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PicoView 550 User Manual

1. ELT ed Figure 2 1 Continuous infusion nanospray Figure 2 2 Nanospray flow injection 33Sf1 A A3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL ID 2 APPLICATIONS ab Figure 2 3 Microspray flow injection mode allows for smaller sample sizes than continuous infusion This type of ex periment is particularly useful for the rapid analysis of desalted samples Voltage application for nanospray flow injection is best accomplished using the coated tip module CTM in the forward position with conductively coated PicoTips SilicaTips in particular are recommended with either a distal coating those with D in their item number or standard coating those with CE in their item number Refer to Section 5 Coated Tip Module in the Forward Position for detailed instructions on setting up the CTM for flow injection Microspray Flow Injection Microspray flow injection as with nanospray flow injection entails injecting small volumes of sample into an established solvent as diagrammed in Figure 2 3 The difference between the two is the flow rate Nanospray flow rates are typically below 300 nL min whereas the higher microspray flow rates are typi cally between 0 5 5 L min Sample is introduced as a plug into the solvent stream from an HPLC pump or syringe at flow rates of 400 5000 nL min Micro Injection Valve can be used for sample injection Microspray flow in
2. Connectors Upchurch P N Upchurch P N Connector Used in System Ferrule Nut P 720 CTM union MicroTight Combined F 125 UTM microtee MicroTight F 172 416 UTM 20 SealTight 92 F194 P 720 Tip holder union MicroTight Combined F 125 Grounding union SealTight F 192 F 194 Injection port adapter MicroTight F 152 416 Sleeves are used within unions to ensure a reliable connection As with the ferrules and nuts it is very important to use the appropriate sleeve for the connection system The table on page 5 3 lists the different sleeves supplied with PicoView and their applications Always use MicroTighf sleeves with MicroTighf unions and SealTight sleeves with 1 16 diameter fittings Sleeves Sleeve Upchurch Sleeve OD Sleeve System Color PartNo inches 10 pm Connector MicroTight Blue F 183x 0 025 280 CTM UTM Tip holder MicroTight Green F 185x 0 025 395 CTM UTM Tip holder SealTight Blue F 240 1 16 280 ZDV Grounding union SealTight Green F 242 1 16 395 ZDV Grounding union The sleeve ID should be approximately equal to the tubing OD Other sleeve sizes are available from Upchurch Scientific 380 ID sleeves are compatible with New Objective SilicaTips vf Always select proper ferrules nuts and sleeves for the union Mismatching of union components will result in spaces with dead volume or a leak
3. differs substantially from conventional ESI that signal levels in the mass spectrometer are more sensitive to solvent composi tion flow stability and applied voltage It may take a little time to learn how to obtain consistent results at low flow rates A little patience and practice early on can pay off greatly in terms of success An effective way to begin working at low flow rates is by starting with larger 15 30 ID tips and using flow rates at the higher end of the recommended range of operation for a given tip style Once you are comfortable working at a certain level of performance you can more easily switch to emitters with smaller diam eters and operate at lower flow rates PicoTip refers to any of New Objective s high quality tips for electrospray ionization such as SilicaTips PicoFrits and TaperTips TaperTips are used for higher flow rate applications and may be a good starting point when learning to work with nanospray Once basic perfor mance levels are established you can switch to SilicaTips with smaller tip sizes For highly aqueous mobile phases however tip sizes of 10 15 and smaller are recommended Choosing a Tip Size and Coating Style New Objective SilicaTips are available in sizes from 2 uim ID to 30 ID in three different coating styles standard CE distal D and uncoated Coated PicoTips have a special enhanced conductive multilayer coating U S Paten
4. NEW OBJECTIVE S PICOVIEW NANOSPRAY SOURCES PICOVIEW NANOSPRAY SOURCE USER MANUAL MODEL PV 550 VERSION 2 NEw OBJECTIVE 2 Constitution Way Woburn Ma 01801 1023 USA 800 220 2998 US toll free 781 933 9560 phone 781 933 9564 fax www newobjective com amp PICOVIEW NANOSPRAY SOURCES PICOVIEW NANOSPRAY SOURCE USER MANUAL MODEL PV 550 VERSION 2 0 XD PV550 2009 New Objective Inc All rights reserved No portion of this document may be repurposed or reprinted without the prior written consent of New Objective Inc Table of Contents Read This First 1 1 Safety Considerations 1 1 Prior to Installation Handling Fused Silica Tubing 1 1 1 1 Tip Adjustment and or Replacement 1 2 The Transfer Line 1 2 Contents amp Components List 1 3 Trademarks 1 4 Limited Warranty 1 4 2 Applications 2 1 Continuous Infusion Nanospray 2 1 Nanospray Flow Injection 2 1 Microspray Flow Injection 2 2 Nanobore LC MS Using a PicoFrit Column 2 2 Nanobore LC MS Using a PicoTip 2 3 with a Separate Column Capillary Bore LC 2 3 Supplying Voltage to the 2 4 Uncoated Tip Module UTM TABLE OF CONTENTS TVANVW 33Sf1 4 OSS Ad PV 550 PICOVIEW USER MANUAL TABLE OF CONTENTS 9 Quick Guide 3 1 PicoView Components 3 1 Tip Modules 3 2 Digitally Controlled XYZ Positioning Stage 3 3 Imaging System 3 3 Injection
5. ing PicoView can be used to generate consistent high quality data for a variety of chemical and biochemical applications 6 TECHNIQUES TO OPTIMIZE PICOVIEW TVANVW 33Sf1 A 3IAODId OSS Ad i PV 550 PICOVIEW USER MANUAL 6 TECHNIQUES OPTIMIZE PICOVIEW 10 7 Mounting an Injection Valve PicoView s optional universal valve mount has been designed to accommodate a variety of valve sizes 6 or 10 port and from different vendors A brief set of instructions are listed below showing the mounting of two popular valve styles the Upchurch manual micro injection valve and the actuated Valco valve Contact New Objective for more information on adding the universal valve mount to you PicoView Mounting an Upchurch 6 Port Manual Valve An adaptor backing plate is used to mount the manual Upchurch valve or similer manual injection valve to PicoView It can be found in the acces sories kit To fasten the plate first place the valve with the port side on the table and the handle pointing away from you Attach the plate as shown in Figure 7 1 using the two 8 32 cap screws with an allen wrench 2 Attach the backing plate with the Upchurch valve to the universal valve mount refer to Figure 3 1 using the two 6 32 cap screws as shown in Figure 7 2 and 7 3 Make sure the valve ports face the opening in the housing 7 MOUNTING INJECTION VALVES Figure 7 1 Upchurch valves require the included bac
6. and B illustrate user side and instrument side views of PicoView showing the general layout of the components The translation stage and stage plate comprise the PicoView hardware used for mounting and aligning tips with respect to the inlet on the mass spectrometer CCD Camera Optical Mount Flange Locking Lever Pan Tilt Focus Interface Flange Fiber Optic Coupler HV Cable Translation Stage Clip X Adjustment Stage Plate eee Universal Valve Mount optional Grounding Clip Component Clamps optional with mount Figure 3 1A User side view of PicoView TVANVW 33Sf1 A A3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 5 7 3 PICOVIEW COMPONENTS The optical stage digital camera and fiber optic illuminator make up the PicoView imaging hardware necessary for optimizing spray conditions Complete descriptions of these components can be found later in this chapter CCD Camera Optic Tube Slip Ring Camera Mount Transparent Cover 1 A Flange Locking d a e Levers 5 5 2 Objective Lens Ne Te E Stage Plate N ON N N 3 Pd N 55 N oupler Illumination a _ Adjustment 7 oe nterface Flange Fiber Optic Bundle Figure 3 1B Instrument side view of PicoView Tip Modules The two tip modules included with the PicoView system afford the user the versat
7. droplet at the tip If it is increase the voltage in 100 volt incre ments until ESI current is observed in the Tune Plus window If solvent is not flowing try increasing the flow rate setting until a solvent droplet comes into view If a solvent droplet does not appear a clog may have formed If neces sary change tips If signal is unstable increase the voltage in 50 volt increments and look for improvements in signal stability If the ion current does not stabilize optimal field strength has been exceed ed Lower the voltage 200 volts below the point at which current was first observed then raise the voltage in 50 volt increments The flow rate should be adjusted if stable ESI current cannot be achieved If no signal is observed turn off the applied voltage and look for droplet for mation at the tip to visually verify solvent flow through If a clog has formed change the tip Do not be concerned if the applied voltage required to generate an electrospray is greater than that normally required for nano electrospray methods e g nano ESI may only require 600 volts for proper operation while PicoTip operation using a 15 um ID may require 2 kV or more The applied voltage should still be well below that required for conventional ESI Voltages above 4 kV are gener ally considered excessive with most PicoTips A corona discharge may occur if the applied voltage is too high even when the distance between the tip and the
8. jection is particularly useful for the rapid analysis of desalted samples Using a TaperTip a type of PicoTip voltage is applied through a liquid junction style contact through either the uncoated tip module UTM in the back position or through the conductive coating distal coating style when using the coated tip module CTM in the forward position For the most robust setup it is recom mended to use uncoated TaperTips and the UTM in the back position Suggested tip sizes for microspray flow injection are 20 to 100 TaperTips Refer to Section 5 Uncoated Tip Module in the Back Position or Coated Tip Module in the Forward Position for detailed instructions on setting up the UTM or the CTM for microspray flow injection Consult Technical Note TT 1 available on our web site www newobjective com to learn more about the use of TaperTips with microspray Nanobore LC MS Using a PicoFrit Column PicoView is ideal for performing nanoscale LC MS and LC MS MS using PicoFrit columns PicoFrit columns a type of PicoTip are nanobore LC MS chromatography columns with an integral tip used at flow rates of 100 500 nL min In this configuration the high voltage is applied to the back of the col umn through a liquid junction in the uncoated tip module UTM placed in the back position as diagrammed in Figure 2 4 Solvent is delivered via an HPLC pump with a flow splitter Suggested tip size is 15 with an uncoated PicoFrit colum
9. majority of operating conditions a distance of 1 2 mm appears to be an optimal compromise between sensitivity and ease of tuning Applied Voltage Applied voltage is perhaps the most important parameter for stable efficient op eration Never use a turn on voltage above 500 volts unless stable ESI has been previously established Direct application of a high voltage e g 2 0 kV can cause a corona discharge or an arc between the tip and inlet This can destroy the conductive coating disrupt the fine structure of the tip and cause the for mation of air bubbles within the tip Once stable operation has been achieved however the voltage can be turned on and off at the same level with no fear of arcing provided no other operating parameters have changed Solvent Flow Rate The optimal flow rate of solvent delivered by PicoView depends on the tubing inner diameter the tip size and the applied voltage A flow rate too low or too high may result in an unstable spray pattern Using a flow rate that is too low leads to large fluctuations in the ion current lon current will likely drop to zero periodically even frequently To improve the spray quality raise the flow rate by 25 percent and look for an improvement in signal and spray stability Using a flow rate that is too high is generally not as problematic ion current will fluctuate but will probably not drop to zero General chemical noise may increase and the quality of the mass s
10. on the PicoView universal valve mount 4 Installing PicoView Preparing the Mass Spectrometer Before installing PicoView the mass spectrometer must be put into standby mode and the standard lon Max source must be removed Placing the Mass Spectrometer into Standby Mode Use the following procedure to put the mass spectrometer into standby mode refer to the LTQ operator manual provided by Thermo Finnigan for additional details 1 2 Wait until data acquisition if any is complete Turn off the flow of sample solution if any from the LC or syringe pump to the lon Max source From the Tune Plus window choose Control from the menu bar then either choose Standby or click the On Standby button to put the MS detector in standby The sheath gas flow will automatically be set to 20 units and the system LED on the front panel of the MS detector will be illuminated orange Disconnecting the High Voltage Cable Gas and Sheath Liquid Lines from the lon Max 1 Disconnect the high voltage cable from the connector labeled on the ESI probe To disconnect the cable turn the locking ring on the cable coun terclockwise and carefully remove Disconnect the sample transfer line from the grounded transfer line fitting on the ESI flange Disconnect the sheath gas line and fitting from the inlet labeled Sheath Gas on the ESI probe by turning the blue fitting counterclockwise until the line and the f
11. point to break the tip Be careful not to touch the tip to any surface the mounting slot on the UTM base with a corresponding tapped hole on the stage plate Refer to Figure 5 31 for positioning 4 Insert the mounting screw through the mounting slot on the UTM base and thread it into a threaded hole on the stage plate and tighten the screw Connecting the High Voltage Thread the high voltage cable from the mass spectrometer through the opening in the left side of the housing Connect the cable by pushing it into the cable jack and turning the locking ring clockwise until it is tight Adjusting the Tip Position By visual inspection adjust the stage plate position using either the manual knob controls see Figure 3 1A or through the PicoView Acquire Stage Control panel until the tip is approximately 2 5 mm from the mass spectrometer inlet Focus the camera using the focus knob on the optical stage see Figure 3 1A Refer to Section 6 for information on optimizing the spray 5 SETUP TVONYW 4350 A 3IAODId OSS Ad 5 SETUP TVONYW 33Sf1 A3IAOOId OSS Ad 6 Techniques to Optimize PicoView This section provides techniques to assure successful use of PicoView along with hints on ways to optimize the spray to consistently generate high quality data Also discussed are the interdependent parameters involved in creating stable electrospray Selecting a PicoTip Low flow rate ESI under 1
12. 00000 PicoView CCD Camera Figure 4 13 Video out from CCD camera to video in connection on monitor Video out from monitor goes to Live Video Capture module back of the monitor 2 Use a BNC RCA adapter to connect the BNC cable to the Hauppauge USB Live Video Capture module USB Live can be directly connected to your computer s USB port Refer to Figure 4 13 for cabling diagram 3 Refer to Happauge instructions for installing the imaging software Turning on the Power to the Mass Spectrometer From the Tune Plus window choose Control from the menu bar then either choose Standby or click the On Standby button to turn on the MS detector The sheath gas flow will automatically be set to 20 units and the system LED on the front panel of the MS detector will be illuminated green Turning Off the Sheath Gas The LTQ source recognition feature will automatically disengage the sheath gas when it recognizes that a nanospray source has been attached Adjusting the Camera Position Optimized operation of PicoView involves adjustments of both the camera and the tip positions Upon installation the imaging system should only require fine adjustment However some course adjustment may be necessary if the lens is changed Fine Adjustment Using your fingertips to move the fine adjustment knobs you can adjust the camera position to optimally image the tip and spray pattern during operation If a tip is not currently lo
13. CHNICAL SUPPORT Contact New Objective For Technical Support questions for your PicoView or regarding columns fit tings valves etc please contact the New Objective Customer Solutions Group Hours Email Telephone Fax Postal Mail Monday Friday 9 a m 5 p m ET TechSupport NewObjective com 1 781 933 9560 1 888 220 2998 U S Toll Free 1 781 933 9564 New Objective Inc 2 Constitution Way Woburn MA 01801 1023 USA NEW OBJECTIVE 2 Constitution Way Woburn MA 01801 1023 888 220 2998 Sales 781 933 9560 Tel 781 933 9564 Fax www newobjective com XD PV550 Rev 2
14. Valves 3 3 4 Installing PicoView 4 1 Preparing the Mass Spectrometer 4 1 Placing the Mass Spectrometer 4 1 into Standby Mode Disconnecting the High Voltage 4 1 Cable Gas and Sheath Liquid Lines Removing the lon Max Source 4 2 Mounting PicoView on the 4 2 Mass Spectrometer Installing the Interface Flange 4 2 Attaching PicoView 4 3 to the Interface flange Installing the Imaging System 4 4 Mounting the Fiber Optic 4 4 Illuminator Grounding Requirements 4 7 3 Setup 5 1 Cutting Fused Silica 5 1 Connectors amp Fittings 5 1 Using the Coated Tip Module CTM 5 3 CTM in the Forward Position 5 3 Loading the MicroTight Union 5 4 Attaching the CTM to the 5 5 Magnetic Stage Plate Connecting High Voltage 5 6 Adjusting Tip Position 5 6 Using the Uncoated Tip Module UTM 5 6 Using PicoFrit Columns 5 7 Removing a PicoFrit Column 5 7 from the FEP Sleeve Attaching the UTM to the 5 8 Stage Plate in the Back Position Loading the MicroTight 5 8 Union and Tip Holder Supplying High Voltage 5 9 Plumbing the MicroTee 5 9 Loading the MicroTee into 5 11 the UTM Connecting High Voltage 5 11 Adjusting Tip Position 5 11 Uncoated Tip Module 5 12 in the Forward Position TABLE OF CONTENTS TVANVW 33Sf1 4 OSS Ad TABLE OF CONTENTS PV 550 PICOVIEW USER MANUAL z Techniques to Optimize 6 1 PicoView Selecting the Right Type of Emitter 6 1 Ch
15. aded in the PicoView source you may use the inlet of the mass spectrometer to adjust the imagainf system The locations of the fine tuning knobs are indicated in Figure 3 1A Move the Tilt knob up and down until the inlet is visible At this point the inlet image will likely be very fuzzy and require focusing Using the Tilt knob center the inlet on the monitor or as viewed using the optional eyepiece 2 Adjust the Focus knob until the inlet comes into focus 3 Move the Pan knob left or right to center the inlet Coarse Adjustment of the Camera Body NOTE Only perform coarse adjustment if the fine adjustments on the opti cal mount and the stage plate will not allow adequate imaging of the tip 1 Using a 9 64 Allen wrench loosen the optical tube fastener at the top of the slip ringholding the optical tube 2 Looking at the monitor slowly slide the optical tube through the slip ring until the inlet is visible and in focus The inlet will appear on the monitor as a polished metal surface at the top of the field of view see Figure 3 5 Tighten the screw on the optical tube slip ring to hold the optical tube in place 4 Usethe fine adjustment method above to focus the image of the inlet further Grounding Requirements In systems where high voltage is applied directly to the ESI tip the liquid sample inside the tip and transfer line tubing are also raised to high voltage To prevent exposure to potentially lethal volt
16. ages a suitable ground point for the liquid inside the line must be provided It is best not to position the ground too close to the tip Two recommended methods for grounding the source are described Grounding Through a Union The ground union Figure 4 13 is a stainless steel low dead volume union During operation this union is placed in the grounding clip on the housing of the PicoView situated just under the valve mount To ground a sample 1 Locate the ground union in the PicoView accessories box 2 Remove the nut and ferrule from one side of the union by turning the fitting counterclockwise 3 Thread fused silica through a green SealTight adapter sleeve Carefully cleave the end of the tubing 4 Insert the sheathed tubing and fitting into the ground union receptacle Finger tighten the fitting by turning clockwise making sure the tubing is firm ly seated into the bottom of the fitting Figure 4 14 4 INSTALLING PICOVIEW Figure 4 14 Grounding union in place TVONYW 33Sf1 A A3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 4 INSTALLING PICOVIEW CTM Figure 4 15A Optimal location for grounding union for continuous flow microspray or nanospray S UTM Figure 4 15B Optimal location for grounding union for nanobore chromatography with a PicoFrit column Repeat steps 1 3 for the tubing leading from the installed tip module Complete descriptions o
17. arn which electrode is best for your application The UTM can operate in modes analogous to the CTM but it is most effectively utilized with microspray flow rates for nanobore chromatography The UTM can be mounted on the stage in the forward Figure 3 3A or the back Figure 3 3B positions In the back position the UTM provides an optimal interface for PicoFrit columns to afford purification concentration and separation of analytes at high sensitivity Mounted in the front position the UTM can connect 3 PICOVIEW COMPONENTS mounting screw Cover screw NI Figure 3 2 Coated tip module mounted in the forward position Figure 3 3A Uncoated tip module UTM mounted in the forward position mounting mi Screw cover Screw Figure 3 3B Uncoated tip Module UTM mounted in the back position with tip holder forward 4350 1 4 IVLIOIG OSS AdG DPV 550 DIGITAL PICOVIEW USER MANUAL 3 PICOVIEW COMPONENTS Figure 3 4 MS inlet and spray as seen through PicoView imaging system an uncoated PicoTip to a nano or capillary bore LC column Turn off or disconnect all power prior to performing any service on Pi coView or any devices attached to it to avoid potentially lethal electrical voltages O Always use chemical and pu
18. ate best at the lower end of the flow rate range and larger diameter tips require higher flow rates to establish a stable spray see Table 6 1 Recommended Flow Rate Ranges for Different Tip IDs SilicaTip Flow Rate Taperlip Flow Rate Tip ID nL min Tip ID pm nL min 5 20 200 20 200 500 8 50 300 75 300 2000 10 100 350 100 400 3000 15 150 500 30 300 1000 Approximate range of flow rates over which the tips will generate a stable electrospray plume without sheath gas assistance Actual performance may differ due to variations in experimental parameters mobile phase composition applied voltage etc Recommended Flow Rate Ranges for Different Coating Styles Tip Coating Recommended Flow Rate Range Standard CE Coated 10 nL min 250 nL min Distal D Coated 100 nL min 5 pL min Uncoated N 100 nL min 5 pL min Operable flow rate is a function of tip size In Line Filtration Loss of spray due to clogging by fine particles is the most common cause of tip failure Installing an in line filter in the solvent transfer line is highly recom mended to minimize this problem even if using HPLC grade solvents Air Bubbles Air bubbles in an emitter have three different possible origins The first is a leaky HPLC fitting A fitting even if under high pressure will leak air into the system if liquid is leaking out Small leaks even those in
19. ate position using either the manual knob controls see Figure 3 1A or through the PicoView Acquire Stage Control panel until the tip is approximately 2 5 mm from the mass spectrometer inlet Focus the camera using the focus knob on the optical stage see Figure 3 1A Instead of focusing on the inlet focus on the tip which may appear as a fuzzy line prior to fine focusing Do not let the tip contact the inlet Uncoated Tip Module in the Back Position Long PicoTips The uncoated tip module uses a microtee to supply high voltage When using PicoView with long PicoTips such as PicoFrits or uncoated TaperTips a tip holder is needed to anchor and position the tip toward the inlet This orientation is ac complished using a MicroTight union within the tip holder Using PicoFrit amp Columns PicoView is ideal for performing nanoscale LC MS and LC MS MS using PicoFrit columns In this configuration the high voltage is applied to the back of the column through a liquid junction in the uncoated tip module The PicoFrit column is packaged as a loop as shown in Figure 5 13 to keep both ends of the column at the same pressure during storage and shipping Do not remove either the orange colored PEEK sleeve or the snug fitting FEP Teflon tubing holding the ends of the loop together until the column is being prepared for installation and use There are some important considerations in the handling and use of PicoFrit col umns that make them v
20. be fastener at the top of the slip ring using a 9 64 Allen wrench Rotate the opti cal tube until the CCD camera is aligned horizontally then tighten the opti cal tube fastener to hold the CCD camera in place Adjusting Camera Gain and Gamma For optimal imaging the CCD camera s gain setting needs to be in the auto A position and the gamma control needs to be turned on The rotary gain switch has three settings auto A fixed F and manual M The gamma setting y is controlled with a dip switch and has on and off positions Both switches are located on the rear of the camera housing just below the video out connector Proper camera settings are shown in Figure 4 2 If your camera settings are cor rect no action needs to be taken 1 Using a jeweler s screwdriver or similar instrument rotate the brown gain control switch clockwise until the slot points to the A position Using the same screwdriver slide the gamma control to the right into the ON position 4 INSTALLING PICOVIEW VIDEO DG IN f OUT SYNC 2 Figure 4 2 camera gain switch set to automatic 4 TVONYW 4350 14 OSS Ad PV 550 PICOVIEW USER MANUAL 4 INSTALLING PICOVIEW 920272 Figure 4 4 LTQ Interface Flange mounted on the LTQ Interface Flange levers locked in Place Ve Figure 4 5 LTQ Interface Flange locked on the LTQ 274772 Figure 4 6 PicoView mo
21. box Fiber optic adapter ring in illuminator box Product literature Inspected by 1 READ THIS FIRST 33Sf1 A 3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 1 READ THIS FIRST Trademarks The following trademarks are found in this instruction manual Teflon is a registered trademark of E l du Pont de Nemours and Co LTQ Orbitrap Deca XP Max Deca XP Plus Max and Xcalibur are trademarks or registered trademarks of Thermo Fisher Scientific MicroTight and SealTight are trademarks or registered trademarks of Upchurch Scientific Inc a division of IDEX Corporation PEEK and PEEKsil are trademarks of Victrex plc PicoView PicoView PicoClear PicoTip PicoFrit SilicaTip TaperTip GlassTip and PicoTip Powered are trademarks or registered trademarks of New Objective Inc All other trademarks are the properties of their respective companies Limited Warranty DISCLAIMER Technical information contained in this publication is for reference purposes only and is subject to change without notice The information is believed to be reliable and accurate however nothing set forth herein constitutes a war ranty of any kind or nature Given the variety of experimental conditions New Objective Inc cannot guarantee performance at a given flow rate the best guide to tip selection and operation is empirical testing The user is solely responsible for complying with any patent s pertaining t
22. coView use an insulated wire with alligator clips on each end for grounding Clip one end to the stainless steel syringe needle Clip the other end to a ground point such as the loop on the injection valve located on the front panel of the mass spectrometer If an HPLC is being used to deliver solvent to PicoView please refer to the manu facturer s directions for grounding the pump WARNING Transfer lines connected to either the tip module or the micro injection valve must not be made from an electrically conductive material such as steel or PEEKsil These transfer lines must be fabricated from an electrically insulating material such as or fused silica Otherwise the operator may be exposed to potentially lethal voltage CAUTION Do not attempt to use the injection valve as grounding point The interior of the valve is fabricated from ceramic materials and is electri cally insulating therefore a proper ground will not result 4 INSTALLING PICOVIEW TVANVW 33Sf1 14 OSS Ad INSTALLING PICOVIEW 4 TVONYW 33Sf1 A3IAOOlId OSS Ad o 5 Setup Cutting Fused Silica Proper cleaving of the fused silica tubing is critical for achieving optimal perfor mance in nanospray applications A flat smooth cut is essential for maintaining low dead volume connections Cleaving is best accomplished with a high qual ity diamond chip scribe available from many chromatography
23. connectors is covered in detail later in this section The two different connector systems differ by size and are not interchangeable The MicroTight system is used with the microtee and MicroTight unions in the CTM and UTM tip modules and 5 SETUP Figure 5 1 A Close up views of standard diamond chip scribe 90 and B high performance diamond bladed scribe 609 Figure 5 2 A Improper cutting angle B Align cleaving tool blade perpendicular to tubing C Press down gently scoring tubing D Too much lownward pressure will crush tubing producing particles that can cause tubing to clog Figure 5 3 Typical cleave using diamond scribe TVANVW 33Sf1 14 OSS Ad PV 550 PICOVIEW USER MANUAL 5 SETUP with the tip holder The SealTight system is used to connect fused silica to any standard 1 16 port such as the grounding union Each union uses specific fer rules nuts and sleeves to seat the fused silica Do not use a MicroTight sleeve with a SealTight fitting and vice versa Table 5 1 lists the different locations of connectors within PicoView and describes which ferrules and nuts are used All of the listed components are found in the PicoView accessory box Replacement unions ferrules nuts and sleeves should be ordered directly from Upchurch Scientific www upchurch com The corresponding Upchurch part numbers are provided
24. croTight union Figure 5 10 MicroTightf union inserted the CTM base TVONYW 33Sf1 A A3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 5 SETUP Figure 5 12 CTM assembly on the stage plate vf The CTM can be mounted so that the PicoTip is pointing directly at the entrance of the inlet This alignment is less desirable because the imaging system cannot adequately image the tip at that angle There is no appar ent loss in sensitivity when spraying at a moderate angle with respect to the inlet Connecting High Voltage If the CTM is mounted so that the PicoTip is directly pointing at the entrance of the inlet thread the high voltage cable through the opening in the rear of the housing 1 Connect the cable by inserting it into the cable jack and turning the locking ring clockwise until it is tight 2 Place the magnetic stage back onto the XYZ stage as shown in Figure 5 12 Adjusting the Tip Position Carefully slide the source into operating position while observing the tip position If it looks like the tip may contact the inlet use the Y axis actuator manual knob control Figure 3 1A or the PicoView Acquire Stage Control panel to adjust the Y axis to prevent contact When certain that contact will not occur lock the bracket latches on the PicoView base securing it to the source mounting flange By visual inspection adjust the stage pl
25. d of the PicoFrit all the way through the union The PicoFrit should freely move within the union 3 When properly installed into the MicroTight union the tip end should extend 15 20 mm past the end of the fitting when it is tight This will afford optimal positioning of the tip within the adjustment range of the stage plate Using a ruler measure and note the distance the tip extends from the fitting Gently move the PicoFrit until the tip extends 15 20 mm past the end of the fitting Finger tighten the compression fitting to hold the tip in place Figure 5 18 shows a correctly loaded union Figure 5 19 Union installed in the tip holder Installing the MicroTight Union into the Tip Holder It is very easy at this point to break the tip Take care not to touch the tip to any surface 1 Being careful not to put mechanical stress on the fused silica tubing insert the MicroTight union into the recessed area of the tip holder cover Press gently until the union snaps into place Figure 5 19 2 Place the top of the tip holder with the loaded union over the base Replace Figure 5 20 Union installed in the tip the cover screw through the hole in the top and thread it through the mating PE hole in the holder base Tighten Supplying High Voltage Do not adjust end cap _ The uncoated tip module uses a modified microtee to supply the high voltage E dm at the liquid
26. e PicoTip does not extend past the tubing sleeve end that will be inserted into the microtee Thread the sleeved PicoTip through the fitting nut and a black MicroFerrule as shown in Figure 5 22 Cleave the end of the PicoTip after the tubing is threaded through the sleeve nut and ferrule Instructions for cleaving fused silica may be found in Section 5 1 on page 21 Slip the end of the tubing through the right post of the mi crotee as viewed in Figure 5 23A until the tubing and sleeve seat against the bottom ledge inside the post as shown in Figure 5 23B Screw the nut finger tight onto the microtee Insertthe distal end of the fused silica transfer line through a green MicroTight sleeve then through the nut and the black MicroFerrule as shown in Figure 5 24 Carefully trim the end of the transfer line After trimming insert the assembly back into the microtee seat the transfer line ferrule and sleeve against the PicoTip and finger tighten the nut as shown in Figure 5 24B Gently pull on the tubing ends to ensure the connection is tight Check for leaks by running solvent through the tubing at the expected operating pres sure Leaks will be apparent if solvent collects at the exposed ends of the sleeves Loading the Assembled Microtee into the UTM 1 Lower the assembled microtee into the cavity in the base of the UTM see Figure 5 28 The setscrews on the black cap should be visible when the mi crotee is properly in place 2 R
27. e needle with an alligator clip as de scribed in Grounding to a Syringe Needle or HPLC on page 4 10 2 Lock PicoView to the mass spectrometer using the mounting brackets Make sure the vacuum LED is illuminated green 3 Turn on the mass spectrometer by accessing the Tune Plus window choos 6 TECHNIQUES TO OPTIMIZE PICOVIEW TVONYW 33Sf1 A 3IAODId OSS Ad 1 PV 550 PICOVIEW USER MANUAL 6 6 TECHNIQUES OPTIMIZE PICOVIEW 10 11 ing Control from the menu bar and selecting On alternatively click the On Standby button In the Tune Plus window click the API Source button and set the initial ESI Voltage to 500 volts In the Tune Plus window set the solvent flow rate by clicking the Syringe Pump icon choosing On next to Flow Control then typing in the desired flow rate next to the Flow Rate parameter If the CTM is being used the flow rate should be set between 50 nL min and 250 nL min If the UTM is em ployed the flow rate should be set to a value greater than 100 nL min Under Type highlight the Hamilton button and under Volume type 250 Click Apply You may observe the ESI current in the Tune Plus window on the mass spec trometer Access the Graph window by clicking the Display Graph View icon Click the Tune icon select the Manual tab and click the ESI Spray Current button Click Start Look on the imaging system monitor to see if solvent is flowing through forming a
28. eplace the UTM module top Place the cover screw through the hole in the module top and align it with the mating hole in the module base and tighten Figure 5 28 shows properly mounted UTM and tip holder vf Alignment of the UTM cover is critical Misalignment of cover may damage the internal spring contact mechanism make sure cover is correctly aligned with base before applying pressure and securing cover Connecting the High Voltage Run the high voltage cable from the mass spectrometer through the opening in the rear housing Connect the cable by pushing it into the cable jack and turning the locking ring clockwise until it is tight Adjusting the Tip Position By visual inspection adjust the stage plate position using the manual knob con trols see Figure 3 1A until the tip is approximately 2 5 mm from the mass spec trometer inlet Focus the camera using the focus knob on the optical stage see Figure 3 1A By focusing on the inlet and then bringing the tip into focus by adjusting the stage position you are assured that the tip is vertically in line with the inlet 5 SETUP Figure 5 25 Plumbed microtee loaded in the UTM Ios Figure 5 26 UTM mounted in rear position with tip holder TVANVW 33Sf1 14 OSS Ad PV 550 PICOVIEW USER MANUAL 5 SETUP 12 Cover screw mounting screw Figure 5 28 UTM mounted in the forward pos
29. er pulling it free of the sleeve or preferably by cleaving the fused silica tubing near the terminus 5 SETUP FEP Teflon Distal end Figure 5 13 PicoFrit column packaged for shipping Figure 5 14 Pushing the PEEK sleeve against the FEP sleeve Figure 5 15 Pushing the FEP sleeve off the tip end of a PicoFrit column TVONYW 33Sf1 14 OSS Ad N PV 550 PICOVIEW USER MANUAL 8 SETUP Figure 5 16 UTM aand tip holder bases attached stage plate UTM mounting holes Rear position groove front positioning groove Figure 5 17 Location of mounting holes for attaching the uncoated tip module and the tip holder Figure 5 18 PicoFri column loaded into MicroTight union Slide the PEEK sleeve toward the tip end until it butts up against the FEP sleeve as in Figure 5 14 Orient the FEP sleeve vertically with the tip facing toward the floor This way when the FEP sleeve slides free it will fall toward the ground without damag ing the tip Carefully push the PEEK sleeve against the FEP sleeve until the FEP sleeve falls off as shown in Figure 5 15 This may or may not take a LOT of force You will have to move the PEEK sleeve about 3 5 mm Grasp the column in one hand while pushing the sleeve off with the other hand Once the FEP sleeve falls away remove the PEEK sleeve by sliding it over the back end Do not l
30. ert the back or distal end of the PicoTip through the trimmed sleeve and through the other compression fitting When properly installed the tip end should extend 15 20 mm past the end of the fitting when it is tight This will afford optimal positioning of the PicoTip within the adjustment range of the stage plate Using a ruler measure and note the distance the tip extends from the fitting Remove the fitting sleeve PicoTip assembly and carefully trim the back end of the PicoTip so the extension of the tip beyond the fitting is 15 20 mm Cleave the remaining portion from the back end of the PicoTip See Cutting Fused Silica earlier in this section for cleaving instructions After trimming reinsert the assembly into the union seat the PicoTip and sleeve against the transfer line or column tubing and tighten the compres sion fitting finger tight Pull gently on the tubing to ensure the connection is tight Figure 5 9 depicts the fully assembled union Always use chemical and puncture resistant gloves and ASNI ap proved safety glasses when handling fused silica tubing Loading the Coated Tip Module 1 Remove the CTM from the PicoView components box Disassemble the CTM by unscrewing and removing the cover screw 2 Lower the assembled MicroTight union and PicoTip into the cavity in the base of the CTM as shown in Figure 5 10 vf The PicoTip must make direct contact with the conductive elastomer inside the cover of
31. ery different from any other chromatography columns on the market today PicoFrit columns are fabricated from 360 um OD polyimide coated fused silica tubing The column has a specially tapered tip with an inte gral high porosity frit Behind the frit is the packed chromatography bed There is no frit at the back end of the bed only unpacked fused silica tubing Mobile phase flow must always be directed toward the tip Reversing the flow may result in partial or complete unpacking of the chromatography bed The column is shipped filled with methanol and needs to be conditioned with an appropriate mobile phase for your gradient Since there is no distal frit the distal end of the column bed may loosen with time this is not a problem as the bed will repack when the column is pressurized vf The label attached to the opaque orange PEEK sleeve that slides along the length of the column The arrow on the label points toward the tip end The tip end may also be identified by the charred section of polyimide coat ing just prior to the tapered region of the tip Removing a PicoFrit Column from the FEP Sleeve Care must be taken to properly remove the column from the clear FEP Teflon sleeve to prevent damaging the fragile tip and frit Use the free sliding orange PEEK sleeve assisted by gravity to push the clear FEP sleeve off the fused silica tubing without damaging the tip 1 Remove the distal end of the column from the FEP sleeve by eith
32. et the PEEK sleeve slide off the tip end as this will damage the tip Retain the column label for identification purposes Attaching the UTM to the Stage Plate in the Back Position Figure 5 16 illustrates proper orientation of the tip holder and the UTM The stage plate is configured like an optical breadboard to provide the user with the great est versatility in tip placement 1 Unlock the PicoView base from the interface flange slide the source away from the mass spectrometer and lift open the cover Remove the magnetic stage plate from the unit by grasping the plate firmly and pulling upward Set the UTM in the rear positioning groove on the stage plate and align the mounting slot on the UTM base with the corresponding tapped holes on the stage plate Refer to Figures 5 16 and 5 17 Insert the mounting screw through the mounting slot on the UTM base and thread it into an appropriate threaded hole on the stage plate as shown in Figures 5 16 and 5 17 and tighten Loading the MicroTight Union 1 A MicroTight union within the tip holder is used as a pass through to position the tip toward the inlet Locate the union in the PicoView accessories box 5 SETUP 2 Unscrew and remove the compression fittings from the end of the union Thread the distal end of the PicoFrit through a green MicroTight sleeve and through a MicroTight fitting Gently screw the fitting into the union but do not tighten Push the distal en
33. f the tip modules can be found in Section 5 Grounding Guidelines with the CTM or UTM For the highest level of safety it is recommended that the grounding union be spliced into the plumbing between the Micro Injection Valve and the CTM or UTM as shown in Figure 4 15A This configuration is recommended for contin uous flow microspray or nanospray operation With nanobore chromatography however the interior volume of the union may deteriorate chromatographic peak shape In this event the optimal place for the grounding union is between the injection valve and the HPLC or syringe pump Figure 4 15B If your mobile phase contains a high concentration of TFA or formic acid the electrical conductivity of the liquid may rise to the point where the electrospray power supply is partially shorted to ground If this occurs it becomes impossible for a stable spray to form because the voltage at the end of the tip is too low and the total spray current displayed on your monitor will read abnormally high in the multi microamp range This can only be corrected by reducing the conduc tivity of the mobile phase or by replacing the tubing between the ground point and the tip module The new splice should be of longer length of smaller inside diameter or both Good performance can be obtained with a 25 30 cm length of fused silica of either 20 or 50 um ID Grounding to a Syringe Needle or HPLC If the syringe pump is being used to deliver solvent to Pi
34. gh a capillary bore chromatography column which is con nected to a TaperTip a type of PicoTip Figure 2 6 Voltage is applied through a liquid junction in the uncoated tip module UTM placed in the forward posi tion Suggested tip size is 50 uim Refer to Section 5 Uncoated Tip Module in the Forward Position for detailed instructions on setting up the UTM for capil lary bore LC using a TaperTip connected to a column 2 APPLICATIONS C O Figure 2 4 Nanobore LC MS using a PicoFrif Column Figure 2 5 Nanobore LC using a PicoTip with a separate column Figure 2 6 Capillary bore LC TVONYW 33Sf1 A A3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 2 APPLICATIONS Figure 2 7 Assembled microtee used for making liquid junction contact in the PicoView UTM Supplying Voltage to the Uncoated Tip Module When using the UTM high voltage is applied directly to the solvent stream in a liquid junction via a microtee made of polymer that contains an integral platinum electrode Figure 2 7 shows the microee which is easy to assemble and is ideal when using PicoFrit columns or other designs that require pre column voltage contact The microtee is unsuitable for low volume post column use however as it has an interior volume 3 PICOVIEW COMPONENTS 3 Quick Guide Components of PicoView Figures 3 1A
35. heated capillary is large greater than 5 mm Optimizing the Spray PicoView is ready for use once the tip or column is in position fluid transfer has been established and electrical contact has been made Bringing the system to stable ESI conditions will involve adjusting the solvent flow rate and the ESI spray voltage and using the imaging system to visualize the resulting spray pattern Selecting a Flow Rate Using the Coated Tip Module The CTM is most effective at flow rates below 250 nL min when used with SilicaTips and above 500nL min with TaperTips The user will need to empirical ly determine the best flow rate based on specific sample conditions and PicoTip placement Use the lowest possible flow rate to generate a stable electrospray when low sample volumes and or extremely dilute samples are analyzed When the pumping element is a syringe pump a 10 100 pL syringe is recommended To set the flow rate using the syringe pump open the Tune Plus window and access the syringe pump parameters by choosing Setup from the menu bar Select either Syringe Pump or click the Syringe Pump icon Choose the appropriate syringe from the pull down menu Set the flow rate to 0 250 L min and click Apply Once spray is established decrease the flow rate while adjusting the voltage and tip position if necessary until a stable spray is established at the desired flow rate Using the Uncoated Tip Module The UTM is used most effectively at flow
36. ideo Capture enables observation of the live spray directly on the system computer screen the video monitor or both Contact New Objective for more information During assembly avoid getting fingerprints on the imaging system lenses Connecting the Optical Tube and Lens 1 Locate the optical tube and the 9 64 Allen wrench in the accessories kit Using the Allen wrench loosen the slip ring refer to Figure 4 1 With the threaded end of the optical tube pointing up slide the optical tube through the slip ring so that approximately 30 mm extends below 2 Tighten the fastener using the Allen wrench Take care to not over tight en Select the low power lens from the accessories box 4 Screw the male threaded end of the lens onto the mating female thread on the instrument side end of the optical tube Turn the lens counterclockwise until it is finger tight see Figure 3 1A Attaching the High Resolution CCD Camera 1 The CCD camera and illuminator are shipped in containers separate from PicoView Remove the CCD camera from its box and remove the protective end cap from the camera Place the optical end of the CCD camera flush with the user end of the opti cal tube While supporting the CCD camera with one hand screw the threaded nut of the CCD camera clockwise onto the threads on the user end of the optical tube until the CCD camera nut is finger tight While supporting the CCD camera with one hand loosen the optical tu
37. ility to design experiments for a variety of flow rates and sen sitivity requirements The modules supply the high voltage for sample ionization through different junction styles as dictated by the experi mental design The tip modules are mounted on the stage plate and each module functions uniquely to accommodate different tip and column styles The coated tip module CTM is depicted in Figure 3 2 and the uncoated tip module UTM in Figures 3 3A and B Detailed descriptions for the experimental configuration of the tip modules are presented in Section 5 Setup Coated Tip Module The coated tip module CTM shown in Figure 3 2 connects the tip to the transfer line or column via a MicroTight union Upchurch P V P 720 High voltage is provided to the coated tip using a conductive elastomer The CTM mounted in the forward position is the module of choice for performing continuous infusion nanospray and microscale flow injection experiments at ultra low flow rates It can also be used with commercial nanobore columns as well as with static or offline nanospray emitters Uncoated Tip Module Within the uncoated tip module UTM the transfer line connects to the emitter via a modified microtee or through a zero dead volume titanium union High voltage is applied to the liquid junction via either the plati num electrode in the microtee or through the body of a zero dead volume titanium union Refer to Section2 Supplying Voltage to le
38. ing how to select the best PicoTip for your applica tion can be found in Section 6 Continuous Infusion Nanospray Continuous infusion is the most straightforward method for sample introduc tion with a syringe pump providing the solvent stream as illustrated in Figure 2 1 Sample is introduced directly from a syringe pump at flow rates typically of 10 300 nL min The high voltage is applied through the coated tip module CTM to the conductive coating on the PicoTip Coated SilicaTips a type of PicoTip are primarily used for ultra low flow rate infusion nanospray to mini mize electrolysis Suggested tip sizes for continuous infusion nanospray 5 10 pm with a distal or standard coating All coated tips should be handled with care since mechanical abrasion can deteriorate the coatings Use a pair of fine twee zers to handle the tips Go to Section 5 3 Coated Tip Module in the Forward Position on page 23 for detailed instructions on setting up the CTM for continu ous infusion Nanospray Flow Injection Nanospray flow injection entails injecting small volumes of sample into an es tablished solvent stream as depicted in Figure 2 2 An aliquot of sample 1 2 pL is injected as a plug into the solvent stream through the micro injection valve sample loop A syringe pump at flow rates of 10 300 nL min is used to deliver the solvent stream that pushes the sample through the PicoTip The flow injection 2 APPLICATIONS
39. ition on the stage plate UTM mounting holes Rear position groove front positioning groove Figure 5 29 Location of mounting holes for attaching the uncoated tip module and the tip holder Uncoated Tip Module in the Forward Position The UTM can be used with uncoated PicoTips in the forward position for con tinuous infusion as shown in Figure 5 28 This configuration minimizes transfer volumes by accommodating a short PicoFrit column 5 cm or shorter or an uncoated PicoTip In this arrangement as with the UTM in the back position high voltage is applied directly to the solvent stream in a liquid junction via an electrode The uncoated tip module uses a special microtee to supply the high voltage Loading the UTM is identical for use in either the forward or back positions See page 5 9 for instructions on loading the UTM Attaching the UTM to the Stage Plate in the Forward Position The stage plate is configured like an optical breadboard to provide the user with the greatest versatility in tip placement The best camera view will be provided when the tip is approximately perpendicular to the camera axis 1 Unlock and slide the PicoView unit away from the mass spectrometer and open the cover 2 Remove the magnetic stage plate from the unit by grasping the plate firmly and pulling upward 3 Set the UTM into the front positioning groove on the stage plate and align It is very easy at this
40. itting pull free from the probe Disconnect the auxiliary gas line and fitting from the inlet labeled Aux Gas on the ESI probe by turning the green fitting counterclockwise until the line and the fitting pull free from the probe 4 INSTALLING PICOVIEW TVONYW 33Sf1 A 3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 4 INSTALLING PICOVIEW Slip ring Figure 4 1 The optical tube should extend approximately 30 mm below the slip ring 5 If the sheath liquid line is attached to the ESI probe disconnect the line and its fitting from the inlet labeled Sheath Liquid on the ESI probe by turning the fitting counterclockwise until the line and the fitting pull free from the probe Removing the lon Max Source 1 Rotate the lon Source Housing Locking Levers to unlock the lon Max source from the MS detector face plate 2 Gently pull the source towards you and away from the mass spectrometer 3 Store the lon Max source in its original foam storage container for protec tion Assembling the PicoView Imaging System The PicoView imaging system consists of an optical stage for fine positioning of the camera a slip ring through which the optical tube is inserted the optical tube a CCD camera a fiber optic illuminator and a lens Installation of the imag ing system involves attaching a lens and the CCD camera or optional eyepiece to the optical tube The optional Hauppauge USB Live V
41. junction This modified microtee has an integrated platinum wire that makes connection with the liquid sample at the point that the two pieces of Inlet Outlet fused silica tubing are joined in the union Figure 5 21 microtee with sleeves Plumbing the Modified Microtee 1 The modified microtee joins the transfer line to the PicoTip and supplies the high voltage Remove the microtee from the PicoView accessory box Orient the microtee as shown in Figure 5 20 so that the platinum electrode TVANVW 33Sf1 4 OSS Ad PV 550 PICOVIEW USER MANUAL 5 SETUP 10 8 is facing away from the user and the setscrews are visible Unscrew the nuts and remove the black ferrules from the posts of the microtee vf Do not loosen the setscrews or remove the electrode cap as this may damage the electrode The solvent will not become charged and an elec trospray will not form Altering will void warranty Figure 5 22 Fitting nut MicroFerrule and sleeve threaded by PicoTip A B Figure 5 23 Assemble the nut ferrule sleeve and PicoTip A and secure the finger tight into the microtee B oie Figure 5 24 A Assemble the nut ferrule sleeve and transfer line and B secure into the microtee Thread the end of the PicoTip tubing through a green MicroTight sleeve Upchurch P N F 185 which is used for assembly with 360 um OD tubing Make sure th
42. kplate be attached prior to mounting Figure 7 2 Attach the backplate to the mount using two mounting screws Figure 7 3 Properly mounted Upchurch valve TVANVW 33Sf1 A 3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 7 MOUNTING INJECTION VALVES Figure 7 4 Attach the Valco valve directly to the mount using two mounting screws as shown Figure 7 6 Component clamps with injection adapter and an IntegraFrit trap column Figure 7 7 Micro injection adapter with mounting nut Mounting a 10 Port Automatic Valco Valve The Valco valves can be attached directly onto the PicoView valve mount The two holes on the valve mount directly match two 8 32 threaded holes on the face of the Valco valve Using two 8 32 cap screws fasten the Valco valve to the valve mount as shown in Figure 7 4 Make sure the valve ports face the opening in the housing Components and Component Clamps PicoView has four 4 component clamps that have been designed to hold an injection adapter included in the accessories kit columns or unions that may be required in your experiment The thumb screws in the component clamps will gently hold items in place Alternately these clamps can be removed and set aside when they are not needed 7 MOUNTING INJECTION VALVES TVANVW 4350 A 3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 7 MOUNTING INJECTION VALVES DPV 550 DIGITAL PICOVIEW USER MANUAL TE
43. n Refer to Section 5 Uncoated Tip Module in the Back Position for detailed instructions on setting up the UTM for a PicoFrit column Nanobore LC Using a PicoTip with a Separate Column PicoView can be used effectively for microscale LC MS and LC MS MS When running nanoscale separations it is best to use a PicoFrit column a combined emitter and a column for increased sensitivity and decreased problems due to less plumbing If experimental parameters do not allow use of a PicoFrit Column PicoView can accommodate a nanobore column connected to a PicoTip New Objective offers IntegraFrit c olumns designed specifically for this purpose In this application it is recommended to use the SilicaTip type of PicoTip Sample flows through the IntegraFrit through the SilicaTip and into the mass spec trometer Figure 2 5 Voltage is applied through a coated tip using the coated tip module CTM in the forward position For a 75 ID column the suggested tip size is 10 uim with a distal coated tip Refer to Section 5 Coated Tip Module in the Forward Position for detailed instructions on setting up the CTM for nano bore LC using a PicoTip connected to a column Capillary Bore LC Capillary bore LC uses a column with a larger diameter than used in nanobore LC Capillary columns usually have an internal diameter of 100 320 and are suggested for use at flow rates of 200 1000 nL min Sample is introduced into a solvent stream throu
44. n the bottom of the PicoView base and gently lock them into place Figure 4 10 Connecting the PicoView Imaging System 1 Remove the monitor from its shipping carton and place it in a convenient viewing area Plug the power cord for the monitor into an electrical outlet preferably one protected by a suitable surge protector Connect the video cable from the monitor to the BNC video out jack on the high resolution CCD camera Connect the camera power supply to the high resolution CCD camera Figure 4 11 Plug the other end of the cable into an electrical outlet again preferably one with a suitable surge protector Connecting the Optional Hauppauge USB Live Video Capture 1 Locate the BNC cable supplied with the Hauppauge system Using a BNC RCA adapter plug the BNC cable into the video output connection on the 4 INSTALLING PICOVIEW 7 2 Figure 4 7 When locking the fiber optic bundle in place do not overtighten thumb screw Figure 4 8 Fiber optic bundle properly installed Figure 4 9 Source pushed firmly against flange E 2117 Figure 4 10 Brackets should line up with latches TVANVW 4350 A 3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 4 INSTALLING PICOVIEW Figure 4 11 Connect the power cable to the camera PicoView 4 y Figure 4 12 Exterior view of assembled Happague USB Live Video Capture Module m ge
45. ncture resistant gloves and ANSI ap proved safety glasses when handling fused silica tubing Digitally Controlled XYZ Positioning Stage Precise alignment of the tip is accomplished using the PicoView PV Acquire software and the actuated X Y and Z translation stage The translation stage moves the stage plate on which a tip module is mount ed Consult the provided PicoView PV Acquire Software Manual for more information regarding the stage operation and specifications Imaging System The imaging system allows the user to quickly and efficiently optimize the electrospray conditions by providing direct observation of the char acter of the spray The system includes a high resolution CCD camera and an objective lens Alignment of the imaging system is accomplished using the Focus Tilt and Pan adjustment screws of the optical stage re fer to Figure 3 1A Injection Valves A micro injection valve typically serves as the inlet for sample intro duction to PicoView The sample is loaded into a sample loop using an injection port adapter or a syringe directly connected to the sample inlet port Sample and solvent from an HPLC pump or syringe are trans ferred to a tip module and subsequently to a column or tip Please refer to the instructions particular to your valve for information on its setup and use as PicoView can accommodate many different valves Section 7 Mounting an Injection Valve illustrates how to mount a valve
46. not be made from an electrically conductive material It must be fabricated from an electrically insulating material such as or fused silica Otherwise the operator may be exposed to potentially lethal voltage In systems where high voltage is applied directly to the ESI tip the liquid sample inside the tip and transfer line tubing is also raised to a high voltage To prevent exposure to potentially lethal voltages a suitable ground point for the liquid inside the line must be provided Instructions for grounding are found in Section 4 4 on page 19 A Do not operate PicoView without the external housing and lid in place A WARNING Do not defeat any mass spectrometer system software or hard ware safety interlocks Contents and Components List PicoView comes partially assembled with system components contained in multiple boxes Model Number and Serial Number Model Number PV 550 Your Serial Number 110V 220V Plug type Components Checklist PicoView base unit translation stage camera mount housing and lid Magnetic stage mounting plate Interface mounting flange Accessories box Uncoated Tip Module UTM Coated Tip Module CTM Tip holder CCD camera 9 B W monitor with cabling Allen wrenches metric amp English Illuminator box with 150 watt illuminator Fiber optic cable in illuminator
47. o applications or methods using the products described or mentioned in this manual New Objective Inc warrants this Product PicoView to be free of defects in materials and workmanship for a period of one 1 year from the date of ship ment Any item believed to be defective within the meaning of the foregoing sentence shall be returned to New Objective Inc and if found by us to be defective shall be repaired or replaced with conforming Product of like kind Please note that a Return Merchandise Authorization Number RMA will be required New Objective Inc will pay return freight on unsatisfactory items New Objective Inc shall have no other liability or obligation with respect to goods alleged to be defective The foregoing shall constitute the sole and exclu sive remedy and New Objective Inc s total liability for any and all losses and damages arising out of any cause whatsoever whether such cause be based in contract negligence strict liability other tort or otherwise shall in no event exceed the purchase price of the Product s in respect of which the cause rose New Objective Inc disclaims and shall not be liable in any event for loss of profits consequential or incidental damages or punitive or exemplary damages in connection with the Product furnished hereunder The foregoing limited warranty i shall be void as to any item of Product which is in any material respect altered by the user and ii does no
48. onnected from the inlet system before installation of the PicoView system Inspect all equipment carefully prior to use Any damaged chipped or cracked components should not be used and must be discarded or repaired Handling Fused Silica Tubing Handling of fused silica or glass tubing and tips can result in serious per sonal injury including eye and skin injury Use safety glasses or goggles meeting ANSI 287 1 1989 requirements or the equivalent Puncture and chemical resistant gloves should also be worn at all times 1 READ THIS FIRST 33Sf1 A 3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 1 READ THIS FIRST Tip Adjustment and or Replacement Do not attempt to adjust or replace the tip unless the ESI high voltage and other applicable voltages are turned off and are at ground potential A Reduce any applicable backing pressure liquid gas etc to ambient be fore loosening ESI tip fittings and removing the fused silica tip or transfer line from the coupling union Prior to pressurization make sure that com ponents are tightened to specifications to prevent separation during use Failure to adhere to this warning could result in projectile like expulsion of the tip from the coupling union which could cause serious personal injury or damage to surrounding apparatus The Transfer Line The transfer line connecting to either tip module or the Micro Injection Valve must
49. oosing Tip Size and Coating Options 6 1 In Line Filtration 6 1 Air Bubbles 6 3 Operating Parameters 6 4 Tip Position 6 4 Applied Voltage 6 4 Solvent Flow Rate 6 4 Electric Field Strength amp 6 5 Flow Rate Interdependence Voltage Tuning 6 5 Optimizing the Spray 6 7 Selecting a Flow Rate 6 7 Using the CTM 6 7 Using the UTM 6 7 Using the Imaging System 6 8 Mounting an Injection Valve 7 1 Mounting a Manual 6 Port Valve 7 1 Mounting a 10 Port Valco Valve 7 2 Components and Component Clamps 7 2 Read This First Safety Considerations WARNING Electrospray ionization involves the use of potentially lethal high voltage electrical current Observe all manufacturers safety recom mendations in the use of such equipment No equipment modifications should be made except by trained personnel using methods approved by the manufacturer in accordance with all safety requirements Installation of equipment should be performed by qualified personnel in accordance with all applicable electrical codes Never use this product with defective damaged or faulty equipment Serious injury or death could result SAFETY PRECAUTIONS Only qualified personnel should use this product Provide a safe workplace equipped with all necessary safety equipment Prior to Installation Follow all safety recommendations of the equipment manufacturer s All sys tem voltages must be brought to ground potential and all high voltage contacts disc
50. ove the MicroTight union Upchurch P N P 720 from the PicoView accessories box Unscrew and remove the compression fittings from both ends of the union Screw the white gauge plug finger tight on to one end of the union Thread the fused silica transfer line through a green MicroTight sleeve The appro priate sleeve size is 0 002 0 003 inches greater than the OD of the capillary tubing Use the green MicroTight sleeves with 360 OD tubing After the transfer line passes through a green sleeve thread it through one of the com pression fittings Figure 5 5 shows the union loaded with the white gauge plug on the right and fused silica tubing threaded through a tubing sleeve and a compression fitting Cleave the end of the tubing and slip it into the union until both the tubing and the sleeve ends seat against the gauge plug inside the union Screw the compression fitting finger tight into the union as shown in Figure 5 6 Remove the gauge plug and return it to the PicoView accessory box Carefully trim a new green MicroTight sleeve to a length of approximately 14 mm as shown in Figure 5 7 The shorter sleeve will allow the coating on the PicoTip to contact the conductive elastomer inside the CTM Choose a PicoTip from the assortment sent with PicoView Although either coating style the standard coating CE or the distal coating D will work if flow rates permit the distal coating is recommended due to its immunity to arcing Ins
51. pectra may be diminished To minimize the ion current fluctuations reduce the flow rate by 25 percent and look for im provements in signal stability Raising the applied voltage can sometimes com pensate for a flow rate that is too high provided the voltage is not above the threshold for corona discharge Electric Field Strength and Flow Rate Interdependence In low flow ESI it is important to recognize the interdependence of flow rate and the applied electric field The optimal value of the flow rate is altered when a parameter is changed that affects the strength of the electric field e g the size of the tip the distance from the tip to the heated capillary inlet or the ap plied voltage For a given tip size stable ESI can occur over a wide range of flow rates but only over a narrow range of field strength 100 volts or less Raising the flow rate requires a higher field strength and vice versa In the static nano electrospray mode where there is no external flow source the system is self regulating in that the electric field actually dictates its own optimal flow rate Voltage Tuning Procedure 1 For a continuous infusion experiment prepare a standard sample and load 100 200 uL into a 250 uL syringe Peptide and protein standards should be prepared at 1 uM in a suitable solvent system such as 1 1 0 5 acetic acid methanol Secure the syringe to the mass spectrometer syringe pump using the syringe holder Ground th
52. ptic bundle Slip one end of the fiber optic bundle through the opening on the right side of the PicoView housing and into the back end of the fiber optic coupler The coupler holds a lens which focuses the illumination on the nanospray emitter and can be adjusted to accommodate for tip position using the Illumination Adjustment knob see Figure 3 1B 4 With the fiber optic bundle securely placed within the coupler lock the bundle in position using the thumb screw Do not over tighten Figure 4 7 Insert the bundle adapter and the other end of the fiber optic bundle into the illuminator faceplate Tighten the set screw to finger tight Figure 4 8 shows the assembled fiber optic bundle and mount Locking the PicoView Housing to the Mounting Brackets Close the transparent top cover on the PicoView source With the PicoView mounted on the Interface Flange base gently slide the assembly toward the face plate of the LTQ A groove located at the top left of the Flange will lock the cover down when the source is is pushed firmly against the flange and interlock switch Figure 4 9 Make sure the lower portion of the mounting brackets line up with the as sociated latches mounted on the base of the PicoView source Figure 4 10 If they do not check to make sure PicoView is properly mounted on the Interface Flange slide base Adjustment of the base may be necessary see Section 4 3 2 To lock the PicoView in place fasten the two latches o
53. rates above 100 nL min using PicoFrit columns at flow rates ranging between 150 and 350 nL min The user will need to empirically determine the best flow rate for each specific experimental design When using a syringe pump for delivery a 100 250 ul syringe is recommended To set the flow rate using a syringe pump open the Tune Plus window and ac cess the syringe pump parameters by choosing Setup from the menu bar Select 6 TECHNIQUES TO OPTIMIZE PICOVIEW TVANVW 33Sf1 A 3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 8 6 TECHNIQUES OPTIMIZE PICOVIEW either Syringe Pump or click the Syringe Pump icon Choose the appropriate syringe from the pull down menu Set the flow rate to approximately 0 350 pL min and click Apply Once the spray is established decrease the flow rate while adjusting the voltage and tip position if necessary until a stable spray is established at the desired flow rate Using the Imaging System to Optimize the Spray The superior imaging capabilities of PicoView allow the user to fine tune the electrospray and easily optimize the distance voltage flow rate parameters The figures below depict characteristics of unstable and stable spray patterns If the applied voltage is slightly low an unstable spray will form with droplets forming periodically at the tip This stream of large droplets normally seen under these conditions is not ideal Figures 6 1 through 6 4 and will result in poor and uns
54. supply houses New Objective carries a high quality scribe as part of a nanospray tool kit stock number TIP KIT Inexpensive carbide or ceramic scribing tools are not recom mended since they generally result in poor quality i e ragged cleaved ends generating fine particles that lead to clogging 1 Place the tubing to be cut on a clean flat surface and position the cleaving tool blade perpendicular to the tubing bore as illustrated in Figure 5 2 2 Press down with a gentle force DO NOT saw or move the blade laterally simply nick the surface of the polyimide coating to score the fused silica Figure 5 2 inset 3 Pull gently on the tubing along its axis it should easily separate at the point of scoring If it does not repeat the procedure with slightly more force Be careful not to force the blade through the tubing as this will create a ragged end vf Be sure to pull the tubing apart do not bend the tubing to induce a snap This will produce a burr along the tubing face and result in less than opti mal connections 4 Itis best to check the cleaved tubing ends for particle contamination prior to use This is most effectively done using a light microscope at 100x magnification Connectors Making leak free connections is important for limiting sample loss and for reduc ing turbulent flow PicoView comes with two different systems of microconnec tors for plumbing fused silica to the various fittings Assembling the
55. t 5 788 166 that provides for excellent electrochemical stability and du rability against ESI solvent exposure All coated tips should be handled with care since mechanical abrasion can deteriorate the coatings Use a pair of fine tweezers to handle the tips 6 TECHNIQUES TO OPTIMIZE PICOVIEW TVANVW 33Sf1 A 3IAODId OSS Ad i PV 550 PICOVIEW USER MANUAL 2 6 TECHNIQUES OPTIMIZE PICOVIEW Coated tips are typically employed for the lowest flow rate applications requir ing the highest degree of performance from the system For lowest flow rate ex periments the standard CE coating is recommended but it may be subject to damage from arcing at high voltage The use of distal D coated tips is encour aged due to their immunity to arcing Use uncoated N PicoTips when a liquid junction is required typically for nanoscale flow injection applications or when using LC columns larger than 100 ID The choice of tip size and coating style will be influenced by the desired flow and application continuous flow nano spray capillary LC CE interfacing etc See Section 2 on page 5 for assistance in choosing tip size and coating style based on the desired application Since the size of the Taylor cone grows with flow rate it is proportional to tip size Use Table 6 1 as a guide in choosing the proper tip size Follow the flowrate guidelines in Table 6 2 to obtain the best results Smaller diameter tips will oper
56. t cover misuse of the Product for example but not limited to dropping or other mishandling of any components of PicoView improper trimming of SilicaTips PicoFrit Columns or TaperTips damage caused by application of or exposure to excessive tem perature pressure or voltage or SilicaTip PicoFrit or TaperTip failure by reason of clogging The video monitor and camera are warranted to be free from defects in material or workmanship for a period of two 1 years from the date of purchase 1 READ THIS FIRST TVANVW 33Sf1 A 3IAODId OSS Ad READ THIS FIRST 1 TVONYW 33Sf1 A3IAOOId OSS Ad 1 2 Applications PicoView supports a variety of operating modes ranging from continuous infu sion at nanospray flow rates to gradient run nanobore LC MS The two tip mod ules the coated tip module CTM and the uncoated tip module UTM included with the PicoView system afford the user the versatility to design experiments for a variety of flow rates and sensitivity requirements With the open design and interchangeable modules you can run the same analysis several different ways This manual provides the most robust configurations for a given appli cation Following the schematic drawings locate the desired application and the most effective PicoView configuration for that experimental design See the referenced section for detailed directions on setting up PicoView Techniques to optimize your spray includ
57. table ion signal for the mass spectrometer This spray mode is most commonly seen in gradient chromatography during highly aque ous portions of the gradient Tuned correctly the spray will stabilize as the gradient becomes more organic Typically the spray should be optimized at 30 organic 7 Figure 6 1 850V Figure 6 2 950V Figure 6 3 1150V Figure 6 4 1400V Drops stick to tip Droplet stream Droplet stream and spray Droplet stream and spray Figure 6 5 1800V Figure 6 6 1900V Figure 6 7 2200V Figure 6 8 2800V Perfectly tuned spray Acceptable spray Multiple sprays formed Mulitple sprays formed When the voltage is correct a stable fine spray will form as shown in Figure 6 5 and the best quality data given the experimental conditions will be generated If the voltage is too high multiple jets can form around the tip as shown in Figures 6 7 and 6 8 Decrease the voltage until a stable spray is formed Throughout an experiment periodically check the imaging system monitor to confirm that the spray is stable Clogs can form in the tip during infusion causing the spray to fail Changes in the organic composition of the solvent gradient may affect the solvent flow rate during micro LC applications and it may become necessary to adjust the position of the tip to maintain an optimal spray It is gen erally not necessary to change any of the other parameters It may take some time to master electrospray at low flow rates With careful tun
58. the CTM 3 Replace the top of the CTM Replace the cover screw through the hole in the top and align it with the mating hole in the base Be careful not to touch the tip of the emitter to any surface Attaching the CTM to the Stage Plate in the Forward Position Proper orientation of the CTM in the forward position is illustrated in Figure 5 9 The stage plate is configured like an optical breadboard to provide the user with the greatest versatility in tip placement The best camera view will be provided when the tip is approximately perpendicular to the camera axis at a 45 angle with respect to the inlet It is very easy at this point to break the tip Be careful not to touch the tip to any surface 1 Unlock and slide the PicoView unit from the mass spectrometer and open the cover 2 Remove the magnetic stage plate from the unit by grasping the plate firmly and pulling upward 3 Set the CTM into the front positioning groove on the stage plate Align the mounting slot on the CTM base with a corresponding tapped hole on the stage plate Refer to Figure 5 9 as necessary 4 Insert the stage mounting screw through the mounting slot on the CTM base and thread it into the threaded hole on the stage plate as shown in Figure 5 9 Tighten the screw 5 SETUP 4 B diis 4 Figure 5 8 PicoTip in fitting before A and after B trimming to length Figure 5 9 A fully assembled Mi
59. unted the LTQ Interface Flange Mounting PicoView Installing the LTQ Interface Flange The LTQ Interface Flange is used to secure the instrument end of PicoView to the LTQ inlet block on the front of the mass spectrometer 1 Locate the LTQ Interface Flange 2 Make sure that the Flange locking levers are open pointing towards the user away from the MS detector 3 Align the two guide pin holes at the base of the Interface Flange with the guide pins on the face of the MS detector and gently push the Interface Flange onto the detector face plate Ensure that the the Interface Flange is flush against the front of the face plate Figure 4 4 4 Rotate the Flange locking levers 90 inward towards the inlet of the mass spectrometer to lock the Flange into place Figure 4 5 Attaching PicoView to the LTQ Interface Flange 1 Carefully align the PicoView base onto the slide base of the Interface Flange 2 With the PicoView mounted on the Interface Flange see Figure 4 6 gently slide the assembly toward the MS detector face plate Mounting the Fiber Optic Illuminator To install the fiber optic illumination pull the PioView source away from the instrument housing The optical fiber provides the intense no heat illumination necessary for the CCD camera 1 Locate the fiber optic bundle bundle adapter and power cord from the il luminator box 2 Remove the plastic sleeve from each end of the fiber o
60. way to completely eliminate the phenomenon is to switch to a pre column high voltage contact with the UTM in combination with a PicoFrit column The even backpressure gradient created by the column will eliminate air bubble formation due to electrolysis If a post column set up must be used fewer air bubbles are produced when using the CTM equipped with coated PicoTips The use of distal coated D PicoTips is recommended due to their immunity to arcing at higher voltages 6 TECHNIQUES TO OPTIMIZE PICOVIEW TVANVW 4350 A 3IAODId OSS Ad 1 Co PV 550 PICOVIEW USER MANUAL 6 TECHNIQUES OPTIMIZE PICOVIEW Operating Parameters Parameters that require optimization for stable electrospray are the position of the tip with respect to the heated inlet the applied voltage and the flow rate Tip Position The transfer of ions created in the electrospray process is maximized when the flow rate is minimized and when the tip is positioned closest to the mass spec trometer inlet The optimal gap between the tip and the inlet varies from 0 5 mm to 5 mm Too close and the tip may arc too far away and the electric field strength drops to a point where the Taylor cone collapses Tuning becomes somewhat difficult at distances less than 0 5 mm due to the large effect on the spray pattern of relatively small changes in voltage Most tips can be used at distances of 2 5 mm with a minimal loss in ion current under 10 percent For the
61. which no liquid is visible can ad mit significant quantities of air into the system Be sure to tighten fittings properly The second possible origin of air bubbles is outgassing of the mobile phase If gasses are forced into the mobile phase at high pressure air bubbles will form as the mobile phase exists the column The use of strictly degassed solvents is strongly recommended If you are connecting a SilicaTip or a TaperTip to the outlet of a conventional LC column air bubbles from outgassing can adversely affect the stability of the electrospray especially at lower flow rates Using a smaller tip size can also reduce this effect since the increase in back pressure generated by the tapered restriction can significantly reduce bubble formation You may end up using a smaller tip than your required flow rate would suggest but stability will improve The third possible cause is outgassing due to electrolysis of mobile phase at the high voltage electrode Since the electrode is at a high potential there are always re dox reactions occurring on its surface Air bubbles may form at the liquid junction interface in the UTM as a result of electrolysis the use of higher flow rates however tends to minimize the effects of air bubbles These relatively higher flow rates used with the UTM match well with the flow rate requirements of most LC delivery systems If air bubbles persist and you believe they are due to electrolysis the most effective
62. y seal vf Always cleave the fused silica either tubing or the back end of a Pico Tip after pushing it through a sleeve The fused silica will collect dirt and particles that were in the sleeve which if left in the fused silica may cause clogs downstream Cleaving the fused silica will help prevent particle contamination Coated Tip Module in the Forward Position This section provides instructions on connecting standard length 5 cm distal or standard coated PicoTips to a transfer line or capillary column PicoTip refers to any of New Objective s high quality tips for electrospray ionization such as SilicaTips PicoFrits and TaperTips vf Take care when tightening the MicroTight fittings making sure to only tighten enough to prevent leaks from occurring Due to the delicate nature of some fused silica tubing it is possible to damage the tubing if the fit tings are overtightened 5 SETUP TVONYW 33Sf1 A 3IAODId OSS Ad PV 550 PICOVIEW USER MANUAL 5 SETUP Figure 5 4 MicroTighf union gauge plug and compression fittings ne mim Figure 5 5 MicroTighf union with gauge plug and compression assembly ready to load Figure 5 6 MicroTighf union with gauge plug and compression assembly loaded dim Figure 5 7 Union compression fitting and MicroTight sleeve cut to 14 Loading the MicroTight Union 1 Rem

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