Waters 1500-Series Operator's Manual
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Waters 1500-Series Operator's Manual

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Waters 1500-Series
HPLC Pump and Options
Operator's Guide
715002013IVD /Revision C
Copyright © Waters Corporation 2014
All rights reserved

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Summary of Contents for Waters 1500-Series

  • Page 1 Waters 1500-Series HPLC Pump and Options Operator’s Guide 715002013IVD /Revision C Copyright © Waters Corporation 2014 All rights reserved...

  • Page 2: Copyright Notice

    Corporation assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with, or arising from, its use.

  • Page 3: Customer Comments

    Contacting Waters ® Contact Waters with enhancement requests or technical questions regarding the use, transportation, removal, or disposal of any Waters product. You can reach us via the Internet, telephone, or conventional mail. Waters contact information Contacting medium Information...

  • Page 4: Considerations Specific To The 1500-Series Hplc Pumps And Column Heater

    Tubing connected to this drain directs the flow to an appropriate waste container. Equipment repair or disposal Direct questions regarding repair or disposal to Waters. Waters carries out equipment disposal in Europe according to the WEEE directive specific to the...

  • Page 5: Safety Advisories

    Waters also accommodates any special requirements for locations outside of Europe. Safety advisories Consult Appendix A for a comprehensive list of warning and caution advisories. Operating the Waters 1500-Series HPLC pump and options When operating these pumps and options, follow standard quality-control...

  • Page 6: Applicable Symbols

    Intended use of the Waters 1500-Series HPLC pump and options Use the Waters 1525, 1525µ, and 1525EF HPLC pumps and the optional 1500-Series Column Heater and its options to deliver a precisely controlled amount of solvent to a column, maintaining a consistent and reproducible mobile phase composition.

  • Page 7: Calibrating

    When analyzing samples from a complex matrix such as soil, tissue, serum/plasma, whole blood, and other sources, note that the matrix components can adversely affect LC/MS results, enhancing or suppressing ionization. To minimize these matrix effects, Waters recommends you adopt the following measures: •...

  • Page 8: Ism Classification

    Class B products are suitable for use in both commercial and residential locations and can be directly connected to a low voltage, power-supply network. EC authorized representative Waters Corporation Stamford Avenue Altrincham Road Wilmslow SK9 4AX United Kingdom...

  • Page 9: Table Of Contents

    Customer comments .................... iii Contacting Waters ....................iii Safety considerations ..................iii Considerations specific to the 1500-Series HPLC pumps and column heater iv Safety advisories ....................v Operating the Waters 1500-Series HPLC pump and options ...... v Applicable symbols ....................v Audience and purpose..................
  • Page 10 Connecting the pump outlet ................2-14 Connecting fluid waste lines ................. 2-16 3 Installing Options and Accessories ........... 3-1 Installing the 1500-series column heater ............ 3-2 Installing a manual injector ................3-4 Connecting to the column or column heater........... 3-5 Connecting the inject start signal (for manual injector)........
  • Page 11 Operating with the integral vacuum degasser..........4-5 Operating with the plunger seal wash system..........4-8 Maximum flow rates for 1500-series pumps ..........4-10 Preparing for Breeze 2 operation ............... 4-10 Priming and purging the pump via Breeze 2 control........4-11 Purging the flow path via Breeze 2 control ..........
  • Page 12 Maintenance considerations ................5-2 Safety and handling..................5-2 Proper operating procedures ................5-2 Spare parts ....................... 5-2 Contacting Waters Technical Service ............. 5-2 Performing pump diagnostic tests ............... 5-3 Retention time stability test ................5-3 Ramp-and-decay test ..................5-3 Replacing and cleaning plunger seals and plungers ........ 5-6 Preparing for plunger seal replacement ............
  • Page 13 Warnings that apply to all Waters instruments ......... A-6 Electrical and handling symbols ..............A-12 Electrical symbols ..................A-12 Handling symbols ..................A-13 B Specifications ..................B-1 C Solvent Considerations ............... C-1 Introduction ...................... C-2 Clean solvents ....................C-2 Solvent quality ....................C-2 Preparation checklist..................
  • Page 14 Table of Contents...
  • Page 15 Introduction This chapter describes key operating principles, pump components, data control configurations, and available options for the 1500-series HPLC pumps. Contents Topic Page HPLC pump operating principles Overview of the 1500-series HPLC pumps Fluid-handling components Electronic components 1-12 Pump control...

  • Page 16: Introduction

    Because the mixer is downstream of the pumps, the gradient is created under high pressure. Other HPLC systems, such as Waters Alliance systems, are designed to mix multiple streams of solvents under low pressure, ahead of a single pump. A gradient proportioning valve selects from the multiple solvent bottles, changing the strength of the mobile phase over time.

  • Page 17: Effects Of Dissolved Oxygen On The Mobile Phase

    Effects of dissolved oxygen on the mobile phase Dissolved oxygen in the mobile phase can be of special concern. It can under certain circumstances interfere with the detection of analytes by UV/Vis, fluorescence, or electrochemical detectors. Effects on UV/Vis detectors Oxygen can form UV-absorbing complexes with solvents such as methanol or tetrahydrofuran (THF).

  • Page 18: Using In-Line Degassing To Remove Gases From Eluents

    Using in-line degassing to remove gases from eluents In-line methods of degassing operate within the chromatographic fluid path. The 1500-series integral vacuum degasser uses this approach. Because degassing occurs close to the pump, this method minimizes reabsorption of ambient gas into the eluent.

  • Page 19: Overview Of The 1500-Series Hplc Pumps

    Overview of the 1500-series HPLC pumps The 1500-series HPLC pumps combine the most important aspects of eluent delivery for HPLC: high precision, reliability, and smooth eluent flow. All pumps perform their intended functions equally well: •...
  • Page 20 The 1525µ binary HPLC pump is designed for precise, reproducible gradient delivery at low flow rates up to 5 mL/minute. In an HPLC system, the 1500-series pump is controlled by Waters Breeze™ 2, Empower™ 2, or MassLynx™ data control software (see page 1-13).

  • Page 21: Fluid-Handling Components

    1525 binary pump with optional column heater Fluid-handling components Before you install a 1500-series pump, familiarize yourself with its fluid-handling components. The following figures identify the fluid-handling components of the 1525/1525EF and 1525µ HPLC pumps. The 1515 pump is an isocratic version of the 1525 binary pump. It shares...
  • Page 22 Fluid-handling components in the 1525/1525EF pumps Draw-off Pump head valve assembly Outlet check valve assembly Indicator rod Gradient mixer Inlet check valve assembly Inlet Manual injector manifold (optional Pressure Pump outlet transducer Reference valve Manual injector waste lines valve Reference valve waste line Pulse dampeners (covered by shroud)
  • Page 23 Fluid-handling components in the 1525µ pump Pump head assembly Draw-off valve Outlet check valve assembly Indicator rod Gradient Inlet check mixer valve assembly Inlet VENT Vent valve manifold Pump outlet Restrictor Pressure transducer PUMP A Solvent inlets Pulse dampeners (behind shroud) Integral vacuum degasser...
  • Page 24 The following table describes the functions of the 1500-series pump’s fluid-handling components. Fluid-handling components Component Description 1500-series column Maintains elevated column temperature to heater (optional) facilitate method reproducibility. Draw-off valve Enables attachment of a syringe for drawing eluent through the eluent reservoir line and into the pump for priming.
  • Page 25 Fluid-handling components (Continued) Component Description Pump outlet Routes eluent to the injector, column, and detector. Reference valve (or Directs flow from the pump to waste for purging, or vent valve for 1525µ) through the injector, the column, and the rest of the system.

  • Page 26: Electronic Components

    Electronic components Before you install the 1500-series pump, familiarize yourself with its electronic components, as illustrated in the figure below. Rear-panel electronic components in the 1500-series pump I/O terminal block IEEE-488 address switches Power switch (side of unit) IEEE-488 port...

  • Page 27: Pump Control

    Fuse holder Contains power fuses. Pump control Using Waters data control software, such as Breeze 2 software, you can control and monitor 1500-series HPLC pumps in isocratic or binary applications. For detailed information about specific data control software versions and requirements, refer to the release notes for the Waters 1500-series HPLC pumps.

  • Page 28: Ethernet Configuration

    IEEE-488 configuration To communicate with the Waters data control system via IEEE-488, an IEEE-488 cable connects the 1500-series pump to an IEEE-488 controller (a busLAC/E™ card in the data control system for Breeze or Empower control or to a NI GPIB card for MassLynx control). For more information, see page 2-6.

  • Page 29: Options And Accessories

    1500-series column heater The 1500-series column heater maintains the column at temperatures from 5 °C above ambient (minimum of 20 °C) to 60 °C. It mounts on the side of the pump, for easy access (see the figure “1525 binary pump with optional column...

  • Page 30: Automated Plunger Seal Wash

    The FlexInject injector can operate with flow rates as high as 150 mL/min and mounts on either side of the 1500-series pump for easy access. Its two sample injectors are pre-mounted along with a selector valve, which diverts the flow to small- or large-scale sample preparations.

  • Page 31: Gradient Mixers

    Gradient mixers The Waters 1500-series pumps support various configurations of gradient mixers that vary in eluent volume capacity. You can order and install one or more of these mixers in a 1500-series pump. For installation instructions, see page 3-8. Options and accessories...
  • Page 32 1-18 Introduction...
  • Page 33 If a pump’s configuration includes options (such as a column heater) or accessories (such as a different eluent mixer), refer to the appropriate sections in Chapter 3 to complete your 1500-series pump installation. Contents Topic Page Site requirements Unpacking...

  • Page 34: Installing The Hplc Pump

    Site requirements Install the 1500-series pump at a site that meets the specifications indicated in the following table. Installation site requirements Factor Requirement Temperature 4 to 40 °C Relative humidity 20 to 80%, noncondensing Bench space Width: 43 cm, including bottle holder (58 cm...

  • Page 35: Unpacking

    30.5 cm 12.7 cm Clearance for TP01723 bottle holder Unpacking A Waters 1500-series HPLC pump is shipped in a single carton that contains the following items: • Waters 1515 Isocratic, 1525 Binary, or 1525µ HPLC Pump • Startup kit Unpacking...

  • Page 36: Connecting Power And Signal Cables

    Waters Licenses, Warranties, and Support for complete information on shipping damages and claims. Connecting power and signal cables For proper operation, the 1500-series HPLC pump requires these items: • A grounded, AC power supply with no abrupt voltage fluctuations •...

  • Page 37: Connecting The Power Supply

    1500-series pump and optional column heater, must communicate with the data system via Ethernet communications. • As with IEEE-488 control, when using an autoinjector, triggering of the inject start signal for the 1500-series pump occurs over the Ethernet Connecting power and signal cables...

  • Page 38: Making Ieee-488 Connections

    I/O cable is needed. For more information, see page 3-5. You cannot use the 1500-series pump’s Ethernet port at the same time you are using its IEEE-488 bus interface for communications. To make the ethernet connections: Connect one end of the Ethernet cable to the Ethernet port on the pump’s rear panel.
  • Page 39 IEEE-488 communications. • When using an autoinjector, triggering of the inject start signal for the 1500-series pump occurs over the IEEE-488 cable, so no external I/O cable is needed. For more information, see page 3-5.
  • Page 40 Connecting IEEE-488 devices The steps in this procedure assume that you have not already connected any of the other HPLC system IEEE-488 devices (the autosampler or detector, for example) to the data control workstation. If you have already connected other devices to the workstation, connect the pump to the existing chain of devices.

  • Page 41: Setting The Ieee-488 Address

    Repeat step 3 for each additional IEEE-488 device. Ensure all IEEE-488 cable connector screws are finger-tight. Setting the IEEE-488 address You must set a unique address for the pump (and for each device connected to the HPLC system on the IEEE-488 bus). Valid IEEE-488 instrument addresses are 2 through 29, and are set using the DIP switches on the rear panel of the pump from 0 to 1 (see the table below).
  • Page 42 IEEE-488 switch settings (Continued) IEEE-488 Switch settings address 2-10 Installing the HPLC Pump...

  • Page 43: Connecting Pump Inlet And Outlet Lines

    Connecting pump inlet and outlet lines This section describes how to make these connections and attachments to the 1500-series pump: • Eluent supply connections • Outlet connection • Fluid waste lines Connecting the eluent supply Follow the instructions in this section to connect the pump inlet to an eluent reservoir.
  • Page 44 Installing the bottle holder To install the bottle holder: Position the rack along the pump’s left-hand side. The two cutouts at the bottom inside of the rack rest must on their corresponding positioning screws on the pump. Holding the rack flush against the surface of the pump, finger tighten the captive screw to secure the rack.
  • Page 45 When using the instrument in combination with a mass spectrometer, refer to recommendations published in the most recent version of “Controlling Contamination in UPLC/MS and HPLC/MS Systems” on the Waters Web site. • Choose 1-L solvent reservoirs that provide a snug fit for the reservoir caps supplied in the startup kit.

  • Page 46: Connecting The Pump Outlet

    5 for the second pump assembly. If you are connecting the pump to a Waters 717plus or 2707 autosampler, Tip: you can use one position of the bottle holder to hold a bottle that contains the needle wash solvent.
  • Page 47 • Circular tubing cutter (part number WAT022384) or knife-edge file • Needle-nose pliers (two pairs if cutting tubing with a knife-edge file) • 5/16-inch open-end wrench (part number WAT022527 (startup kit) Cutting the tubing to length To cut the pump outlet tubing you will need: Measure the length of stainless steel tubing required to connect the pump outlet to the injector or other instrument.

  • Page 48: Connecting Fluid Waste Lines

    Use the 5/16-inch wrench to tighten the screw another quarter turn. This seats the ferrule against the tubing. Unscrew the assembled fitting, and verify that the length of tubing extending beyond the ferrule is 3 mm. Repeat step 1 step 4 for the other end of the stainless steel tubing.
  • Page 49 Connecting pump waste tubing To connect the tubing: Obtain a piece of convoluted tubing and stretch it apart. ® Feed a piece of smooth Teflon tubing through the convoluted tubing until ends of both pieces are flush with one another. Tip: This dual tubing prevents the waste line from developing kinks.
  • Page 50 2-18 Installing the HPLC Pump...

  • Page 51: Installing Options And Accessories

    This chapter describes how to install the various options and accessories for 1500-series HPLC pumps. For the current list of 1500-series pump options and accessories, as well as ordering information and prerequisites, visit the Waters Web site or contact Waters.

  • Page 52: Installing The 1500-Series Column Heater

    Installing the 1500-series column heater You can mount the column heater on the right-hand side of 1500-series pumps using three screws. Connectors on the rear panel of the column heater provide power to the device and enable its control. Installation considerations •...
  • Page 53 To avoid damage during shipment, transport the device Caution: in its original shipping container with the custom foam insert. Special shipping considerations are printed on the shipping container. Store the device according to the guidelines given in Appendix Installing the 1500-series column heater...

  • Page 54: Installing A Manual Injector

    For additional information, see Controlling Contamination in UltraPerformance LC/MS and HPLC/MS Systems on the Waters Web site. Installing a manual injector If you are installing a 1500-series manual injector or FlexInject dual manual injector, refer to the installation instructions and startup list included with the injector.

  • Page 55: Connecting To The Column Or Column Heater

    Connecting the inject start signal (for manual injector) For 1500-series pumps with an optional 1500-series manual injector or FlexInject dual manual injector, you make an inject-start output signal connection from the I/O terminal block, on the pump, to the appropriate inputs on the detector.
  • Page 56 Insert the red stripped wire into position 1 of the detector’s terminal block, as indicated in the table below (Inject Start + on the 2489 or Chart Mark + on the 2414), and then tighten the screw to secure the wire. I/O terminal block connections Function Pin number...
  • Page 57 For instructions on operating the FlexInject Dual Manual Injector with a detector, refer to the Waters FlexInject Dual Manual Injector Module Installation Guide. Installing a manual injector...

  • Page 58: Installing Different Eluent Mixers

    Waters FlexInject Dual Manual Injector Module Installation Guide. Installing different eluent mixers You can use various eluent mixer configurations in the 1500-series pumps. This section describes these configurations, including how to install a new mixer, or replace an existing mixer, in your pump.
  • Page 59 These hole pairs are designated 1 to 4, top to bottom (see below). Position of mixer mounting hole pairs on pump chassis face 1500-series mixer options Standard Pump model...
  • Page 60 1525 pump showing different mixer configurations (labeled 1, 2, and 3) Outlets Configuration 1 – Single GM150 mixer Configuration 2 – Dual GM150 mixers* Configuration 3 – Large-volume mixer *Oriented horizontally top to bottom. Inlets Pressure Reference valve transducer Installing a single GM150 gradient mixer This mixer is standard on the 1525 pump.
  • Page 61 To install the single GM150 gradient mixer on the 1515 pump: Acquire a mixer (part number WAT055847). Remove the mounting screws and mixer clamps from the upper-left corner of the chassis face. Orient the mixer between the clamps as shown in configuration 1 of the figure “1525 pump showing different mixer configurations (labeled 1, 2, and 3)”...
  • Page 62 Installing dual GM150 gradient mixers To install the dual GM150 gradient mixers in the 1515 pump: Acquire two mixers (part number WAT055847). Remove the mounting screws and the mixer clamps from their positions on the upper left-hand corner of the chassis face. Put the mixers inside the mounting clamps, as shown in configuration 2 of the figure “1525 pump showing different mixer configurations (labeled...
  • Page 63 Put both mixers inside the mounting clamps, as shown in configuration 2 of the figure “1525 pump showing different mixer configurations (labeled 1, 2, and 3)” on page 3-10, using hole pairs 2 and 4. Firmly tighten the mounting screws. Reattach the pressure transducer outlet tubing to the inlet of the bottom mixer.

  • Page 64: Installing The Integral Vacuum Degasser

    These fittings are labeled on—and accessible from—the pump’s front panel. The degasser installation procedure involves removing the 1500-series pump’s top and side panels to incorporate the degasser assembly, along with its wiring and plumbing (see the installation instructions included with the Integral Vacuum Degasser Kit (P/N 205000253).

  • Page 65: Connecting Tubing To The Degasser Inlet And Outlet

    1525 binary pump with integral vacuum degasser Integral vacuum degasser Required materials • A tubing cutter that suits the tubing type: razor knife or razor blade, or a file with a cutting edge • Tubing, 1/8-inch, OD, thick-walled Tefzel (startup kit) or 1/16-inch OD stainless steel •...

  • Page 66: Installing The Degasser Vent Line

    Requirements: • You connect the eluent supply tubing from the eluent reservoirs to the degasser’s pump A and pump B (In) inlet fittings, instead of directly to the inlet connectors on the pump manifolds (see page 2-11). • You connect the pump inlet tubing between the degasser’s pump A and pump B (Out) outlet and the pump inlet connectors on the corresponding pump manifolds (see page...

  • Page 67: Using The Degasser

    Installing the plunger seal wash system The plunger seal wash system is an optional accessory for all of the 1500-series pumps. The seal wash solvent lubricates the plunger and flushes away any solvent or dried salts forced past the plunger seal from the high-pressure side of each piston chamber.

  • Page 68: Preparing The Instrument

    Required materials • Plunger seal wash system kit. Use the appropriate kit for your model pump: – 1515 – part number 205000251 – 1525 – part number 205000251 – 1525µ – part number 205000250 – 1525EF – part number 205000252 •...

  • Page 69: Removing Pump Head Components

    Removing pump head components To remove pump head components for all 1500-series pumps: When installing the seal wash system, work on one pump head support at Tip: a time. Complete the procedure for one pump head before removing another. In your data control software, set the flow to 0.3 mL/min.
  • Page 70 loosen the screws 1/2-turn at a time for the first two turns (see the figure, below). To avoid breaking the plunger, pull the pump head off Caution: straight toward you. Removing head bolts (1515, 1525 and 1525EF pumps) Pump head support Pump head Carefully remove the head from its support.

  • Page 71: Installing Head Support Components (1515/1525 Pump Only)

    Installing head support components (1515/1525 pump only) Replace the high pressure seal while the head is removed. See page 5-6 more information. Perform these steps only if you have the 1515 or 1525 pump: Lay the head support on a hard surface, back side up. You can identify the back side of the pump head support by its Tip: larger wash seal cavity.
  • Page 72 Using the wash seal installation tool, press the wash seal spacer until it bottoms in the wash seal cavity. Wash seal installation tool Rotate the wash seal spacer with a screwdriver until the slots line up with the holes in the edge of the head support. Doing so creates a fluid path for the seal wash solvent.
  • Page 73 Wash fitting installation tool 10. Hold the wash fitting installation tool upright, and carefully lower the head support onto the tool so that the retaining ring fits into the center of the wash seal cavity and the thin end of the tool slides through the hole in the plunger wash seal.
  • Page 74 16. From the back side of the head support, slide the plunger indicator rod through the hole in the head support. 17. Lubricate the plunger rod and seals with methanol. 18. Align the head support on the pump housing face. The back side of the head support must contact the pump housing face.

  • Page 75: Installing Head Support Components (1525Ef Pump Only)

    Installing head support components (1525EF pump only) Replace the high pressure seal while the head is disassembled. See page 5-6 for more information. Perform these steps only if you have the 1525EF model: Lay the head support on a hard surface, back side up. The back side of the pump head support is identified by the larger Tip: wash seal cavity.
  • Page 76 Inspect the fluid path for obstructions by holding the head support up to a light source and looking through the holes in the edge of the head support. The fluid path must be unobstructed. If it is not, blow through the hole, or use a thin wire to clear any obstructions.

  • Page 77: Installing Head Components (1525Μ Pump Only)

    The back side of the head support must contact the pump housing face. When viewed from the front of the pump, the indicator rod must be oriented to the upper right of the head support. To avoid damaging the support head, support it securely Caution: at all times while installing and tightening the screws.
  • Page 78 1525µ head support components Wash seal Metal seal retainer Head support assembly O-ring Spring side Press the seal retainer O-ring into the front of the seal retainer. After starting the O-ring into the groove, turn over the retainer and Tip: O-ring, and press them onto a flat surface to seat the O-ring into the O-ring groove.

  • Page 79: Installing The Solenoid

    13. Repeat the process for the remaining pump heads, and then complete the steps in “Installing the solenoid”, below. Installing the solenoid Perform these steps for all 1500-series pumps: Install the solenoid bracket on the pump chassis with the two TORX head screws provided with the kit.
  • Page 80 Push the metal body of the solenoid into the bracket until it snaps into position. Correct solenoid positioning Solenoid cable connector Solenoid cable Solenoid pump Pump solenoid Positioning bracket arrow Seal wash warning label Solenoid valve outlet fitting Pulse dampener shroud Ensure that the solenoid bracket does not contact the label on the solenoid.

  • Page 81: Installing Seal Wash Tubing

    Installing seal wash tubing Perform these steps for all 1500-series pumps: ® Cut the PharMed tubing, provided with the kit, into the lengths described in the following table. ® PharMed tubing lengths Length Number of pieces 8.0 cm 20.0 cm 41.0 cm...
  • Page 82 Seal wash tubing routing 20.0 cm Upper-left pump Upper-right pump head head Seal wash inlet 41.0 cm) tubing 8.0 cm Lower-left pump Lower-right pump head head 20.0 cm Seal wash waste tubing Route the ETFE tubing to a suitable waste receptacle, and cut the tubing to length.

  • Page 83: Using The Seal Wash System

    10. Firmly seat the tubing end into the inlet on the left-hand side of the solenoid pump, and then hold the tubing in place while finger tightening the compression screw. To avoid damaging the ferrule, do not overtighten the Caution: compression screw.
  • Page 84 3-34 Installing Options and Accessories...

  • Page 85: Preparing For Operation

    Preparing for Operation This chapter describes how to prepare the 1500-series pump for making chromatographic runs via Waters data control software. Contents Topic Page Startup and initial preparation Preparing for Breeze 2 operation 4-10 Preparing for Empower 2 operation 4-15...

  • Page 86: Startup And Initial Preparation

    Startup and initial preparation Before you use your data control software to ready the pump for making chromatographic runs, familiarize yourself with these procedures and recommendations: • Powering-on the pump • Recommendations for preparing the pump for operation • Dry priming the pump •...
  • Page 87 Priming, purging, and equilibrating procedures are fully described later in this chapter. Eluent guidelines When you prepare a 1500-series pump for operation, follow these guidelines: • Use methanol to prime a new pump, or one that has been stored for an extended period.

  • Page 88: Dry Priming The Pump

    • Dedicate an eluent supply tube and filter assembly for each eluent. If doing so is not possible, purge the tube and filter of any remaining eluent before you use a new eluent. • To prevent cross contamination of eluents, dedicate a set of glassware for preparing, storing and supplying each eluent.

  • Page 89: Operating With The Integral Vacuum Degasser

    Insert the syringe into the Luer fitting at the center of the draw-off valve handle. Priming the pump Rotate the draw-off valve handle counterclockwise, 1/2-turn, to open the valve. Withdraw several milliliters of eluent with the syringe, and then close the valve handle.
  • Page 90 These events take place when the degasser is enabled: • The LED on the 1500-series pump front panel illuminates. • The vacuum pump begins its evacuation. Normal operation begins when the vacuum pump reaches its vacuum target and changes from high to low RPM.
  • Page 91 Degasser operating statuses The LED on the 1500-series pump front panel indicates the status of the degasser as described in the table, below. LED indications LED state Description Unlit Pump is powered-off. Steady yellow Degasser is operating with pump at high RPM, vacuum level above 60 mmHgA/1.16 psiA.

  • Page 92: Operating With The Plunger Seal Wash System

    Operating with the plunger seal wash system If your HPLC pump includes the plunger seal wash system, during operation, plunger seal wash solvent flows from a reservoir to the solenoid wash pump, which pumps the solvent through the fluid path. •...
  • Page 93 The seal wash solvent lubricates the plunger. It also flushes away solvent or precipitated salts forced past the plunger seal from the high-pressure side of the solvent piston chamber. Tips: • Once you install the seal wash option, use it at all times during instrument operation to protect plungers and seals.

  • Page 94: Maximum Flow Rates For 1500-Series Pumps

    Maximum flow rates for 1500-series pumps To prevent the pump from stalling, do not exceed the pump’s maximum flow rate. When specifying flow rates via Waters data control software, do not exceed the maximum flow rate that is specified for the pump. •...

  • Page 95: Priming And Purging The Pump Via Breeze 2 Control

    In the Select Project and System dialog box, verify that your chromatographic system, with a 1500-series pump, is configured and online. If you did not yet create a chromatographic system with the 1500-series pump, do so now. • Ensure the pump is properly configured in the Node Properties,...

  • Page 96: Purging The Flow Path Via Breeze 2 Control

    In the Run Samples window, click the Start Flow icon , and enter appropriate values for the pump’s flow rate and gradient settings. To quickly prime and purge just the pump, Tip: • for the 1515 pump, set the rate to 2.0 mL/min. •...

  • Page 97: Equilibrating The System Via Breeze 2 Control

    Before purging • Make sure the eluent is filtered and degassed. • Prime and purge the pump with fresh eluent as described in the section above. • Ensure the pump’s reference valve knob is turned to the left, directing the eluent flow to the system (for the 1525µ pump, position the vent valve knob to the system).
  • Page 98 Before equilibrating • Make sure the eluent is filtered and degassed. • Allow the eluent and the system to reach proper temperatures before pump operation. • Prime the pump, as described in the section above. • Ensure the pump’s reference valve knob is turned to the left, directing the eluent flow to the system (for the 1525µ...

  • Page 99: Preparing For Empower 2 Operation

    Preparing for Empower 2 operation Use this section to ready the 1500-series pump for performing runs under Empower 2 software control. It describes simple techniques for accomplishing these tasks: • Priming and purging the pump • Purging the system •...

  • Page 100: Purging The Flow Path Via Empower 2 Control

    If you did not yet create a chromatographic system with the 1500-series pump, do so now. • Ensure the pump is properly configured in the Empower Node Properties, which allows you to scan for available instruments (including the pump), and set the pump-head size in the Instruments tab (for 1525µ...
  • Page 101 When bringing new instruments online or troubleshooting a solvent Tip: problem, you can restrict the flow path to only those sections of the system that require purging by disconnecting the flow path at an inlet or outlet junction, such as the injector outlet or column outlet, and routing the flow to waste.

  • Page 102: Equilibrating The System Via Empower 2 Control

    Equilibrating the system via Empower 2 control Equilibrate the system to achieve a final state of system readiness for running samples (see page 4-2). Before equilibrating • Make sure the eluent is filtered and degassed. • Allow the eluent and the system to reach proper temperatures before pump operation.

  • Page 103: Preparing For Masslynx Operation

    Preparing for MassLynx operation Use this section to ready the 1500-series pump for performing runs under MassLynx software control. It describes simple techniques for accomplishing these tasks: • Priming and purging the pump • Purging the system • Equilibrating the system For additional information and instructions on setting up the HPLC system and creating inlet methods, see the MassLynx online Help.

  • Page 104: Purging The Flow Path Via Masslynx Control

    If you did not yet create an inlet method with the 1500-series pump under Waters Pump Control, do so now. Ensure the pump is properly configured in the Waters Pump Control Configuration, which allows you to scan for available instruments (including the pump), and set the pump-head size (for 1525µ...
  • Page 105 Before purging • Make sure the eluent is filtered and degassed. • Prime and purge the pump with fresh eluent, as described in the section above. • Ensure the pump’s reference valve knob is turned to the left, directing the eluent flow to the system (for the 1525µ pump, position the vent valve knob to the system).

  • Page 106: Equilibrating The System Via Masslynx Control

    Equilibrating the system via MassLynx control Equilibrate the system to achieve a final state of system readiness for running samples (see page 4-2). Before equilibrating • Make sure the eluent is filtered and degassed. • Allow the eluent and the system to reach proper temperatures before pump operation.

  • Page 107: Powering Off The Pump

    Powering off the pump To power-off the pump: If you have been using a buffer, purge it from the pump and other HPLC system components with HPLC-grade water. If the pump will be idle for more than one day, purge with a methanol/water solution to prevent the growth of microorganisms.
  • Page 108 4-24 Preparing for Operation...

  • Page 109: Maintaining The Hplc Pump

    Maintaining the HPLC Pump This chapter provides important safety and handling considerations for the Waters 1500-series pump, describes how to run diagnostic tests and set pump calibration parameters, and explains how to replace pump components. Contents Topic Page Maintenance considerations...

  • Page 110: Maintenance Considerations

    4-2. Spare parts For a comprehensive catalog of spare parts, see the Waters Quality Parts Locator on the Waters Web site’s Services & Support page. Contacting Waters Technical Service If you encounter any problems replacing parts in the 1500-series pump, contact Waters Technical Service.

  • Page 111: Performing Pump Diagnostic Tests

    Performing pump diagnostic tests The following diagnostic tests can help you track system performance and prevent or identify potential problems before they interfere with operation: • Retention time stability test • Ramp-and-decay test Retention time stability test Observing retention time stability during system performance monitoring tests is useful for determining the performance of your HPLC system and its components, including the pump.
  • Page 112 Requirements: • If your eluent is not miscible with methanol, use an intermediate eluent. • Move the eluent reservoir below the pump head level to prevent gravity flow of the eluent. For binary pumps, disconnect Pump B from the tee by removing the outlet tube of the pulse dampener;...
  • Page 113 • If there is a bad outlet check valve, pressure can possibly increase, and then immediately decrease as the plunger recedes. • If the pressure does not rise to the high-pressure limit, try repriming the pump or increasing the flow rate parameter setting. Allow the pump to reach the high-pressure limit.

  • Page 114: Replacing And Cleaning Plunger Seals And Plungers

    • Removing and installing plungers for cleaning and replacement The plungers in a 1500-series pump are ultrasmooth, chemically inert sapphire rods. Salt crystals that precipitate from the eluent can form on the plunger and cause wear on the plunger seals and on the plunger itself. The result is a slow leak and a very slight cyclic pressure fluctuation, with a possible increase in retention time.
  • Page 115 To remove a pump head: Purge the pump with methanol (see Chapter Requirements: • If your eluent is not miscible with methanol, use an intermediate eluent. • Move the eluent reservoir below the pump head level to prevent gravity flow of the eluent. Insert the priming syringe into the Luer fitting at the center of the draw-off valve handle, and then turn the handle counterclockwise about 1/2-turn to open the valve.
  • Page 116 For the 1525µ pump only, remove the plunger bearing from the pump head. For all 1500-series pumps, use the seal insertion tool or other nonmetalic tool to pry the seal out of the pump head. Twist the plastic screw end of the tool clockwise into the seal, and Tip: then pull the seal to remove it from the pump head.

  • Page 117: Cleaning And Replacing The Plungers

    Press the tool firmly into the pump head, and then remove it. For the 1525µ pump only, refit the plunger bearing on top of the seal. Make sure the grooves in the bearing are facing outward and are visible as you look at the pump head. 10.
  • Page 118 Required materials • Open-end wrench, 5/16-inch (startup kit) • Allen wrench, 5/32-inch (startup kit) • Allen wrench, 9/64-inch (startup kit) • Adjustable wrench (startup kit) • Snap-ring pliers (for 1515/1525 pumps only) (part number WAT025263) • Plunger insertion tool and seal extraction tool (startup kit) •...
  • Page 119 When the indicator rod is fully extended from the pump head, use the software to stop the flow, turning off the pump. Carefully remove the plunger: • For a 1525µ pump, slide the plunger assembly out of the back of the support assembly.
  • Page 120 Cleaning the plunger To clean the plunger: Remove the plunger from the plunger assembly. Clean the plunger by sonicating it in 50:50 methanol/water for a few minutes. Assembled plunger 1515/1525 plunger assembly 1525µ plunger assembly Washer Snap-ring Plunger Plunger Piston Retainer seal Spring Spring...
  • Page 121 Reinstall the plunger assembly • For 1525µ pump, reinstall the plunger assembly into the head support assembly. • For 1515/1525 pumps, use the plunger insertion tool to push the plunger assembly into the pump piston as far as it will go, and then use the snap-ring pliers to insert the snap ring.

  • Page 122: Replacing Check Valves

    Carefully slide the pump head over the plunger and onto the pump. To avoid breaking the plunger, hold the pump head Caution: securely against the pump. Holding the pump head securely against the pump, alternately tighten the two screws. 10. Check for even head alignment by observing the gap between the pump head and the pump head support assembly.

  • Page 123: Replacing 1515/1525 Check Valves

    Requirements: • Replace each check valve and check valve housing as a single unit. Do not separate the check valve from the housing. • Before replacing a check valve, move the eluent reservoir to a level below the pump head to prevent the gravity flow of eluent. Required materials •...
  • Page 124 Disconnecting the outlet tubing (1515/1525 pump) Using forceps, remove the cartridge from the housing and replace it with the new cartridge. Requirements: • The arrow printed on the check valve cartridge indicates the direction that liquid can flow. For both inlet and outlet check valve assemblies, the arrow must point upward when installed in the pump head.

  • Page 125: Replacing 1525Ef Check Valves

    With the check valve held upright to prevent the cartridge from falling out, hand-tighten the check valve into the pump head. Tighten the check valve another 1/2-turn with the open-end wrench. Reconnect the eluent supply line to the inlet manifold on the pump. Prime the pump as described in Chapter 4.
  • Page 126 Replacing the 1525EF outlet check valve To replace the outlet check valve: Purge the pump with methanol (see Chapter 4). If methanol is not miscible with your current eluent, use an intermediate eluent. Use the data control software to run the pump until the indicator rod fully retracts into the pump head, and then stop the flow, turning off the pump.

  • Page 127: Replacing 1525Μ Check Valves

    Carefully slide the pump head assembly off the pump (see page 5-6). Pull the pump head off straight to avoid breaking the Caution: plunger. Hold the pump head upside-down in one hand with the outlet check valve housing facing downward. Remove the entire outlet check valve housing and replace it with a new one.
  • Page 128 While holding the check valve housing with the adjustable wrench, use the 5/16-inch open-end wrench to loosen the compression screw, and remove the tubing assembly from the inlet check valve housing. Use the adjustable wrench to remove the inlet check valve housing from the pump head.
  • Page 129 Replacing the 1525µ outlet check valve Before you replace the outlet check valve, move the eluent reservoir to a level below the pump head to prevent the gravity flow of eluent. To replace the outlet check valve: Purge the pump with methanol (see Chapter 4).

  • Page 130: Replacing A Draw-Off Valve

    With the outlet check valve housing held upright to prevent the cartridge from falling out, finger-tighten the housing into the pump head. Turn the pump head over and carefully slide it over the plunger and onto the pump. To avoid damaging the plunger, ensure that the pump Caution: head is straight and the indicator rod is properly aligned with the pump head.

  • Page 131: Removing The Draw-Off Valve

    • Priming syringe (startup kit) • Phillips screwdriver • Replacement draw-off valve • HPLC-grade methanol Removing the draw-off valve To remove the draw-off valve: Purge the pump with methanol (see Chapter 4). If methanol is not miscible with your current eluent, use an intermediate eluent. Before you continue with this procedure, lower the eluent reservoir to halt gravity flow of eluent.

  • Page 132: Installing A Draw-Off Valve

    Use the 5/16-inch open-end wrench to disconnect the two stainless steel compression screws from the inlet manifold. (These screws connect the inlet manifold to the inlet check valves.) Use the 7/64-inch Allen wrench to remove the two Allen screws that hold the draw-off valve assembly and bracket to the pump.

  • Page 133: Replacing Fuses

    For continued protection against fire hazard, replace fuses only with those of the same type and rating. The fuse holder is located on the pump’s rear panel. The 1500-series pump is shipped with two factory-installed 3.15 A fuses.

  • Page 134: Replacing The Rear Panel Fuses

    Replacing the rear panel fuses Required materials • Screwdriver, flat-blade, small • Replacement fuses To replace a blown or faulty fuse: • Power-off the pump, and then remove the power cord from its connector. • Use the small flat-blade screwdriver to remove the fuse holder located just above the power cord connector.

  • Page 135: Troubleshooting

    This chapter describes how to troubleshoot pump problems based on error messages and malfunctions, identify and correct noises, and identify pump-related chromatographic problems. To prevent injury, always observe Good Laboratory Warning: Practices when you troubleshoot the 1500-series pump Contents Topic Page Troubleshooting pump problems...

  • Page 136: Troubleshooting Pump Problems

    Verify eluent viscosity (the observed pressure may be normal for the column/eluent blend). If necessary, change to a less viscous eluent. Defective pressure Contact Waters Technical transducer Service. Blocked outlet tubing Locate the source of blockage. or fluid path (in pump...
  • Page 137 5-22). Pump low-pressure Set to the correct low-pressure limit set higher than limit. operating pressure Flow rate set to zero Set the desired pump flow rate. Pressure transducer Contact Waters Technical out of adjustment or Service. defective Troubleshooting pump problems...
  • Page 138 Determining pump malfunctions (Continued) Symptom Possible cause Corrective action Pump not Pump not primed Prime the pump (see delivering eluent Chapter (continued) Immiscible eluents in Purge pump with appropriate pump head eluents. Verify miscibility of eluents being used and change to more miscible eluents.
  • Page 139 Replace filter. inlet filter Volatile eluents in Prime pump (see Chapter pump head Use correct tubing. Tubing ID too small Ruptured Contact Waters Technical high-pressure noise Service. filter Defective pump motor Contact Waters Technical Service. Defective circuit board Contact Waters Technical Service.
  • Page 140 Determining pump malfunctions (Continued) Symptom Possible cause Corrective action Leak from pump Worn pump plunger Replace defective plunger seals head seals (see page 5-6). Worn plunger Replace the plunger (see page 5-6). Loose pump head Tighten the two pump head screws.
  • Page 141 Pump cavitation Refer to the Corrective Action(s) under the “Pump not delivering eluent” symptom listed above. Pump electronics Contact Waters Technical failure Service. High system Pump Flow parameter Set the correct flow parameter pressure due to set too high value.

  • Page 142: Identifying And Correcting Noises

    Identifying and correcting noises The following table is a guide to troubleshooting and correcting noises in the Waters 1500-series pump. Identifying noises Symptom Possible cause Corrective action Click or loud Binding plunger seal If click does not stop and cannot be...

  • Page 143: Identifying Chromatographic Problems

    Identifying chromatographic problems The following table is a guide to troubleshooting and correcting chromatographic problems. Correcting chromatographic problems Symptom Possible cause Corrective action Erratic retention Air bubble in pump head Degas all eluents and times prime pump (see Chapter Malfunctioning check Replace pump check valves valves (see...
  • Page 144 Correcting chromatographic problems (Continued) Symptom Possible cause Corrective action Reduced retention Incorrect flow rate Change flow rate. times Incorrect eluent Change composition. composition High column temperature Reduce column temperature. Incorrect eluent Use correct eluent. Column contaminated Clean or replace column. Incorrect column Use correct column.
  • Page 145 Correcting chromatographic problems (Continued) Symptom Possible cause Corrective action Baseline noise Fluctuating flow rate Degas eluent. Try cycling, short-term repriming the pump(s). If (30 to 60 sec) necessary, check flow accuracy of the pump(s). Inadequate eluent Install a mixer as shown blending page 3-8.
  • Page 146 Correcting chromatographic problems (Continued) Symptom Possible cause Corrective action Baseline noise, Eluents not properly Degas eluents. random degassed Flow erratic, pump not Prime the pump (see primed Chapter Purge the pump to dislodge any air bubbles, inspect for failing seals. Eluents contaminated Use fresh eluent.
  • Page 147 Safety Advisories Waters instruments display hazard symbols designed to alert you to the hidden dangers of operating and maintaining the instruments. Their corresponding user guides also include the hazard symbols, with accompanying text statements describing the hazards and telling you how to avoid them.

  • Page 148: A Safety Advisories

    Heed all warnings when you install, repair, and operate Waters instruments. Waters assumes no liability for the failure of those who install, repair, or operate its instruments to comply with any safety precaution.

  • Page 149: Specific Warnings

    The following warnings can appear in the user manuals of particular instruments and on labels affixed to them or their component parts. Burst warning This warning applies to Waters instruments fitted with nonmetallic tubing. Pressurized nonmetallic, or polymer, tubing can burst. Warning: Observe these precautions when working around such tubing: •...
  • Page 150 Also ensure a gas-fail connection is connected to the LC system so that the LC solvent flow stops if the nitrogen supply fails. Mass spectrometer shock hazard This warning applies to all Waters mass spectrometers. To avoid electric shock, do not remove the mass spectrometer’s Warning: protective panels.

  • Page 151: Caution Symbol

    Biohazard warning This warning applies to Waters instruments that can be used to process material that might contain biohazards: substances that contain biological agents capable of producing harmful effects in humans. Waters instruments and software can be used to analyze or...

  • Page 152: Warnings That Apply To All Waters Instruments

    Warnings that apply to all Waters instruments When operating this device, follow standard quality control procedures and the equipment guidelines in this section. Attention: Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user’s authority to operate the equipment.
  • Page 153 • Keine Schläuche verwenden, die stark geknickt oder überbeansprucht sind. • Nichtmetallische Schläuche nicht für Tetrahydrofuran (THF) oder konzentrierte Salpeter- oder Schwefelsäure verwenden. • Durch Methylenchlorid und Dimethylsulfoxid können nichtmetallische Schläuche quellen; dadurch wird der Berstdruck des Schlauches erheblich reduziert. Warnings that apply to all Waters instruments...
  • Page 154 Attenzione: fare attenzione quando si utilizzano tubi in materiale polimerico sotto pressione: • Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero pressurizzati. • Spegnere tutte le fiamme vive nell'ambiente circostante. • Non utilizzare tubi eccessivamente logorati o piegati. •...
  • Page 155 농축 질산 또는 황산과 함께 사용하지 마십시오. • 염화 메틸렌(Methylene chloride) 및 디메틸술폭시드(Dimethyl sulfoxide)는 비금속 튜브를 부풀려 튜브의 파열 압력을 크게 감소시킬 수 있으므로 유의하십시오. 警告：圧力のかかったポリマーチューブを扱うときは、注意してください。 • 加圧されたポリマーチューブの付近では、必ず保護メガネを着用してください。 • 近くにある火を消してください。 • 著しく変形した、または折れ曲がったチューブは使用しないでください。 • 非金属チューブには、テトラヒドロフラン(THF)や高濃度の硝酸または硫酸などを流 さないでください。 • 塩化メチレンやジメチルスルホキシドは、非金属チューブの膨張を引き起こす場合が あり、その場合、チューブは極めて低い圧力で破裂します。 Warnings that apply to all Waters instruments...
  • Page 156 Warning: The user shall be made aware that if the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired. Attention: L’utilisateur doit être informé que si le matériel est utilisé d’une façon non spécifiée par le fabricant, la protection assurée par le matériel risque d’être défectueuses.
  • Page 157 警告 : 為了避免火災， 更換保險絲時， 請使用與儀器保險絲蓋旁面板上所印刷之相同類 型與規格的保險絲。 警告 : 为了避免火灾，应更换与仪器保险丝盖旁边面板上印刷的类型和规格相同的 保险丝。 경고: 화재의 위험을 막으려면 기기 퓨즈 커버에 가까운 패널에 인쇄된 것과 동일한 타입 및 정격의 제품으로 퓨즈를 교체하십시오. 警告 : 火災予防のために、ヒューズ交換では機器ヒューズカバー脇のパネルに記 載されているタイプおよび定格のヒューズをご使用ください。 Warnings that apply to all Waters instruments A-11...

  • Page 158: Electrical And Handling Symbols

    Electrical and handling symbols Electrical symbols These can appear in instrument user manuals and on the instrument’s front or rear panels. Electrical power on Electrical power off Standby Direct current Alternating current Protective conductor terminal Frame, or chassis, terminal Fuse Recycle symbol: Do not dispose in municipal waste.

  • Page 159: Handling Symbols

    Handling symbols These handling symbols and their associated text can appear on labels affixed to the outer packaging of Waters instrument and component shipments. Keep upright! Keep dry! Fragile! Use no hooks! Electrical and handling symbols A-13...
  • Page 160 A-14 Safety Advisories...

  • Page 161: B Specifications

    Specifications This appendix provides the following types of specifications for the Waters ® 1500-series pumps: • Physical • Environmental • Electrical • Performance • Degasser • Instrument control and communication Physical specifications for 1500-series pump Item Specification Height 43 cm Depth 61.0 cm...
  • Page 162 Physical specifications for 1500-series column heater Item Specification Height 43 cm Depth 35.5 cm Width 15.2 cm Weight 5.9 kg Environmental specifications Item Specification Operating Temperature 4 to 40 °C Humidity 20 to 80%, noncondensing Audible Noise <70 dBA at operator position...
  • Page 163 Electrical specifications (Continued) Item Specification 120/140 VAC Line voltage Frequency 50/60 Hz, single phase Fuses 3.15 A, 250 V, two a. Protection Class I – The insulating scheme used in the instrument to protect you from electri- cal shock. Class I identifies a single level of insulation between live parts (wires) and exposed conductive parts (metal panels), in which the exposed conductive parts are connected to a grounding system.
  • Page 164 Performance specifications for the 1515 and 1525 pumps (Continued) Item Specification Gradient Accuracy (1525) ±0.5% of setting at 1 mL/min and 6894.75 kPa (68.9 bar, 1000 psi) backpressure (Methanol: methanol with 5.6 mg/L propyl paraben) with one GM-150 mixer Gradient Precision (1525) <0.5% RSD at 1 mL/min and 6894.75 kPa (68.9 bar, 1000 psi) backpressure (Methanol: methanol with 5.6 mg/L propyl paraben)
  • Page 165 Performance specifications for the 1525µ pump (Continued) Item Specification Gradient precision <0.5% RSD (typical) between 10 to 90%, 0.1 mL/min, and 6894.75 kPa (68.9 bar, 1000 psi) backpressure (methanol: water with octanophenone and one 50-µL mixer) Gradient delay volume <30 µL (without mixer, no manual injector and no column heater) <100 µL (with one 50-µL mixer, no manual injector, and no column heater)
  • Page 166 Performance specifications for the 1525EF pump (Continued) Item Specification Gradient precision <0.5% RSD between 10 to 90%, 1 mL/min, and 6894.75 kPa (68.9 bar, 1000 psi) backpressure (methanol: methanol with 5.6 mg/L propylparaben > 05 AUFS @257 nm and one GM150 mixer) Gradient ripple <1.5% normalized to full scale between 10 to...
  • Page 167 High-density polyethylene b. polytetrafluoroethylene c. ethylenetetrafluoroethylene ® The following table lists Teflon AF chemical compatibility test results from Waters. Additional materials in the flowpath are PPS and PTFE. Tip: Teflon AF chemical compatibility test results Compatible Incompatible Acetonitrile ®...
  • Page 168 Teflon AF chemical compatibility test results (Continued) Hexane Isopropanol Methanol Methylene chloride N-methylpyrrolidone Tetrahydrofuran Toluene Trichlorobenzene Water a. Compatible solvents are not so soluble that they change the appearance or weight of Teflon AF after prolonged exposure at 23 °C. b.

  • Page 169: C Solvent Considerations

    Solvent Considerations Contents Topic Page Introduction Solvent compatibility Solvent miscibility Buffered solvents Head height Solvent viscosity Mobile phase solvent degassing Wavelength selection C-12...

  • Page 170: Introduction

    Introduction Clean solvents Clean solvents provide these benefits: • Reproducible results • Operation with minimal instrument maintenance A dirty solvent can cause these issues: • Baseline noise and drift • Blockage of the solvent filters with particulate matter Solvent quality Use HPLC-grade solvents to ensure the best possible results.

  • Page 171: Buffers

    Solvents to use Materials of construction used in a 1500-series HPLC pump do not react with most acids, bases, salts, and organic solvents. The solvents listed in the tables on...
  • Page 172 Seek information on the use of a specific solvent or concentration not listed in this manual directly from Waters. Aqueous buffers for use with the 1500-series HPLC pump Aqueous buffers Acetate Perfluorobutyric acid Fe(CN) Ca(OCl) Fe(CN) Phosphate CaCl NaCl Tartrate...
  • Page 173 Bases for use with the 1500-series HPLC pump Bases Ba(OH) NaOH, up to 10 OH, up to 3 LiOH Tetramethylam- monium hydroxide pentahydrate Organic solvents for use with the 1500-series HPLC pump Organic solvents 4-cyanophenol Chloroform Ethylene glycol Methylene chloride Acetone Cyclohexane Formaldehyde...

  • Page 174: Solvent Miscibility

    Solvent miscibility Before you change solvents, refer to the table below to determine the miscibility of the solvents to be used. When you change solvents, be aware that: • You can make changes involving two miscible solvents directly. Changes involving two solvents that are not totally miscible (for example, from chloroform to water), require an intermediate solvent (such as methanol).
  • Page 175 Solvent miscibility (Continued) Boiling Miscibility λ Cutoff Polarity Viscosity Solvent point °C number index CP, 20 °C (nm) (@1 atm) Ethylene chloride 0.79 83.5 –– Butyl alcohol 3.00 117.7 –– –– Butanol 3.01 177.7 –– Tetrahydrofuran 0.55 66.0 Ethyl acetate 0.47 77.1 1-propanol...

  • Page 176: How To Use Miscibility Numbers (M-Numbers

    How to use miscibility numbers (M-numbers) Use miscibility numbers (M-numbers) to predict the miscibility of a liquid with a standard solvent (see the table above). To predict the miscibility of two liquids, subtract the smaller M-number value from the larger M-number value. •...

  • Page 177: Head Height

    Head height Position the solvent reservoirs in the bottle holder supplied with the 1500-series HPLC pump (with adequate spill protection). Solvent viscosity Generally, viscosity is not important when you are operating with a single solvent or under low pressure. However when you are running a gradient, the viscosity changes that occur as the solvents are mixed in different proportions can result in pressure changes during the run.

  • Page 178: Eluent Degassing Methods

    Changes in the composition, temperature, or pressure of the mobile phase can lead to outgassing. Effects of intermolecular forces Nonpolar gases (N , CO , He) are more soluble in nonpolar solvents than in polar solvents. Generally, a gas is most soluble in a solvent whose intermolecular attractive forces are similar to those in the gas (“like dissolves like”).
  • Page 179 This method provides an automatic, continuous method of removing dissolved gasses, and allows for quick eluent changeover. Waters makes two in-line degassers: part numbers WAT079700 (2 Eluent) and WAT079800 (4 Eluent). Contact Waters for details. The longer a solvent is exposed to a vacuum, the more dissolved gases are removed.

  • Page 180: Wavelength Selection

    Conclusions With any of the above techniques, the eluent reequilibrates to air saturation in 12 to 24 hours, depending on the eluent. Degassing by vacuum or sonication or both is often performed for improved pump performance in multipump gradient applications. Wavelength selection The tables in this section provide UV cutoff values for: •...

  • Page 181: Mixed Mobile Phases

    UV cutoff wavelengths for common chromatographic solvents (Continued) UV cutoff UV cutoff Solvent Solvent (nm) (nm) Cyclohexane n-Pentane Cyclopentane n-Propanol Diethyl amine n-Propyl chloride Dioxane Nitromethane Ethanol Petroleum ether Ethyl acetate Pyridine Ethyl ether Tetrahydrofuran Ethyl sulfide Toluene Ethylene dichloride Xylene Mixed mobile phases The following table provides approximate wavelength cutoffs for some other...

  • Page 182: Refractive Indices Of Common Solvents

    202, 212 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, Waters PIC Reagent B-6, monobasic, 10 mM...
  • Page 183 Refractive indices for common chromatographic solvents (Continued) Solvent Solvent Ethanol 1.361 Cyclohexane 1.427 Methyl acetate 1.362 Ethylene glycol 1.427 Isopropyl ether 1.368 N,N-Dimethyl formamide 1.428 (DMF) Ethyl acetate 1.370 N,N-Dimethyl acetamide 1.438 (DMAC) 1-Pentene 1.371 Ethyl sulfide 1.442 Acetic acid 1.372 Chloroform 1.443...
  • Page 184 C-16 Solvent Considerations...
  • Page 185 1525µ pump pump inlet 2-12 performance specifications connections 1525EF pump electrical performance specifications guidelines contacting Waters Technical Service addresses DIP switch settings contamination setting IEEE-488 contamination, eluent air bubbles cooling fan 1-12 audience and purpose cyclic pressure difference...
  • Page 186 replacing 5-25 EC Authorized Representative viii electrical connections gas solubility inject start signal gradient mixer 1-10 power supply guidelines electrical symbols A-12 connection eluent contamination eluents eluent filter eluent level. See head height eluent reservoir handling symbols A-13 connecting 2-13 head height positioning 2-13...
  • Page 187 leaks inspecting 5-12 replacing 5-12 worn manual injector 1-10 power entry module 1-13 waste line 1-10 power supply, connecting mass spectrometer shock hazard power switch 1-13 methanol powering-off the pump 4-23 miscibility numbers pressure transducer 1-10 miscibility of eluents pressure, relieving miscibility of solvents priming the pump M-numbers.
  • Page 188 See reservoirs warning symbols solvents. See eluents waste line sonication C-11 connecting drip tray 2-17 specifications connecting pump 2-17 electrical Waters Technical Service, contacting performance symbols wavelength selection C-12 caution electrical A-12 handling A-13 warning system pressure, relieving...

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