Mariner Biospectrometry Workstation

Mariner Biospectrometry Workstation TurboIonSpray Module Supplement DRAFT August 28, 2001 6:53 pm TurboIonSprayfmRevB.fm

Copyright 1999, 2001, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems 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 Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. Applied Biosystems and TurboIonSpray are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. AB (Design), Applera, Biospectrometry, and Mariner are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. Teflon is a registered trademark of E.I. Du Pont de Nemours and Co. All other trademarks are the sole property of their respective owners. Printed in the USA, 07/2001 Part Number V900083 Rev. C DRAFT August 28, 2001 6:53 pm TurboIonSprayfmRevB.fm

WARNING For continued protection against fire hazard, replace fuses with those of the same type and rating. AVERTISSEMENT Remplacez les fusibles par ceux de même type et puissance pour éviter les risques d incendie. WARNING Always wear eye protection when working with solvents. AVERTISSEMENT Porter toujours des protections pour les yeux lorsque vous manipulez des solvants. WARNING Use the Mariner Biospectrometry Workstation only as specified in this document. Using this system in a manner not specified may result in injury or damage to the system. AVERTISSEMENT Veuillez suivre avec attention les indications figurant dans ce document lorsque vous utilisez la Station de Travail de Biosptectrométrie Mariner. Un usage différent de la station pourrait causer un accident ou endommager le système. DRAFT August 28, 2001 6:53 pm TurboIonSprayfmRevB.fm

WARNING Before handling any chemicals, refer to the Material Safety Data Sheet provided by the manufacturer, and observe all relevant precautions. AVERTISSEMENT Avant de manipuler des produits chimiques, veuillez consulter la fiche de sécurité du matériel fournie par le fabricant, et observer les mesures de précaution qui s imposent. WARNING Exhaust from the external mechanical pump contains chemical vapors. Vent the mechanical pump to a fume hood. AVERTISSEMENT L échappement de la pompe mécanique externe contient des vapeurs chimiques. Connecter la pompe mécanique à une hotte d aspiration. DRAFT August 28, 2001 6:53 pm TurboIonSprayfmRevB.fm

Table of Contents Table of Contents Section 1 Overview of the... TIS-1 Section 2 Installing the... TIS-5 2.1 Overview of the TurboIonSpray Spray Chamber... TIS-6 2.2 Installing the TurboIonSpray Spray Chamber... TIS-8 2.3 Installing the Exhaust Trap Line and Bottle... TIS-11 2.4 Connecting, Configuring, and Adjusting Nebulizer, Auxiliary, and Curtain Gases... TIS-13 2.4.1 Connecting the Gases... TIS-14 2.4.2 Configuring the Gases... TIS-16 2.4.3 Adjusting the Gases... TIS-17 2.5 Connecting and Adjusting the Compressed Air Supply... TIS-18 2.6 Connecting the Solvent Inlet Tubing... TIS-19 2.7 Positioning the IonSpray Probe... TIS-22 2.8 Removing the TurboIonSpray Spray Chamber... TIS-26 Section 3 TurboIonSpray Operation... TIS-29 3.1 Overview of TurboIonSpray Operation... TIS-29 3.2 Sample, Solvent, and LC Column Guidelines... TIS-30 3.3 Optimizing Signal... TIS-30 3.4 Shutting Down the TurboIonSpray Chamber When Not in Use... TIS-34 Section 4 Maintenance... TIS-35 4.1 Replacing the Spray Tip and Union... TIS-35 4.2 Replacing PEEK Tubing... TIS-40 4.3 Replacing the Lamp Bulb... TIS-43 Section 5 TurboIonSpray Troubleshooting... TIS-45 Appendix A Spare Parts... TIS-47 Index Mariner Biospectrometry Workstation TIS Module Supplement v

Table of Contents vi Applied Biosystems

Overview of the 1 Overview of the Purpose The Mariner (Figure 1-1) is an optional electrospray ionization (ESI) spray chamber and associated parts that allow operation on the Mariner Biospectrometry Workstation at higher flow rates than the standard spray chamber (up to 1.0 ml/min). TurboIon Spray module PB100741 Figure 1-1 Mariner Workstation with Installed Probe function The TurboIonSpray module includes an IonSpray probe that delivers sample and solvent, and a heated Turbo probe that directs a jet of heated gas at the sample stream. The heated gas increases the rate of droplet evaporation, aids desolvation, and increases ion signal. Mariner Biospectrometry Workstation TIS Module Supplement TIS-1

Section 1 Overview of the Parts of the TurboIonSpray module Auxiliary gas flow meter The TurboIonSpray module is a modified version of the Applied Biosystems TurboIonSpray Source. It has been physically adapted to fit onto the Mariner Biospectrometry Workstation. Basic operation and performance is unchanged. The TurboIonSpray module includes: TurboIonSpray spray chamber Exhaust trap line and bottle IonSpray probe Heated Turbo probe Exhaust trap line to bottle (bottle not shown) PB100732 Compressed air inlet Figure 1-2 Parts of the TurboIonSpray Chamber TIS-2 Applied Biosystems

Overview of the Features The TurboIonSpray source provides the following features: Increased ionization efficiency leading to improved sensitivity at higher flow rates than conventional electrospray, particularly for compounds with extremely high polarity and low surface activity. Accommodates flow rates from 10 µl/min to 1.0 ml/min, with optimum performance at approximately 200 µl/min. NOTE: Below 50 µl/min, you may see decreased sensitivity. Use the standard electrospray spray chamber for applications that require low flow rates and optimum sensitivity. Is mild enough to be used with labile compounds such as peptides, proteins, and thermally-labile pharmaceuticals. Vaporizes 100 percent aqueous to 100 percent organic solvents. Designed to handle common LC and high-throughput flow rates (>200 µl/min, and up to 1 ml/min) without flow splitting. Stable performance for a variety of LC conditions and solvent buffers. Mariner Biospectrometry Workstation TIS Module Supplement TIS-3

Section 1 Overview of the TIS-4 Applied Biosystems

Installing the 2 Installing the In this section This section includes: Overview of the TurboIonSpray spray chamber Installing the TurboIonSpray spray chamber Installing the exhaust trap line and bottle Connecting and configuring nebulizer, auxiliary and curtain gases Connecting and adjusting the compressed air supply Connecting the solvent inlet tubing Positioning the IonSpray probe Removing the TurboIonSpray spray chamber Materials and supplies needed In addition to the materials and supplies needed to install and run the Mariner Biospectrometry Workstation, you need the following to install and run the TurboIonSpray module: Compressed air Powers the exhaust pump in the exhaust system. Compressed air source must be: Oil and water-free Have a pressure range of 80 to 100 psi Be regulated to a minimum flow rate of 15 liters/min. External exhaust Exhaust bottle must be vented to a fume hood or other external exhaust source, to remove chemical vapors present in the exhaust. Mariner Biospectrometry Workstation TIS Module Supplement TIS-5

Section 2 Installing the 2.1 Overview of the TurboIonSpray Spray Chamber Safety interlocks The Mariner Workstation includes safety interlocks that prevent you from turning on high voltages if: Spray chamber is not installed Mass spectrometer top cover panel is not in place If any of the above conditions are true, interlocks are considered open, and: Interlocks LED on the front panel is yellow and blinking. See Figure 1-10 in the Mariner Biospectrometry Workstation User s Guide. Interlocks indicator on the System Status page in the Instrument Control Panel is yellow. See the appropriate section on system status in the Mariner Biospectrometry Workstation User s Guide. To reset the safety interlocks, install or close the spray chamber and replace the top cover panel. If the fault does not clear, press the Reset button on the right side panel of the system near the back. See Figure 2-1 in the Mariner Biospectrometry Workstation User s Guide. TIS-6 Applied Biosystems

Installing the Spray chamber precautions Read the following warnings before handling the spray chamber. WARNING When the spray chamber is removed, the curtain gas plate is exposed and may be hot. Do not touch. Curtain gas plate opening Curtain gas plate PB100716 Figure 2-1 Curtain Gas Plate Location with Spray Chamber Removed Right Side View of System Mariner Biospectrometry Workstation TIS Module Supplement TIS-7

Section 2 Installing the 2.2 Installing the TurboIonSpray Spray Chamber Before you begin Adjusting spray tip distance from nozzle Installing the spray chamber Before installing the TurboIonSpray chamber, make sure that either an integrated syringe pump or the front and side panels are installed on the workstation. Do not install the TurboIonSpray chamber if there is no support below the spray chamber platform. If unsupported, the spray chamber may not properly seal at the nozzle area, and can cause liquid or gas leaks. To adjust the distance of the spray tip from the nozzle opening, use the positioning knob and gauge located on the back of the IonSpray probe. The spray tip lateral positioning knob and gauge are difficult to see when the spray chamber is installed on the system. You can adjust before installing the spray chamber, if desired. For instructions, see Adjusting spray tip distance from nozzle opening on page 23. To install the TurboIonSpray spray chamber: 1. Read Spray chamber precautions on page 7 before proceeding. 2. Make sure the spray chamber lever is pulled out (see Figure 2-14 on page 27). 3. Hold the TurboIonSpray spray chamber with two hands, one under the base, one on the Turbo probe (Figure 2-2). The Turbo probe is the front probe when you are looking at the spray chamber with the probes pointing to the right. NOTE: Do not hold the chamber by the IonSpray probe (the back probe). Doing so can change probe position. TIS-8 Applied Biosystems

Installing the Base Turbo probe PB100731 Figure 2-2 Installing the TurboIonSpray Chamber 4. Position the spray chamber at the top front right corner of the workstation. 5. Push the spray chamber to the back wall of the workstation and slide the spray chamber to the left into place. Watch to make sure that the electrical and gas connectors on the spray chamber and the workstation are aligned. 6. Press the spray chamber firmly into place and push in the lever so that it is flush with the side of the mass spectrometer. When the spray chamber is properly installed, the Interlock LED turns green. 7. Turn on the spray chamber light using the switch on the rear of the spray chamber. Mariner Biospectrometry Workstation TIS Module Supplement TIS-9

Section 2 Installing the 8. Perform the procedures below as described in the following sections to complete the installation: Section 2.3, Installing the Exhaust Trap Line and Bottle Section 2.4, Connecting, Configuring, and Adjusting Nebulizer, Auxiliary, and Curtain Gases Section 2.5, Connecting and Adjusting the Compressed Air Supply Section 2.6, Connecting the Solvent Inlet Tubing Section 2.7, Positioning the IonSpray Probe TIS-10 Applied Biosystems

Installing the 2.3 Installing the Exhaust Trap Line and Bottle WARNING Make certain there is no restriction in the tubing between the TurboIonSpray spray chamber vent outlet and the exhaust bottle. If there is any restriction in the tubing, liquid can back up into the spray chamber and pose an explosion hazard. To install the exhaust trap line and bottle: 1. Connect the two lengths of 1/2-inch OD plastic tubing to the connectors on the exhaust trap bottle cap and screw the cap onto the bottle. 2. To one length of 1/2-inch OD tubing, connect the adapter, then connect the exhaust trap inlet tubing (1/4-inch OD plastic tubing with metal connector) to the adapter. 3. Check the length of the combined tubing. It should be just long enough to reach between the spray chamber and the exhaust trap bottle cap, without any extra tubing that might kink and cause a restriction in the line. If it is longer than necessary, shorten the 1/2-inch OD tubing. 4. Remove the end cap from the compressed air fitting on the TurboIonSpray module. Save the end cap. You can replace it on the fitting when the spray chamber is not in use. 5. Connect the exhaust trap inlet tubing (short tubing with metal connector) to the spray chamber by slipping the connector onto the fitting until it locks in place (Figure 2-3). Mariner Biospectrometry Workstation TIS Module Supplement TIS-11

Section 2 Installing the Compressed air fitting PB100734 Exhaust trap inlet tubing Figure 2-3 Connecting the Exhaust Trap Line to the Spray Chamber 6. Connect the exhaust trap outlet tubing (the other length of tubing connected to the exhaust trap bottle cap) to a fume hood or other ventilation source. TIS-12 Applied Biosystems

Installing the 2.4 Connecting, Configuring, and Adjusting Nebulizer, Auxiliary, and Curtain Gases In this section Gas functions This section includes: Connecting the gases Configuring the gases Adjusting the gases Gases perform the following functions: Nebulizer gas Breaks solvent into droplets. Auxiliary gas Delivers gas to the Turbo probe. This gas blows directly at the spray to aid desolvation, especially at higher flow rates where the gas can also be heated. Curtain gas Prevents large solvent droplets and uncharged particles from entering into the nozzle. Also aids desolvation. Gas requirements Use one of the following for nebulizer, curtain, and auxiliary gases: Compressed dry nitrogen, grade 4.8 or better Liquid nitrogen in a dewar (requires drying if you see water clusters or water adducts on proteins) Using one gas supply If you want to use one gas supply for all gases, you can connect one line from the regulated nitrogen supply and install a quick-connect tee (provided in the Mariner Workstation startup kit) just before the Gas 1 and Gas 2 connections on the right side panel of the workstation. Mariner Biospectrometry Workstation TIS Module Supplement TIS-13

Section 2 Installing the 2.4.1 Connecting the Gases Connecting the compressed gas supply To connect the compressed gas supply lines: 1. Using Teflon tubing (1/16-inch ID, 1/8-inch OD), connect the gas sources to the right side of the mass spectrometer (see Figure 2-1 of the Mariner Biospectrometry Workstation User s Guide): Gas Connector Nebulizer 1 Auxiliary 2 Curtain 2 Push the tubing on to the push-on fittings on the mass spectrometer. 2. Regulate the gases at the source between 80 and 100 psi. CAUTION Do not regulate gas above 100 psi. Doing so can damage the gas seals in the Mariner Workstation. Regulating the gas below 80 psi may not generate high enough gas pressure at the flow meters. TIS-14 Applied Biosystems

Installing the Internal gas connections on the spray chamber Internal gas tubing between the spray chamber base and the probes may be disconnected when the spray chamber is provided to you. If this tubing is disconnected, connect the internal gas tubing to the probes (Figure 2-4): Nebulizer Connect from the left fitting on the base to the fitting at the base of the IonSpray probe Auxiliary Connect from the right fitting on the base to the fitting at the base of the Turbo probe Turbo probe IonSpray probe PB100755 Auxiliary gas Nebulizer gas Figure 2-4 Connecting the Internal Gas Tubing Mariner Biospectrometry Workstation TIS Module Supplement TIS-15

Section 2 Installing the 2.4.2 Configuring the Gases To configure the gases: 1. In the Instrument Control Panel, select System Settings from the Instrument menu, then click the Gas Flow tab to display the Gas Flow page (Figure 2-5). Figure 2-5 Gas Flow Page 2. Select Nebulizer, Auxiliary, and Curtain. 3. Select the gas mode: Automatic Gas flow starts automatically when you set the instrument state to On. NOTE: You can override automatic control mode by clicking in the toolbar during operation. Manual Gas flow does not start automatically when you set the instrument state to On. Click in the toolbar to start gas flow during operation. TIS-16 Applied Biosystems

Installing the 2.4.3 Adjusting the Gases To adjust the gases: 1. Regulate the gases at the supply between 80 and 100 psi. CAUTION Do not regulate gas above 100 psi. Doing so can damage the gas seals in the Mariner Workstation. Regulating the gas below 80 psi may not generate high enough gas pressure at the flow meters. 2. Turn gas on using toolbar buttons in the Instrument Control Panel: If the gas flow is in automatic mode, click in the toolbar to turn on. If the gas flow is in manual mode, click in the toolbar to turn on. 3. Turn gas off using toolbar buttons in the Instrument Control Panel: If the gas flow is in automatic mode, click in the toolbar to turn off. If the gas flow is in manual mode, click in the toolbar to turn off. CAUTION Do not turn gas off using the flow meters on the front panel of the mass spectrometer. Doing so will damage the valve in the flow meters. Turn off gas using toolbar buttons or at the gas supply. Mariner Biospectrometry Workstation TIS Module Supplement TIS-17

Section 2 Installing the 2.5 Connecting and Adjusting the Compressed Air Supply Requirements Connecting Compressed air drives the exhaust pump in the exhaust system. The compressed air source must: Be oil and water-free Have a pressure range of 80 to 100 psi Be regulated to a minimum flow rate of 15 liters/min To connect: 1. Connect the quick-disconnect fitting provided to 1/8-inch air inlet tubing. 2. Connect the air inlet tubing to the spray chamber by pulling back on the quick-disconnect fitting, inserting the fitting into the compressed air port on the bottom right corner of the spray chamber, and releasing the connector (Figure 2-6). Quick-disconnect fitting Compressed air adjustment knob PB100735 Figure 2-6 Connecting the Compressed Air to the Spray Chamber TIS-18 Applied Biosystems

Installing the Adjusting After you start solvent flow, turn the compressed air adjustment knob (Figure 2-6) one to one and a half turns until you hear the flow. This generates 15 liters/min of flow. Adjust the flow to optimize signal intensity. If the compressed air flow is: Too high Gas may disturb the ion-producing spray. Too low Exhaust pump does not work effectively and moisture condenses in the spray chamber. NOTE: If you do not hear flow when you turn the compressed air knob, the fitting may not be connected properly. Push the fitting on to the connector and pull away from the connector to seat. 2.6 Connecting the Solvent Inlet Tubing Fitting and tubing requirements Connecting Use plastic or stainless steel fittings to connect solvent inlet tubing to the TurboIonSpray spray chamber. Use the smallest diameter tubing possible between the sample injection device and the spray chamber to minimize dead volume. To connect tubing: 1. Slip a compression fitting over the end of the tubing (Figure 2-7). 2. Slip a ferrule over the end of the tubing, with the tapered end of the ferrule pointing away from the compression fitting (Figure 2-7). Mariner Biospectrometry Workstation TIS Module Supplement TIS-19

Section 2 Installing the Figure 2-7 Connecting Plastic Fittings to Tubing 3. Bottom the tubing in the top LC inlet to the probe (Figure 2-8). Finger-tighten the compression fitting. LC inlet block Compression fitting Ferrule LC inlet to probe PB100607 IonSpray probe LC inlet connector Flow split inlet Split ratio adjuster PB100755 Figure 2-8 Connecting the Solvent Inlet Tubing TIS-20 Applied Biosystems

Installing the Flow splitting To use the built-in flow splitter: 1. Connect the solvent inlet tubing to the flow split inlet, the bottom connector on the LC inlet block (Figure 2-9). 2. Connect the PEEK tubing to the flow split outlet (Figure 2-9). NOTE: The LC inlet block provides a grounded liquid junction connection. Do not bypass the LC inlet block. 3. Connect a length of tubing to the bottom connector on the base of the LC inlet block to adjust the split ratio. For information on determining split ratio, see the Mariner Biospectrometry Workstation User s Guide. LC inlet block LC inlet to probe IonSpray probe Flow split inlet PB100774 Split ratio adjuster Flow split outlet Figure 2-9 Connecting the Solvent Inlet Tubing for Flow Splitting Mariner Biospectrometry Workstation TIS Module Supplement TIS-21

Section 2 Installing the 2.7 Positioning the IonSpray Probe Overview Position the probe so that the spray tip is: 1.2 to 2 cm away from the nozzle opening Spray is directed to the left of the nozzle opening Figure 2-10 illustrates the spray chamber without the housing, to show relative position of spray tip and nozzle opening. Nozzle opening Spray directed to left of opening PB100725 IonSpray probe Heated Turbo probe Figure 2-10 Positioning the IonSpray Probe and Spray Tip Figure 2-11 illustrates where the spray from the IonSpray probe strikes the curtain gas plate, and its relative position to the nozzle opening at different flow rates. Nozzle opening in curtain gas plate Nozzle opening in curtain gas plate Turbo probe Top view IonSpray probe 1 ml/min 200 µl/min 40 µl/min Front view Figure 2-11 Orientation of Spray and Nozzle Opening TIS-22 Applied Biosystems

Installing the Adjusting spray tip distance from nozzle opening To adjust the spray tip distance from the nozzle opening: 1. Locate the black knurled positioning knob and distance gauge on the back of the IonSpray probe (Figure 2-12). Distance gauge 15 10 Positioning knob 5 0 PB100723 Rear view of IonSpray probe Figure 2-12 Adjusting Spray Tip Distance from Nozzle Opening NOTE: The positioning knob and gauge are difficult to see when the spray chamber is installed on the system. You can adjust before installing the spray chamber, if desired. 2. Turn the positioning knob until the gauge is set between 5 and 10. These settings correspond to a 1.2 to 2 cm distance between the spray tip and the nozzle opening. Mariner Biospectrometry Workstation TIS Module Supplement TIS-23

Section 2 Installing the Adjusting lateral spray tip position To adjust the spray tip position over the nozzle opening: 1. Locate the adjustment wheel on the right side of the spray chamber and the gauge on the top right of the chamber (Figure 2-13). Right side view of spray chamber 5 0 5 Lateral position gauge Locking nut Adjustment wheel Turn wheel to the left PB100722 Figure 2-13 Adjusting Lateral Spray Tip Position 2. Loosen the locking nut (Figure 2-13). 3. Turn the adjustment wheel clockwise (to the left) until the gauge is set between 0 and the 5 to the right of (or below) the 0. A setting closer to 0 positions the IonSpray probe slightly to the left of the nozzle opening. TIS-24 Applied Biosystems

Installing the Use the following settings as a start, then optimize signal by fine-tuning the setting: For flow rate 20 to 200 µl/min 0 2 200 to 500 µl/min 2 3 500 to 1,000 µl/min 3 5 Set lateral position gauge to NOTE: Do not adjust so the gauge is set between 0 and the 5 to the left of (or above) the 0. Doing so positions the spray directly over the nozzle opening, and can decrease performance and contaminate the nozzle. 4. Tighten the locking nut (Figure 2-13) when the probe is properly positioned. Mariner Biospectrometry Workstation TIS Module Supplement TIS-25

Section 2 Installing the 2.8 Removing the TurboIonSpray Spray Chamber Removing To remove the TurboIonSpray spray chamber: 1. Read Spray chamber precautions on page 7 before proceeding. 2. Stop solvent flow and close the shutter. CAUTION Always turn off solvent flow before turning off high voltages to prevent liquid from accumulating in the spray chamber. Accumulated liquid can cause degradation of gas seals. 3. In the Instrument Control Panel, set Spray Chamber Temperature 2 to 0 C. 4. To facilitate cooling, set the Nozzle Temp to 100 C. 5. Wait until Spray Chamber Temperature 2 decreases below 50 C. NOTE: To facilitate cooling, leave the gases and compressed air flowing until temperature decreases below 50 C. 6. In the Instrument Control Panel, select Instrument-Off from the Instrument menu. CAUTION Removing the spray chamber when the instrument is in Standby or On state (and gas and internal heaters are on) can damage the gas seals and cause fuses to blow. TIS-26 Applied Biosystems

Installing the NOTE: Do not leave the instrument state set to Off for any longer than necessary. Setting the state to Off turns off instrument heaters. Internal elements of the system can take up to an hour and a half to equilibrate. High voltages, integrated syringe pump, and gases are turned off. The gas seals release when the gases are turned off. 7. Disconnect the compressed air by pulling back on the fitting and removing the tubing. See Figure 2-6 on page 18. NOTE: It is not necessary to turn off the compressed air before disconnecting. 8. In the Instrument Control Panel, check the status bar to make sure the High Voltage status is Off. 9. Disconnect the following from the spray chamber: Exhaust trap inlet tubing (Figure 2-14) Solvent inlet tubing (Figure 2-14) 1. Set Spray Chamber Temp 2 to 0 C and wait until temp decreases below 50 C. 3. Pull lever to right. 2. Disconnect exhaust trap inlet, compressed air inlet, and solvent inlet. PB100742 4. Grab the Turbo probe and pull the chamber to the right. Figure 2-14 Removing the TurboIonSpray Chamber Mariner Biospectrometry Workstation TIS Module Supplement TIS-27

Section 2 Installing the 10. Pull the spray chamber lever to the right (Figure 2-14). Reconfiguring gas for use with standard spray chamber 11. Remove the spray chamber by grasping the Turbo probe and pulling the spray chamber to the right, away from the workstation (Figure 2-14). 12. Hold the TurboIonSpray spray chamber with two hands, one under the base, one on the Turbo probe (see Figure 2-2 on page 9) and lift it out of the way. If you are removing the spray chamber and replacing it with the standard spray chamber, disable the auxiliary gas. See Section 2.4.2, Configuring the Gases. The standard spray chamber does not use auxiliary gas. If auxiliary gas is enabled and turned on when a standard spray chamber is installed, you may hear a hissing sound as gas leaks from the auxiliary gas line. TIS-28 Applied Biosystems

TurboIonSpray Operation 3 TurboIonSpray Operation This section includes: Overview of TurboIonSpray operation Sample, solvent, and LC column guidelines Optimizing signal Shutting down the TurboIonSpray chamber when not in use 3.1 Overview of TurboIonSpray Operation Basic operation After optimizing signal as described in this section, operation is identical to standard electrospray operation. Refer to the sections of the Mariner Biospectrometry Workstation User s Guide listed below to perform the following tasks: Tuning and Calibrating the Mass Analyzer Acquiring Data Refer to the Data Explorer User s Guide for information on processing data. For additional information, refer to the following sections in this document: Section 3.4, Shutting Down the TurboIonSpray Chamber When Not in Use Section 4, Maintenance Section 5, TurboIonSpray Troubleshooting Mariner Biospectrometry Workstation TIS Module Supplement TIS-29

Section 3 TurboIonSpray Operation 3.2 Sample, Solvent, and LC Column Guidelines For more information on sample, solvent, and LC guidelines, or on sample cleanup, refer to the following sections in the Mariner Biospectrometry Workstation User s Guide: Sample, Solvent, and LC Column Guidelines Sample Cleanup 3.3 Optimizing Signal Preparing the workstation For more information on the following procedure, please see the Mariner Biospectrometry Workstation User s Guide. To prepare the workstation before optimizing signal: 1. Read Spray chamber precautions on page 7 before proceeding. 2. Check the exhaust trap bottle daily and empty as needed. 3. Load an instrument settings file. 4. In the Instrument Control Panel, select Instrument-Standby from the Instrument menu. 5. Turn on the compressed air until you hear the flow, approximately 1 to 1 and 1/2 turns of the compressed air knob to generate a 15 liters/min flow (see Figure 2-6 on page 18). NOTE: If you do not hear flow when you turn the compressed air knob, the fitting may not be connected properly. Push the fitting on to the connector and pull away from the connector to seat. TIS-30 Applied Biosystems

TurboIonSpray Operation 6. If gases are in manual mode, click in the toolbar to turn on. 7. In the Instrument Control Panel, set Spray Chamber Temperature 2 to the temperature range appropriate for your flow rate: Flow Rate (µl/min) <40 0 200 40 200 200 350 200 500 350 400 500 1,000 400 450 Temperature ( C) 8. In the Instrument Control Panel, set Nozzle Temperature to 180 to 200 C and wait for the temperature to stabilize. NOTE: Temperatures closer to 200 C may provide increased sensitivity. 9. Allow the probe to warm up for 5 minutes to minimize condensation in the probe. 10. Adjust the gas flow rates as indicated in Table 3-1 using the flow meters on the front panel of the mass spectrometer and spray chamber. Mariner Biospectrometry Workstation TIS Module Supplement TIS-31

Section 3 TurboIonSpray Operation Gas Nebulizer (right flow meter on the front panel) Curtain (left flow meter on the front panel) Auxiliary (flow meter on TurboIonSpray spray chamber) Table 3-1 Gas Flow Rates Flow Rate (liter/min) Comment 0.2 to 1.0 Use lower nebulizer gas flow for lower solvent flow rates. If nebulizer gas flow is too high, liquid can be blown off the tip and a stable spray does not form. 0.5 to 2.0 Use higher curtain gas flow for samples with high water content. 3 to 8 Use lower auxiliary gas flow for lower solvent flow rates. Optimizing signal To optimize signal: 1. In the Instrument Control Panel, select Instrument-On from the Instrument menu. 2. In the Instrument Control Panel, set Spray Tip Potential between 4,500 and 5,500 V. If you are operating in negative ion mode, set the Spray Tip Potential between 3,500 and 4,500 V. 3. Open the shutter. 4. Start solvent flow at a flow rate appropriate for your application. 5. Make a loop injection of a standard. 6. Observe signal intensity in the Instrument Control Panel. 7. Increase the compressed gas flow until the signal drops off, then decrease the flow rate slightly. TIS-32 Applied Biosystems

TurboIonSpray Operation Condition to Adjust Lateral probe position as described on page 24 Spray Chamber Temperature 2 8. To optimize signal, adjust the following conditions in the order listed below: Comments This condition has the greatest impact on signal, and is usually all you need to adjust to optimize signal. Increase in 25 C increments until you no longer see an increase in signal response. Note the effects of setting too high or low: Too high Causes thermal decomposition of sample. Too low Evaporation of droplets does not occur quickly enough to allow desorption of organic molecules. Auxiliary, nebulizer, and curtain gases as described on page 17 Spray tip distance from nozzle opening as described on page 23 Increase the flow rate until the signal drops off, then decrease the flow rate slightly. Move closer to the 5 setting for lower flow rates, and closer to the 10 setting for higher flow rates. Example TIC data Figure 3-1 represents typical TIC data for TurboIonSpray applications. Figure 3-1 Example TurboIonSpray TIC Data Mariner Biospectrometry Workstation TIS Module Supplement TIS-33

Section 3 TurboIonSpray Operation 3.4 Shutting Down the TurboIonSpray Chamber When Not in Use When not acquiring data: 1. Close the shutter. 2. Stop solvent flow. 3. In the Instrument Control Panel, set Spray Chamber Temperature 2 to 0 C. 4. Leave the compressed gas flowing for at least 15 minutes after stopping solvent flow to vent all solvent vapors, particularly if you have used highly combustible solvent mixtures. TIS-34 Applied Biosystems

Maintenance 4 Maintenance This section includes: Replacing the spray tip and union Replacing PEEK tubing Replacing the lamp bulb 4.1 Replacing the Spray Tip and Union When to replace Removing the spray chamber from the base This section describes: When to replace Removing the spray chamber from the base Replacing the spray tip and union Aligning spray tip and Turbo probe Reassembling Replace the spray tip if performance decreases. Replace the union if you observe contaminant peaks you cannot eliminate. To remove the spray chamber from the base: 1. Remove the spray chamber as described in Section 2.8, Removing the TurboIonSpray Spray Chamber. 2. Disconnect the nebulizer gas from the Turbo probe and the auxiliary gas from the IonSpray probe. 3. Remove the 9/64-inch hex screw holding the spray chamber to the base (Figure 4-1). Mariner Biospectrometry Workstation TIS Module Supplement TIS-35

Section 4 Maintenance 9/64-inch hex screw Phillips screw PB100743 Phillips screw Figure 4-1 Removing the Spray Chamber from the Base 4. Remove the two Phillips screws from the left side of the spray chamber that hold the spray chamber to the base. 5. Carefully remove the chamber from the base without pulling on the high voltage wire that is connected between the base and the chamber. Carefully place the spray chamber next to the base. Replacing the spray tip and union To replace the spray tip: 1. Grasp the white Teflon end of the IonSpray probe with a lint-free tissue and unscrew and remove it (Figure 4-2). TIS-36 Applied Biosystems

Maintenance White Teflon sleeve Figure 4-2 Disassembling the Ion Spray Tip 2. Remove the nebulizer sheath from the spray tip union (Figure 4-3) with two wrenches: Hold the 1/4-inch stainless steel union with a 1/4-inch wrench PB100718 Unscrew and remove the nebulizer sheath with another 1/4-inch wrench Union Spray tip Nebulizer sheath PB100719 PB100720 Figure 4-3 Removing the Nebulizer Sheath From the Spray Tip Union Mariner Biospectrometry Workstation TIS Module Supplement TIS-37

Section 4 Maintenance 3. If the nebulizer sheath is discolored, sonicate it in methanol for 10 minutes. WARNING Before handling methanol, consult the MSDS provided by the manufacturer. CAUTION Do not sonicate in acid. Acid can degrade the solder on the nebulizer sheath. 4. Remove the spray tip from the union by grasping the PEEK sleeve at the base of the spray tip with forceps and pulling out (Figure 4-4). Spray tip PB100721 Figure 4-4 Removing the Spray Tip From the Union 5. With forceps, grasp a new tip by the PEEK sleeve and insert it into the existing union, or a new union if you are replacing. 6. Screw the nebulizer sheath on until finger tight. TIS-38 Applied Biosystems

Maintenance 7. Tighten the nebulizer sheath 1/4-turn with two wrenches: Aligning spray tip and Turbo probe Hold the 1/4-inch stainless steel union with a 1/4-inch wrench Tighten the nebulizer sheath with another 1/4-inch wrench 8. Replace the Teflon sheath and tighten until finger tight. To align: 1. Hold the spray chamber with the opening up, so you can see the alignment of the IonSpray probe in relation to the Turbo probe (Figure 4-5). Turbo probe Spray tip Figure 4-5 Adjusting Spray Tip Position over Turbo Probe 2. Grasp the high voltage wire nut with a wrench, and adjust the position of the spray tip so that it points directly at the opening in the Turbo probe. PB100728 Reassembling NOTE: If the spray tip is not aligned properly over the turbo probe, you may see decreased sensitivity. Reassemble the base and the spray chamber. Reconnect the nebulizer gas to the Turbo probe and the auxiliary gas to the IonSpray probe. Replace the spray chamber as described in Section 2.2, Installing the TurboIonSpray Spray Chamber. Mariner Biospectrometry Workstation TIS Module Supplement TIS-39

Section 4 Maintenance 4.2 Replacing PEEK Tubing When to replace Removing the PEEK tubing Replace the PEEK tubing if: You observe contaminant peaks you cannot eliminate Tubing is blocked To remove the PEEK tubing: 1. Remove the spray chamber as described in Section 2.8, Removing the TurboIonSpray Spray Chamber. 2. Remove the spray chamber from the base as described in Removing the spray chamber from the base on page 35. 3. Grasp the white Teflon end of the IonSpray probe with a lint-free tissue and unscrew and remove it (see Figure 4-2 on page 37). 4. Remove the spray tip union (Figure 4-6) with two wrenches: Hold the high voltage wire nut with one wrench Unscrew and remove the 1/4-inch stainless steel union with another wrench TIS-40 Applied Biosystems

Maintenance Hold high voltage wire nut with wrench Unscrew and remove union with wrench Figure 4-6 Removing the Spray Tip Union PB100757 5. Disconnect the PEEK tubing from the LC inlet block (Figure 4-7). 6. Cut off the end of the tubing at the ferrule. LC inlet PEEK compression fitting at LC inlet to probe LC inlet block PB100755 Figure 4-7 Removing PEEK Tubing Mariner Biospectrometry Workstation TIS Module Supplement TIS-41

Section 4 Maintenance Installing new PEEK tubing 7. Loosen the PEEK compression fitting at the LC inlet to the probe (Figure 4-7). Do not remove the fitting. 8. Pull the other end of the PEEK tubing (at the tip-end of the probe) out of the assembly and discard. To install new PEEK tubing: 1. Insert the a 20-inch length of 0.005-inch ID red PEEK tubing into the spray tip end of the probe until it extends out of the other end of the probe. NOTE: To reduce dead volume at flow rates below 200 µl/min, you can use smaller diameter PEEK tubing. However, smaller diameter tubing may plug more easily. 2. Slip a compression fitting over the LC inlet end of the PEEK tubing, with the threads of the fitting toward the end of the tubing. 3. Slip a tapered ferrule on the tubing, with the taper toward the end of the tubing. 4. Insert the tubing, ferrule, and fitting into the LC inlet (see Figure 4-7 on page 41) and bottom the tubing. Tighten the fitting with a wrench to secure the tubing in place. Do not overtighten. NOTE: The LC inlet block provides a grounded liquid junction connection. Do not bypass the LC inlet block. 5. Slip a tapered ferrule over the other end of the tubing (tubing extending from tip end of probe), with the taper toward the end of the tubing. 6. Hold the union over the tip end of the tubing. Push on the PEEK tubing at the LC inlet to bottom the tubing and ferrule in the union. 7. Tighten the union with a wrench to secure the tubing in place. Do not overtighten. TIS-42 Applied Biosystems

Maintenance Reassembling 8. When the union is tightened, gently pull back on the PEEK tubing at the LC inlet, then finger-tighten the PEEK compression fitting at the LC inlet to the probe. 9. Check the alignment of the IonSpray probe. Adjust if needed as described in Aligning spray tip and Turbo probe on page 39. Reassemble the base and the spray chamber. Reconnect the nebulizer gas to the Turbo probe and the auxiliary gas to the IonSpray probe. Replace the spray chamber as described in Section 2.2, Installing the TurboIonSpray Spray Chamber. 4.3 Replacing the Lamp Bulb Replacing the bulb To replace the lamp bulb: 1. Remove the spray chamber from the base as described in Removing the spray chamber from the base on page 35. 2. Snap the bulb out of the holder and replace with a new bulb. 3. Reassemble the base and the spray chamber. 4. Replace the spray chamber as described in Section 2.2, Installing the TurboIonSpray Spray Chamber. Mariner Biospectrometry Workstation TIS Module Supplement TIS-43

Section 4 Maintenance TIS-44 Applied Biosystems

TurboIonSpray Troubleshooting 5 TurboIonSpray Troubleshooting This section includes troubleshooting information for the TurboIonspray module. For additional troubleshooting information, refer to the Technical Support section of the Mariner Biospectrometry Workstation User s Guide. Troubleshooting information is organized according to likelihood of possible cause, from most likely to least likely possible cause. If you are unable to solve your problem using the information in the following tables, call Applied Biosystems Technical Support. To reach Applied Biosystems Technical Support, refer to the Technical Support section of the Mariner Biospectrometry Workstation User s Guide. Table 5-2 TurboIonSpray Electrospray Troubleshooting Symptom Possible Cause Action Sample tailing, band broadening, poor chromatographic resolution with LC/MS Auxiliary gas flow rate not optimized between 3 to 8liter/min Adjust Auxiliary gas. See Section 2.4.3, Adjusting the Gases. Background ions Exhaust backed up Empty exhaust bottle, uncrimp exhaust tubing. Spray tip contaminated Clean. See Section 4.1, Replacing the Spray Tip and Union. Low sensitivity Spray tip not properly aligned with Turbo probe Align. See Aligning spray tip and Turbo probe on page 39. Mariner Biospectrometry Workstation TIS Module Supplement TIS-45

Section 5 TurboIonSpray Troubleshooting Table 5-2 TurboIonSpray Electrospray Troubleshooting (Continued) Symptom Possible Cause Action You do not hear flow when you turn the compressed air knob Unstable background signal No peaks observed above 300 m/z. Corona discharge observed at spray tip. Quick-disconnect fitting not connected properly Gases not optimized Spray Tip Potential too high Push the quick-disconnect fitting on to the connector and pull away from the connector to seat. Optimize nebulizer and auxiliary gas flow rates. See Preparing the workstation on page 30. Decrease Spray Tip Potential. No peaks observed above 300 m/z. No corona discharge observed at spray tip. Phthalate peaks at many masses Solvent, sample, or tubing contaminated Leaching from LC tubing Use fresh solvent, clean up sample, change tubing. No action. Normal occurrence. TIS-46 Applied Biosystems

TurboIonSpray Troubleshooting A Spare Parts Table A-1 Mariner TurboIonSpray Consumables Kit Item Where used Quantity Part Number Mariner TurboIonSpray Consumables Kit, includes: 1 kit V700736 Ferrule Spray tip, LC inlet 1 Fitting/ferrule, 1 piece, PEEK Spray tip 1 Sheath, nebulizer (electrode) Spray tip 1 Spray tip Spray tip 1 Tubing, PEEK, red, 0.005-inch ID LC inlet block to spray tip and tubing to LC inlet block 5 feet Union Spray tip 1 Table A-1 Mariner TIS Spare Parts Item Where used Quantity Part Number Exhaust bottle kit, includes 1/2-inch OD and 1/4-inch OD plastic tubing, adapter, exhaust bottle and cap Exhaust outlet 1 kit V700710 Bulbs, lamp APCI spray chamber 10/pkg V700672 Mariner Biospectrometry Workstation TIS Module Supplement TIS-47

Section 5 TurboIonSpray Troubleshooting TIS-48 Applied Biosystems

Index A Air, compressed, see Compressed air Applied Biosystems Technical Support TIS-45 Applied Biosystems TurboIonSpray Source TIS-2 Auxiliary gas adjusting TIS-17 auto and manual mode TIS-16 configuring TIS-16 connecting TIS-13 disabling before installing standard spray chamber TIS-28 flow rate TIS-32 mode, selecting TIS-16 parameters TIS-16 pressure TIS-17 removed from system TIS-13 requirements TIS-13 turning on and off in automatic mode TIS-17 turning on and off in manual mode TIS-17 B Background peaks, troubleshooting C TIS-46 Cautions turning off gases with front panel controls TIS-17 turning off solvent flow before high voltages TIS-26 Column, LC, guidelines TIS-30 Compressed air adjusting TIS-19 connecting TIS-18 keeping on for 15 minutes after solvent flow TIS-34 no flow TIS-19 pressure TIS-18 requirements TIS-5 shutting off TIS-34 Curtain gas adjusting TIS-17 auto and manual mode TIS-16 configuring TIS-16 connecting TIS-13 flow rate TIS-32 mode, selecting TIS-16 parameters TIS-16 pressure TIS-17 requirements TIS-13 turning on and off in automatic mode TIS-17 turning on and off in manual mode TIS-17 Curtain gas plate distance from IonSpray probe spray tip TIS-22 location TIS-7 E Exhaust gas, see Compressed air Exhaust pump TIS-18 Exhaust trap bottle emptying daily TIS-30 installing TIS-11 Exhaust trap line, installing TIS-11 I D E X N Mariner Biospectrometry Workstation TIS Module Supplement Index-1

I N D E X F Ferrules, integrated injection valve or syringe pump TIS-19 Fittings, connecting plastic TIS-19 Flow rate, solvent and probe position TIS-25 and temperature TIS-31 range TIS-1, TIS-3 Flow splitter connecting TIS-21 split ratio TIS-21 Fume hood requirements TIS-5 G Gas see also Auxiliary, Curtain, Nebulizer gas reconfiguring for use with standard spray chamber TIS-28 seal, degradation TIS-26 H Heater, IonSpray probe TIS-31, TIS- 33 Help, see Applied Biosystems Technical Support I Installing compressed air TIS-18 exhaust trap line and bottle TIS-11 gases TIS-13 materials needed TIS-5 solvent inlet tubing TIS-19 TurboIonSpray spray module TIS-8 Instrument settings file TIS-30 Interlocks description TIS-6 resetting TIS-6 IonSpray probe aligning with Turbo probe TIS-39 cleaning tip TIS-35 distance from curtain gas plate TIS-22 distance from nozzle opening, adjusting TIS-22 heater temperature, final TIS-33 heater temperature, initial TIS-31 lateral position and flow rate TIS-25 lateral position, adjusting TIS-24 warm up period TIS-31 L Lamp bulb, replacing TIS-43 LC column guidelines TIS-30 LC inlet block, grounded liquid junction TIS-21 M Maintenance lamp bulb, replacing TIS-43 removing base from spray chamber TIS-35 solvent inlet PEEK tubing, replacing TIS-40 spray tip and union, replacing TIS-35 spray tip, aligning with Turbo probe TIS-39 Mariner Workstation, TurboIonSpray module TIS-1 Index-2 Applied Biosystems

N Nebulizer gas adjusting TIS-17 auto and manual mode TIS-16 configuring TIS-16 connecting TIS-13 flow rate TIS-32 mode, selecting TIS-16 parameters TIS-16 pressure TIS-17 requirements TIS-13 turning on and off in automatic mode TIS-17 turning on and off in manual mode TIS-17 Nozzle Temperature increasing sensitivity TIS-31 maximum, TurboIonSpray TIS-32 O Optimizing signal P TIS-30 PEEK tubing, solvent inlet, replacing TIS-40 Plastic fittings TIS-19 Pressure auxiliary gas TIS-17 compressed air TIS-18 curtain gas TIS-17 nebulizer gas TIS-17 Probe, IonSpray aligning with Turbo probe TIS-39 cleaning tip TIS-35 distance from curtain gas plate TIS-22 distance from nozzle opening, adjusting TIS-22 heater temperature, final TIS-33 heater temperature, initial TIS-31 lateral position and flow rate TIS-25 lateral position, adjusting TIS-24 warm up period TIS-31 Probe, Turbo, description TIS-1 Probe, TurboIonSpray, see Probe, IonSpray R Requirements compressed air TIS-5 fume hood TIS-5 gas TIS-13 S Safety warnings TIS-38 Sample guidelines TIS-30 preparing TIS-29, TIS-30 Shutting down module when not in use TIS-34 Signal, optimizing TIS-30 Solvent flow rate TIS-1, TIS-3 flow rate, and probe position TIS-25 flow rate, and temperature TIS-31 guidelines TIS-30 Solvent inlet tubing connecting TIS-19 flow splitter, connecting TIS-21 replacing TIS-40 Spare parts TIS-47 Split ratio, flow splitter TIS-21 Spray Chamber Temperature 2 setting final TIS-33 I D E X N Mariner Biospectrometry Workstation TIS Module Supplement Index-3

I N D E X setting initial TIS-31 Spray chamber, TurboIonSpray see also TurboIonSpray module installing TIS-8 overview TIS-6 precautions TIS-7 removing TIS-26 removing base TIS-35 warm up period TIS-31 Spray Tip Potential, maximum, TurboIonSpray TIS-32 Spray tip, IonSpray probe aligning with Turbo probe TIS-39 distance from curtain gas plate TIS-22 replacing TIS-35 System settings, Gas Flow TIS-16 T Technical support, contacting TIS-45 Temperature IonSpray probe, final TIS-33 IonSpray probe, initial TIS-31 TIC, Instrument Control Panel, TurboIonSpray example TIS-33 Troubleshooting background peaks TIS-46 compressed air, no flow TIS-46 TurboIonSpray TIS-45 Tubing, solvent inlet, PEEK, replacing TIS-40 TurboIonSpray module see also TurboIonSpray probe features TIS-3 flow rate, solvent TIS-1, TIS-3 grounded liquid junction TIS-21 heated TurboProbe TIS-1 installing TIS-5, TIS-8 maintenance TIS-35 materials needed TIS-5 operation TIS-29 overview TIS-1, TIS-3, TIS-6 parts of TIS-2 precautions TIS-7 removing TIS-26 removing base TIS-35 removing, caution TIS-26 shutting down TIS-34 TIC example TIS-33 troubleshooting TIS-45 warm up TIS-31 TurboIonSpray probe, see Probe, IonSpray TurboIonSpray Source, Applied Biosystems TIS-2 TurboProbe, see Probe, Turbo U Union, replacing W TIS-35 Warm up period TIS-31 Warnings, safety chemical handling TIS-38 spray chamber may be hot TIS-7 spray chamber, TurboIonSpray TIS-7 Waste bottle, see Exhaust bottle Index-4 Applied Biosystems