caplc guide issue2

Micromass CapLC SystemUser’s Guide

71500021802Revision A

Micromass CapLC System User’s Guide

Safety WarningsThese warnings appear in this manual where care is required to avoid personalinjury.

Des avertissements sont donnés dan ce manuel aux endroits où l’utilisateur doitêtre particulierement prudent pour eviter les blessures.

HighVoltage

ToxinHazard

ChemicalHazard

FlammableMaterial

GeneralHazard

HeuteTension

Risqued’empoi-sonement

ChimiquesDangereux

ProduitsInflammables

HazardGénéral

If the instrument is used in a manner not specified by the manufacturer, theprotection provided by the equipment can be impaired.

Dans la cas où appareil serait utilisé de maniere non specifice par la fabricant, leniveau de protection de l’appareil pourrait être altèré.

Safety SécuritéThe instrument bears thissymbol where highvoltages are present.

Ce pictogramme indique lapresence d’haut tension.

The instrument bears thissymbol where the usershould referto this User's Guide forinstructions which mightprevent damage to theinstrument.

Ce pictogramme indique lanecessité de se réferer aumanuel d'utilisation afind'eviter d'endomagerl'appareil.


Attention: Always follow generally accepted procedures forquality control and methods development when you use thisinstrument.

Caution: To protect against fire hazard, replace fuses with thoseof the same type and rating.

Attention: Changes or modifications to this unit not expresslyapproved by the party responsible for compliance could void theuser’s authority to operate the equipment.

Caution: Always follow generally accepted procedures for qualitycontrol and methods development when you use this instrument.


vTable of Contents

Table of ContentsSafety Warnings ...................................................................................................................... iTable of Contents ................................................................................................................... v

Chapter 1: Introduction.........................................................................................................1-1

System Description .............................................................................................................1-1Pump ...................................................................................................................................1-4

Controls and Indicators.................................................................................................1-4Components ..................................................................................................................1-5

Autosampler ........................................................................................................................1-6Autosampler Injection Modes.......................................................................................1-8

Stream Select Module .........................................................................................................1-8Chapter 2: Installation ...........................................................................................................2-1

Unpacking ...........................................................................................................................2-3Installing the Autosampler ..................................................................................................2-4

Preparing the Autosampler ...........................................................................................2-6Attaching the Autosampler Wash Solvent Bracket ......................................................2-6

Connecting Tubing..............................................................................................................2-7Connecting Tubing to the Autosampler Inject Valve ...................................................2-8Connecting the Waste Tubing and Waste Container ....................................................2-8Connecting the Micromass CapLC Waste Ports...........................................................2-9Connecting the Vent Valve Waste Tubing .................................................................2-10Attaching the Stream Select Module ..........................................................................2-10Connecting Tubing to the Stream Select Module.......................................................2-14Assembling low-pressure connectors .........................................................................2-16Stream Select Valve Flow Patterns.............................................................................2-16Installing Precolumns and Analytical Columns on the Stream Select Module ..........2-18

Reconfiguring the Micromass CapLC System..................................................................2-20Changing the System Configuration...........................................................................2-20Changing the Autosampler Configuration..................................................................2-21

Connecting Cables and Power...........................................................................................2-22Connecting the Computer (PC) ..................................................................................2-22Connecting the Autosampler ......................................................................................2-23Connecting Analog Signals ........................................................................................2-23Contact Closure Connection Cable.............................................................................2-26Connecting the Air Supply .........................................................................................2-26Connecting to the Power Supply ................................................................................2-27

Chapter 3: System Configuration .........................................................................................3-1

Starting the System..............................................................................................................3-2Configuring the Software ....................................................................................................3-3Preparing the Solvent Reservoirs ........................................................................................3-7

Filtering Buffers ...........................................................................................................3-7Degassing Solvents.......................................................................................................3-7Priming the Pumps........................................................................................................3-8Dry Priming the Pumps ................................................................................................3-8Wet Priming................................................................................................................3-10

Preparing the Autosampler................................................................................................3-11Setting Up the Wash Solvent ......................................................................................3-11Preparing Transport Vials...........................................................................................3-13

Configuring Sample Plates................................................................................................3-14Guidelines ...................................................................................................................3-14Loading Sample Plates ...............................................................................................3-15


viTable of Contents

Generating Plate Types and Setting Vial References .................................................3-16Configuring Bed Layouts ...........................................................................................3-20Changing Bed Layouts ...............................................................................................3-21

Chapter 4: Operation .............................................................................................................4-1

Creating a Project ................................................................................................................4-2Creating an Inlet Method.....................................................................................................4-3

Setting Initial Conditions..............................................................................................4-3Setting Stream Select Module Parameters: Precolumn-only .......................................4-6Setting Stream Select Module Parameters: Precolumn-Plus Analytical Column......4-10Setting Initial Event Conditions..................................................................................4-16Setting Timed Events..................................................................................................4-17Setting Chart Recording Parameters...........................................................................4-19Choosing a Method Type............................................................................................4-21Setting Autosampler Parameters.................................................................................4-22Setting Mix Method Parameters .................................................................................4-24Saving the Method......................................................................................................4-27Acquiring Data ...........................................................................................................4-27Equilibrating the System.............................................................................................4-27Tuning the Mass Spectrometer ...................................................................................4-28Injecting Samples........................................................................................................4-28Creating a Sample List................................................................................................4-30Starting an Acquisition ...............................................................................................4-30Stopping a Run ...........................................................................................................4-31

Chapter 5: Maintenance ........................................................................................................5-1

Maintenance Considerations ...............................................................................................5-2Proper Operating Procedures ........................................................................................5-2Routine Maintenance Protocols ....................................................................................5-3

Maintaining the Pump .........................................................................................................5-3Replacing the Solvent Bottle Filters .............................................................................5-4Replacing the Pump Tray Frit Filters ...........................................................................5-5Replacing the Pump Fuse .............................................................................................5-6

Maintaining the Autosampler..............................................................................................5-8Replacing the Sample Loop........................................................................................5-10Replacing the Syringe or Plunger Tip.........................................................................5-14Replacing the Sample Needle .....................................................................................5-17Replacing the Puncture Needle...................................................................................5-19Replacing the Buffer Tubing ......................................................................................5-20Replacing the Autosampler Fuse ................................................................................5-22Replacing Precolumns and Columns ..........................................................................5-26Connecting Liquid Pathways......................................................................................5-27Cutting PEEK Tubing.................................................................................................5-27Connecting Tubing to Pump Tray Manifold Outlets ..................................................5-28Connecting Tubing to Vent, Inject, and Stream Select Valves...................................5-29

Chapter 6: Specifications .......................................................................................................6-1

Chapter 7: Spare Parts ..........................................................................................................7-1


1-1Introduction

Chapter 1: IntroductionThis chapter describes the system and how its components function.

The Micromass® CapLC™ system combines isocratic or gradient solvent delivery,automated sample management, and mass spectrometer interfacing with acapillary LC system controlled by Micromass MassLynx NT™ software.Micromass CapLC system configurations include the following features:

• Isocratic, binary, and ternary gradient

• Binary gradient with post-column reagent addition

• Sample focusing

• Column bypass

System DescriptionThese major components compose the Micromass CapLC system:

• Pump

• Autosampler

• Stream select module

• Any Micromass mass spectrometer fitted with a ZSpray™ source

• Trolley

• MassLynx NT software (Version 3.4 or later) running on a PC (notshown)


1-2Introduction

Figure 1-1 shows the major components of the Micromass CapLC system.

Figure 1-1 Micromass CapLC System Components

Trolley

Stream Select Module

Mass Spectrometer

Pump


1-3Introduction

Table 1-1 describes the primary functions of each system component.

Table 1-1 Micromass CapLC System Components

Component Function

Autosampler Processes samples in 1.5-mL vials, 1.5-mLEppendorf tubes, and 96- or 384-wellmicrotiter plates. A wash station rinses thebuffer tubing and sample needle to preventcross-contamination. A 6-port, 2-positioninjection valve with an interchangeablesample loop injects sample onto thecolumn. A sample heater/cooler regulatesthe temperature of the sample plate on theplate holder.

Stream select module Connects the Micromass CapLC system tothe nanospray interface of a MicromassZMD™, or Micromass LCT™, Quattro LC™,or Q-ToF™ mass spectrometer.

Pump Delivers low flow rates as required forcapillary analysis. Configurable forgradient and isocratic operation, the systemsupports binary or ternary gradientoperation. As an alternative to ternarygradient operation, the third pump canfunction as an auxiliary for post-columnaddition or sample focusing.

Trolley Provides a stable support for the pump andautosampler. Retractable supports keep thetrolley stationary.

Software interface(not shown)

MassLynx NT software controls andmonitors all components in the MicromassCapLC system and manages all LC/MSdata acquisition functions.


1-4Introduction

PumpFigure 1-2 shows pump controls, indicators, and components.

Figure 1-2 Pump Components (Front View)

Controls and Indicators

The pump is a self-contained unit with its own electrical power supply. Table1-2 describes the functions of each control or indicator.

Table 1-2 Pump Controls

Control or Indicator Function

Power switch Select position I to switch on the pump.Select position O to switch it off.

ON Green LED illuminates when you switchon the pump system.

RUN Green LED illuminates when you start arun.

ON and RUNIndicators

Power Switch10-Port Valve


1-5Introduction

Components

The Micromass CapLC system includes three pump trays, each containing thevalves, pumps, manifolds, and other components that provide the low-volumesolvent delivery needed for capillary-scale analysis.

Table 1-3 briefly describes pump components and their functions.

Table 1-3 Pump Components

Component Function

Pump traymanifold

Connects each solvent reservoir and its correspondingvalve/pump-syringe pair on the intake stroke, and thevalve/pump-syringe pair and the vent valve on thedelivery stroke. A stainless steel frit on the outlet ferrulefilters delivered solvent.

Four-portvalves

Operate in phase with the pump syringes, switchingbetween three positions: delivery, intake, andprecompression or decompression of solvent.

Pumpsyringes

Aspirate and deliver solvent. The pump syringes workreciprocally to provide a smooth, continuous flow: oneaspirating solvent, the other delivering it.

Transducer(one perpumpsyringe)

Senses the pressure in the pump syringe.

Stainlesssteel frit(located ineach pumptray manifoldoutlet)

Filters the solvent as it flows from the pumps to theautosampler.


1-6Introduction

AutosamplerThe Micromass CapLC autosampler accepts samples from 1.5-mL vials, 1.5-mLEppendorf tubes, and 96-well or 384-well microtiter plates. It can use bothstandard and deep-well 96-position microtiter plates. Buffer tubing addssufficient line volume between the sample and the syringe to prevent syringecontamination, and a wash station rinses the buffer tubing and sample needleafter each injection. The needle assembly contains a fused silica sample needle(for sample transport) within a hollow outer needle (for puncturing). A six-position injection valve with an interchangeable sample loop injects sample ontothe column. A heater/cooler regulates the temperature of the sample plate on theplate holder. Figure 1-3 shows the components and Table 1-4 describes theirfunctions.

TP01673

Figure 1-3 Autosampler Components

SampleLoop

WashStation

DrainOutlets

Syringe

NeedleAssembly

SamplePlate

BufferTubing

Inject Valve

TransportVial


1-7Introduction

Table 1-4 Autosampler Components

Component Function

Wash solvent bottle Contains up to 100 mL of degassed wash solventfor cleaning buffer tubing and the sample needleafter each injection

Syringe Aspirates sample from the plate, or mobile phasefrom the transport vial, as required

Buffer tubing Provides line volume between the syringe andinjector, thus preventing syringe contamination

Inject valve Provides the switching functions by whichsamples (and transport liquids in Microliter-Pickup mode) are loaded into the sample loopand injected onto the column

Needle assembly Outer needle punctures the septum, capmat, orsealer, and can apply headspace pressure to thesample or transport vial to assist in sampleaspiration

Transport/reagent vials In Microliter-Pickup mode, vials hold the mobilephase that transport the sample into the sampleloop or hold the reagents used in mix methods

Sample plate Racks for 1.5-mL vials or 1.5-mL Eppendorftubes (with sleeves); microtiter plates for 96 or384 positions (96-position microtiter platesinclude standard and deep-well styles)

Plate holder Holds and positions the sample plate

Wash station Bathes the sample needle and buffer tubing withwash solvent

Drain outlet Outlet for wash station effluent

Condensed water outlet Outlet for condensate and/or leakage


1-8Introduction

Autosampler Injection Modes

You can set the autosampler to any of the following injection modes:

• Full-Loop – The sample loop completely fills with sample.

• Partial-Loop – The sample loop partially fills with sample, resultingin little sample loss.

• Microliter-Pickup – Sample is aspirated then transported into theloop with mobile phase, resulting in zero sample loss but diminishedreproducibility.

Stream Select ModuleThe stream select module includes components for low flow ratechromatography, which uses a short precolumn to trap and desalt protein digestsand rapidly elute them (Figure 2-10). The module also works with a gradientmethod, using the precolumn plus a longer, analytical column. Though slower,this second method yields better resolution than the first (Figure 2-11).

Figure 1-4 shows the Stream Select Module components.

Figure 1-4 Stream Select Module Components

Mixing Tee

Frit Filter

Module InterfaceConnector

Nano Tee

Column Cartridge

Stream SelectValve

Mixing Tee


1-9Introduction

Table 1-5 describes how the stream select module components work.

Table 1-5 Stream Select Module Components

Component Description

Stream select valve A 10-port switching valve controlled byautosampler timed events. It employs auser-serviceable rotor seal.

Column cartridge A small column that concentrates asample before elution.

Frit filter A filter element that filters flow from theautosampler.

Nano tee A tee whose design allows no deadvolume within it

Mixing tee (2) A tee that mixes solvents from pumptrays A and B.

Module interfaceconnector

A 15-pin connector that carries signalsbetween the module and the autosampler.


1-10Introduction


2-1Installation

Chapter 2: InstallationFigure 2-1 sequentially depicts the installation procedures in this chapter.

Figure 2-1 Installation Flowchart

Unpacking

ConnectingLiquid

Pathways

Reconfiguring theMicromass CapLC

System

ConnectingCables and

Power

InstallationComplete

Connecting theWaste Tubing

Connecting the StreamSelect Module

Connecting theWaste Ports

Installing a Precolumnand Column

Connecting OtherWaste Tubing

Preparing theAutosampler

Attaching theWash Solvent Bracket

Connecting to thePower Supply

System

Autosampler

Connecting theComputer (PC)

Connecting theAutosampler

Connecting AnalogSignals

Connecting theAir Supply

Installing theAutosampler

Installing theAutosamplerFuse Holder

Connecting Tubing tothe Autosampler

Inject Valve

Installation

Pump

Autosampler InjectValve

Trolley

Connecting theContact Closure

Cable


2-2Installation

Table 2-1 shows the system requirements.

Caution: Allow at least 2 inches (5 cm) of space at the rear andsides of the unit.

Table 2-1 System Requirements

Item Requirement

Temperature 4 to 40 °C (39 to 104 °F)

Relative humidity 20 to 80%, noncondensing

Bench space Width: 19 in. (50 cm)Depth: 23 in. (60 cm)Height: 28 in. (70 cm)Weight: 216 lb. (98 kg)

Clearance At least 2 in. (5 cm) at rear and sides forventilation and cable connections

Power requirements 100 to 120 VAC or 220 to 240 VAC, 50to 60 Hz, single phase; 10 A

Air supply 35 to 45 psi (2.4 to 3.1 bar) regulatednitrogen or clean filtered ambient air

Electromagnetic fields No nearby source of electromagneticnoise, such as arcing relays or electricmotors

Static electricity Negligible

Vibration Negligible


2-3Installation

UnpackingMicromass® ships the Micromass CapLC� system in six parts:

• Pump with the Startup Kit

• Autosampler with the Autosampler Startup Kit

• Steam select module

• Waste bottle assembly

• Stream select cable

• Trolley

Included in the shipment are the Micromass CapLC System User’s Guide andDeclaration of Conformity.

Caution: The pump is heavy and requires at least two people to liftit from its shipping pallet.

Required Materials

• Utility knife or scissors

• Trolley

Before You Begin

Inspect the shipping containers for damage or evidence of mishandling. If youdetect such evidence, notify the carrier before opening the container.

Procedure

1. Cut and remove the straps that secure the large carton (pump).

2. Lift the carton off the pallet.

3. Remove the foam packing material from the top of the pump.

4. Remove the Startup Kit.

5. Raise the pump from its foam support, and set it carefully on the trolley.

6. Anchor the trolley in place by adjusting its feet.

Note: The trolley must be level for solvents to properly drain.

7. Confirm that all items are included in the Startup Kit, checking themagainst the accompanying parts list.

9. Save the Declaration of Conformity.


2-4Installation

10. Inspect all items for damage. Immediately report any shipping damageto both the shipping company and your Micromass representative.

Installing the AutosamplerCaution: Do not connect power or signal wires to the autosampler atthis time.

Complete the following procedures to prepare the autosampler for operation:

• Installing the autosampler fuse holder

• Preparing the autosampler

• Installing the autosampler bracket

Installing the Autosampler Fuse Holder

Perform this procedure when:

• Installing the system

• Moving the system to a location at which the voltage is higher orlower than the original voltage

Required Materials

• Small, flat-blade screwdriver

• Fuses

Before You Begin

Caution: Do not connect power or signal wires to the autosampler atthis time.

Establish the input voltage source rating.


2-5Installation

Procedure

1. Use the flat-blade screwdriver to pry the fuse holder out of its assembly(Figure 2-2).

Figure 2-2 Fuse Holder (Top View)

2. Install a fuse in the fuse holder spring clips. It must be compatible with thevoltage source, 110 to 120 VAC or 220 to 240 VAC (Table 2-2).

Attention: The fuse holder position determines the autosampler voltagesetting. If you set the fuse holder to the lower voltage range (110 to 120VAC), and the input voltage source is 220 to 240 VAC, the higher voltagecould damage the autosampler. Conversely, if you set the fuse holder to thehigher voltage range (220 to 240 VAC), and the input voltage source is 110to 120 VAC, the instrument will not operate properly.

3. Position the fuse holder to the proper voltage setting. The selected voltagesetting appears at the bottom of the fuse holder label.

4. Reinstall the fuse holder in the power switch assembly.

5. Set the autosampler power switch to On.


2-6Installation

Table 2-2 Autosampler Fuses

Voltage Source Fuse

110 to 120 VAC 5 A

220 to 240 VAC 2.5 A

Preparing the Autosampler

Required Materials

• 3-mm hex wrench

• Wash-solvent bracket (Autosampler Startup Kit)

• Plate holder (Autosampler Startup Kit)

Before You Begin

Make sure electrical power is not connected to the system.

Procedure

1. Set the autosampler on top of the pump. Make sure the autosamplerdrain outlet fits into its corresponding drain receptacle on the pump.

2. Use the 3-mm hex wrench to remove the shipping screw from the leftside of the autosampler cover, then open the cover.

3. Locate the plate holder in the Autosampler Startup Kit, and position it onthe autosampler. The gear wheel must fit into the gear-strip on thebottom of the plate holder.

Attaching the Autosampler Wash Solvent Bracket

Required Material

Wash solvent bracket (Autosampler Startup Kit)

Before You Begin

Make sure electrical power is not connected to the system.


2-7Installation

Procedure

1. Connect the red (0.005-inch) PEEK™ tubing from the 10-port valve toport 1 of the inject valve (Figure 2-3).

TP01702

Figure 2-3 Attaching the Wash Solvent Bracket

2. Connect the brown (0.0025-inch) PEEK tubing to port 6 of the injectvalve.

3. Close the autosampler cover.

Connecting TubingConnect tubing as follows:

1. Connect line port 8 on the vent valve to port 1 on the autosampler injectvalve with PEEK tubing.

2. Connect the pump outlet to the stream select module with two PEEKlines.

3. Connect the column, inlet and outlet.

4. Connect the waste tubing to a waste container.


2-8Installation

Connecting Tubing to the Autosampler Inject Valve

Each PEEK tubing connection at the inject valve includes a ferrule, a stainlesssteel lock ring, and a stainless steel compression screw (Figure 2-4).

Figure 2-4 Tubing Assembly for Valves

Required Materials

• 1/4-inch open-end wrench (Autosampler Startup Kit)

• PEEK tubing (attached to system)

• Compression screw with ferrule and lock ring (attached to tubing)

Before You Begin

Make sure the stainless steel compression screw, lock ring, and ferrule areattached to the valve-end of the PEEK tubing, as shown Figure 2-4.

Procedure

1. While pressing the tubing assembly into the appropriate valve port,finger-tighten the compression screw.

Note: Make sure the tubing is inserted fully into the valve port.

2. To securely seat the ferrule against the tubing, tighten the compressionscrew a quarter-turn beyond finger tight with the 1/4-inch wrench.

Refer to Chapter 5, Maintenance, for details on connecting tubing in theMicromass CapLC system.

Connecting the Waste Tubing and Waste Container

Connect waste tubing from the following waste outlets:

• Two ports, for sample and condensate waste streams at the lower leftof the pump

• One 1/4-inch corrugated waste tube from the vent valve

• One pump-waste drain, right side, bottom (Figure 2-5)

TubingLock Ring Ferrule

Compression Screw


2-9Installation

Connecting the Micromass CapLC Waste Ports

Required Materials

• Tubing, 3/16-inch ID

• Connectors

• Y-connector (part number 607305)

• Solvent waste container (chemically compatible with solvents andsamples)

• Condensate waste container

Before You Begin

Check that the autosampler drain outlets are aligned with the drain receptacle onthe pump.

Procedure

1. Place the pump on the trolley, so that the drain tubes align with the holesin the trolley's top plate.

2. Place the waste bottles in their holders at the front of the trolley.

3. Push the tapered connectors through the holes in the table.

4. Cut lengths of tubing to go from the connectors to the barbed fittings onthe waste bottle lids. Push the tubing onto each fitting.

5. Dress the Y-connector along the trolley's right side.

6. Cut tubing to extend from the stream select module drip tray to theY-connector.

7. Cut tubing to extend from the drain on the left side of the pump to theY-connector.

8. Connect the tubing to the Y-connector.

9. Cut tubing to extend from the Y-connector to the solvent waste bottle(Figure 2-5).


2-10Installation

Figure 2-5 Micromass CapLC Waste Port Connections

Connecting the Vent Valve Waste Tubing

Procedure

1. Find three lengths of waste tubing, each secured at one end to valve ports5, 6, and 10. The free ends are coiled above the pump trays.

2. Route each free end through a convoluted waste tube to the solvent wastecontainer.

Attaching the Stream Select Module

The stream select module attaches to the nanoflow flange. It assumes one ofthree orientations, depending on the mass spectrometer model. Two of theseorientations fix the module parallel to the nanoflow flange (Figure 2-6 andFigure 2-7). The third orientation fixes it perpendicular to the nanoflow flange(Figure 2-8).

Trolley

Tubing to Stream Select Module

Y-Connector

Connectors (2)


2-11Installation

Orientation 1 (For use with a Q-ToF™ Mass Spectrometer)

Required Materials

• Stream Select Module

• Angle bracket (provided)

• Drip tray

• One M6 × 16 screw (provided)

• Two M5 × 16 screws (provided)


• Knurled thumbscrew

Procedure

1. Attach the angle bracket to the nanoflow flange on the mass spectrometerwith the two M5 x 16 screws.

2. Attach the stream select module to the shorter side of the angle bracket(Figure 2-6). Use the M6 x 16 screw and the M4 x 5 screw provided.The M4 x 5 screw prevents the module from rotating.

3. Attach the drip tray with the knurled thumbscrew.

Figure 2-6 Orientation 1: Parallel to Nanoflow Flange Using Angle Bracket(Q-ToF Model)

ThumbscrewFront Flange

Drip Tray

NanoLCSprayer

Source


Nanoflow FlangeAngle Bracket


2-12Installation

Orientation 2 (For use with an alternative Q-ToF Mass Spectrometer)

Required Materials

• Stream Select Module

• 2 dowels (provided)

• 2 M5 × 16 screw

• 2 M6 × 16 screw

• 2 setscrews (provided)

• Earth plate (provided)


• Drip tray (provided)

• Thumb nut (provided)

Procedure

1. Insert the two dowels into the right end (as viewed from the front) of thestream select module front flange (Figure 2-7).

2. Fit the flange onto the nanoflow source.

3. Secure the flange with the two setscrews.

4. Align the earth plate with two holes on the nanoflow flange and twoholes on the module.

5. Align holes in the support bracket with those in the earth plate, andsecure the parts with the two M6 x 16 screws.

6. Secure the nanoflow flange to the angle bracket with the two M5 x 16screws.

7. Secure the drip tray with the thumbscrew.


2-13Installation

Figure 2-7 Orientation 2: Parallel to Nanoflow Flange (Alternative Q-ToFModels)

Orientation 3 (For use with Quattro LC™, Ultima, and LCT™ MassSpectrometer models)

Required Materials

• Stream select module


• Drip tray


• Two M5 × 16 screw (provided)


• Knurled thumbscrew

SupportBracket

Earth Plate


Front Flange

Drip Tray

Thumbscrew NanoLCSprayer

Source

Dowels


2-14Installation

Procedure

1. Attach the angle bracket to the nanoflow flange (Figure 2-8) with the twoM5 x 16 screws.

2. Attach the module to the angle bracket with the M6 x 16 screw and theM4 × 5 screw.

3. Attach the drip tray with the thumbscrew.

Figure 2-8 Orientation 3: Perpendicular to Nanoflow Flange (Quattro, Ultima,and LCT Models)

Connecting Tubing to the Stream Select Module

PEEK tubing extends from the autosampler outlet to the stream select module. Itcarries solvent from pumps A and B through the pump vent valve to themodule’s mixing tee. PTFE tubing encloses the terminal ends of fused silicalines as a shim/sleeve.

Note: The lower the eluent flow, the more critical is the need to minimize linevolume.

Source

Front Flange

DripTray

Thumbscrew

NanoLCSprayer

Angle Bracket



2-15Installation

Connecting Fused Silica Tubing

Required Materials

• 375-µm OD × 25-µm ID fused silica tubing



• PTFE tubing (for fashioning the sleeve)

Procedure

Note: Refer to Figure 2-10 and Figure 2-11 to identify the stream selectmodule flow pattern required.

1. Cut two approximately 30-mm lengths of PTFE tubing.

2. Slide the end of the fused silica line through the PTFE sleeve.

3. Tighten the fitting, ensuring the sleeve rests against the bottom of theport by pushing it inward as you tighten. The end of the fused silica mustbe flush with the end of the PTFE sleeve.

4. Make sure all fused silica waste tubing passes through the guide and intothe waste container at the rear of the plate.

Attention: Minimize tubing lengths when connecting fluid lines on thestream select module.

Connecting PEEK Tubing

Required Materials

• PEEK tubing, 1/16-inch × 64-µm ID

• PEEK tubing, 1/16-inch × 125-µm ID (from pumps)


2-16Installation

Procedure

1. Identify the correct ID tubing from the flow path diagrams (Figure 2-10and Figure 2-11).

2. Cut the lengths of tubing as needed.

3. Insert the tubing fully into the port, taking care to avoid creating deadvolumes.

Assembling Low-pressure Connectors

Low-pressure connectors are fittings that use 375 OD fused silica to connect thestream select module output to the nanoLC sprayer (Figure 2-9).

Required Materials

• 375-µm OD fused silica tubing (any ID)

• PTFE tubing (part number 6060786)

• Low-pressure PTFE connectors (part number 6060786)

Procedure

1. Cut the following approximate tubing lengths:

• 3/4 inch (20-cm) of PTFE tubing

• 8 inch (20-cm) of 375 OD of fused silica (any ID)

2. Insert the fused silica through the PTFE tubing until it emerges at theopposite end.

Note: This is often a difficult operation due to the friction of forcing one tubeinside another.

Note: You should plumb the nanoLC sprayer so that a short tail of fusedsilica is positioned at its rear (Figure 2-9).

Figure 2-9 NanoLC Sprayer

Stream Select Valve Flow Patterns

Figure 2-10 and Figure 2-11 illustrate two alternative stream select valve flowpatterns. Regardless of which flow pattern you choose, you must configurepump C in the MassLynx software as an auxiliary gradient. To do this, go to theCapLC Diagnostics window (see Figure 3-10). Select Control from the menu

FromFrom StreamSelect Module

Low Pressure PTFEConnector

Short Tail ofFused Silica


2-17Installation

bar, then User Diagnostics and, finally, Configuration. Refer to “SettingStream Select Module Parameters” in Chapter 4 for details.

Flow Pattern for Precolumn Use

Figure 2-10 shows the stream select module plumbed for a short precolumn only.This configuration affords fast chromatography when a highly resolvedseparation is not needed.

Figure 2-10 Stream Select Module Configured for Precolumn-only Use

Red Peek 64 µm ID PEEK 50 µm ID Fused Silica

25 µm ID Fused Silica75 µm ID Fused Silica

Holder and CartridgeFilter

Nano Tee

FromAutosampler

Nano Tee Side Port

PTFE Low PressureConnector

Mixing Tee

A

B


2-18Installation

Flow Pattern for Precolumn Plus Analytical Column

Figure 2-11 shows the stream select module plumbed for a precolumn and ananalytical column. This configuration affords excellent resolution when usedwith a gradient method.

Figure 2-11 Stream Select Module Configured for Precolumn Plus AnalyticalColumn

Installing Precolumns and Analytical Columns on theStream Select Module

The stream select module holds precolumns and columns. Depending on youranalytical needs, you can configure the system to use only the precolumn or botha precolumn and an analytical column.

Installing a Precolumn

The precolumn is a cartridge that fits inside a holder. The holder has liquid sealsat each end, which you secure with finger-tight fittings and PTFE sleeves over afused silica capillary (Figure 2-12).

A

B

Red Peek 64 µm ID PEEK 50 µm ID Fused Silica

25 µm ID Fused Silica75 µm ID Fused Silica

Holder and CartridgeFilter

FromAutosampler

Mixing Tee

Analytical Column


2-19Installation

Required Materials

• Precolumn cartridge (part number 6062925)

• Precolumn holder (part number M950609AD1)

• Two finger-tight fittings (6062940)

• Fused silica, 50 micron ID (6490506)

• PTFE sleeve

Procedure (refer to Figure 2-10)

1. Cut about 16 inches (40 cm) of PTFE tubing for use as sleeves to placeover the fused silica capillary.

2. Pass the fused silica through the sleeve, and pass the sleeve through thefinger-tight nuts.

3. Place a precolumn cartridge in the holder.

Note: The fused silica on one side of the cartridge holder can attach to thefused silica of the nano tee, depending on the application.

4. Be sure to press the sleeve and fused silica firmly into the fitting.

5. Tighten the two nuts (only finger tightness is required) until they grip thefused silica. Their exposed thread lengths must be equal in length.

Figure 2-12 Precolumn

Installing an Analytical Column (Refer to Figure 2-13).

Required Materials

Microtight union, P770 (part number 6072213)

Microtight sleeve, F185 (part number 6072210)

Note: For longer analytical columns, Micromass supplies a microtight union,which connects the fused silica to a 1/16- inch male fitting. Micromass alsosupplies fittings for 360-micron OD fused silica.

Precolumn Cartridge

Fused Silica

Finger-tight NutCartridge Holder

Finger-tight Nut


2-20Installation

Procedure (refer to Figure 2-11)

1. Insert the inlet side of the column into the microtight union.

2. Pass the fused silica from port 2 through the microtight sleeve

3. Press the fused silica and sleeve into the fitting.

4. Tighten the nut to grip the fused silica

Note: Only finger tightness is required.

5. Secure the column to the front flange of the stream select module with anappropriate clip.

Figure 2-13 Analytical Column

Reconfiguring the Micromass CapLC SystemYou can configure the Micromass CapLC system to perform optimally invarious applications. This often means changing the following system features:

• System configuration, such as changing from the standard precolumnconfiguration to a precolumn plus analytical column configuration

• Autosampler configuration, such as sample loop size, syringe size,sample needle size, and buffer tubing size

Changing the System Configuration

Adopting a new system configuration involves making new tubing connections.Consult Chapter 5, “Maintenance,” for instructions on how to connect tubingbetween the various system components. The optional Tubing Kit (part number186000684) contains the tubing and fittings you need to reconfigure the system.

Note: Some Micromass CapLC configurations require optional components.Make sure you have these components before starting the reconfiguration.

Note: Changing the system configuration might require editing the systemconfiguration settings in the MassLynx NT™ Micromass CapLC DiagnosticsConfiguration page.

Microtight Sleeve

Fused Silica

Microtight Union

Analytical Column with Male Fitting

Microtight Nut


2-21Installation

Changing the Autosampler Configuration

Sample Loop

You can change the sample loop from the standard 10-µL to 5-, 20-, 50-, or100-µL.

Sample Syringe

You can change the sample syringe from the standard 25-µL to 100- or 250-µL.

Sample Needle

You can change the sample needle from the standard 2.4-µL to 5.4-µL.

Buffer Tubing

You can change the buffer tubing from the standard 50-µL to 500-µL.

Note: For instructions on how to change the loop, syringe, needle, and buffertubing, see Chapter 5, “Maintenance.”


2-22Installation

Connecting Cables and Power

Caution: Avoid electrical shock and damage to instruments: do notconnect cables while the system power is on.

Figure 2-14 shows the cables you must connect before providing power to thesystem.

USE ONLY WITH 250VFUSES/EMPLOYERUNIQUEMENT AVEC

DESFUSIBLES DE 250V

110-120 V

220-240V

AUTO SAMPLER POWER

V - 115/230

AMAX 2.5

Hz 50/60

NITROGEN OR FILTEREDSHOP AIR CONNECTION

310 KPA (45psi) MAX PRESSURE240 KPA (35psi) MIN PRESSURE

B

IEEE 488

V- 120/240 Hz 50/60

- - F10A-250A

MAX6.5

A

1 2 3 4 5 6 7 8 9 10 11 12

Figure 2-14 Micromass CapLC Cable Connections (Rear View)

Connecting the Computer (PC)

Required Materials

• 6-ft (1.8-m) IEEE-488 cable

• RS-232 communications cable

Before You Begin

• Turn off the power to the Micromass CapLC system.

• Turn off the power to the computer.

Procedure

1. Connect the IEEE-488 cable from the IEEE-GPIB board in the PC to thetop IEEE-488 port on the pump.

2. Connect the RS-232 communications cable from the RS-232 serialconnector in the PC to the RS-232 communications port on theautosampler.

To Power Supply


To Air Supply

Autosampler

AnalogInputs/Outputs

RS-232 Communications Cable

1.8-m CableIEEE-488

to PC

Pump


2-23Installation

Connecting the Autosampler

Required Material

Autosampler/stream select cable (Startup Kit)

Before You Begin

Turn off the power to the Micromass CapLC system.

Procedure

Connect the stream select cable from the stream select port on the autosampler tothe stream select port on the stream select module.

Connecting Analog Signals

Two removable connectors are mounted on rear panel of the pump. Thesecontain screw-in terminals for analog signals, event signals, and the contactclosure (inject start) signal to the mass spectrometer.

Table 2-3 identifies the individual I/O signal connections on the analog signalconnectors terminal.

Note: To prevent external electrical disturbances from adversely affectinginstrument performance, do not connect cables longer than 9.8 feet (3-m) to theterminals. Also, to ensure adequate grounding, always attach the shield of eachcable to the chassis of one instrument only.


2-24Installation

I/O Signals

Table 2-3 describes input and output signals on the Micromass CapLC systemI/O connectors terminal.

Table 2-3 Signal Connections

Terminal Description

Inject Start Contact closure output signals to themass spectrometer that an injection hasbegun. Connect a wire pair from thetwo Inject Start terminals to the contactclosure input connections on the massspectrometer.

Switch 1

Switch 2

See the “Event Signals” section, below.

Chart Out 1

Chart Out 2

An output signal (selected in the ChartRecording page of the Waters CapLCInlet Editor) to an integrator or stripchart recorder. Select one of thefollowing parameters as the Chart Outsignal:Flow rateSystem pressurePercent composition (A, B, C)Column temperature

Stop Flow An input signal from LC or MScomponents that stops the MicromassCapLC pumps. Connect the positiveinput wire to the Stop Flow (+) terminaland the negative input wire to the StopFlow (–) terminal.


2-25Installation

Chart Out Signal Conditions

Table 2-4 describes input and output signals on the Micromass CapLC systemI/O connectors terminal.

Table 2-4 Chart Out Signal Connections

Chart Out Signal Parameter Setting at0.0 V (Minimum)

Parameter Setting at2.000 V (Maximum)

Off 0 0

Flow rate 0.000 µL/min 40.000 µL/min

System pressure 0.0 psi (0.0 bar, 0.0 kPa) 5000 psi (345 bar,34,474 kPa)

Percent composition(A, B, C)

0.0% 100.0%

Column temperature 20 °C 70 °C


2-26Installation

Event Signals

You can use terminal switches 1 and 2 as contact closure switches to control acolumn selection valve, fraction collector, or similar external device. Each pairof switch terminals (the two Switch 1 terminals, for example) connects to anexternal device. To program a switch as an initial or timed event, use the WatersCapLC Inlet Editor. Table 2-5 describes each switch position and function.

Table 2-5 Event Switch Positions

Position Function

On Closes the switch

Off Opens the switch

Toggle Changes the current state of the switch

Pulse Closes / opens the switch for a user-defined period

No Change Leaves the switch in its current state

Contact Closure Connection Cable

You must connect the contact closure cable (part number 441000252) from themass spectrometer contact closure input (Init 1) to the Inject Start terminals (11and 12) on the Micromass CapLC system I/O-connector terminal block.

Note: Systems that use Version 3.5 of MassLynx NT, including massspectrometer models ZQ and Quattro, require you to configure contact closureswithin the 3.5 application software. To do this, select Inlet Method from thetoolbar, and proceed to Tools/Instrument Configuration. See Chapter 3,“System Configuration.”

Connecting the Air Supply

Required Materials

• Supply of nitrogen or clean, filtered house air

• Regulator (Startup Kit)

• Tee connector (Startup Kit)

Before You Begin

Verify that the compressed air or N2 to the mass spectrometer is shut off.

Procedure

1. Cut the 6-mm line that connects the air or N2 supply to the massspectrometer (Figure 2-15).


2-27Installation

2. Reconnect the line using two legs of the tee.

3. Connect the regulator to the tee's third leg with a short piece of 6-mmline.

4. Connect the remaining regulator port to the pump air/N2 supply inlet atlower-right corner of the pump.

5. Adjust the pump air/N2 pressure to 25 to 45 psi (2.4 to 3.1 bar).

Figure 2-15 Air Supply Connection

Connecting to the Power Supply

For proper operation, the system requires a grounded AC power supply with noabrupt voltage fluctuations. The Micromass CapLC system automatically adjustsfor AC input voltages in two ranges:

• 100 to 120 VAC, 50 to 60 Hz

• 220 to 240 VAC, 50 to 60 Hz

Caution: To avoid electrical shock, do not connect cables while thesystem power is on.

MassSpectrometer

SolventManager

Compressed Air/N2 Supply Line

Regulator

Tee


2-28Installation

Required Materials

• Autosampler power cord

• Main power cord

Before You Begin

Verify that:

• The input voltage setting on the autosampler matches the sourcevoltage.

• The power cord is removed from the power supply.

Procedure

1. Connect the autosampler power cord from the autosampler to the leftpower connector on the pump (Figure 2-16).

USE ONLY WITH 250VFUSES/EMPLOYERUNIQUEMENT AVEC

DESFUSIBLES DE 250V

110-120 V

220-240V

AUTO SAMPLER POWER

V - 115/230

AMAX 2.5

Hz 50/60

NITROGEN OR FILTEREDSHOP AIR CONNECTION

310 KPA (45psi) MAX PRESSURE240 KPA (35psi) MIN PRESSURE

B

IEEE 488

V- 120/240 Hz 50/60

- - F10A-250A

MAX6.5

A

1 2 3 4 5 6 7 8 9 10 11 12

Figure 2-16 Electrical Power Connections

2. Connect the female end of the main power cord to the right powerconnector on the pump.

3. Insert the male end of the main power cord into a grounded power outlet.

Pump

Autosampler

To Power Supply


3-1System Configuration

Chapter 3: System ConfigurationThis chapter explains how to configure MassLynx NT™ and otherwise preparethe system for operation. Figure 3-1 shows the configuration and preparationtasks.

Figure 3-1 System Configuration Flowchart

System Configuration

Starting the System

Configuring theSoftware

Preparing theSolvent Reservoirs

Priming the Pumps

Generating PlateTypes andSetting VialReference

Setting Up theWash Solvent

Preparing TransportVials

Loading SamplePlates

Configuring BedLayouts

Preparing theAutosampler

Configuring SamplePlates

Dry Priming thePump

ConfigurationComplete



Starting the System

Requirements

• User name

• Password

Before You Begin

Verify that:

• The system is connected to an electrical power supply.

• MassLynx NT software is installed on the computer.

Procedure

1. Start the Micromass CapLC unit, moving the power switch to the On (I)position.

2. Start the MassLynx NT PC, observing status LEDs (Table 3-1).

Table 3-1 Status LEDs

LED On (Green) Off Flashing

Power The system isstarted and readyto run.

The power isoff.

The system failedstart-up diagnostics.

Run The system isperforming a run.

The system isnot performinga run.

An error occursduring the run, orcommunication islost.

3. Enter your user name and password to access the Windows NToperating system.

4. Double-click the MassLynx NT icon on the desktop. The MassLynx NTLogin window appears.

5. Enter your logon information and click OK. The MassLynx NT softwarestarts.



Configuring the SoftwareYou must configure the MassLynx NT software to use the Micromass CapLCsystem as an inlet before initially operating the system.

Required Materials

• Priming syringe (part number WAT271053)

• Syringe adapter (part number WAT025559)

Before You Begin

Start the Micromass CapLC system.

Procedure

1. Double-click the MassLynx NT icon on your desktop to start MassLynxNT.

2. Enter your logon name and password, then click OK. The MassLynxMain Window appears (Figure 3-2).

Figure 3-2 MassLynx Main Window



3. Do one of the following to open the Acquisition Control Panel(Figure 3-3):

• If you are using MassLynx NT Version 3.4 or 3.5, which connects thePC to the mass spectrometer via an EPCAS (Ethernet) interface, youmust configure contact closures within the application. (This appliesto Waters® mass spectrometer models ZQ™ and Quattro™). To do this,

click (Inlet Editor) on the MassLynx Main Window (see Figure3-2), then go to step 7.

• If you are using MassLynx NT Version 3.4, and the PC connects withthe mass spectrometer via a T-DAT interface, select Control Panelfrom the Run menu on the MassLynx Main Window (see Figure 3-2),then go to step 4.

Figure 3-3 Acquisition Control Panel

4. Select Interface from the Configure menu. The Select Interface dialogbox appears (Figure 3-4).

Figure 3-4 Select Interface Dialog Box

5. Select GC or LC system (ACE), then click OK. The MicromassCapLC icon appears in the Acquisition Control Panel (Figure 3-5).



Figure 3-5 Acquisition Control Panel with Micromass CapLC Icon

6. Select Inlet from the Methods menu. The Solvent Status page for theWaters CapLC Editor appears (Figure 3-6).

Figure 3-6 Waters CapLC Editor Solvent Status Page

7. Select Instrument Configuration from the Tools menu. TheInstruments page of the Inlet Configuration dialog box appears(Figure 3-7).

Note: MassLynx NT displays the Contact Closure page only when thePC - mass spectrometer connection is an EPCAS (Ethernet) interface(Waters ZQ or Quattro model).



Figure 3-7 Inlet Configuration Dialog Box

8. Click GPIB Communication. The GPIB (general purpose interface bus)Communication page appears (Figure 3-8).

Figure 3-8 GPIB Communication Page

9. Enter the IEEE-488 address for the pump in the ID drop-down list.

Note: Set the pump address to 4.

10. Select 3 s from the I/O Timeout drop-down list.



11. Click OK to save your settings.

12. Select Communications from the Cap LC Tools menu of the WatersCapLC Editor. The Waters CapLC Communications dialog box appears(Figure 3-9).

Figure 3-9 Waters CapLC Communications Dialog Box

13. Choose the communications port the PC uses to connect with theautosampler. This is usually the COM1 port. However, if COM1 isused by another device, select whatever communications port theautosampler uses.

14. Click OK.

The MassLynx NT software is now configured for the Micromass CapLC systemand ready for operation.

Preparing the Solvent ReservoirsCaution: Always observe safe laboratory practices whenoperating your system to avoid chemical or electricalhazards,.

Note: To avoid spilling solvents, do not overfill the solvent reservoirs. Themaximum capacity of each solvent reservoir is 100 mL.

Filtering Buffers

Filter buffers with a 0.5-µm (or smaller) filter. Always filter buffers after mixingthem. Afterward, store them in closed, particulate-free containers.

Degassing Solvents

Always degas solvents, exposing them to vacuum or ultrasonic sound beforeattaching them to the system.



Priming the Pumps

Note: Micromass recommends using methanol when priming the pumps forinitial operation.

Prime the Micromass CapLC pumps as follows:

• Dry prime when the solvent lines are dry.

• Wet prime after a dry prime.

• Wet prime when changing miscible solvents.

Note: You must dry prime each of the three pumps before you operate them forthe first time.

Dry Priming the Pumps

Dry prime for initial operation of the system or whenever a pump is dry.

Required Materials

• Priming syringe (part number WAT271053)

• Syringe adapter (part number WAT025559)

Before You Begin

• Add filtered, degassed solvent to each reservoir

• Connect the three reservoirs to the pump

• Disconnect the waste tube from the vent valve of Pump A, exposingvalve port 10.

• Connect an empty syringe to valve port 10, using the syringe adapter.

Procedure

1. Select Diagnostics from the Cap LC Tools menu of the Waters CapLCEditor. The CapLC Diagnostics window appears (Figure 3-10).



Figure 3-10 CapLC Diagnostics Window

2. Select Dry Prime from the Control menu. The Dry Prime dialog boxappears (Figure 3-11).

Figure 3-11 Dry Prime Dialog Box

3. Specify how many priming cycles you desire. (Micromass recommendsat least 10 cycles.)

4. Select Pump A, then click Start.

5. Retract the syringe plunger as the dry prime method runs. Often,considerable force is needed to move all the air and solvent in the systemtoward the syringe. Continue to retract the plunger for the pump's entiredry prime sequence (Figure 3-12).



Figure 3-12 Dry Priming Ports

6. Repeat steps 4 and 5 for pumps B and C, connecting the syringe to valveports 5 and 6, respectively.

7. Disconnect the syringe and reconnect the waste tubing to the three ventvalve ports.

8. Wet prime as described below.

Wet Priming

Attention: Wet prime only when solvent is in the lines. Solvent mustbe present to prevent damage to pump seals.

A wet prime replaces the solvent in the solvent lines with fresh solvent from thereservoir. Wet prime when changing reservoirs or solvents, or after the pumpshave been idle, which causes the system to partially lose its prime.

Note: If the solvent lines are dry, dry prime first.

Before You Begin

Make sure the solvent reservoirs are connected to the front of the pump andcontain filtered, degassed solvent.

Note: When changing between buffers and nonpolar solvents, always use anintermediate solvent, like water, to prevent buffer precipitation.

Port 10

Port 6Port 5



Procedure

1. Select Diagnostics from the Cap LC Tools menu of the Waters CapLCEditor (see Figure 3-6). The CapLC Diagnostics window appears (seeFigure 3-10).

2. Select Wet Prime from the Control menu. The Wet Prime dialog boxappears (Figure 3-13).

Figure 3-13 Wet Prime Dialog Box

3. Select the pump you want to prime.

4. Specify how many priming cycles you desire. (Micromass recommendsat least 10 cycles, which will require 30 to 35 minutes to complete.)

5. Click Start. The selected pump starts to operate. When the Wet Primemethod ends, the system idles.

6. Repeat steps 2 to 5, as needed, for each solvent. Alternatively, you cancheck all three boxes to wet prime the three pumps simultaneously.

Preparing the AutosamplerSetting Up the Wash Solvent

Caution: Keep hands clear of the autosampler needle during operation.

To prepare the autosampler, you must add filtered, degassed wash solvent to thewash solvent bottle and, if you are operating in Microliter-Pickup mode, preparetransport vials.

Required Materials

• Wash-solvent bottle assembly (bottle holder, bottle, and cap)

• Filtered, degassed wash solvent



Before You Begin

Make sure the monitor displays the MassLynx Main Window (see Figure 3-2).

Procedure

1. Remove the wash-solvent bottle and its holder from the autosampler bylifting and pulling it outward (Figure 3-14).

Figure 3-14 Connecting the Bottle Holder

2. Unscrew the bottle from the cap, which is an integral part of the bottleholder.

3. Fill the bottle with filtered, degassed solvent.

4. Screw the bottle onto the cap.

5. Reattach the bottle holder.

6. Insert the wash solvent tube into the wash solvent.

7. Click (Inlet Editor) in the CapLC Binary status area of the MassLynxMain Window. The Waters CapLC Editor Solvent Status page appears(Figure 3-6).


9. Select User Diagnostics from the Control menu. TheAutosampler/Detector page of the User Diagnostics window appears(Figure 3-15).

1 2



Figure 3-15 User Diagnostics Window

10. Click Wash to perform a standard wash routine.

Note: You need freshly degassed solvent and a bubble-free syringe for good areaprecision. Check the syringe for air bubbles. If you see any, repeat theprocedure until none remain.

Preparing Transport Vials

If you operate in Microliter-Pickup mode, you must also prepare a transport vial.

Required Materials

• Transport vial

• Filtered, degassed solvent

Before You Begin

Ensure that the monitor displays the MassLynx Main Window (see Figure 3-2).

Procedure

1. Fill the transport vial with filtered, degassed solvent.


3. Click Exchange in the Sample Plate section. The plate holder moves tothe left.



4. Insert the transport vial in position 1 on the plate holder, immediately tothe right of the wash station, at the rear of the plate holder (Figure 3-16).

TP01706

Figure 3-16 Autosampler Plate Holder

5. Click Exchange. The plate holder moves to the operating position.

Configuring Sample PlatesGuidelines

Caution: Keep hands clear of the autosampler needle duringoperation.

Compatible Plates

The Micromass CapLC autosampler can accommodate the following plate types:

• 96-well microtiter plate

• 96-well deep microtiter plate

• 384-well microtiter plate

• 48-vial plate

Headspace Pressure Considerations

Depending on programming, the autosampler might apply headspace pressurewhile injecting samples. Observe the following precautions when you preparesamples:

• Do not fill vials to the rim. Pressure could force the sample into theprepuncturing needle. This could contaminate the sample needle andcross-contaminate the samples.

• Fill the standards vials with a narrow-end pipette that allows for airdisplacement.

Transport Vial



• Be sure the seals are sufficiently gastight to prevent bubbles in thesample and, where the samples are volatile, evaporation.

• Do not use vials with limited-volume inserts (LVI).

• Do not apply headspace pressure to microtiter plates.

Loading Sample Plates

Caution: Keep hands clear of the autosampler needle duringoperation.

Required Materials

Sample plate containing samples

Before You Begin

Make sure the monitor displays the MassLynx Main Window (see Figure 3-2).

Note: If you are using wells or vials that are not gastight, be sure that theHead Space Pressure check box is not selected in the AutoSampler page onthe Waters CapLC Editor (Figure 3-17).

Figure 3-17 AutoSampler Page

Procedure

1. Click (Inlet Editor) in the CapLC Binary area of the MassLynx MainWindow. The Waters CapLC Editor Solvent Status page appears (seeFigure 3-6).

2. Select Diagnostics from the Cap LC Tools menu. The CapLCDiagnostics window appears (see Figure 3-10).



3. Click Exchange in the Sample Plate section. The plate holder moves tothe left.

4. Position the plate on the plate holder.

5. Click Exchange. The plate holder moves to the operating position.

6. Confirm that the correct plate type is selected in the MassLynx NT BedLayout page (see Figure 3-22).

7. Confirm that the correct vial reference is selected in the MassLynx NTPlate Generator window (see Figure 3-18).

Generating Plate Types and Setting Vial References

You can establish up to 12 vial-referencing schemes for the plates. Eachreferencing scheme uniquely identifies a plate's vials or wells.

Before You Begin


Procedure

1. Click (Inlet Editor) in the CapLC Binary area of the MassLynx MainWindow. The Waters CapLC Editor Solvent Status page appears (seeFigure 3-6).

2. Select Plate Generator from the Cap LC Tools menu. The PlateGenerator window appears (Figure 3-18).

Figure 3-18 Plate Generator Window



3. Click (New Plate) from the menu bar to create a new plate. To

modify an existing plate, click (Next Plate) to scroll through the listof defined plates until the plate appears in the Plate Name text box.

4. Set the appropriate parameters for the plate. Table 3-2 describes vial-referencing information.

Table 3-2 Micromass CapLC Vial Reference Parameters

Parameter Description

Horizontal Specifies the horizontal axis positions aseither alphabetic (ABC) or numeric (123)when using XY referencing. Default:numeric

Vertical Specifies the vertical axis positions as eitheralphabetic (ABC) or numeric (123) whenusing XY referencing. Default: alphabetic

Priority (See below, XY Referencing.)

XY Referencing With Horizontal first selected, specifies thatthe horizontal axis position appears firstwhen referencing a vial (1,A). WhenHorizontal first is not selected, specifiesthat the vertical axis position appears firstwhen referencing a vial (A,1).

Sequential Referencing With Horizontal first selected, specifies thatvial numbering proceeds horizontally, fromleft to right. When Horizontal first is notselected, specifies that vial numberingproceeds vertically, from top to bottom.

From the menu bar, click (Save Plate) to save the layout.

For more information on creating or modifying plate layouts, see the MassLynxNT Guide to Data Acquisition, Chapter 6, “Controlling Inlet Systems andAutosamplers.”



Vial Referencing Examples

Table 3-3 shows four examples of vial referencing for a simplified 4 × 3 vialplate.

Table 3-3 Vial Referencing Examples

Example 1

1 2 3 4 Horizontal: 1 2 3 ...

A 1,A 2,A 3,A 4,A Vertical: A B C ...

B 1,B 2,B 3,B 4,B Referencing: XY

C 1,C 2,C 3,C 4,C Priority: Horizontalfirst selected

Example 2

1 2 3 4 Horizontal: 1 2 3 ...

A A,1 A,2 A,3 A,4 Vertical: A B C ...

B B,1 B,2 B,3 B,4 Referencing: XY

C C,1 C,2 C,3 C,4 Priority: Horizontalfirst not selected

Example 3

Horizontal: N/A

1 2 3 4 Vertical: N/A

5 6 7 8Referencing:SequentialDiscontinuous

9 10 11 12Priority: Horizontalfirst selected



Example 4

Horizontal: N/A

1 6 7 12 Vertical: N/A

2 5 8 11Referencing:SequentialContinuous

3 4 9 10Priority: Horizontalwas not selected

For more information on vial referencing, see the MassLynx NT Guide to DataAcquisition, Chapter 6, Controlling Inlet Systems and Autosamplers.

Figure 3-19 and Figure 3-20 show a sequential processing order for the last twoexamples above, on a 96-well microtiter plate.

Star t

TP01657

1 2 3 4 5 6 7 8 9

ABCDEFGH

10 11 12

Figure 3-19 Sequential Discontinuous Horizontal First Vial Referencing



1 2 3 4 5 6 7 8 9

ABCDEFGH

Star t

TP01660

10 11 12

Figure 3-20 Sequential Continuous Nonhorizontal First Vial Referencing

Configuring Bed Layouts

The Micromass CapLC bed layout specifies the type of well plate installed in theautosampler. Configure or change bed layouts in the Bed Layout dialog box.

Before You Begin


Procedure

1. Click (Inlet Editor) in the CapLC Binary area of the MassLynx MainWindow (see Figure 3-2). The Waters CapLC Editor Solvent Status pageappears (see Figure 3-6).

2. Select Bed Layout from the Cap LC Tools menu. The Bed Layoutdialog box appears (Figure 3-21), displaying the Current Layout and alist of defined bed layouts.



Figure 3-21 Bed Layout Dialog Box

3. Select a bed layout from the Current Layout drop-down list, then clickOK.

Changing Bed Layouts

To change the plate, modify a previously defined layout.

Before You Begin

Confirm that all plates used in Bed Layout are defined.

Procedure

1. Select Bed Layout from the Cap LC Tools menu of the Waters CapLCEditor (see Figure 3-6). The Bed Layout dialog page appears, displayingthe Current Layout and a list of other defined bed layouts.

2. Select the layout you want to modify in the Bed Layouts list box, thenclick Modify Bed Layout. The Modify Bed Layout page appears (Figure3-22), displaying the selected bed layout.



Figure 3-22 Modify Bed Layout Page

3. Click the plate you want to change. The Plate Position and Type dialogbox appears (Figure 3-23).

Figure 3-23 Plate Position and Type Dialog Box

4. Select the plate type you intend to replace the selected bed layout platewith, then click OK.

For more information on creating or modifying bed layouts, see the MassLynxNT Guide to Data Acquisition, Chapter 6, Controlling Inlet Systems andAutosamplers.


4-1Operation

Chapter 4: OperationFigure 4-1 shows the operational procedures in this chapter.

Figure 4-1 Operation Flowchart

Creating a Project

Operation

Injecting Samples

Setting Initial EventConditions

Creating an InletMethod

Tuning the MassSpectrometer

Setting InitialConditions

Setting Mix MethodParameters

Setting ChartRecording Parameters

Setting AutosamplerParameters

Setting Timed Events

Equilibrating theSystem

Saving the Method

Creating a Sample List

Starting an Acquisition

Stopping a Run

Acquiring Data

Setting GradientParameters

Choosing a MethodType

Using the System

Setting Stream SelectModule Parameters


4-2Operation

Creating a ProjectThis section describes how to create a MassLynx™ NT project using the CreateProject wizard. A project stores inlet methods, calibration curves, raw data, peaklists, sample lists, tune-page settings, and other run parameters. For moreinformation on projects, refer to the MassLynx NT User’s Guide.

1. Select Project Wizard from the File menu of the MassLynx MainWindow. The Create Project page appears (Figure 4-2).

Figure 4-2 Create Project Page

2. Enter the following information:

• Project name

• Description

• Location for saving the project. (You can accept the default locationor type a new one.)

3. Click Next to go to the second page.

4. Select one of the following types of project creation:

• Create new project – Creates an empty project (without copying anyfiles).

• Create using current project as template – Creates a project basedon the currently active project (copies acquisition, method, andsample list files from the active project to the new project).

5. Click Finish.


4-3Operation

Creating an Inlet MethodAn inlet method directs the MassLynx NT software to acquire specific LC data.For example, a normal inlet method specifies when the system injects sampleand which data it acquires. Besides the normal inlet method, you can create aprerun or post-run method. These methods condition the column, equilibrate thesystem, and/or prime it without injecting sample.

To create a Micromass CapLC inlet method, first specify the method type thenprogram conditions for the pumps and autosampler.

Setting Initial Conditions

For gradient runs, set initial pump conditions. For isocratic runs, set initial andoverall pump conditions.

Before You Begin

Make sure the monitor displays the MassLynx Main Window (Figure 4-3).

Figure 4-3 MassLynx Main Window


4-4Operation

Procedure

1. Click (Inlet Editor) in the CapLC Binary area. The Waters CapLCEditor Solvent Status page appears (Figure 4-4).

Figure 4-4 Solvent Status Page

2. Click (Inlet Parameters) in the toolbar. The Initial Conditions pageappears (Figure 4-5).

Figure 4-5 Initial Conditions Page

3. Set parameter values for the application. Table 4-1 describes the initialcondition parameters.


4-5Operation

Table 4-1 Initial Conditions Page Parameters


Select Solvent A1 Specifies the default solvent.

Solvent B and C Specifies the percentage of solvent flow frompump B and/or C (if pump C is installed).

Solvent Name Specifies the solvent in the corresponding solventreservoir. Default names: Solvent A1, B, and C.

Flow Specifies the total flow rate for the system inµL/min. Range: 0.000 to 40.000 µL/min. Default:1.000 µL/min.

Run Time* Specifies how much time the Micromass CapLCpumps run until the system injects again. Range:0.00 to 600.00 min. Default: 5.00 min.

PumpSynchronization

When selected, specifies injection always at thesame moment in the pump’s stroke cycle. Thiscan improve reproducibility. Default: Notselected.

Low Pressure Specifies the system’s low-pressure limit. Ifsystem pressure falls below this limit, flow stops,and the OK lights (LC Status Error) on the SolventStatus page and in the Micromass CapLC area ofthe MassLynx Main Window show red. Range: 0to 4500 psi. Default: 0.0 psi.

High Pressure Specifies the system’s high-pressure limit. Ifsystem pressure exceeds this limit, the flow stops,and the OK lights on the Solvent Status page andin the Micromass CapLC area of the MassLynxMain Window show red. Range: 0 to 5000 psi.Default: 5000 psi.

Name Specifies the name of the installed column.Default: Column.

Attention: When setting timed events, set the initial conditions RunTime to a value that equals or exceeds the largest Time value in theTimed Events table. Event table rows that contain Time valuesgreater than the Run Time set for initial conditions do not operate.

Attention: Selecting Pump Synchronization causes an injectiondelay of a few minutes as the pumps synchronize. This delay cancause sample diffusion, which can adversely affect chromatographicresults for isocratic separations.


4-6Operation

Setting Stream Select Module Parameters: Precolumn

Use the Waters CapLC Editor window pages to configure pump C as anauxiliary gradient and to establish parameters for precolumn-only use.

Before You Begin

Ensure that the monitor displays the Waters CapLC Editor.

Procedure

View Figure 4-6 through Figure 4-12, a 7-screen sequence that suggestsparameters for precolumn use.

Note: The parameters shown are suggestions. Alter them to fine-tune yoursystem.

Figure 4-6 shows the Initial Conditions page of the Waters CapLC Editorwindow. The suggested settings are for precolumn-only use.

Figure 4-6 Initial Conditions Page of the Waters CapLC Editor Window

Figure 4-7 shows the Gradient page of the Waters CapLC Editor window. Thesuggested settings are for precolumn-only use.


4-7Operation

Figure 4-7 Gradient Chart Recording Page of the Waters CapLC Editor Window

Figure 4-8 shows the Auxiliary Pump page of the Waters CapLC Editor window.The suggested settings are for precolumn-only use.

Figure 4-8 Auxiliary Pump Page of the Waters CapLC Editor Window


4-8Operation

Figure 4-9 shows the Initial Events page of the Waters CapLC Editor window.The suggested settings are for precolumn-only use.

Figure 4-9 Initial Events Page of the Waters CapLC Editor Window

Figure 4-10 shows the Timed Events page of the Waters CapLC Editor window.The suggested settings are for precolumn-only use.

Figure 4-10 Timed Events Page of the Waters CapLC Editor Window


4-9Operation

Figure 4-11 shows the Chart Recording page of the Waters CapLC Editorwindow. The suggested settings are for precolumn-only use.

Figure 4-11 Chart Recording Page of the Waters CapLC Editor Window

Figure 4-12 shows the Method Type page of the Waters CapLC Editor window.The suggested settings are for precolumn-only use.

Figure 4-12 Method Type Page of the Waters CapLC Editor Window


4-10Operation

Setting Stream Select Module Parameters: Precolumn-Plus-Analytical Column

Use the Waters CapLC Editor window pages to configure pump C as anauxiliary gradient and establish parameters for precolumn plus analyticalcolumn use.

Before You Begin

Ensure that the monitor displays the Waters CapLC Editor window.

Procedure

View Figure 4-13 through Figure 4-19, a 7-screen sequence that suggestsparameters for precolumn-plus-analytical column use. The parameters define arun in which a 180-micron analytical column was used in conjunction with a1 µL/min. flow rate.

Note: The parameters shown are suggestions. Alter them to fine-tune yoursystem.

Figure 4-13 shows the Initial Conditions page of the Waters CapLC Editorwindow. The suggested settings are for precolumn-plus-analytical column use.

Figure 4-13 Initial Conditions Page of the Waters CapLC Editor Window


4-11Operation

Figure 4-14 shows the Gradient page of the Waters CapLC Editor window. Thesuggested settings are for precolumn-plus-analytical column use.

Figure 4-14 Gradient Page of the Waters CapLC Editor Window

Figure 4-15 shows the Auxiliary Pump page of the Waters CapLC Editorwindow. The suggested settings are for precolumn-plus-analytical column use.

Figure 4-15 Auxiliary Gradient Page of the Waters CapLC Editor Window


4-12Operation

Figure 4-16 shows the Initial Events page of the Waters CapLC Editor window.The suggested settings are for precolumn-plus-analytical column use.

Figure 4-16 Initial Events Page of the Waters CapLC Editor Window

Figure 4-17 shows the Timed Events page of the Waters CapLC Editor window.The suggested settings are for precolumn-plus-analytical column use.

Figure 4-17 Timed Events Page of the Waters CapLC Editor Window


4-13Operation

Figure 4-18 shows the Chart Recording page of the Waters CapLC Editorwindow. The suggested settings are for precolumn-plus-analytical column use.

Figure 4-18 Chart Recording Page of the Waters CapLC Editor Window

Figure 4-19 shows the Method Type page of the Waters CapLC Editor window.The suggested settings are for precolumn-plus-analytical column use.

Figure 4-19 Method Type Page of the Waters CapLC Editor Window


4-14Operation

Setting Gradient Parameters

Use the Gradient table to specify timed-based changes in mobile phasecomposition.

Before You Begin


Procedure

1. Click Gradient. The Gradient page appears (Figure 4-20).

Figure 4-20 Gradient Page

2. Specify time, percent composition, flow rate, and curve values in the

Gradient Entry section, then click (Add Row) to insert a row in theGradient table to receive data entries. You can enter up to 15 rows in thetable. Table 4-2 describes the gradient parameters.

Note: For isocratic runs, set run parameters only in the Initial Conditions page.The Gradient table must remain blank for isocratic operation.

Note: Percent composition and flow values set for a 0.00 Time row in theGradient table supersede the percent composition and flow values set in theInitial Conditions page.


4-15Operation

Table 4-2 Gradient Parameters


Time (mins) Specifies when the conditions (%A,B,C; Flow and Curve)specified in the row take effect. A row that sets Time to0.00 overrides the values set on the Initial Conditions page(see Figure 4-5). Range: 0.00 to 600.0 minutes.

B% and C%1 Specifies the percentage of solvent flow from eachreservoir. For each row, the total of all solvents mustequal 100%. Range: 0 to 100%.

Flow(µL/min)

Specifies the total flow rate for the system. Range: 0.000to 40.000 µL/min.

Curve2 Specifies the rate profile that describes timed changes insolvent composition and flow. The gradient curve youindicate in each row of the Gradient Table represents therates of these changes. The Gradient Curves Diagramrepresents these curves, which number 1 to 11 (Figure4-21).

1C% is displayed only if pump C is used for ternary gradient. Usually, pump Cfunctions as an auxiliary pump, which provides a flow of carrier solvent betweenthe syringe and the injector. It has its own gradient table, and you can program itto start at a predetermined time and stop after a sample has been injected.Percent flow for reservoir A is not displayed. Calculate it as 100%-(B% + C%)

2You cannot set a curve for time 0 (see Figure 4-21).


4-16Operation

Figure 4-21 Gradient Curves Diagram

Setting Initial Event Conditions

Use the Initial Events page (Figure 4-22) to set the initial condition for the twocontact-closure output switches and the initial positions of the stream selectvalve and the vent valve.

Before You Begin


Procedure

1. Click Initial Events. The Initial Events page appears (Figure 4-22).

Figure 4-22 Initial Events Page

2. Select the appropriate initial conditio for each switch and valve. Table4-3 describes the initial events parameters.

Table 4-3 Initial Events Page Parameters

Segment Start Time

Final Conditions

Eluent Compositionor Flow Rate

Profile

Initial Conditions

1

Run Time Segment End Time


4-17Operation


Switch 1 Specifies the initial state of contact-closure Switch 1,located on the terminal strip. Possible values: On, Off,and No Change. Default: Off.

Switch 2 Specifies the initial state of contact-closure Switch 2.Possible values: On, Off, and No Change. Default: Off.

Stream Select Specifies the initial state of the stream select valve(Position 1 or 2). The stream select valve is configurablefor post-column addition, sample focusing, columnbypass, or as a diverter valve. Default: Position 1.

Vent Valve Specifies the initial state of the vent valve (System orVent). Default: System.

Setting Timed Events

Use the Timed Events page (Figure 4-23) to control the activity of switches,valves, and alarms during the run.

Before You Begin

Make sure the monitor displays the Solvent Status page of the Waters CapLCEditor window (Figure 5-7).

Procedure

1. Click Timed Events. The Timed Events page appears (Figure 4-23).

Figure 4-23 Timed Events Page


4-18Operation

2. Select an event item in the left section of the Timed Events page.

Note: Enter switch and valve position changes during a run in the Eventtable.

3. Set the time and action for the event, then click (Insert) to insert theentries into the first row in the Event table. You can enter up to 15different events in the table. Table 4-4 describes the timed eventsparameters.

Note: Set the initial state of switches and valves in the Initial Eventspage.

Note: If you program timed events, you must select the Run Event Tablecheck box on the Method Type page. Timed events do not run unless youselect the Run Event Table check box.

Note: The Run Time value set in the Initial conditions page must begreater than or equal to the greatest Time value in the Event table. Eventtable rows with Time values greater than the initial conditions Run Timedo not run.

Table 4-4 Timed Events Page Parameters


Time Specifies how much time passes between an injection andthe start of an event. Event rows are sorted automatically,by time. Note that you can program different events tooccur simultaneously. Range: 0.00 to 600.0 min.

Specifies the type of event signal: one of the two contact-closure output switches (S1 or S2), or one of the internalevents (stream select valve position, vent valve position).Choose from these event types to program up to 16 totalevents. Note that you can program the same event morethan once. Available choices are:

Switch 1 and 2 Specifies terminal stripposition S1 or S2.

Stream select Specifies the position (1 or 2)of the stream select valve.

Event

Vent valve Specifies the position (Systemor Vent) of the vent valve.

Switch States Specifies the state of the switch when you specify a Switchevent. This information appears in the Action column ofthe Event table. Available switch states are:


4-19Operation


On Actuates a contact closure thattriggers an external or internalevent. The contact closure remainsclosed until an Off function is sent.

Off Switches off the contact closure forthe event. With this switch state,the contact closure is broken.

Toggle Changes the current state of theswitch.

Pulse Transmits a single On/Off pulse.The contact closure is maintainedfor the number of seconds youspecify in the Width (min) field.Range: 0.01 to 100.00 minutes.

Setting Chart Recording Parameters

Use the Chart Recording page to specify up to two analog output signals toexternal devices such as an integrator or strip-chart recorder. You can specifytwo of the following signals:

• Flow

• Pressure

• Percent A, B, or C

Before You Begin


Procedure

1. Click Chart Recording. The Chart Recording page appears(Figure 4-24).


4-20Operation

Figure 4-24 Chart Recording Page

2. Select a chart-recording signal from the Chart Record 1 and/or ChartRecord 2 drop-down list.

Note: To record a signal, you must connect each external device to theappropriate Chart Out terminal pair on the rear of the unit.


4-21Operation

Choosing a Method Type

You can define one of four inlet method types:

• Normal method – Creates an inlet method that makes and acquiresdata.

• Column Condition method – Creates a prerun or post-run inletmethod that runs a solvent gradient through the column withoutperforming an injection. Use a Column Condition method beforerunning a new gradient method or when changing the column.

• Equilibrate method – Creates a prerun or post-run inlet method thatequilibrates the system with the solvent conditions specified in theInitial Conditions page (or with the conditions specified at 0.00 Timein the Gradient table, if applicable). No injection is performed in anEquilibrate method.

• Wet Prime method – Creates a prerun or post-run inlet method thatprimes the system with the selected solvent (useful when changingsolvents). No injection is performed in a Wet Prime method. Specifythe solvent line you want to prime and the number of system volumesyou want to use.

Before You Begin

Ensure that the monitor displays the MassLynx NT Micromass CapLC Editorpage.

Procedure

1. Click Method Type. The Method Type page appears (Figure 4-25).

Figure 4-25 Method Type Page


4-22Operation

2. Choose the appropriate method type. For information on the fields in thispage, refer to the MassLynx NT Guide to Data Acquisition.

3. If you program events on the Timed Events page, make sure the RunEvent Table check box is selected. Timed events do not run unless thischeck box is selected.

Attention: Do not wet prime the system if the solvent lines are dry. Toprevent damage to the system, dry prime first.

Note: You must run Column Condition, Equilibrate, and Prime methods as aprerun or post-run inlet method (not as a normal inlet method) in the sample list.Refer to the MassLynx NT Guide to Data Acquisition for information onformatting the sample list.

Note: Parameters set for the Autosampler do not affect Column Condition,Equilibrate, and Wet Prime methods.

Setting Autosampler Parameters

Before You Begin

Verify that:

• You know the height of the sample containers.

• The monitor must show the Solvent Status page of the Waters CapLCEditor (see Figure 3-6).

Procedure

1. Select Micromass CapLC Autosampler from the View menu. TheAutoSampler page appears (Figure 4-26).

Figure 4-26 AutoSampler Page


4-23Operation

2. Enter values in the Injection, Wash, and Temperature fields. Table 4-5describes the parameters in the AutoSampler page.

Table 4-5 AutoSampler Page Parameters


DrawHeight

Specifies the depth of the needle tip, thus adjusting forsample containers of different depths or for sedimentedsamples. A value of 0 corresponds to the bottom of thevial/well for the plate selected in the Bed Layout Editor.Range: 0 to 40 mm in 1-mm increments. Default: 5.

Note: Make sure the Draw Height parameter is set highenough to accommodate your sample containers. If DrawHeight is set too low, you could damage the sample needle.

The plate size and the vial depth are set in the PlateGenerator. Refer to “Generating Plate Types and SettingVial References” in Chapter 3 for information onconfiguring plate types.

Refer to Configuring Bed Layout for information onselecting the plate type for the method.

DrawSpeed

Specifies the draw rate of the syringe for samples of varyingviscosity. The rate for each selection depends on the size ofthe installed syringe. Valid selections: Fast, Normal, andSlow. Default: Normal.

InjectionType

Specifies one of the following injection types:

Full Loop The sample loop is completelyfilled. Provides maximumreproducibility.

Partial Loop The sample loop is partially filledwith a user-defined injectionvolume (up to 50% of the volumeof the sample loop). Providessample conservation withreproducibility slightly less thanFull Loop mode.

µL Pickup The sample loop is filled with onlythe amount of sample to be injected(resulting in no sample waste).Mobile phase from the transportvial moves the sample into theloop. Reproducibility is typicallyless than Full or Partial Loopmodes.


4-24Operation


FlushVolume

Specifies the volume (in microliters) of sample taken from avial before the sample loop fills with sample. Range: 0 to999 µL. Default: 10 µL.

Head SpacePressure

Specifies that the prepuncturing needle apply approximately0.5 bar pressure in the headspace above the sample toprevent air or vapor bubbles from forming in the sample.Enable this parameter only when using sample vials withgastight caps.

Air SpaceSegment

Specifies adding an air plug to the front of the flush volumeto minimize dilution and bandspreading and reduce theamount of flush volume. In Full and Partial Loop modes,the air segment is flushed to waste. In µL Pickup mode, theair segment is injected.

Note: Micromass does not recommend using an air spacesegment in Micoliter pickup.

HighEfficiencyMode

Specifies the sample loop's removal from the flow streamafter the sample has been flushed, but before the gradientfront reaches the injection valve. This can reduce peakbroadening of the sample.

WashVolume

Specifies the volume (in microliters) of wash solvent forcleaning the needle and buffer tubing. Range: 0 to 999 mL.Default: 10 µL.

SampleTemp

Specifies the target operating temperature for the optionalsample heater/cooler. Range: 4.0 to 40.0 °C.Default: 20.0 °C.

Temp Limit Specifies the maximum allowable temperature deviationfrom the value set for the Sample Temperature. If thesample temperature deviates beyond the specified range, therun stops and the OK lights on the Solvent Status page andin the Micromass CapLC area of the MassLynx MainWindow show red. Range: ±1.0 to ±20.0 °C.Default: ±20.0 °C.

Setting Mix Method Parameters

A mix method aspirates reagent from the vial positions on the plate holder. Itthen dispenses the reagent into the sample position specified in the sample set.Finally, it mixes the sample and reagents. Thus the system performs precolumnderivatization, dilution, adds an internal standard.

Before You Begin

Make sure the monitor displays the MassLynx Main Window, then click inthe window’s CapLC Binary area to open the Waters CapLC Editor window.


4-25Operation

Procedure

1. Click Mix Method. The Mix Method page appears (Figure 4-27).

Figure 4-27 Mix Method Page

2. Enter values for each field. Table 4-6 describes the parameters in theMix Method page.

Note: Each mix cycle draws an entire syringe volume from the sample vial/well.This volume may exceed the sample quantity.

Attention: Do not run more than three mix cycles. Mix cycles can movethe sample/reagent mixture through the buffer loop and into the syringe,which can contaminate it.

Table 4-6 Mix Method Page Parameters


Mix Delay Specifies the amount of time the system waits betweenmix cycles and before injection. Range: 0.0 to 99.9 min.Default: 0.

Mix Cycles Specifies the number of times to mix the sample andreagents (using syringe draw) before injection. Each mixcycle aspirates and dispenses one syringe volume fromthe sample position specified in the sample set. Range: 0to 99. Default: 1.

Reagent APosition

Specifies the position of the reagent vial on the platecarrier. A value of 0 indicates reagent A is not used.Range: 0 to 4. Default: 0.

Reagent AVolume

Specifies the amount of reagent A added to the sample.The maximum reagent volume is the volume of the


4-26Operation


autosampler syringe. Range: 0.0 to 99.9 mL.Default: 1 mL.

Reagent BPosition

Specifies the position of the reagent vial on the platecarrier. A value of 0 indicates reagent B is not used.Range: 0 to 4. Default: 0.

Reagent BVolume

Specifies the volume of reagent B added to the sample.The maximum reagent volume is the volume of theautosampler syringe. Range: 0.0 to 99.9 mL.Default: 1 mL.


4-27Operation

Saving the Method

Before You Begin

Make sure the monitor displays the MassLynx Main Window (Figure 3-2), then

click in the CapLC Binary area to open the Waters CapLC Editor window.

Procedure

1. Select Save As from the File menu.

2. Type a name for the method, then click Save. For more information onthe fields in the Waters CapLC Editor, refer to the MassLynx NT Guideto Data Acquisition.

Acquiring Data

Complete the following tasks before acquiring data:

• Equilibrating the system

• Calibrating the mass spectrometer

• Injecting samples

• Creating a sample list

• Starting an acquisition

• Stopping a run

Equilibrating the System

Before you begin a run, equilibrate the Micromass CapLC system by runningsolvent through it without making injections. This flushes out any solvent orsample in the system and prepares it for the run.

Before You Begin

Verify that:

• The system is primed.

• The monitor displays the MassLynx Main Window (see Figure 3-2).

Procedure

1. Click (Inlet Editor) in the CapLC Binary area of the MassLynx MainWindow. The Waters CapLC Editor appears with the last opened inletmethod. To open a different inlet method, select Open from the Filemenu. Select the desired method, then click Open.


4-28Operation

2. Select Load Method from the CapLC Control menu. Solvent flowstarts, using the flow rate, percent composition, and column temperatureset in the Initial Conditions page (see Figure 4-5). No injection occurs.

Note: Percent composition and flow values set for a 0.00 Time row in theGradient table of the Gradient page supersede the percent composition and flowvalues set in the Initial Conditions page.

Tuning the Mass Spectrometer

You must tune the mass spectrometer from the MassLynx NT Tune page beforeacquiring data for the first time and thereafter periodically. For information ontuning the mass spectrometer, refer to the MassLynx NT Guide to DataAcquisition and to the documentation that accompanies your mass spectrometermodel.

Injecting Samples

After you configure, equilibrate, calibrate, and tune the Micromass CapLCsystem and mass spectrometer, you can make injections and acquire data.

MassLynx NT lets you create a new sample list, open an existing sample list,print a sample list, format sample lists using sample list formats, and so on. Formore information on sample lists and sample list formats, refer to the MassLynxNT User’s Guide.

Sample Lists

The MassLynx Main Window includes a Sample List Editor for definingmultiple samples. In the sample list, define the following:

• Number of samples to inject

• Sample vial numbers

• Number of injections from each vial

• Injection volume, inlet (LC), and MS methods to use

• Other run parameters

Figure 4-28 shows the sample list in the MassLynx Main Window.


4-29Operation

Figure 4-28 Sample Lists, MassLynx Main Window

Table 4-7 describes the parameters in the Sample List Editor.

Table 4-7 Sample List Editor Parameters


File Name Specifies the name of the file to which data are saved.

File Text Provides a description of the sample.

MS File Specifies the file containing the MS method to runwith the sample. Double-click the MS File cell todisplay a drop-down list of configured MS methods.

Inlet File Specifies the file containing the Micromass CapLCinlet method to run with this sample. Double-click theInlet File cell to display a drop-down list of configuredLC instrument methods.

Bottle Specifies the vial (or well) position from which asample is drawn.

Inject Volume Specifies the volume of sample to inject.

Inlet Prerun Specifies the condition column, equilibrate, or primingmethod to run before running the specified inletmethod in the sample list row.

Inlet Post-Run Specifies the condition column, equilibrate, or primingmethod to run after running the specified inlet methodin the sample list row.


4-30Operation

Creating a Sample List

Before You Begin

Make sure the monitor displays the MassLynx Main Window (Figure 4-3).

Procedure

1. Select New from the File menu. A sample list with one empty defaultrow appears.

2. Key data into sample list fields as required. The Sample Lists windowdefines some of the most frequently used sample list fields.

3. Select Number of Injections from the Samples menu. The Number ofInjections dialog box appears.

4. In the Enter No. field, enter the desired number of injections per vial (orbottle), then click OK.

5. Select Number of Samples from the Samples menu. The Samplesdialog box appears.

6. Type the total number of rows you want to appear in the sample list.Each row corresponds to an injection. For example, were you to acquiredata for two injections per vial (Number of Injections = 2), and you havesix vials, you would enter 12 in the Number of Samples field.

7. Click OK. The sample list adds the correct number of rows (based onthe Number of Injections and Number of Samples entries).

8. Change sample list fields as needed.

9. Run an inlet prerun and/or post-run method, if necessary, to equilibratethe system, condition the column, or prime the system.

10. Customize the sample list to include Inlet Prerun and/or Inlet Postruncolumn(s). In the sample list rows that require a prerun or post-run, clickthe Inlet Pre-run and/or Inlet Post-run field, then select a method from thedrop-down list: equilibrate, condition column, or prime method. (Refer tothe MassLynx NT User’s Guide, Chapter 2, Sample Lists.)

Note: An Inlet prerun method runs before the row's specified inlet method.An inlet post-run method runs after the specified inlet method for the row.

11. Save the sample list by selecting Save As from the File menu, then namethe sample list.

Starting an Acquisition

1. Select the rows in the sample list corresponding to the samples you wantto run. If you do not select any rows, all samples run.


4-31Operation

2. Select Start from the Run menu. The Start Sample List Run dialog boxappears.

3. Select Acquire Sample Data and/or Auto Process Samples.

4. Type values in the Run From Sample and To Sample text boxes. Thedefault values correspond to the rows you selected in the sample list(step 1).

5. Click OK. The Micromass CapLC system runs the samples and acquiresdata.

For more information on making injections and acquiring data, refer to theMassLynx NT Guide to Data Acquisition.

Stopping a Run

Procedure

1. From the MassLynx Main Window (Figure 4-3), click (Stop SampleRun) or select Stop from the Run menu.

2. Click OK at the prompt to delete the currently running process. Dataacquisition stops, and the MS status clock resets to 0.00.

3. Click (Inlet Editor) in the CapLC Editor Binary area to open theSolvent Status page (Figure 4-4).

4. Click (Stop Method) in the toolbar.

5. Click Yes, at the prompt, to stop the currently running inlet method andany queued post-run methods, or click No to stop only the inlet method(any queued post-run methods will run). The inlet method stops and theWaters CapLC status clock resets to 0.00.

For More Information

For general information on MassLynx NT software, detailed information aboutspecific features, and installation instructions, refer to the MassLynx NT User’sGuide.

For more detailed information on configuring MassLynx NT software for usewith Micromass instrumentation, refer to the MassLynx NT Guide to DataAcquisition.


5-1Maintenance

Chapter 5: MaintenanceThis chapter describes routine maintenance procedures you can perform toensure the system delivers accurate, precise results. Figure 5-1 shows theindividual maintenance tasks.

Figure 5-1 Maintenance Flowchart

Replacing StreamSelect ModuleValve Seals

Replacing VentValve Seals

Maintaining theSolvent Manager

MaintenanceConsiderations

Maintenance

Replacing thePump Tray Frit Filters

Replacing theSolvent Bottle Filters

Maintaining RotaryValves

Maintaining theAutosampler

Replacing Fuses

ReplacingInjector Valve Seals

Replacing the PunctureNeedle

Replacing the SampleNeedle

Replacing the Fuse

Replacing the Sample Loop

Replacing the Syringeor Plunger Tip

Replacing Precolumnsand Columns

Replacing the BufferTubing


5-2Maintenance

Maintenance Considerations

Safety and Handling

Observe these safety considerations before performing system maintenance:

Caution: Do not open the power supply cover. Hazardousvoltages within the power supply present a shock hazard.

Note: The power supply does not contain user-serviceableparts.

Caution: Always observe good laboratory practices when youhandle solvents, change tubing, or operate the system. Knowthe physical and chemical properties of the solvents you use.Refer to Material Safety Data Sheets for solvent information.

Attention: The autosampler is not attached to the MicromassCapLC base unit. Avoid pushing or pulling the autosamplerwhen attempting to move the trolley.

Attention: To avoid damaging electrical parts, neverdisconnect an electrical assembly while current flows to thesystem. After you turn off the power, wait ten seconds beforedisconnecting an assembly.

Attention: To prevent circuit damage from static charges, donot touch integrated circuit chips or other components that donot specifically require manual adjustment.

Proper Operating Procedures

To maintain peak system performance, follow all recommended operatingprocedures and guidelines.

Spare Parts

For a complete list of spare parts, contact your local Micromass CustomerService Center.


5-3Maintenance

Routine Maintenance Protocols

Table 5-1 lists maintenance tasks you must perform periodically to ensureoptimal system operation. If you use the system for extended periods (forexample, nights and weekends), or if you use aggressive solvents, like buffers,you might need to perform these routine tasks more frequently.

Table 5-1 Routine Maintenance Protocols

Maintenance Procedure Frequency

Replace solvent bottle filters Yearly

Replace pump tray frit filters Yearly

Inspect rotor seals Yearly

Clean unit with cloth As needed

Maintaining the Pump

Caution: To prevent injury, always observe good laboratorypractices when you handle solvents, change tubing, or operate thesystem. Know the physical and chemical properties of the solvents.Refer to Material Safety Data Sheets for solvent characteristics.

Attention: Before you operate the system, make sure the pump traysare in place with screws to ensure drip protection.

Maintaining the pump involves three procedures:

• Replacing solvent bottle filters

• Replacing the pump tray frit filters

• Replacing the pump fuse


5-4Maintenance

Replacing the Solvent Bottle Filters

Each solvent reservoir on the system has a filter. Consider replacing the filterwhen any of the following problems occur:

• Difficulty in dry or wet priming

• Poor retention time precision

Required Materials

• Solvent reservoir filter (part number 700000995)

• Compression screw (part number 700000991)

• Lock ring (packaged with compression screw)

• Ferrule (packaged with compression screw)

Before You Begin

Set the system flow to 0.0 µL/min.

Procedure

1. Unscrew the solvent reservoir bottle from its cap on the pump tray.

2. Remove the compression screw from the solvent reservoir cap.

3. Remove the old filter assembly.

4. Over the end of the new filter tubing, slide new parts in the followingorder: compression screw, lock ring, and ferrule. Note that the flattenedend of the lock ring must face the compression screw, and the narrow endof the ferrule must face the lock ring (Figure 5-2).

Figure 5-2 Tubing Assembly for the Solvent Reservoir Filters

5. Insert the tubing assembly into the reservoir cap fitting. Seat the tubingfully.

6. While pressing the tubing snugly against the fitting, finger-tighten thecompression screw.

Filter

Compression Screw

Lock Ring

Ferrule


5-5Maintenance

Replacing the Pump Tray Frit Filters

Each pump tray manifold has a frit filter. Consider replacing the frit filter whenany of the following problems occur:

• High backpressure when no column is connected to the system

• Unstable pressure

• Poor retention time precision

Figure 5-3 shows the frit filter location.

Figure 5-3 Frit Filter in Pump Tray Manifold

Required Materials

• Upchurch Scientific® removal tool (part number 700001012)

• Replacement filter, 2-µm frit in a ferrule (part number 700000992)

Before You Begin

Set the system flow rate to 0.0 µL/min.

Procedure

1. Remove the top, rear compression screw from the pump tray manifold.

2. Use the Upchurch removal tool to pry the lock ring away from the fritfilter (Figure 5-4).

Top, RearCompression

Screw


5-6Maintenance

Figure 5-4 Removing the Frit Filter

3. Discard the frit filter, but keep the lock ring.

4. Slide the new frit filter onto the tubing.

5. Press the frit filter onto the lock ring.

6. Place the filter so that it lies flat inside the manifold. Carefully tightenthe compression screw while gently pushing down on the PEEK™ tubing.

7. Make sure the filter is secure by gently pulling on the PEEK tubing.

8. Pressurize the system and check the connection for leaks.

Replacing the Pump Fuse

The pump power connector includes a fuse holder that contains two fuses.Replace the fuses if the system fails to start. Figure 5-5 shows the fuse assemblyand its location.

Figure 5-5 Main Line Fuse Assembly


5-7Maintenance

Required Materials


• Long-nose pliers

• Two 10-A replacement fuses (part number 700001003)

• Electrical continuity tester (optional)

Before You Begin

Verify that:

• The system is switched off.

• No power cables are plugged into outlets.

Procedure

1. Pry the cover of the fuse holder with the flat-blade screwdriver.

2. Use the long-nose pliers to remove the fuse holder from the assembly.Repeat this step for the second fuse.

3. Carefully remove each fuse from its holder.

4. Check each fuse for evidence of an open circuit. If the filament is eitherbroken or burnt, discard the fuse. If you question the fuse's integrity,check it for electrical continuity.

5. Insert a replacement fuse in the holder for each damaged one.

6. Reinstall each fuse holder in the switch.

7. Close the fuse holder cover.


5-8Maintenance

Maintaining the Autosampler

Caution: To prevent injury, always observe good laboratory practices whenyou handle solvents, change tubing, or operate the system. Know the physicaland chemical properties of the solvents you use. Refer to Material Safety DataSheets for solvent characteristics.

Caution: Be careful when moving the system with the autosampler atop thepump. The autosampler is not attached to the pump and can slide off it ifpushed or pulled.

Maintaining the autosampler can involve:

• Replacing the syringe or plunger tip

• Replacing the sample needle

• Replacing the puncture needle

• Replacing the buffer tubing

• Replacing the sample loop

• Replacing the inline filter

• Replacing the autosampler fuse

Before you reconfigure the autosampler by changing syringe, buffer loop,sample needle, or sample loop sizes, consult Table 5-2 to determine thecompatibility of the new configuration’s components. The table also lists otherparameters for each autosampler syringe, such as injection volume range andspeed.

Note: Microliter pickup Injections require a 10-µL or larger sample loop. Usingmicroliter pickup with a sample loop smaller than 10 µL results in zero sampleinjected.


5-9Maintenance

Table 5-2 Autosampler Component Compatibility

Syringe VolumeComponent/Parameter

25 µL 100 µL 250 µL

Buffer Tubing Volume (µL) 50 500 500

Needle Volume (µL) 0.0–99.0

Default: 2.4

1.0–200.0 1.0–200.0

Sample Loop Volume (µL) 0.0–20

Default:10.0

5–100 5–100

Flush Volume (µL) 0–25 0–999 0–999

Wash Volume (µL) 10–9999 300–9999 300–9999

Injection Volume Range(µL)

0.000–10.00

0.00–100.00 0.00–100.00

Fast 94 375 940

Normal 63 250 625

SyringeSpeed(µL/min)

Slow 32 125 310

Wash Speed (µL/min) 34 1370 3430

Flush Volume must be at least twice the needle volume.

Full-Loop Mode injection volume is equal to the sample loop volume.

Partial-Loop Mode injection volume is 0.0 µL to (sample loop volume/2).

Microliter-Pickup Mode injection volume is 0.0 µL to[sample loop volume – (3 × needle volume))/2)].


5-10Maintenance

Replacing the Sample Loop

Change the sample loop when you encounter any of the following conditions:

• The application requires a sample loop with a different volume.

• The peak shape is distorted (tailing).

• The ends of the sample loop are damaged.

Figure 5-6 shows the sample loop and its location.

Figure 5-6 Sample Loop

Sample Loop


5-11Maintenance

Required Materials

• Two 10-A replacement fuses (part number 700001003)

• Replacement sample loop

• 1/4-inch slotted wrench (part number 71500001015)

Note: Refer to Table 5-3 for information on the compatibility of sample loopswith other autosampler components.

Table 5-3 Replacement Sample Loops

Description Part Number

Fused Silica, 5-µL 700000676

Stainless Steel, 5-µL 700000683

Fused Silica, 10-µL 700000677





PEEK, 100-µL 700000684

Before You Begin

Verify that:

• The system flow rate is 0.0 µL/min.

• The system pressure is at 0 psi.

• No injections are programmed.

• The new sample loop is compatible with other autosampler components.

Procedure

1. Lift the autosampler cover.

2. Use the 1/4-inch slotted wrench (part number 700001015) to remove thefittings from ports 2 and 5 of the inject valve.

3. Install the replacement sample loop, and tighten the fittings.

4. Lower the autosampler cover into place.


5-12Maintenance

5. Click (Inlet Editor) in the CapLC Binary area of the MassLynx MainWindow (see Figure 3-2). The Waters CapLC Editor Solvent Status pageappears (Figure 5-7).

Figure 5-7 Solvent Status Page

6. Select Diagnostics from the Cap LC Tools menu. The CapLC Diagnosticswindow appears (Figure 5-8).

Figure 5-8 CapLC Diagnostics Window

7. Select User Diagnostics from the Control menu. The User Diagnosticsdialog box appears (Figure 5-9).


5-13Maintenance

Figure 5-9 User Diagnostics Dialog Box

8. Click the Configuration tab. The Configuration page appears (Figure5-10).

Figure 5-10 Configuration Page Showing Normal (Auxiliary Gradient) Operation

Note: Micromass suggests auxiliary gradient operation for pump C and hasconfigured the pump and autosampler accordingly. Nevertheless, youcan customize your system, reconfiguring the pump and autosampler


5-14Maintenance

tubing connections for ternary gradient operation by diverting theoutflow from pumps A, B, and C from the pump vent valve to theautosampler inject valve. Specifically, remove the three red PEEKlines from the vent valve, and connect them to a four-way fitting.Connect the fitting's remaining leg to port 1 of the autosampler injectvalve via a fourth PEEK line. Finally, specify Ternary Gradient on theuser Diagnostics configuration page as shown (Figure 5-11).

9. Enter the volume of the new sample loop in the Sample Loop field.

10. Click OK. The new sample loop is now configured in the system.

11. Check connections for leaks after pressurizing the system.

Figure 5-11 Configuration Page Showing Custom Ternary Gradient Operation

Replacing the Syringe or Plunger Tip

Replace the autosampler syringe when you encounter any of the followingconditions:

• The syringe plunger tip is worn.

• The application requires a syringe that delivers a different volume.

• The syringe leaks.

• The syringe contains air bubbles that cannot be removed.


5-15Maintenance

Note: You can remove the autosampler syringe and manually fill it to remove anair bubble trapped in it.

Figure 5-12 shows the syringe assembly and its location.

Figure 5-12 Autosampler Syringe Assembly

Attention: For good peak area precision, use freshly degassed solvent.

Required Materials

• Replacement syringe

• Freshly degassed solvent

• Replacement plunger tip, if the plunger is worn. See Table 5-5 forreplacement syringe and plunger tip part numbers.

Syringe Adapter (for SyringeVolumes >25 µL)

Plunger


5-16Maintenance

Table 5-4 Replacement Syringes

Size (µL) Part Number

25 700000668

100 *700000860

250 *700000861

*100- and 250-µL syringes require a syringe adapter (part number 700000664).

Table 5-5 Replacement Plunger Tips


25 700000618

100 700000856

250 700000858

Before You Begin

Verify that:

• The system flow is set to 0.0 µL/min.

• The system pressure is at 0 psi.


• The new syringe is compatible with other autosampler components.

• The MassLynx Main Window is open.

Procedure

1. Click (Inlet Editor) in the CapLC Binary area of the MassLynx MainWindow. The Waters CapLC Solvent Status page appears (Figure 5-7).

2. Select Diagnostics from the Cap LC Tools menu. The CapLCDiagnostics window appears (Figure 5-8).

3. Select User Diagnostics from the Control menu. The User Diagnosticsdialog box appears (Figure 5-9).

4. Click Fill (in the Sample Syringe section) to move the autosamplersyringe to the fill position.

5. Remove the autosampler cover.

6. Remove the wash bottle (Figure 5-13).


5-17Maintenance

Figure 5-13 Removing the Wash Bottle

7. Unscrew the syringe cap.

8. Carefully press down on the syringe, and remove it.

9. Fill the replacement syringe with the appropriate wash solvent. Makesure no air bubbles are in the syringe.

10. Connect the bottom of the new syringe to the autosampler, and reinstallthe syringe cap.

11. Reinstall the wash bottle.

12. Close the autosampler cover.

13. Click the Configuration page of the User Diagnostics dialog box. TheConfiguration page appears (see Figure 5-10).

14. Select the volume of the new autosampler syringe from the Syringe Sizedrop-down list.

15. Click the Autosampler/Detector page of the User Diagnostics dialogbox. The Configuration page appears (see Figure 5-10).

16. Click Empty (in the Sample Syringe section) to return the syringe to itshome position.

17. Click Wash to perform a standard wash routine.

18. Check the syringe for air bubbles. If bubbles appear, repeat step 17.

19. Click OK. The system is now configured for the new autosamplersyringe.

Replacing the Sample Needle

Replace the sample needle when any of the following conditions occur:

• The needle is damaged.


5-18Maintenance

• The application requires a needle with a different volume.

Figure 5-14 shows the components of the needle holder.

Figure 5-14 Needle Holder Components

Required Materials

• 1/4-inch slotted wrench (part number 700001015, AutosamplerStartup Kit)

• Replacement sample needle

Table 5-6 gives the replacement numbers for sample needles.

Table 5-6 Replacement Sample Needles


2.4 700000621

5.4 700000634

Before You Begin

Verify that:

• System flow is set to 0.0 µL/min.

• System pressure is at 0 psi.


Connection Nut

Inject Valve

Sample Needle

Air Outlet Nut

Puncture Needle(Do Not Remove)


5-19Maintenance

• The new sample needle is compatible with other autosamplercomponents.

Procedure

1. Lift the autosampler cover up.

2. Remove the connection from port 4 of the injection valve with the slottedwrench (part number 700001015).

3. Loosen the needle connection nut with the open-end wrench.

4. Withdraw the sample needle tubing carefully.

5. Remove the air outlet nut, and replace it with the one accompanying thenew sample needle. (Do this if you are installing a sample needle that isa different size than the previous needle.)

6. Insert the replacement needle tubing through the needle holder.

7. Retighten the connection nut.

8. Reconnect the compression screw to port 4 of the injection valve.

9. Lower the autosampler cover.

10. Click (Inlet Editor) in the CapLC Binary area of the MassLynx MainWindow. The Waters CapLC Editor Solvent Status page appears.

11. Select Diagnostics from the Cap LC Tools menu. The CapLCDiagnostics window appears (Figure 5-8).

12. Select User Diagnostics from the Control menu. The User Diagnosticsdialog box appears. (See Figure 5-9).

13. Click the Configuration tab. The Configuration page appears (seeFigure 5-10).

14. Enter the volume of the new sample needle in the Needle field.

15. Click OK. The system is now configured for the new sample needle.

Replacing the Puncture Needle

Replace the puncture needle if you damage it. Figure 5-14 shows the needleholder components.

Required Materials

• Small Phillips screwdriver

• 1/4-inch open-end wrench (part number 022527, Startup Kit)


5-20Maintenance

• Replacement puncture needle (part number 700000641)

Before You Begin

Verify that:


• System pressure equals 0 psi.


Procedure


2. Pull the needle holder forward to the limit of its travel on the track.

3. Loosen the sample needle connection nut with the 1/4-inch wrench.

4. Withdraw the sample needle tubing carefully.

5. Remove the leakage plate, using the Phillips screwdriver to loosen thetwo screws that attach it to the side of the train assembly.

6. Remove the puncture needle connection nut from the train assembly withthe 1/4-inch wrench.

Caution: Keep your hands clear of the sharp end of the punctureneedle.

7. Pull the puncture needle to the right, through the slot in the trainassembly gently, then lift the puncture needle out of the needle unit.

8. Insert the replacement needle through the hole in the vial stripper, thengently push it to the left, through the slot in the train assembly.

9. Slide the puncture needle up to the hole in the train assembly, andreinstall it using the 1/4-inch wrench.

10. Reattach the leakage plate to the side of the train assembly with the twoPhillips screws.

11. Insert the sample needle tubing through the needle holder.

12. Retighten the sample needle connection nut.

13. Push the needle holder back to its home position gently.


Replacing the Buffer Tubing

Change the buffer tubing when you encounter any of the following conditions:


5-21Maintenance

• The application requires a buffer loop with a different volume.

• The ends of the buffer tubing are damaged.

Note: See Table 5-2 for information on the compatibility of buffer tubing withother autosampler components.

Required Materials


• Replacement buffer tubing (Table 5-7)

Table 5-7 Replacement Buffer Tubing


50 700001026

500 700000859


5-22Maintenance

Before You Begin

Verify that:


• System pressure equals 0 psi.


• The new buffer tubing is compatible with other autosamplercomponents.

Procedure


2. Use the 1/4-inch slotted wrench (part number 700001015) to remove thefitting from port 3 of the inject valve.

3. Remove the wash bottle.

4. Remove the buffer tubing from the syringe assembly.

5. Install the replacement buffer tubing and tighten the fittings to port 3 ofthe inject valve and to the syringe assembly.

6. Reattach the wash bottle.


Replacing the Autosampler Fuse

The autosampler power switch (see Figure 1-2) includes a fuse holder within theswitch assembly. Replace the fuse if one of the following conditions apply:

• The autosampler does not start.

• You change the voltage setting.

Required Materials


• Replacement fuse (Autosampler Startup Kit)

• 110–120 VAC, 5 A (part number 700001005)

• 220–240 VAC, 2.5 A (part number 700001004)

• Electrical continuity checker (optional)


5-23Maintenance

Before You Begin

Verify that:

• Power to the system is switched off.

• The power cable is disconnected from the autosampler.

Procedure

1. Observe the position of the switch assembly, either 110–120 VAC or220–240 VAC.

2. Use the flat-blade screwdriver to pry the fuse holder out of the switchassembly.

3. Carefully remove the fuse from the spring clips (Figure 5-15).

Figure 5-15 Fuse Holder (Top View)

4. Visually inspect the fuse for an open circuit. If the filament is eitherbroken or burnt, discard the fuse. If you are unsure of the fuse's integrity,check it for electrical continuity.

5. Insert the replacement fuse into the spring clip.

Attention: The position of the fuse holder determines the voltagesetting of the autosampler. If the holder is set to the lower voltagerange (110 to 120 VAC) and the input voltage source is 220 to 240VAC, the autosampler might be damaged.

6. Return the fuse holder to the position in the switch assembly youpreviously observed. Note that the selected voltage setting appears at thebottom of the fuse holder label.


5-24Maintenance

Maintaining Rotary Valves: Micromass CapLC Pumpand Autosampler

Three valves control direction and flow inside the liquid pathway:

• Inject valve

• Vent valve

• Stream select valve

Each valve has a rotor seal that can wear.

Note: Although these valves look similar, their rotor seals are notinterchangeable.

Inject Valve Rotor Seal Symptoms

Replace the inject valve rotor seal when you encounter any of the followingconditions:

• Poor peak area relative standard deviation (RSD)

• Leaks

• Low backpressure

• Drops of fluid appearing on the end of the sample needle

Vent Valve Rotor Seal Symptoms

Replace the vent valve rotor seal when you encounter any of the followingconditions:

• System fails to build pressure

• Leaks

• Low backpressure

Stream Select Valve Rotor Seal Symptoms

Replace the stream select valve rotor seal when you encounter any of thefollowing conditions:

• Unstable pressure

• Leaks


5-25Maintenance

Required Materials

• 9/64-inch hex wrench

• Replacement rotor seal

• Magnifier (optional)

Before You Begin

Ensure that the system power is switched off.

Procedure

1. Observe and record the tubing connections to each numbered valve port.You will need this information when reconnecting the valve.

2. Remove all connections to the valve.

3. Use the 9/64-inch hex wrench to alternately loosen the two hex screwsthat secure the valve.

4. Carefully remove the valve cover.

5. Remove the rotor seal from the valve.

6. Inspect the backside of the rotor seal for wear. (Use the magnifier, ifpossible.) If you see scratches on the rotor seal, replace it. Also inspectthe valve body and valve cover for scratches. If you see scratches on thebody or cover, contact a Micromass service engineer to replace the valve.

Figure 5-16 shows a valve body and rotor seal.

Figure 5-16 Valve Body and Rotor Seal

7. Install the new rotor seal in the valve, matching the rotor tabs with thedepressions on the valve body.

Note: The smooth side of the seal must face inward, toward the valve body.

8. Reinstall the valve cover by matching the alignment pins with the cover.

9. Alternately tighten the two hex screws.

10. Remake all connections to the valve according to the information yourecorded in step 2.


5-26Maintenance

11. Start flow in the system, and check the valve for leaks.

Replacing Precolumns and Columns

The stream select module holds precolumns and columns. Depending on youranalytical needs, you can configure the system to use both a precolumn and ananalytical column, or solely the precolumn. The precolumn is a cartridge thatfits inside a holder. The holder has liquid seals at each end, secured with finger-tight fittings and Teflon® sleeves over the fused silica line. For longer, analyticalcolumns, Micromass supplies a micro-tight union, which connects the fusedsilica to a 1/16-inch male fitting, and also supplies fittings to accommodate360-micron OD fused silica.

Replace precolumns and columns when you encounter any of the followingconditions:

• The application requires a different precolumn or column

• Erratic retention time

• Poor peak shape

• Baseline noise

• Sensitivity loss

• Excessive backpressure

Required Materials

• Precolumn cartridge (part number 6062925)

• PTFE sleeve (part number 6060743)

• Analytical column, if needed

Note: Always replace Teflon sleeves and ferrules when you change precolumnsand columns. Reusing such items can broaden peaks and cause liquid seals tofail. Note also that sleeves must fit flush on the fused silica line to minimizedead volume.

Before You Begin

Verify that:


• The system pressure is 0 psi.

Procedure

To replace a precolumn, loosen the finger-tight nuts, replace the cartridge, andremake the connection (Figure 5-17).


5-27Maintenance

Figure 5-17 Replacing the Precolumn

To replace the column, place a Teflon sleeve over the fused silica line, and insertit into one end of the micro-tight union and tighten it with the micro-tight nut.Connect the column to the other end of the union (Figure 5-18).

Figure 5-18 Replacing the Column

Connecting Liquid Pathways

Attention: To prevent damage, do not place detectors, solvents, orany item atop the system.

Cutting PEEK Tubing

This section describes the recommended procedure for cutting PEEK tubing.

Required Materials

• Upchurch Scientific tubing cutter (part number 700001012)

Note: To avoid angled cuts, which create dead volume at connectionpoints and thus cause bandspreading, always use the Upchurch tubingcutter.

• PEEK tubing

• Magnifier

Fused Silica Tubing

Teflon SleevePrecolumn Cartridge

Finger-Tight Nutand FerruleCartridge Holder

Micro-Tight Sleeve

Fused Silica

Micro-Tight Union

Analytical Column with Male Fitting

Micro-Tight Nut


5-28Maintenance

Before You Begin

Determine the length of tubing you need to connect system components. Allowenough length so the tubing does not pull tightly around sharp corners.

Procedure

1. Insert the tubing through the smallest possible hole in the cutter. Thisensures a snug enough fit to prevent the tubing from flexing when cut.Extend the tubing from the cutter to the required length.

2. Rotate the tubing cutter around the tubing at least 10 times, ensuring asquare cut.

3. Remove the tubing from the cutter, and break it at the score line.

4. View the cut end with the magnifier, ascertaining it is square and freefrom burrs and scratches.

Connecting Tubing to Pump Tray Manifold Outlets

The connection at the outlet of each pump tray manifold (to the vent valve)includes a ferrule with a 2-µm stainless steel frit, a stainless steel lock ring, and acompression screw, which does not have a flange.

Required Materials

• PEEK tubing



• Ferrule with frit (packaged with compression screw)

Before You Begin

Verify that:


• System pressure is 0 psi.


5-29Maintenance

Procedure

1. Slide (in order) the compression screw, lock ring, and ferrule/ frit overthe manifold-end of the PEEK tubing. The flattened end of the lock ringmust face the compression screw (Figure 5-19).

Figure 5-19 Tubing Assembly for the Pump Tray Manifold

2. Insert the tubing assembly into the manifold outlet fitting.

3. With the tubing pressed against the ferrule, finger-tighten thecompression screw.


Connecting Tubing to Vent, Inject, and Stream SelectValves

Each PEEK tubing connection at the vent, inject, and stream select valvesincludes a ferrule, a stainless steel lock ring, and a stainless steel compressionscrew.

Required Materials


• PEEK tubing



• Ferrule (packaged with compression screw)

Before You Begin

Verify that:


• The system pressure is 0 psi.

Flattened End

Tapered End

Tubing Compression Screw LockRing


5-30Maintenance

Procedure

1. Slide a stainless steel compression screw, lock ring, and ferrule (in thatorder) over the valve-end of the PEEK tubing. The flattened end of thelock ring must face the compression screw; the narrow end of the ferrulemust face the lock ring (Figure 5-20).

Figure 5-20 Tubing Assembly for Valves

2. Press the tubing assembly into the appropriate valve port, as you finger-tighten the compression screw.

Note: Make sure the tubing is fully seated in the valve port.

3. Seat the ferrule securely against the tubing with the 1/4-inch wrench.Tighten the compression screw another 1/4-turn.


Tubing

Compression Screw

LockRing

FlatEnd

TaperedEnd


5-31Maintenance


6-1Specifications

Chapter 6: SpecificationsThis chapter includes the following specifications:

• Physical (Table 6-1)

• Environmental (Table 6-2)

• Electrical (Table 6-3)

• Pump (Table 6-4)

• Autosampler (Table 6-5)

Table 6-1 Physical Specifications

Item Specification

Autosampler Weight

Height

Width

Depth

57 lb. (26 kg)

17.3 in. (44 cm)

11.0 in. (28 cm)

15.7 in. (40 cm)

Pump Weight

Height

Width

Depth

180 lb. (82 kg)

17.5 in. (45 cm)

11.5 in. (30 cm)

23 in. (58 cm)

Wetted surfacematerials

Teflon®, 316 stainless steel, titanium, GFP (BAL), UPC-10 (BAL), UPC-30 (BAL), Valcon H, PEEK™, fusedsilica, glass, and Teflon AF


6-2Specifications

Table 6-2 Environmental Specifications

Item Specification

Operating temperature range 4 to 40 °C (39 to 104 °F)

Storage temperature range –40 to 70 °C (–40 to 158 °F)

Operating humidity range 20 to 80%, noncondensing

Storage humidity range 10 to 90%

Acoustic noise <65 dB(A)

Pneumatic air supply Pump: 35 to 45 psi bottled nitrogen orfiltered ambient air.Maximum flow rate: 0.002 SCFM.

Solvent compatibility Solvents consistent with materials ofconstruction. Salts and buffers can reduceseal life, especially at high pressure.


6-3Specifications

Table 6-3 Electrical Specifications

Item Specification

Voltage range 85 to 132 or 180 to 264 VAC (must be switched)

Frequency 47 to 63 Hz

Operating current 6.5 A, RMS

Fuse 10 A, slow-blow, 110 VAC nominal, ANSI10 A, slow-blow, 220 VAC nominal, IEC

Time or user-controllableswitch closures (twooptional event outputs)

Two controllable contact closuresMaximum allowable current = 0.5 A per contact

Maximum allowable voltage = 30 VDC

Contact resistance = 0.2 ohms

Outputs can be controlled from the MassLynxNT™ software Chart Recording page.

Ground terminals Connected to signal ground and used as referencefor outputs.


6-4Specifications

Table 6-4 Pump Specifications

Item Specification

Number of solvents Three to pumps A, B, and C.

Solvent containers Standard 100-mL bottles with screw tops.

Maximum operatingpressure

5000 psi (345 bar) at all flow rates. Programmableupper and lower limits.

Programmable flowrate range

0.00 to 40.00 µL/min in 0.01-µL/min increments.

Typical operatingflow rate range

1.00 to 40.00 µL/min in 0.01-µL/min increments.

System delay volume <15 µL without sample loop, inline filter, and mixer.Measured at 3.0-µL/min, methanol/methanol acetonemeasured at liftoff.

Flow precision ±0.1 minutes SD, at 5.0 µL/min with 70% water:30% methanol, premixed mobile phase from anypump using uracil/caffeine at a temperature of40 °C ±0.5 °C, N ≥=6=with Symmetry® C18,100-A, 5-µm (0.32 mm × 150 mm).

Compositionprecision

±0.15 minutes SD, at 5.0 µL/min with degassed 70%water: 30% methanol Dial-a-Mix (premixed mobilephase in A1 and B bottles each running at 50% flowrate), using uracil/caffeine at a temperature of 40 °C±0.5 °C, N ≥=6=with Symmetry C18, 100-A, 5-µm(0.32 mm × 150 mm).

Gradient profiles Eleven gradient curves (including linear, step [2],concave [4], and convex [4].


6-5Specifications

Table 6-5 Autosampler Specifications

Item Specification

Number of samples One sample plate (48-vial plate or 24-, 96-, or384-well microtiter plate)

Residual samplevolume

<2 µL with total recovery 2-mL vial

Syringe sizes 25-µL standard, 100-µL and 250-µL optional

Sample loop sizes 5- and 10-µL standard, up to 100-µL optional

Needle wash reservoirvolume

100 mL

Needle wash volume User-programmable

Injection modes Full loop, partial loop, and microliter pickup

Injection volume range 0.02 µL to 100 µL, depending on sample loopvolume and injection mode

Injection areaprecision

2.0% RSD for 0.2-µL injection volume usingpartial loop injection mode, at 5.0 µL/min with70% water: 30% methanol Dial-a-Mix in A1 andB bottles, using uracil/caffeine at a temperatureof 40°C ±0.5 °C, N ≥=6=with Symmetry C, 100-A, 5-µm (0.32 mm × 150 mm).

Injection linearity ≥0.995% correlation coefficient, 0.1 to 1.0 µL

Sample carryover ≤0.1% measured using a caffeine standard

Sample platetemperature control

Programmable 4 °C to 40 °C, ±2 °C (ambient to20 °C)


6-6Specifications


7-1Spare Parts

Chapter 7: Spare Parts

Item Part Number

Sample loop: fused silica 5 µL 6063034

Sample loop: stainless steel 5 µL 6063035

Sample loop: fused silica 10 µL 6063036





Sample loop: PEEK 100 µL 6063041

Syringe (25 µL) 6062934

Syringe (100 µL) 6063042

Syringe (250 µL) 6063043

Syringe adapter (for 100- and 250-µL syringes) 6063044

Syringe plunger tip, 10-pack (25 µL) 6062985



Sample needle assembly (2.4 µL) 6062936

Sample needle assembly (5.4 µL) 6063047

Puncture needle 6062937

Buffer tubing (50 µL) 6063048

Buffer tubing (500 µL) 6063049

Tubing, syringe to waste 6063050

Tubing, syringe to wash 6063051

Filter element for inline filter (0.5 µm) 6063052

Inject valve rotor seal 6062938

Valve fittings (5) 6063053

Larger volume injection kit S100848DC1

Pump head 6062942

Rotor seal, vent valve 6062943

Pump tray assembly 6062944


7-2Specifications

Item Part Number

Solvent reservoir filter 6062946

Face seal 6062947

Rotor 4-port pump valve 6062953

4-port pump valve 6062954

Sensor wheel PCB 6062955

Pump tray PCB 6062956

Motor driver PCB 6062957

Pump interconnect PCB 6062958

CPU PCB 6062959

Power supply 6062960

Line fuse 6062961

Power entry module with line fuse 6062962

Rotor point port pump valve 6062954

Rotor seal, Valco 10 port 6062941

Filter ferrule, 2 µm Super Flangless (10 pk) 6070189

SSI compatible male nut 1/16”-1/4” (10 pk) 6060803

PEEK tubing 1/16”X 0.025” bore (5 ft pk) 6436011

Filter ferrule, 2 µm Super Flangless (10 pk) 6070189

SSI compatible male nut 1/16”-1/4” (10 pk) 6060803

PEEK tubing 1/16”X 0.025” bore (5 ft pk) 6436011

NanoTee 6070336

PEEK tubing sleeve (green) 381 (0.015”) 6070190

Sealtight ferrules 6070191

Headless sealtight nut, short, including ferrule 6070250

Tubing cutter 5871072

Column cartridge 6062925

Micro-fingertight nut 6070193

caplc guide issue2

Documents