Column 2 Column 3 Column 4       Waste In from Out to Alliance e2695 detector February 27, 2018, 715003794 Rev. C Page 205...
Page 206: Connect The Detector
The 2-column regeneration valve is shipped separately with a coil of 0.010-inch PEEK tubing, a 0.0025-inch ID restrictor tube, and SealTight fittings. Connect as appropriate for your application. (See figure “Column regeneration valve connections (example):” on page 66.) Contact Waters Technical Service for more information. Figure B–11: Column regeneration valve connections (example) In from...
Page 207: Signal Connections
Signal connections The rear panel of the separations module provides connection terminals and communication ports for operation with external devices. (See figure “Rear panel of the separations module:” on page 48.) The signal connections you need to make to your separations module depend on these considerations: •...
Page 208: I/O Signal Connections
Figure B–12: Connect signal cables Start Connect to non-IEEE, Install event and non-Ethernet detector I/O cable(s) or an external auto- sampler? Connect to Install RS-232 non-Waters data cable system? Install IEEE-488 Connect to cable IEEE-488 bus? Connect to Install Ethernet Ethernet bus?
Page 209 Figure B–13: I/O signal connectors Connector A Connector B Inject Start Switch 1 Ground Ground Stop Flow Switch 2 Hold Inject 1 Switch 3 Hold Inject 2 Switch 4 Ground Ground Chart Out Run Stopped B.3.1.1 I/O signals The following table describes the signals sent via the I/O connectors of the separations module. Appendix C for information about the electrical specifications of each signal.
Page 210 Table B–4: I/O Signals (continued) Signal Description Chart out Outputs the analog signal (specified in the current separation method, or default separation method) to a device such as an integrator or strip chart recorder. (See “Set I/O parameter values” on page 162.) You can select one of the following signals as the chart out signal: •...
Page 211: Digital Signal Connections
Alternatively, when the separations module is in the System Controller mode, you can use the RS-232 port to transmit data to external devices (a Waters 746 Data Module or serial printer, for example). You can transmit two types of data over this interface: •...
Page 212 Where the separations module and data system communicate via IEEE-488, and include one or more detectors that can only communicate with the data system via Ethernet (for example, Waters 2998, 2489, 2475, 2424, e-SAT/IN module-connected, or 3100 Mass Spectrometer Detector.
Page 213: Add Hardware Options
37.) Tip: For help with HPLC Ethernet connections, you can order from Waters the Ethernet Switch Communications Kit (700004123). The kit includes an 8-port Ethernet switch, cables, and a mounting bracket, for mounting the switch on the rear panel of the separations module or 1500 series HPLC pump.
Page 214: Install An Optional Sample Loop
B.4.1 Install an optional sample loop To inject sample volumes greater than 100 µL, install an optional sample loop. To do so, you must perform these tasks: • Remove the right-hand side panel • Install the sample loop • Refit the right-hand side panel Required materials •...
Page 215 B.4.1.1 To install a sample loop: Use the 5/16-inch wrench to disconnect the sample loop from the pressure transducer, and the needle tee on the injector assembly. (See figure “Disconnect the sample loop:” on page 78.) Connect one end of the sample loop to the needle tee. Connect the open end of the new sample loop to the pressure transducer.
Page 216: Install The Column Heater And Column Heater/Cooler
A connector on the rear panel of the separations module provides power and signal connections to the column heater or column heater/cooler. See the Waters Alliance Series Column Heater and Column Heater/Cooler Operator’s Guide for procedures explaining the installation, operation, maintenance, and basic troubleshooting of the Waters Alliance series column heater and column heater/cooler instruments.
Page 217: C Specifications
Alliance e2695 Separations Module Site Preparation Guide, or contact the Waters Technical Service organization for additional information about specifications. Physical specifications The following table lists the physical specifications for the Alliance e2695 Separations Module. Table C–1: Physical specifications Attribute Specification Height 57.1 cm (22.5 inches)
Page 218: Environmental Specifications
Solvents consistent with materials of construction. Salts and buffers can reduce seal life, especially at pressures in excess of 3000 psi. Electrical specifications The following table lists the electrical specifications for the Alliance e2695 Separations Module. Table C–3: Electrical specifications Attribute Specification Power requirements...
Page 219 Table C–3: Electrical specifications (continued) Attribute Specification Hold inject (input) Two sets of (+, -) terminals that allow other LC devices to delay an injection Boolean operator selects whether one or both inputs delay the injection Input voltage range: ±30 Vdc Logic high = >...
Page 220: Solvent Management Specifications
Solvent management specifications The following table lists the solvent management specifications for the Alliance e2695 Separations Module. Table C–4: Solvent management system specifications – e2695 XE configuration Attribute Specification Number of solvents One to four Solvent conditioning Vacuum degas, four chambers ≈...
Page 221: Sample Management Specifications
Sample management specifications The following table lists the sample management specifications for the Alliance e2695 Separations Module. Table C–5: Sample management specifications Item Specification Number of sample vials 120 vials configured in 5 carousels of 24 vials each Number of sample injections 1 to 99 injections per sample vial Sample delivery precision Typically <...
Page 222 February 27, 2018, 715003794 Rev. C Page 222...
Page 223: D Solvent Considerations
• When using the instrument for general chromatography (that is. reverse and/or normal phase and gel permeation [GPC]), Waters recommends the use of high-quality lab glassware made of borosilicate glass for all reservoirs (solvent, seal wash & needle wash). •...
Page 224: Preparation Checklist
D.1.4 Preparation checklist The following solvent preparation guidelines help to ensure stable baselines and good resolution: • Filter solvents with a 0.45-µm filter. • Degas the solvent. • Stir the solvent. • Keep solvents in a location that is free from drafts and shock. D.1.5 Water Use water only from a high-quality water purification system.
Page 225: Solvent Compatibility
To prevent premature plunger seal failure when using toluene, o-dichlorobenzene (o-DCB), or 1,2,4 trichlorobenzene (TCB), Waters recommends the use of the optional Alliance Clear Seals, the removal of installed seal wash seals, and no seal wash solvent. The use of graphite-filled PTFE (black) seals is not recommended when any salts are present in the solvent.
Page 226: Solvents To Use
Information on the use of a specific solvent or concentration that is not listed in this manual can be obtained by contacting Waters. Table D–2: Aqueous buffers for use with the separations module...
Page 227 Phenol Amyl acetate Dibutyl phathalate Hexane Tetrahydrofuran (THF) Benzaldehyde Dimethyl formamide iso-Octane Toluene Benzene Dimethyl sulfoxide iso-Propanol Waters PIC Reagents Benzyl alcohol Ethanol Lysine hydrochloride Xylene Butanol Ethyl acetate Methanol Carbon Ethylene dichloride Methyl ethyl ketone tetrachloride February 27, 2018, 715003794 Rev. C...
Page 228: Solvent Miscibility
Solvent miscibility Before you change solvents, refer to the following table to determine the miscibility of the solvents to be used. When you change solvents, be aware of these issues: • Changes involving two miscible solvents are made directly. Changes involving two solvents that are not totally miscible (for example, from chloroform to water), require an intermediate solvent (such as methanol).
Page 229: How To Use Miscibility Numbers (M-Numbers)
Table D–6: Solvent miscibility Boiling  cut-off Polarity Viscosity Miscibility Solvent point °C index CP, 20 °C number (M) (nm) (@1 atm) Dioxane 1.54 101.3 Ethanol 1.20 78.3 Pyridine 0.94 115.3 Nitroethane 0.68 114.0 –– –– Acetone 0.32 56.3 15, 17 Benzyl alcohol 5.80 205.5...
Page 230: Buffered Solvents
Buffered solvents When using a buffer, use a good quality reagent and filter it through a 0.45-µm filter. Do not leave the buffer stored in the system after use. Flush all flow pathways with HPLC-quality water before shutting down the system and leave distilled water in the system (flush with 90% HPLC-quality water/10% methanol for shutdowns scheduled to be more than one day).
Page 231: Gas Solubility
D.7.1 Gas solubility Only a finite amount of gas can be dissolved in a given volume of liquid. This amount depends on these factors: • The chemical affinity of the gas for the liquid. • The temperature of the liquid. •...
Page 232: Wavelength Selection
D.7.2.1 Vacuum degassing considerations The longer a solvent is exposed to a vacuum, the more dissolved gases are removed. Two factors affect the amount of time the solvent is exposed to the vacuum: • Flow rate – At low flow rates, most of the dissolved gas is removed as the solvent passes through the vacuum chamber.
Page 233: Mixed Mobile Phases
TRIS HCl, 20 mM, 202, 212 pH 7.0, pH 8.0 HEPES, 10 mM, pH 7.6 Triton X-100, 0.1% Hydrochloric acid, 0.1% Waters PIC Reagent A, 1 vial/liter MES, 10 mM, pH 6.0 Waters PIC Reagent B-6, 1 vial/liter Potassium phosphate,...
Page 234: Refractive Indices Of Common Solvents
D.8.3 Refractive indices of common solvents The following table lists the refractive indices for some common chromatographic solvents. Use this table to verify that the solvent you intend to use for your analysis has a refractive index (RI) significantly different from the RIs of the sample components. Table D–9: Refractive indices for common chromatographic solvents Solvent Solvent...

Waters Alliance e2695 Operator's Manual

1 Getting Started with the Alliance E2695 Separations Module

2 Install the E2695 Separations Module

3 Prepare the E2695 Separations Module for Operation

4 Front Panel Control

5 Automatic Runs

6 Create Methods, Sample Sets, and Sample Templates

7 Maintenance Procedures

8 Diagnostic Testing and Troubleshooting

A Safety Advisories

B External Connections

C Specifications

D Solvent Considerations

Quick Links

Troubleshooting

Need help?

Questions and answers

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Summary of Contents for Waters Alliance e2695