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Waters MicromassQuattro Premier

NanoFlow ElectroSprayOperator’s Guide

34 Maple StreetMilford, MA 01757

71500082402 Revision A

NOTICE

The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation. Waters Corporation assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with, or arising from, the use of this document.

© 2004 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA AND IRELAND. ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.

Micromass and Waters are registered trademarks, and MassLynx, NanoFlow ElectroSpray, ZSpray, and Quattro Premier are trademarks of Waters Corporation.GELoader Tip is a registered trademark of Eppendorf.PEEK is a trademark of Victrex Corporation.PicoTip and PicoFrit are registered trademarks of New Objective.Swagelok is a registered trademark of Swagelok Inc. Valco is a registered trademark of Valco Instruments Corporation Inc.Viton is a registered trademark of Dupoint.Windows is a registered trademark of Microsoft Corporation.

All other trademarks or registered trademarks are the sole property of their respective owners.

Safety Information

GeneralThe Waters® Micromass® Quattro Premier™ NanoFlow ElectroSpray™ is designed solely for low flow rate in electrospray. NanoFlow is used to create a mass spectrum to aid identification, when low amounts of sample are used.

The Quattro Premier NanoFlow ElectroSpray conforms to European standard EN61010-1:2001, Safety requirements for electrical equipment for measurement, control, and laboratory use - Part 1: General requirements.

The component has been designed and tested in accordance with recognized safety standards. If the instrument is used in a manner not specified by the manufacturer, the protection provided by the instrument may be impaired.

Whenever the safety protection of the instrument has been compromised, disconnect the instrument from all power sources, and secure the instrument against unintended operation. This instrument must be installed so that the operator can easily isolate the instrument from the mains power at all times.

Biological HazardWhen you analyze physiological fluids, take all necessary precautions and treat all specimens as potentially infectious. Precautions are outlined in “CDC Guidelines on Specimen Handling,” CDC – NIH Manual, 1984.

Chemical HazardGood Laboratory Practice should be adhered to when using potentially toxic, caustic, or flammable solvents and analytes.

Safety SymbolsWarnings in this guide or on the instrument must be observed during all phases of service, repair, installation, and operation of the instrument. Failure to comply with these precautions violates the safety standards of the design and intended use of the instrument.

Waters Corporation assumes no liability for the user’s failure to comply with these requirements.

The following safety symbols may be used in this guide or on the instrument. A Warning is an instruction that draws the user’s attention to the risk of injury or death; a Caution is an instruction that draws attention to the risk of damage to the instrument.

Consignes de sécurité

GénéralitésL' option de source Waters® Micromass® NanoFlow Electrospray™ est conçue pour l'usage exclusivement avec le Waters Micromass Quattro Premier Mass Spectrometer. Tout usage détourné du l'option de source Quattro Premier Combined APPI and APCI risquerait d’endommager l’instrument et invaliderait sa garantie.

L'option de source NanoFlow Electrospray est conforme à la norme européenne EN61010-1:2001, Règles de sécurité pour appareils électriques de mesurage, de régulation et de laboratoire - Partie 1: prescriptions générales.

Cet équipement a été conçu et testé dans le respect de normes de sécurité approuvées. Toute utilisation de l’équipement non conforme aux instructions du fabricant risque de remettre en cause la protection assurée par l’équipement.

Dans le cas où la sécurité de l’utilisateur se trouverait compromise, débranchez le cordon d’alimentation de l’équipement et assurez-vous qu’il ne pourra être mis en marche par mégarde.

L’instrument doit être installé de façon à permettre à l’opérateur, dans tous cas, de le débrancher facilement de toute alimentation électrique.

Risques biologiquesLorsque vous analysez des fluides physiologiques, faîtes preuve d'une extrême prudence et considérez tous les spécimens comme potentiellement infectieux. Une liste des précautions à prendre figure dans le guide de recommandations élaboré par le CDC,« CDC Guidelines on Specimen Handling », CDC – NIH Manual, 1984.

Risques chimiquesL'usage de solvants et analytes potentiellement toxiques, caustiques ou inflammables doit s'effectuer dans le respect des bonnes pratiques de laboratoire.

Pictogrammes de sécuritéLes avertissements présents dans le manuel de l’utilisateur ou sur l’instrument-même doivent être scrupuleusement pris en considération, et ce à tout moment, que ce soit pendant l’entretien, la réparation, l’installation ou le fonctionnement de l’instrument. Tout défaut d’application de ces règles de sécurité serait considéré comme une violation des normes de sécurité relatives à la conception et à l’usage prévu de l’instrument.

Waters Corporation ne saurait voir sa responsabilité engagée en cas de manquement de l’utilisateur à respecter les consignes de sécurité.

Vous pourrez rencontrer les pictogrammes qui suivent dans le manuel de l’utilisateur ou sur l’instrument. L’Avertissement s’applique à toute instruction destinée à attirer l’attention de l’utilisateur sur l’existence d’un risque de blessure ou de mort. L’Attention s’applique à toute instruction destinée à informer l’utilisateur de la présence d’un risque d’endommagement de l’instrument.

Warning: General warning indicating a potential health or safety hazard. See the operator’s guide for instructions.

Avertissement: Risque générale des blessures ou d’endommagement de l’instrument. Consultez les instructions dans le manuel de l’utilisateur.

Warning: Hazardous voltages may be present.

Avertissement: Tensions dangereuses - Risque de choc électrique.

Warning: Danger of hot surfaces.

Avertissement: Danger des surfaces chaudes.

Warning: Danger from corrosive substances.

Avertissement: Danger de la présence des substances corrosives

Warning: Biological agents present that may constitute a serious health threat.

Attention: Présence des agents biologiques qui peuvent constituer un menace à la santé.

Warning: Danger from toxic substances.

Avertissement: Danger des substances toxiques.

Warning: Danger of flammable substances.

Avertissement: Danger des substances inflammables.

Warning: Danger from laser radiation.

Avertissement: Risque de rayonnements laser.

Warning: Danger from UV radiation.

Avertissement: Risque de rayonnements UV.

Caution: Care must be taken to avoid the possibility of damaging the instrument, or affecting its operation.

Attention: Veillez bien à eviter la possibilité d’endommagement de l’instrument ou de nuire à son fonctionnement.

NanoFlow ElectroSpray Information

Intended UseThe Quattro Premier NanoFlow ElectroSpray is used to introduce samples into the electrospray source at flow rates of between 5nL/min and 1µL/min. For a given concentration of sample, the absolute ion currents produced at these reduced flow rates are comparable to the ion currents produced at a flow rate of 10µL/min using the standard electrospray probe.

This great reduction in the flow rate leads to the high sensitivity gains. The reduced flow rate, and, hence reduced sample consumption, results in ion signals persisting for long periods of time even when relatively small volumes are introduced. This allows you to perform MS, MS/MS and high resolution accurate mass analyses on a single loading of 1 to 2 µL of solution.

CalibrationFollow acceptable methods of calibration with pure standards to calibrate methods. Use a minimum of five standards to generate a standard curve. The concentration range should cover the entire range of quality-control samples, typical specimens, and atypical specimens.

Table of Contents

Chapter 1 NanoFlow ElectroSpray .......................................................................................... 1

1.1 Overview ........................................................................................................... 11.2 Installing the NanoFlow Source........................................................................ 31.3 Installing the NanoFlow Sprayer....................................................................... 7

Chapter 2 Borosilicate Glass Capillary NanoFlow (Nanovials) ..................................... 9

2.1 Fitting the Borosilicate Glass Capillary ............................................................ 92.2 Positioning the Borosilicate Glass Capillary Tip ............................................ 12

2.2.1 Adjusting the Borosilicate Glass Capillary Tip Position .................. 132.3 Restarting the ElectroSpray............................................................................. 14

Chapter 3 NanoFlow Nebulizer Sprayer (Nano-LC) ....................................................... 15

3.1 Nano-LC Sprayer Assembly ........................................................................... 153.2 Assembling the Fused Silica Capillary and PTFE Support Sleeve ................. 173.3 Fitting the Capillary in the Nano-LC Chamber............................................... 203.4 Fitting the Nebulizer Tip ................................................................................. 213.5 Using the Nano-LC Sprayer ............................................................................ 22

Chapter 4 PicoTip Sprayer ........................................................................................................ 23

4.1 PicoTip Sprayer Assembly.............................................................................. 234.2 Installing the PicoTip ...................................................................................... 24

Table of Contents ix

4.2.1 Attaching the PicoTip to the PicoTip Sprayer .................................. 244.2.2 Attaching the Nebulizer Gas ............................................................. 254.2.3 Attaching the PicoTip Sprayer .......................................................... 254.2.4 Removing the PicoTip ...................................................................... 25

Chapter 5 PicoFrit Tip Sprayer ............................................................................................... 27

5.1 Installing the PicoFrit Tip................................................................................ 275.1.1 Attaching the PicoFrit Tip to the PicoFrit Sprayer ........................... 28

5.2 Mounting the Column ..................................................................................... 29

Chapter 6 CE/CEC Sprayer ..................................................................................................... 31

6.1 Converting from Nano-LC.............................................................................. 326.1.1 Plumbing the Sprayer ........................................................................ 32

6.2 Attaching the Sprayer...................................................................................... 346.3 Operating the CE Sprayer ............................................................................... 34

x Table of Contents

Chapter 1NanoFlow ElectroSpray

1.1 Overview

NanoFlow is the name used for a number of techniques that use low flow rate electrospray ionization (ESI). The NanoFlow source (Figure 1-1) allows electrospray ionization to be performed in the flow rate range 5 to 1000 nanoliters per minute.

Figure 1-1 NanoFlow Source

Overview 1

The following options are available for the spraying capillary:

• Borosilicate Glass Capillary NanoFlow (Nanovials), see Chapter 2, Borosilicate Glass Capillary NanoFlow (Nanovials).Metal-coated glass capillaries allow the lowest flow rates. They are used for one sample only and must then be discarded.

• NanoFlow Nebulizer Sprayer (Nano-LC), see Chapter 3, NanoFlow Nebulizer Sprayer (Nano-LC).This option is for flow injection or for coupling to nano-HPLC and uses a pump to regulate the flow rate down to 100 nL/min. If a syringe pump is used, a gas-tight syringe is necessary to obtain correct flow rates without leakage. A volume of 250 µL is recommended.

• NanoFlow Pico Sprayer, see Chapter 4, PicoTip Sprayer.The PicoTip™ sprayer is for use with non-standard tips from New Objective.

• NanoFlow PicoFrit™, see Chapter 5, PicoFrit Tip Sprayer.This option allows the PicoTip sprayer to accommodate the use of a 5-mm New Objective PicoFrit column.

• NanoFlow Capillary Electrophoresis/Capillary Electrochromatography sprayer, see Chapter 6, CE/CEC Sprayer.This option uses a make-up liquid at the CE capillary tip which allows a stable electrospray to occur. The make-up flow rate is less than 1 microliter per minute.

For a given sample concentration, the ion currents observed in NanoFlow are comparable to those seen in normal flow rate electrospray. Great sensitivity gains are therefore observed for similar experiments due to the great reductions in sample consumption.

The NanoFlow source enclosure consists of a sprayer (either Borosilicate Glass Capillary, Nano-LC, PicoTip, PicoFrit or CE/CEC) mounted on a three-axis manipulator.

The combined unit is mounted on the NanoFlow stage which runs on a pair of guide rails with two defined positions. Pull on the spring-loaded knob on the base of the NanoFlow stage to allow it to slide.

A light within the source provides illumination for the spray, which can be observed using the microscope mounted on top of the source housing.

2 NanoFlow ElectroSpray

1.2 Installing the NanoFlow Source

Removal of the source enclosure from the instrument is described in the Waters Micromass Quattro Premier Operator’s Guide.

1. Fit and secure the Nanoflow source enclosure using the three hexagonal head screws.

2. Remove the two protective end caps from the microscope.

Warning: When fitting the NanoFlow source the ion block will be exposed. This may be hot. Ensure that the source heater has been turned off and the block has cooled before opening the source.

NanoFlow Source

Enclosure

Installing the NanoFlow Source 3

3. Fit the NanoFlow microscope and stage assembly to the NanoFlow source enclosure and secure using the side clips.

4. Install the lamp through the hole in the side of the NanoFlow source enclosure. A rubber grommet holds the lamp in place.

5. Fix the NanoFlow gas regulator and pressure gauge assembly to the source enclosure using the two screws provided.

Side Clip

NanoFlow Microscope and Stage Assembly

NanoFlow Gas Regulator

NanoFlow Gas Regulator

NanoFlow Pressure Gauge

Lamp


6. Connect the larger diameter PTFE tube (4 mm) between the instrument’s NEBULISER gas connection and the NanoFlow gas regulator’s input.

7. Connect the smaller diameter PTFE tube (1/16 inch) between the NanoFlow gas regulator’s output and the sprayer assembly, and tighten the connection. For details regarding how to fit each sprayer, see the corresponding chapter.

Nebulizer Gas Connection

NanoFlow Gas Regulator Input

NanoFlow Gas Regulator Output

Sprayer Assembly (Borosilicate)

Installing the NanoFlow Source 5

8. Connect the probe cable to the instrument’s PROBE connection.

Note: The NanoFlow stage contains a high voltage interlock so that unless the sprayer is pushed fully forward in the source, the capillary voltage (the voltage applied to the sprayer assembly) and the sampling cone voltage are disabled.

9. Connect the high voltage cable to the instrument’s HV connection.The installation of the NanoFlow source enclosure, NanoFlow stage, and microscope is now complete.

Probe Cable Connection

High Voltage Cable Connection


1.3 Installing the NanoFlow Sprayer

1. Loosen the NanoFlow stage retaining screw.2. Pull the stop screw to release the stage.

3. Pull the NanoFlow stage out of the NanoFlow source enclosure.4. Remove the sprayer cover by removing the two securing screws.

Stop Screw

Retaining Screw

Sprayer Cover

Securing Screws

Installing the NanoFlow Sprayer 7

5. Fit the required sprayer, securing it with the captive screw on the underside of the baseplate.

6. Replace the sprayer cover, ensuring that the PTFE back pressure tubing (Borosilicate Glass Capillary option) or the fused silica transfer line (Nano-LC option) is fed through the slot in the cover.

7. Refit the two securing screws.8. Pull the stop screw to release the NanoFlow stage.

9. Push the NanoFlow stage into the NanoFlow source enclosure.10. Secure the NanoFlow stage with the retaining screw.

Caution: Adjust the probe position before you push the sprayer into the source to ensure that the probe does not collide with the cone.

Captive ScrewPTFE Tubing

Borosilicate Sprayer


Chapter 2Borosilicate Glass Capillary NanoFlow (Nanovials)

2.1 Fitting the Borosilicate Glass Capillary

1. Loosen the stage retaining screw.2. Pull the stop screw to release the stage.3. Slide the stage out of the NanoFlow source enclosure.

Warning: Do not touch the sharp end of the capillary. As well as the risk of injury by a sliver of glass, the capillary may contain toxic and biohazardous samples.

Caution: Capillaries are extremely fragile and must be handled with great care.

Caution: Always handle using the blunt end of the capillary. The needle may be damaged if the sharp end is touched.

Fitting the Borosilicate Glass Capillary 9

4. Unscrew the union from the end of the sprayer assembly.

5. Remove the existing capillary from the sprayer.6. Carefully remove the new Borosilicate Glass Capillary from its case by lifting

vertically while pressing down on the foam with two fingers (Figure 2-1).

Figure 2-1 Removing the Borosilicate Glass Capillary from its Case

Capillary

Union

Capillary

Foam

10 Borosilicate Glass Capillary NanoFlow (Nanovials)

7. Load sample into the capillary using either a fused silica syringe needle or a GELoader™ tip, minimizing any bubbles between the capillary tip and the sample.

8. Thread the knurled nut and approximately 5-mm of conductive elastomer over the blunt end of the capillary

9. Fit the capillary into the holder (probe).10. Finger tighten the nut so that 5 mm of glass capillary is protruding from the end of

it. This distance is measured from the end of the nut to the shoulder of the glass capillary.

Figure 2-2 Sprayer Assembly

11. Screw the sprayer back into the assembly.12. Replace the sprayer cover.13. On the MassLynx™ Tune Page, ensure that the Capillary (kV) parameter on the

ES+/- Source tab is set to 0.

Fitting the Borosilicate Glass Capillary 11

14. Push the stage back into the NanoFlow source enclosure using the stop and handle, taking care that the tip of the capillary does not collide with the cone or the side of the source.

15. When using a GELoader tip, it may be beneficial to break the glass capillary in half, by scoring with a fused silica cutter so that the GELoader can reach the tip of the glass capillary.

2.2 Positioning the Borosilicate Glass Capillary Tip

Once a signal is obtained, the tip position needs to be adjusted to maximize the signal. Using the three-axis manipulator, the tip position can be adjusted up and down, left and right, forwards and backwards. As a starting point, the tip should be set so that it is on the center line of the sampling cone and at a distance between two and three times the diameter of the cone orifice (Figure 2-3). Typically this is approximately 2 mm.

Figure 2-3 Capillary Tip Position

Caution: Adjust the sprayer tip position before you push the sprayer inside the NanoFlow source enclosure, to ensure that the tip does not collide with the cone or the side of the source.


2.2.1 Adjusting the Borosilicate Glass Capillary Tip Position

Figure 2-4 Gas Regulator Operation

1. Position the sprayer assembly so that the microscope can view the capillary tip.

2. On the MassLynx Tune Page, click to turn the API gas on.3. Using the NanoFlow pressure regulator, apply pressure to the capillary until a drop

of liquid is seen at the tip.4. Reduce the back pressure to its original value.

5. On the ES+/- Source tab, set the Capillary (kV) parameter between 0.6 and 1.0. 6. On the Tune Page, click Press for Operate.

An ion beam should now be visible on the Tune Page.7. Adjust the three-axis manipulator, the Tune Page source voltages, and gas flow to

give maximum ion current.

Note: The ion current may change dramatically with very slight changes in position.

8. On the ES+/- Source tab, optimize the Cone Gas (V) parameter. This can have a significant effect on the signal intensity (typically 5 to 200 L/h).

Caution: Set the gas regulator so that there is no gas flow to the probe, by turning counterclockwise (Figure 2-4).

Caution: Do not exceed 1.5 kV on the capillary, as this will shorten the lifetime of the tip.

Off (-)Off (-)On (+)

Positioning the Borosilicate Glass Capillary Tip 13

2.3 Restarting the ElectroSpray

Should the spray stop, it is possible to restart it by adjusting the three-axis manipulator so that, viewed under magnification, the capillary tip touches the sample cone and a small piece of the Borosilicate Glass Capillary shears off. Set the Tune Page Capillary (kV) parameter to 0 when doing this.

It may also be necessary to apply some NanoFlow gas pressure to force a drop of liquid from the capillary. Up to 1.4 bar (20 psi) can be applied and, with this pressure, a drop should be visible unless the capillary is blocked.


Chapter 3NanoFlow Nebulizer Sprayer (Nano-LC)

3.1 Nano-LC Sprayer Assembly

The Nano-LC sprayer assembly contains a microvolume insert that couples the flow from an injector, syringe pump, or nano-HPLC system to a length of fused silica capillary that acts as the spray nozzle at the end of the nebulizer tip (Figure 3-1).

A length of PTFE tubing supports the fused silica capillary within the chamber. The PTFE fits into the microvolume insert and is retained using a metal Valco ferrule, along with the thread on the end of the chamber.

Nano-LC Sprayer Assembly 15

Figure 3-1 Nano-LC Sprayer Assembly

1/16 Metal Valco Ferrule


Nebulizer Tip

Nebulizer Gas via NanoFlow Gas

Regulator

PTFE Support Sleeve

MicrovolumeUsing Alternate

3-way Microvolume T-Insert

Fused Silica Capillary

Chamber

Low Pressure PTFE Connector

16 NanoFlow Nebulizer Sprayer (Nano-LC)

3.2 Assembling the Fused Silica Capillary and PTFE Support Sleeve

1. Cut a 50-mm length of PTFE support sleeve tubing (1/6 × 0.12).2. Cut a 70-mm length of capillary (TSP020090).3. Thread the capillary through the prepared length of support sleeve.4. Align the support sleeve and capillary flush at one end.

5. Thread the supplied plug cap nut and 1/16 metal Valco ferrule over the support sleeve.

Support Sleeve and

Capillary Aligned Flush


Plug Cap Nut

Plug Cap

Assembling the Fused Silica Capillary and PTFE Support Sleeve 17

6. Fit the plug cap over the end of the support sleeve.

7. Push the support sleeve tubing to the end of the plug cap nut to set the “pilot” depth (see Figure 3-2).

8. Engage the plug cap nut in the plug cap thread and tighten so that the 1/16 metal Valco ferrule just grips the support sleeve.

Note: Do not fasten the ferrule too tight at this point as you are required to rethread the silica capillary through the PTFE support sleeve later in the procedure.

9. Unscrew the plug cap nut and remove from the support sleeve.


10. Remove the capillary and plug cap from the support sleeve, leaving the ferrule behind.

11. Cut the support sleeve so that only 10 mm protrudes through the back of the ferrule, (Figure 3-2).

Figure 3-2 Fused Silica Capillary and PTFE Support Sleeve Assembly

Nebulizer Tip

Microvolume T-Insert

“Pilot Depth”

Assembling the Fused Silica Capillary and PTFE Support Sleeve 19

12. Rethread the capillary through the 10-mm length of support sleeve and align flush with end of the tubing.

13. Using the plug cap and nut again, tighten the ferrule so that the capillary is firmly gripped within the support sleeve.

14. Remove the plug cap and nut from the support sleeve.

3.3 Fitting the Capillary in the Nano-LC Chamber

1. Thread the capillary and the PTFE support sleeve through the Nano-LC chamber.2. Fit the microvolume insert into the Nano-LC body. Rotate the insert so that the

ferrule seats line up with the column and chamber connections.3. Fit the Nano-LC chamber into the body and tighten (see Figure 3-1).4. Pull gently on the PTFE support sleeve to confirm that the support sleeve is gripped

firmly.


3.4 Fitting the Nebulizer Tip

1. Slide the nebulizer tip over the capillary and tighten into the chamber, (see Figure 3-1).

2. Continue tightening until approximately 3 mm of tip protrudes (Figure 3-3).

Figure 3-3 Fitting the Nebulizer Tip

3. Using a silica cutter cut the exposed silica capillary flush with the end of the nebulizer tip (Figure 3-3).

4. Continue tightening until approximately 1 mm of silica capillary protrudes through the tip (Figure 3-4).

Figure 3-4 Fitting the Silica Capillary

Fitting the Nebulizer Tip 21

3.5 Using the Nano-LC Sprayer

For tuning purposes, it may be useful to infuse a known sample in 95% water using a Harvard syringe pump.

1. Connect the syringe pump to the sprayer assembly using a fused silica capillary (see Figure 3-1).

2. Connect the PTFE tube between the NanoFlow gas regulator and the Nano-LC sprayer (see Figure 3-1).

3. Remove the stage from the NanoFlow source enclosure.4. Attach the sprayer to the stage using the captive screw on the underside of the

baseplate.It may be necessary to reposition the retaining screw to the alternative locating hole.

5. Place the cover over the sprayer ensuring that the tubing coming from the sprayer is threaded correctly through it.

6. Secure the cover with the two screws provided.7. Push the NanoFlow stage back into the NanoFlow source enclosure and adjust the

tip so that the capillary is approximately 5 mm from the cone.

8. On the MassLynx Tune Page, click to turn the API gas on.9. Click Press for Operate.10. On the ES+/- Source tab, ensure that the Capillary (kV) parameter is set to 3-3.5.

An ion beam should now be present.11. By viewing under magnification, the spray emanating from the capillary may be

examined and tuned by adjusting the nebulizer tip so that a fine spray is observed. Altering the nebulizer gas to approximately 5 psi may also help the tuning process.

12. Optimize the ion beam by altering the position of the spray using the translation controls stage. The sprayer can now be connected to the HPLC system.

Warning: Without the sprayer cover fitted, high voltages are exposed when the instrument is in operate mode and the sprayer stage is pushed into the NanoFlow source enclosure.


Chapter 4PicoTip Sprayer

4.1 PicoTip Sprayer Assembly

The PicoTip sprayer (Figure 4-1) is for use with non-standard tips manufactured by New Objectives. The tips are 7-cm long and have a 4-cm distal coating. The distal coating allows electrical potential to contact the spray liquid.

The PicoTip capillary uses a capillary voltage of approximately 3 kV and nebulizer pressure of approximately 5 psi.

It is recommended that you infuse a known sample and optimize the sprayer by adjusting the position of the capillary with the NanoFlow stage and source voltages.

Figure 4-1 PicoTip Sprayer

Stainless Steel Nebulizer Capillary

PTFE Sleeve

Nebulizer Gas Fused Silica Adapter

Conductive Elastomer

Modified Valco Nut

Valco T-PieceBase PlatePicoTip

PicoTip Sprayer Assembly 23

4.2 Installing the PicoTip

4.2.1 Attaching the PicoTip to the PicoTip Sprayer1. Insert the PicoTip through the nebulizer capillary and through the back of the Valco

T-piece, distal-end first (see Figure 4-1).2. Fit a fused silica adapter and the modified Valco nut over the distal-end of the

capillary.3. Position the spray tip as in Figure 4-2.

Figure 4-2 Tip Position

4. Tighten the modified Valco nut until the fused silica adapter is fastened into place.5. Place a 5-mm length of conductive elastomer over the PicoTip and press into the

modified Valco nut.6. Attach the column or other output to the nebulizer capillary using low pressure

connectors to the PicoTip Sprayer.

Caution: Fit the PicoTip before attaching the PicoTip sprayer to the NanoFlow stage.

Caution: Handle the PicoTips with great care as they are extremely fragile.

Caution: To avoid damage to the PicoTip, insert the tip into the nebulizer distal-end first.

Spray TipNebulizer Capillary

24 PicoTip Sprayer

4.2.2 Attaching the Nebulizer Gas1. Attach the PTFE nebulizer tube to the side port with a Valco nut and ferrule.2. Attach the Swagelok™ coupling to the nebulizer gas output on the instrument front

panel.

4.2.3 Attaching the PicoTip Sprayer1. Remove the NanoFlow stage from the NanoFlow source enclosure.2. Attach the PicoTip sprayer to the NanoFlow stage using the captive screw on the

underside of the base plate. 3. Push the NanoFlow stage back into the NanoFlow source enclosure to activate the

interlocks.

4.2.4 Removing the PicoTip1. Remove the NanoFlow stage from the NanoFlow source enclosure so that the

capillary voltage is turned off.2. Disconnect any column from the rear of the sprayer by pulling the fused silica from

the PTFE low pressure connector.3. Loosen the modified nut until the fused silica adapter no longer grips the fused

silica.4. Touching the distal-end of the tip only, push the fused silica out of the sprayer.

For details of how to obtain a beam, see Section 3.5, Using the Nano-LC Sprayer.

Installing the PicoTip 25

26 PicoTip Sprayer

Chapter 5PicoFrit Tip Sprayer

The PicoFrit tip can accommodate uncoated 5-cm long columns. Electrical potential contacts the liquid via a low dead volume union, hence the extension on the rear of the tip option.

Figure 5-1 PicoFrit Tip

5.1 Installing the PicoFrit Tip

Caution: Fit the PicoFrit tip before attaching the PicoFrit sprayer to the NanoFlow stage.

Caution: Handled the PicoFrit tips with great care as they are extremely fragile.

Caution: To avoid damage to the PicoFrit tip, insert the tip into the nebulizer distal-end first.

Sealtight Nut and Ferrule

M3 x16 Screws

Valco Nut and Ferrule

PicoFrit ColumnUnion

Sleeves

Union Clamp Mounting Bracket

Installing the PicoFrit Tip 27

5.1.1 Attaching the PicoFrit Tip to the PicoFrit Sprayer1. Remove the sprayer from the stage if it is attached.2. Remove any tip that may be attached to the sprayer.3. Attach the mounting bracket (see Figure 5-1).4. Loosely attach the two components of the tip clamp to the mounting bracket using

two M3 × 16 screws (see Figure 5-1).5. Insert the union into the clamp.6. Tighten the screws to secure the union.7. Prepare a sleeve for the fused silica capillary using the green sleeve, a Valco nut,

and a Valco ferrule.8. Push the sleeve into the union, while tightening the nut to secure the ferrule onto the

sleeve.9. Prepare another sleeve using another sleeve, nut and ferrule and attach in the same

way as described above.

28 PicoFrit Tip Sprayer

5.2 Mounting the Column

Note: Refer to the instructions that come with the column before attempting to mount the column.

1. Carefully remove the column from its packaging.2. Hold the tip towards the light to identify the start of the packaging and cut the fused

silica approximately 25 mm from this point (Figure 5-2). This will give you a total length of 75 mm for the tip for a 50-mm packed bed.

Figure 5-2 Column Packing

3. Insert the column distal-end first through the nebulizer capillary fused silica adapter and modified nut.

4. Push the fused silica through the sleeve and ensure the silica is flush with the end of the sleeve.

5. Attach the column to the union by tightening the Valco nut.6. Move the union so that the tip is positioned as in Figure 5-1.

25 mm Packing

Mounting the Column 29

30 PicoFrit Tip Sprayer

Chapter 6CE/CEC Sprayer

The Capillary Electrophoresis/Capillary Electrochromatography (CE/CEC) sprayer is designed for use on the ZSpray™ NanoFlow source enclosure, and utilizes the body and chamber components of the Nano-LC sprayer.

Figure 6-1 CE/CEC Sprayer

The sprayer uses a make-up liquid at the CE capillary tip which allows a stable electrospray to occur. The make-up flow rate is less than 1 microliter per minute.

Figure 6-2 Sprayer

Capillary Adjustment

CE Capillary CE Extension Capillary

Chamber

Nebulizer Tip

Nebulizer Gas Make-up

Flow

Sprayer Body

Microvolume T-Insert

Microtight Union

31

The sprayer consists of the following components:

• A T-piece to allow the addition of the make-up solvent.• A fused silica CE extension capillary.• A stainless steel capillary to direct the make-up liquid to the sprayer tip.

At the rear of the sprayer is a capillary adjuster mechanism which allows spray stability to be optimized.

The sprayer allows conversion between Nano-LC and CE/CEC.

6.1 Converting from Nano-LC

1. Remove the sprayer from the stage.2. Remove all fused silica capillaries from the sprayer.3. Remove the microvolume union from the sprayer.4. Store these components safely for future use.5. Locate the components for the CE sprayer.

Continue with the procedure described in Section 6.1.1, Plumbing the Sprayer.

6.1.1 Plumbing the Sprayer1. Detach the sprayer from the stage.2. Insert the microtight union into the space in the adjuster block. 3. Secure it in position using a M2 × 6 mm screw.4. Screw the blanking plug for the union into the back end of the union.5. Attach the adjuster assembly to the sprayer using a M2 × 6 mm screw.6. Place the Valco microvolume T-insert into the sprayer’s main body.

The two sizes of fused silica CE extension, the line from the pump delivering make-up liquid, and the stainless steel make-up capillary all require sleeves of 1/16 inch to fit in the Valco parts of the sprayer.

Locate the appropriate sleeve material for each component as listed:

• CE extension capillary 50 micron i.d., TSP050192. use TTF110 PTFE.• CE extension capillary 75 micron i.d., TSP075200, use TTF 110 PTFE.

32 CE/CEC Sprayer

• Fused silica TSP075375 (make-up liquid from the pump to the sprayer), use TTF115 PTFE.

• Stainless steel make-up capillary, use 0.020 inch i.d. PEEK™.

To make a sleeve:

1. Locate the plug cap, a Valco nut and a Valco ferrule.2. Cut a 25-mm length of the appropriate sleeve material.3. Set the ferrule to the correct depth using the plug cap by tightening with a pair of

spanners such that the ferrule grips the sleeve.4. Remove the sleeve from the plug cap, then the nut from the sleeve and cut the sleeve

so that approximately 10 mm protrudes from the rear of the ferrule.5. Locate the correct fused silica for your application as the CE extension capillary and

cut a 15-cm length.6. Thread this fused silica from the rear of the sprayer through the sleeve and T-piece.7. Sleeve the make-up capillary and place in the end of the Nano-LC chamber.8. Thread the fused silica into the capillary, taking care to ensure that the fused silica

does not bend excessively.9. Screw the chamber into the Nano-LC body until the make-up capillary is secure.10. Attach the fused silica to the microtight union in the adjuster assembly using the

correct sleeve and ensure that dead volumes are eliminated by correct use of the blanking nut which comes with the union.

Note: This microtight union is designed to require only finger tightness on the nut.

11. Using the adjuster screw, alter the position of the microtight union so that the union sleeve touches the back end of the sprayer sleeve.This will reduce the length of the extension capillary to a minimum.

12. Cut the extension capillary so that approximately 2 mm of fused silica protrudes from the stainless steel make-up capillary.

13. Thread the make-up capillary through the probe tip and screw the tip into the chamber such that approximately 1 mm of make-up capillary can be seen.

14. Attach the make-up liquid to the port at the top of the sprayer and set to flow at 1 µL/min.

15. Tighten the nut supporting the PTFE sleeve for the CE extension capillary.

Converting from Nano-LC 33

This can be deemed as at the correct tightness when it is still possible to adjust the fused silica at the tip using the adjuster screw but with no liquid leakage through the PTFE sleeve.It is best to tighten the sleeve in stages while checking the movement at the tip regularly. Make-up flow liquid should now be visible at the tip on the outside of the CE extension capillary and on the inside of the make-up capillary.

6.2 Attaching the Sprayer

The sprayer is now ready to be attached to the stage and to the CE column.

If necessary:

• Alter the position of the thumbscrew underneath the stage.• Replace the nanoflow HV cable attached to the stage with the CE HV cable.

1. Ensure that, for cables incorporating an earth lead, the earth lead is attached to the tag on the NanoFlow flange.

2. Secure the sprayer cover to the stage using the two screws.

6.3 Operating the CE Sprayer

1. Set the position of the sprayer to be approximately 7 mm from the sample cone and about 3 mm off the axis of the apex.

2. For positive ion requirements, use a make-up liquid of 50:50 methanol:water + 1% acetic acid, at a flow of 0.8 µL/min.

3. For negative ion work, use a make-up liquid of 80:20 IPA: H2O + 2 mM ammonium acetate, at a flow of 0.8 µL/min.For initial tuning purposes, it is recommended that a known sample is included in the make-up liquid composition.

Warning: High voltages are present when operating the sprayer coupled to CE apparatus. See the CE apparatus manual for additional instructions.

Caution: A high CE current can lead to heating effects in the capillary which can cause an unstable electrospray beam.

34 CE/CEC Sprayer

4. Thread the capillary from the CE instrument through some PTFE tubing.5. Attach the fused silica into the rear of the microtight union in the adjuster block.

This is done using F-185 sleeve for 375-µm o.d. fused silica.6. Fill the column with buffer solution.7. Turn on the CE voltage and check that a current is reading back.8. Switch on the capillary voltage and set it to 4.9 kV (positive ion mode) or -4 kV

(negative ion mode).9. Turn on the nebulizer backpressure and set it to approximately 20 psi.

Note: For improved CE performance, it is recommend that you turn off the nebulizer gas and capillary voltage during the injection cycle.

10. Once the CE voltage is turned on after each injection, immediately turn back on the capillary voltage and nebulizer gas and proceed as follows:

11. While viewing under magnification, take the CE capillary forward using the adjuster screw until a spitting effect is observed.

12. Pull the capillary back and, just before the fused silica goes into the make-up capillary, observe a fine spray.

13. Optimize the spray if necessary, using the various source and analyzer parameters on the MassLynx Tune Page.

Operating the CE Sprayer 35

36 CE/CEC Sprayer

Index
Aassembling
fused silica capillary 15PTFE support 15

attachingthe silica to the chamber 20

Bborosilicate 9

CCapillary Electrochromatography

sprayer 31Capillary Electrophoresis sprayer 31CE. See Capillary Electrophoresis sprayerCEC. See Capillary Electrochromatography

sprayerchamber, attaching the silica 20consignes de sécurité iv

Eelectrospray, restarting 14

Ffitting

NanoFlow ElectroSpray 7the nebulizer tip 21

fused silica capillary, assembling 15

Gglass capillary 9

Iinstalling, NanoFlow source enclosure 3intended use vii

NNanoFlow ElectroSpray

fitting 7NanoFlow nebulizer sprayer 15NanoFlow source enclosure 2

installing 3NanoFlow sprayer

operating 15Nano-LC 15Nano-LC Sprayer

operating 22Nanovials 9Nanovials. See Borosilicate glass capillarynebulizer tip, fitting 21

Ooperating, the NanoFlow sprayer 15operating, the Nano-LC sprayer 22option, glass capillary 9

PPicoFrit tip sprayer 27PicoTip

capillary voltage 23

Index 37

nebulizer pressure 23PicoTip tip sprayer 23PTFE support, assembling 15

Rrestarting, electrospray 14risques biologiques ivrisques chimiques iv

Ssource enclose NanoFlow 2sprayer tips

PicoFrit 27PicoTip 23

Ttips

nebulizer tip 21PicoFrit 27PicoTip 23

38 Index

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