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Tuning and Calibrating Automatically in the ESI/MS Mode

Testing the Operation of the MS Detector in the ESI/MS Mode

Thermo Electron Corporation

LTQ XL Getting Started

Figure 22.

Spectrum view of the Tune Plus window, showing ionization time (IT) and normalization level (NL) of the
calibration solution

IT=ionization time

NL=normalization level

Summary of Contents for LTQ XL

Page 1: ...For Research Use Only Not for use in Diagnostic Procedures LTQ XL Getting Started 97355 97042 Revision A June 2006 ...

Page 2: ...sions of this document and those contained in Thermo Electron Corporation s Terms and Conditions the provisions of the Terms and Conditions shall govern System Configurations and Specifications supersede all previous information and are subject to change without notice 2004 Thermo Electron Corporation All rights reserved Microsoft and Windows are registered trademarks of Microsoft Corporation Tefl...

Page 3: ...g a part or adding components options or peripherals not specifically authorized and qualified by Thermo Electron To ensure continued compliance with EMC and safety standards replacement parts and additional components options and peripherals must be ordered from Thermo Electron or one of its authorized representatives EN 55011 1999 EN 61000 4 3 2002 EN 55011 1998 IEC 61000 4 3 A1 1998 EN 61000 3 ...

Page 4: ...n compliance with international regulations If this instrument is used in a manner not specified by Thermo Electron San Jose the protection provided by the instrument could be impaired THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES OPERATION IS SUBJECT TO THE FOLLOWING TWO CONDITIONS 1 THIS DEVICE MAY NOT CAUSE HARMFUL INTERFERENCE AND 2 THIS DEVICE MUST ACCEPT ANY INTERFERENCE RECEIVED INCLUD...

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Page 7: ...r 8 What Types of Buffers Should I Use What Types Should I Avoid 10 How Should I Set Up the MS Detector for Various LC Flow Rates 11 What is Tuning and Calibration of the MS Detector All About 13 What Types of Experiments Can I Perform with the LTQ XL MS Detector 16 General MS or MSn Experiments 16 Data Dependent Experiments 17 Ion Mapping Experiments 20 Ion Tree Experiments 23 Chapter 2 Setting U...

Page 8: ...tor Tune Automatically with Your Analyte 77 Saving the ESI MS Tune Method 81 Chapter 5 Acquiring ESI Sample Data Using the Tune Plus Window 83 Setting Up to Acquire MS MS Data in the Full Scan Type 84 Optimizing the Isolation Width and Setting Up to Optimize the Collision Energy 84 Optimizing the Collision Energy Automatically for an MS MS Experiment 89 Setting Up to Introduce Sample by Loop Injec...

Page 9: ...can Mode 134 Appendix A Sample Formulations 139 Caffeine MRFA and Ultramark 1621 Stock Solutions 140 Stock Solution Caffeine 141 Stock Solution MRFA 142 Stock Solution Ultramark 1621 142 ESI Calibration Solution Caffeine MRFA Ultramark 1621 143 Reserpine 144 Stock Solution Reserpine 144 Reserpine Tuning Solution and Reserpine APCI Sample Solution 144 Appendix B LTQ XL High Mass Range Calibration 1...

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Page 11: ...dition to this guide Thermo Electron provides the following documents for the LTQ XL system LTQ XL Preinstallation Guide LTQ XL Getting Connected LTQ XL Hardware Manual Help is also available from within the software Safety and Special Notices Make sure you follow the precautionary statements presented in this guide The safety and other special notices appear in boxes Safety and special notices in...

Page 12: ...00 532 4752 Fax 1 561 688 8731 International contacts for ordering information Visit www thermo com finnigan for the current listing Changes to the Manual and Online Help To suggest changes to this guide or to the Help use either of the following methods Fill out a reader survey online at www thermo com lcms techpubs Send an e mail message to the Technical Publications Editor at techpubs finnigan ...

Page 13: ...olumn The sample is then separated into its various components The components elute from the LC column and pass into the MS detector where they are analyzed Analysis by direct infusion or flow injection provides no chromatographic separation of components in the sample before it passes into the MS detector The data from the MS detector is then stored and processed by the Xcalibur data system This ...

Page 14: ...with the compound of interest Single stage mass analysis allows for the identification of analytes of interest Atmospheric pressure ionization typically produces mass spectra that provide molecular mass information Two stage mass analysis allows for even more positive compound identification MS MS analysis monitors how a parent ion fragments when exposed to an additional stage of ionization There ...

Page 15: ...ed Wideband Activation The Wideband Activation option allows the LTQ XL MS detector to apply collision energy to ions during MS MS fragmentation over a fixed mass range of 20 u This option allows the LTQ XL MS detector to apply collision energy to both the parent ion as well as to product ions created as a result of non specific losses of water 18 u or ammonia 17 u for example or to product ions f...

Page 16: ...tion into the gas phase Many samples that previously were not suitable for mass analysis for example heat labile compounds or high molecular mass compounds can be analyzed by ESI ESI can be used to analyze any polar compound that makes a preformed ion in solution The term preformed ion can include adduct ions For example polyethylene glycols can be analyzed from a solution containing ammonium acet...

Page 17: ...ds and bases are extremely detrimental to the instrument The rules for a good electrospray are as follows Keep non volatile salts and buffers out of the solvent system For example avoid the use of salts containing sodium or potassium and avoid the use of phosphates If necessary use ammonium salts instead Use organic aqueous solvent systems and volatile acids and bases If possible optimize the pH o...

Page 18: ...more basic nitrogen or other basic atoms Molecules which generally produce strong negative ions with acidic sites such as carboxylic acids and acid alcohols are an exception to this general rule Although in general fewer negative ions are produced than positive ions negative ion polarity can be more specific This is because the negative ion polarity mode sometimes generates less chemical noise tha...

Page 19: ...ind the sweep cone and can result in solvent declustering and adduct reduction When you are analyzing complex matrices such as plasma or nonvolatile salt buffers Sweep gas is required for ruggedness In full scan MS or data dependent scan experiments the signal to noise ratio can be improved by application of Sweep gas In some cases signal intensity can be increased by using Auxiliary gas particula...

Page 20: ...duce samples from a syringe into the loop of the injector valve You can then use the divert valve to introduce the sample into an LC flow which then enters the MS detector This technique is used in ESI or APCI to introduce pure analytes into the MS detector in a slug It is useful when you have a limited quantity of pure analyte You can also use an LC autosampler to introduce samples into an LC flo...

Page 21: ...XL Getting Started Figure 4 1 ESI Figure 6 1 APCI Loop injection into LC flow ESI or APCI analysis of a pure analyte solution LTQ XL Getting Started Figure 5 6 ESI Figure 8 1 APCI Autosampler injection into LC flow one or multiple injections ESI or APCI analysis of a pure analyte solution LTQ XL Getting Connected Figure 11 5 ESI Figure 11 8 APCI LC Flow With Chromatographic Separation Autosampler ...

Page 22: ...nonvolatile buffers with the MS detector because they can cause the following problems Blocking the capillary in the probe Causing salt buildup on the spray head and thus compromising the integrity of the spray Use volatile buffers when you use the MS detector Many volatile buffer solutions are available that can be used instead of nonvolatile ones Volatile buffer solutions can include the followi...

Page 23: ...w rates for the Sheath Auxiliary and or Sweep gas For APCI you need to adjust the capillary temperature and vaporizer temperature and adjust the gas flow rates for the Sheath Auxiliary and or Sweep gas In general an increase in the rate of liquid flowing into the MS detector requires a higher temperature of the ion transfer capillary and vaporizer and the higher gas flow rate Table 2 provides guid...

Page 24: ...n conditions Typical setting 0 to 20 units LC at flow rates from 100 to 500 μL min 2 to 3 mm ID Typical setting 250 to 350 C Required Typical setting 40 to 60 units Not required but usually helps to reduce solvent background ions Typical setting 0 to 20 units LC at flow rates from 0 4 to 1 mL min 4 6 mm ID Typical setting 300 to 400 C Required Typical setting 60 to 100 units Required Typical setti...

Page 25: ...re that the calibration parameters complete automatic calibration successfully The Calibrate dialog box in Tune Plus provides a readback of the status of the calibration parameters both during the automatic calibration and when calibration is complete Lastly if you want to maximize the detection of one or more particular ions you can optimize the tune of the MS detector with your analyte of intere...

Page 26: ... Analyte in LC ESI MS Mode Use the first method for tuning if you intend to use an experiment type at a low flow rate involving the syringe pump The second method is useful if you intend to use an experiment type at a higher flow rate involving the LC However the second method of introduction puts a comparatively large amount of analyte into the MS detector Therefore before you can perform an anal...

Page 27: ...r each of the calibration and tuning procedures Table 4 Summary of methods of sampleintroduction for calibration and tuning Calibrating Tuning Sample Sample Intro Check Auto Semi auto Auto Semi auto Manual Collision Energy Calibration solution Syringe pump 9 9 9 9 9 9 9 Your tune solution Syringe pump 9 9 9 9 Your tune solution Syringe pump into LC flow by using Tee union 9 9 9 9 ...

Page 28: ...r MSn experiment template see Figure 1 In a General MS experiment you need to specify the mass range of your analyte s of interest In a General MS MS experiment you need to specify a parent precursor ion that fragments into distinctive product ions In a General MSn experiment you need to specify the mass to charge ratios of all the parent ions of interest The LTQ XL MS detector can then collect da...

Page 29: ...ector can be used to confirm structures of compounds generated with other LTQ XL systems Data Dependent Experiments A Data Dependent experiment is best used for the qualitative analysis of unknown compounds for structure elucidation or confirmation The LTQ XL MS detector uses the information in a data dependent experiment to make decisions about the next step of the experiment automatically withou...

Page 30: ... decisions about what the next step in the experiment should be to collect even more data For example in a data dependent Triple Play experiment for a mixture of compounds the LTQ XL MS detector can decide which parent ion to isolate the charge state of the parent ion and the molecular mass of the compound Ion Mapping experiments can be data dependent The Total Ion Map Neutral Loss Ion Map and Par...

Page 31: ...about your compound you can set up the parameters of a data dependent experiment so that if the intensity of the ion signal is above a specified threshold the LTQ XL MS detector generates product spectra Parameters that you might specify for example include threshold values for the intensity of the MS or MSn ion signal Whatever threshold values you choose should accomplish the isolation of your pa...

Page 32: ...h purity compounds Data Dependent MS MS Identify metabolites in a complex mixture Chromatographic Separation with Data Dependent MS MS Build a custom library of composite MSn spectra Ion Tree You can use a Data Dependent MSn experiment to identify process impurities In the quality assurance process for aspirin for example the LTQ XL MS detector can identify impurities of less than 0 1 A Data Depen...

Page 33: ...uct ion scans The LTQ XL MS detector includes the following Ion Mapping templates in Instrument Setup so you can get started with an Ion Mapping experiment Total or full scan Ion Map Neutral Loss Ion Map Parent Ion Map These Ion Mapping experiments in general require that sample solution enter the MS Detector at a composition that is constant throughout Therefore you use infusion to introduce your...

Page 34: ...ermine which parent ions are related to specific product ions For example you can map the spectral peaks in a mass range from m z 400 to m z 2000 and specify to scan for MS MS product ions in incremental steps of every mass to charge ratio every fifth mass to charge ratio or every tenth mass to charge ratio A Neutral Loss Ion Mapping Experiment collects scans for masses that have lost neutral frag...

Page 35: ...Dependent Zoom Map is an Ion Mapping experiment that collects ZoomScan data on every scan interval in a mass range that you specify as well as Data Dependent MS MS product spectra on every mass above an intensity threshold The results of any of the Ion Mapping experiments can be viewed in the Xcalibur Qual Browser window Ion Tree Experiments In an Ion Tree experiment the LTQ XL MS detector can col...

Page 36: ...ptions that prioritize how the LTQ XL MS detector gathers information depth focus and breadth focus Depth focus characterizes an ion by performing a series of MSn level fragmentations for example MS MS MS3 MS4 etc before characterizing the next most intense ion in the MSn series Breadth focus characterizes all ions to the same MSn level before advancing to the next MSn level For example if you spe...

Page 37: ...vel of MS3 from this parent ion This process is then repeated for the second most intense ion in the MS spectrum If you chose the breadth focus after the most intense ion of the MS spectrum is fragmented producing an MS MS spectrum the LTQ XL MS detector selects and fragments the second most intense ion of the same MS spectrum The fragmentation of parent ions continues to the Max Breadth level tha...

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Page 39: ...and calibrating your LTQ XL MS detector You tune and calibrate the MS detector in the ESI mode before you acquire data in either the ESI or APCI mode This chapter contains the following topics Placing the LC MS System in Standby Removing the APCI Probe Removing the Ion Max Ion Source Housing optional Installing the Ion Sweep Cone optional Installing the Ion Max Ion Source Housing Installing the ES...

Page 40: ... Direct Control dialog box e In the Direct Control dialog box click the Pump Off button to stop the MS pump 2 If Tune Plus is not already open choose Start Programs Xcalibur LTQ XLTune from the taskbar to open Tune Plus You can determine the state of the MS detector by observing the state of the On Standby button on the Control Scan Mode toolbar The three different states of the On Standby button ...

Page 41: ...ve the auxiliary gas line green colored fitting from the APCI probe Figure 5 4 Remove the sheath gas line blue colored fitting from the APCI probe Note The following procedures assume that you are familiar with your instrument and software If you need additional guidance refer to LTQ XL online Help LTQ XL Getting Connected Ion Max API Source Hardware Manual or the LTQ XL Hardware Manual CAUTION AV...

Page 42: ...pin on the probe manifold will prevent you from rotating the probe until the pin is aligned with the slot in the API interlock block Once the probe is all the way back and aligned with the slot turn the probe 45 degrees counter clockwise to free the probe from the alignment notch d Pull the probe straight out to remove it from the ion source housing e Store the APCI probe in its original shipping ...

Page 43: ...g the 8 kV cable from the corona needle high voltage receptacle 7 Remove the corona needle as follows a Unlock the ion source housing door by turning the locks 90 degrees so that the knobs are horizontal b Open the ion source housing door c Using pliers grasp the corona needle and pull it straight out of the corona needle contact See Figure 7 ESI Interlock Socket API Interlock Block Probe Locking ...

Page 44: ...al shipping container The APCI probe and the corona needle are now properly removed from the Ion Max ion source housing If you want to install the optional ion sweep cone go to the next section Removing the Ion Max Ion Source Housing If you do not want to install the ion sweep cone go to Installing the ESI Probe on page 39 Figure 7 Corona needle view from rear Corona Needle Contact Corona Needle g...

Page 45: ...ng locking levers 90 degrees to release the ion source housing from the ion source mount assembly 3 Remove the ion source housing by pulling the housing straight off of the ion source mount assembly 4 Place the ion source housing in a safe location for temporary storage The Ion Max ion source housing is now properly removed Figure 8 Ion Max ion source housing detail of components Note If an ion so...

Page 46: ...be free of contaminants The net result is a significant increase in the number of samples that can be analyzed without a loss of signal intensity In addition keeping the ion transfer tube entrance cleaner reduces the need for frequent MS detector maintenance Install the ion sweep cone 1 Remove the ion sweep cone from its storage container Inspect and clean it if necessary 2 Note the location of th...

Page 47: ...into position 4 If necessary to achieve a proper ion sweep cone installation you might adjust the set screws around the perimeter of the ion sweep cone The ion sweep cone is now properly installed on the MS detector Gas Inlet CAUTION AVOID BURNS At operating temperatures the ion transfer tube can severely burn you The ion transfer tube typically operates between 200 and 400 C Always allow the ion ...

Page 48: ...ng Reinstall the Ion Max ion source housing 1 Carefully align the two guide pin holes on the rear of the ion source housing with the ion source housing guide pins on the MS detector and carefully press the ion source housing onto the ion source mount See Figure 11 and Figure 12 Figure 11 Rear view of the Ion Max ion source housing Guide Pin Holes Ion Source Housing Drain Ion Source Housing Locking...

Page 49: ...ing ion source housing guide pins 2 Rotate the ion source housing locking levers 90 degrees to lock the ion source housing onto the ion source mount assembly 3 Reinstall the ion source drain tube as follows Ion Source Housing Guide Pins CAUTION Prevent solvent waste from backing up into the ion source and MS detector Always ensure that liquid in the drain tube is able to drain to a waste container...

Page 50: ...on the MS detector CAUTION Do not vent the API source drain tube or any vent tubing connected to the waste container to the same fume exhaust system to which you have connected the forepumps The analyzer optics can become contaminated if the API source drain tube and the blue forepump exhaust tubing are connected to the same fume exhaust system CAUTION Your laboratory must be equipped with at leas...

Page 51: ...the guide pin on the probe body at a minus 45 degree angle from the API interlock block See Figure 14 Figure 13 Ion Max ion source housing probe locking lever open Note If your ESI probe does not already have a sample tube fused silica capillary or metal needle and safety sleeve attached follow the procedure for installing a sample tube and PEEK safety sleeve that is outlined in Installing a New F...

Page 52: ...the pin with the alignment notch at the rear of the port push the probe straight in until the guide pin stops at the bottom of the alignment notch 6 Lock the probe in place by rotating the probe locking lever towards the front of the housing closing the probe locking lever towards the rear of the ion source housing might make it difficult to unlock You might first need to tighten the locking lever...

Page 53: ...ure 15 10 Connect the auxiliary gas fitting green to the auxiliary gas inlet A on the probe See Figure 15 11 Connect the 8 kV cable to the ESI needle high voltage receptacle on the ESI probe Tighten the locking ring on the 8 kV connector 12 Connect the sample transfer tubing to the grounding union The ESI probe is now properly installed in the Ion Max ion source housing Leave the LC MS system in S...

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Page 55: ...MS detector automatically in the ESI MS mode you do the following Infuse a low concentration calibration solution containing caffeine MRFA and Ultramark 1621 into the ESI source by using the syringe pump Refer to the section Setting Up the Syringe Pump for Tuning and Calibration Test the efficiency and stability of the spray of calibration solution into the MS detector You can observe the followin...

Page 56: ...ESI MS Mode 44 LTQ XL Getting Started Thermo Electron Corporation Tuning the MS Detector Automatically in the ESI MS Mode Saving Your ESI MS Tune Method Calibrating the MS Detector Automatically Cleaning the MS Detector after Tuning and Calibrating ...

Page 57: ...nd a brown ferrule to the black LC union See Figure 16 Figure 16 Plumbing connections for the syringe 2 Load a clean 500 μL Unimetrics syringe with 450 μL of the calibration solution Refer to Appendix A Sample Formulations on page 139 for a procedure for making the calibration solution 3 Insert the syringe needle into the segment of Teflon tube 4 Place the syringe into the syringe holder of the sy...

Page 58: ...nd a brown ferrule to the free end of the LC union b Connect the other end of the infusion line with a red fingertight fitting and a brown ferrule to the grounding union The syringe pump is now properly set up for infusing solution into the MS detector Go to the next section Setting Up the MS Detector in the Xcalibur Data System for Tuning and Calibration Figure 17 ESI MS plumbing connections for ...

Page 59: ...dows XP task bar as follows a Choose Start Programs Xcalibur Xcalibur to display the Xcalibur Home Page Roadmap view b Click the Instrument Setup button to display the Instrument Setup window c Click the Finnigan LTQ XL button to display the New Method page d Click the Tune Plus button to display the Tune Plus window See Figure 18 2 In the Tune Plus window on the Control Scan Mode toolbar click th...

Page 60: ...ron Corporation Off mode and turn it On When you turn the MS detector to On you initiate the following events The MS detector begins scanning Nitrogen flows into the ESI probe The LTQ XL MS detector applies high voltage to the ESI probe The Xcalibur data system shows a real time display in the Spectrum view Figure 18 Tune Plus window showing the MS detector in the Standby mode ...

Page 61: ... Plus downloads the Tune Method parameters to the MS detector 4 Examine the pre tune ESI source settings as follows a From the Instrument Setup toolbar click the API Source button to open the ESI Source dialog box Verify that the settings in your dialog box are the same as those shown in Figure 19 b Click OK to return to the Tune Plus window Figure 19 ESI Source dialog box showing the settings to ...

Page 62: ...on b In the Scan Description group box in the Mass Range list box select Normal to allow for a selection of mass ranges between m z 150 to 2000 c In the Scan Rate list box select Normal to specify a normal scan rate d In the Scan Type list box select Full specify a full scan e In the Scan Time group box in the Microscans spin box enter 1 to set the total number of microscans to 1 f In the Max Inje...

Page 63: ...set the last mass for the scan range to m z 2000 k Ensure that the settings in your Define Scan dialog box are the same as those shown in Figure 20 l Click OK to apply the MS detector scan parameters and to close the Define Scan dialog box 6 On the Control Scan Mode toolbar click the Centroid Profile button to toggle the data type to profile The picture on the button should be the same as that sho...

Page 64: ... detector Test the operation of the MS detector in the ESI MS mode 1 Click the Syringe Pump button to display the Syringe Pump dialog box See Figure 21 Figure 21 Syringe Pump dialog box 2 Turn on the syringe pump and set an infusion flow rate of 5 μL min as follows a In the Flow Control group box click the On option button to make active the Flow Rate spin box b Type 5 in the Flow Rate spin box to...

Page 65: ...yringe parameters start the syringe pump and close the Syringe Pump dialog box 3 On the File Display toolbar click the Display Spectrum View button to ensure that the Spectrum view is displayed 4 Monitor the data for the calibration solution as follows a In the Spectrum view of the Tune Plus window observe the mass spectra of the singly charged ions of calibration solution The ions are as follows ...

Page 66: ... step 4 d then your MS detector is operating properly If you answered no to either of these questions try the following troubleshooting measures Ensure that the fused silica sample tube does not extend beyond the tip of the ESI needle Ensure that the entrance to the ion transfer capillary is clean and is not covered with a piece of septum Ensure that the solution entering the probe is free of air ...

Page 67: ...peration of the MS Detector in the ESI MS Mode Thermo Electron Corporation LTQ XL Getting Started 55 Figure 22 Spectrum view of the Tune Plus window showing ionization time IT and normalization level NL of the calibration solution IT ionization time NL normalization level ...

Page 68: ...I MS mode to optimize important parameters including heated capillary voltage and tube lens voltage Tune the MS detector automatically 1 On the Control Scan Mode toolbar click the Tune button to display the Tune dialog box 2 If necessary click the Automatic tab to display the Automatic tuning page See Figure 23 3 In the What to Optimize On group box select the Mass option button to make active the...

Page 69: ...automatic tuning is in progress the LTQ XL MS detector displays various tests in the Spectrum and Graph views in Tune Plus and displays various messages in the Status group box in the Tune dialog box Your Tune Plus window should now look similar to the one shown in Figure 24 8 Click the ESI Source dialog box to examine the ESI source parameters after tuning Compare the settings shown in Figure 25 ...

Page 70: ...Automatically in the ESI MS Mode Tuning the MS Detector Automatically in the ESI MS Mode 58 LTQ XL Getting Started Thermo Electron Corporation Figure 24 Tune Plus window showing the results of a typical automatic tune procedure ...

Page 71: ...e ESI MS Mode Thermo Electron Corporation LTQ XL Getting Started 59 Figure 25 ESI Source dialog box showing typical parameters after automatic tuning Figure 26 Ion Optics dialog box showing examples of voltages of lenses and Intermultipoles which are optimized by the LTQ XL automatic tuning procedure ...

Page 72: ...btained using calibration solution You can recall the Tune Method and use it as a starting point for optimizing the MS detector on a different analyte of interest or at a different flow rate Save your ESI MS Tune Method for low flow operation when automatic tuning is complete 1 Choose File Save As to display the Save As dialog box See Figure 27 Figure 27 Save As dialog box showing files in the fol...

Page 73: ...tion LTQ XL Getting Started 61 3 Click the File Name text box and type ESImyTune to name the Tune Method ESImyTune LTQTune 4 Click Save to save the Tune Method and return to the Tune Plus window Note that the Tune Method is named ESImyTune LTQTune Once you have tuned the MS detector you are now ready to calibrate ...

Page 74: ...tically from the Tune Plus window 1 Choose Control Calibrate to display the Calibrate dialog box 2 If necessary click the Automatic tab to display the Automatic calibration page See Figure 28 Figure 28 Calibrate dialog box showing the Automatic calibration page 3 Start the automatic calibration procedure as follows a Click Start A message box displays the following message Please ensure that the 5...

Page 75: ...calibrating on that item again after you ensure the following the spray is stable the solution flow rate is sufficient and all the ions in the calibration solution are present with adequate signal to noise ratios If the sensitivity of the ions is low increase the solution flow rate somewhat and then use the semi automatic calibration procedure to calibrate the specific parameter that failed See Fi...

Page 76: ...in the ESI MS Mode Calibrating the MS Detector Automatically 64 LTQ XL Getting Started Thermo Electron Corporation Figure 29 Tune Plus window with Calibrate dialog box showing the results of a successful semi automatic calibration procedure ...

Page 77: ...ge pump handle to free the syringe b Remove the syringe from the holder c Disconnect the tip of the syringe needle from the Teflon tubing 3 Clean the syringe thoroughly as follows a Clean the syringe with a solution of 5 formic acid in water b Rinse the syringe with a solution of 50 50 methanol water c Use acetone to rinse the syringe Repeat this step several times 4 To gain access to the ion tran...

Page 78: ...hly with a solution of 5 formic acid in water or with another appropriate solvent as follows a Fill a clean 250 μL Unimetrics syringe with a solution an appropriate solvent b While holding the plunger of the syringe in place carefully insert the needle of the syringe into the free end of the Teflon tube c Flush the sample transfer line sample tube and ESI probe with the solution by slowly depressi...

Page 79: ...ray shield c Use the spray bottle to flush contaminants from the exterior surface of the spray shield d Remove the Kimwipe you used to absorb the solution Swab the surface of the spray shield with a dry Kimwipe e Repeat step 14 a through step 14 d with acetone to remove the high molecular weight Ultramark 1621 15 Being careful not to touch the ion transfer capillary with your hand remove the septu...

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Page 81: ...t parameters that interact with the ESI interface and signal quality are as follows Electrospray voltage Heated capillary temperature voltage Tube lens voltage Capillary voltage Sheath gas flow rate Auxiliary gas flow rate Sweep gas flow rate The settings for these parameters depend on the solvent flow rate and target analyte composition In general you should fine tune your MS detector whenever yo...

Page 82: ...C with a Tee union Optimize the MS detector parameters for your analyte while the solution flows into the MS detector This chapter contains the following topics Setting Up to Introduce Sample by Syringe Pump into Solvent Flow from an LC Setting Up to Tune the MS Detector with Your Analyte Optimizing the MS Detector Tune Automatically with Your Analyte Saving the ESI MS Tune Method Note 1 If your e...

Page 83: ... fits readily into the opening in the free end of the Teflon tubing If necessary you can enlarge the opening in the end of the tubing slightly 4 Place the syringe into the syringe holder of the syringe pump 5 While squeezing the blue release buttons on the syringe pump handle push the handle forward until it just contacts the syringe plunger 6 Connect the fused silica infusion line from the black ...

Page 84: ...le inlet as described in the section Installing a New Stainless Steel Needle in the ESI Probe and Installing a New Fused Silica Sample Tube and PEEK Safety Sleeve in the Ion Max API Source Hardware Manual 9 Connect an appropriate length of PEEK tubing transfer line from the divert inject valve from the divert inject valve to the free end of the black LC Tee union as follows a Connect a length of P...

Page 85: ...ows a Connect a length of PEEK tubing with a stainless steel nut and a stainless steel ferrule to port 2 of the divert inject valve b Connect the other end of the PEEK tubing with a proper fitting and a ferrule to the outlet of the LC 11 Connect an appropriate length of PEEK tubing waste line from the divert inject valve to a waste container as follows a Connect a length of PEEK tubing with a stai...

Page 86: ...tton to take the MS detector out of Standby mode and turn it On The MS detector begins scanning the LTQ XL MS detector applies high voltage to the ESI probe and the LTQ XL MS detector shows a real time display in the Spectrum view 3 Open the ESImyTune LTQTune Tune Method the Tune Method you saved in Chapter 3 as follows a On the File Display toolbar click the Open File icon to display the Open dia...

Page 87: ...x type 200 000 to specify a 200 ms maximum injection time g In the Scan Ranges group box in the Input list box select Center Width to make available the Center Mass and Width text boxes in the Scan Ranges table h In the Source Fragmentation group box confirm that the On check box is not selected to specify that the ion source fragmentation option is turned off i In the Scan Ranges group box Scan R...

Page 88: ...quiring reserpine data of the SIM type 5 On the Control Scan Mode toolbar click Centroid Profile to toggle the data type to profile The picture on the button should be the same as that shown here 6 Click Positive Negative to toggle the ion polarity mode to positive The picture on the button should be the same as that shown here You have completed setting up to tune your MS detector with your analy...

Page 89: ...e Refer to Table 2 for guidelines about setting flow rates and temperatures Optimize the MS detector Tune Method 1 On the Control Scan Mode toolbar click the Tune button to display the Tune dialog box 2 If necessary click the Automatic tab to display the Automatic tuning page See Figure 33 3 In the What to Optimize On group box select the Mass option button to make active the Mass spin box 4 In th...

Page 90: ... Thermo Electron Corporation Ensure the syringe pump contains at least 450 μL of the 125 fg μL reserpine tuning solution b Click OK to close the message box and return to the Tune Plus window 7 In the File Display toolbar click the Graph View button to display the Graph view Figure 33 Tune dialog box showing the Automatic tuning page ...

Page 91: ...Status group box in the Tune dialog box Your Tune Plus window should now look similar to the one shown in Figure 35 You have now successfully tuned the MS detector in ESI MS mode for the compound reserpine or your analyte of interest Note The most important parameters that affect the signal quality during ESI MS operation are electrospray voltage ion transfer capillary temperature heated capillary...

Page 92: ...yte in LC ESI MS Mode Optimizing the MS Detector Tune Automatically with Your Analyte 80 LTQ XL Getting Started Thermo Electron Corporation Figure 35 Tune Plus window with the Tune dialog box showing the Automatic tuning page ...

Page 93: ...he Save As dialog box See Figure 36 Figure 36 Save As dialog box showing files in the folder C Xcalibur methods 2 Select the C Xcalibur methods folder 3 Click the File Name text box and enter reserpine or the name of your analyte of interest 4 Click Save to save the Tune Method and return to the Tune Plus window Note that the Tune Method is named reserpine LTQTune Note Save the Tune Method while t...

Page 94: ...The Tune Method is now properly saved and you are ready to acquire data on your analyte of interest ...

Page 95: ...p to Acquire MS MS Data in the Full Scan Type Setting Up to Introduce Sample by Loop Injection into Solvent Flow from an LC Acquiring MS Data in the SIM Scan Note The following procedures assume that you are familiar with your LTQ XL instrument and the Tune Plus window If you need information refer to the LTQ XL online Help LTQ XL Getting Connected and or the LTQ XL Hardware Manual Ensure that you...

Page 96: ...imize the Collision Energy Optimizing the Collision Energy Automatically for an MS MS Experiment Optimizing the Isolation Width and Setting Up to Optimize the Collision Energy Optimize the isolation width and set up to optimize the collision energy for an MS MS experiment 1 If you have not already done so from the Tune Plus window click the On Standby button to take the MS detector out of Standby ...

Page 97: ...me you might want to turn on your LC pump and specify a flow rate of 0 4 mL min for example to ensure that your system does not leak 5 Click the Syringe Pump button to display the Syringe Pump dialog box See Figure 38 6 Turn on the syringe pump and set an infusion flow rate of 5 μL min as follows a In the Flow Control group box click the On option button to make active the Flow Rate spin box b Typ...

Page 98: ...e list to set the proper syringe volume Note that if you are using a Unimetrics syringe the LTQ XL MS detector automatically sets the syringe ID to its proper value of 3 260 mm d If you are not using a Unimetrics or Hamilton syringe set up the syringe parameters as follows i In the Type group box click the Other option button to make active the syringe ID spin box ii Enter the inner diameter of yo...

Page 99: ...ialog box in the MSn Settings group box in the Normalized Collision Energy box type 20 to specify an initial value of 20 for the collision energy Click Apply 9 In the Tune Plus window observe the mass spectrum of the product ions of reserpine or your analyte of interest If necessary increase the value for the Normalized Collision Energy in increments of 5 to cause the clear display of product ion ...

Page 100: ...e button to display the Tune dialog box 11 In the Tune dialog box click the Collision Energy tab to display the page See Figure 40 Figure 39 Define Scan dialog box showing typical settings for acquiring MS MS data in the Full scan type on reserpine 12 Click the Product Ion Mass option button to make active the spin box Type 397 2 to specify the product ion at m z 397 2 for reserpine The LTQ XL MS ...

Page 101: ... optimum relative collision energy is the one that produces the maximum product ion intensity Automatically optimize the relative collision energy for the ESI MS MS analysis of reserpine or your analyte of interest 1 In the Tune dialog box on the Collision Energy page Figure 40 click Start to start the optimization procedure A message box displays the following message Ensure that the 500 microlit...

Page 102: ...pine or that of your analyte of interest 4 When the collision energy is optimized the Accept Optimized Value dialog box appears See Figure 42 5 Click Accept to accept the new collision energy value and return to Tune Plus The new value is displayed in the Define Scan dialog box 6 In the Syringe Pump dialog box select the Off option button to turn off the syringe pump Click Close to close the Syrin...

Page 103: ... Window Setting Up to Acquire MS MS Data in the Full Scan Type Thermo Electron Corporation LTQ XL Getting Started 91 Figure 41 Tune Plus window showing MS MS product ions of reserpine in the Spectrum view Figure 42 Accept Optimized Value dialog box ...

Page 104: ...pillary tube from the grounding union to the ESI probe sample inlet as described in the section Installing a New Stainless Steel Needle in the ESI Probe and Installing a New Fused Silica Sample Tube and PEEK Safety Sleeve in Ion Max API Source Hardware Manual 2 Connect an appropriate length of red PEEK tubing transfer line from the divert inject valve from the divert inject valve to the stainless ...

Page 105: ...a length of the PEEK tubing with a stainless steel nut and stainless steel ferrule to port 2 of the divert inject valve b Connect the other end of the PEEK tubing with a proper fitting and a ferrule to the outlet of the LC 5 Connect an appropriate length of PEEK tubing waste line from the divert inject valve to a waste container as follows a Connect a length of PEEK tubing with a stainless steel n...

Page 106: ...n the Spectrum view 2 Verify that the Centroid data type is selected The picture on the button should be the same as that shown here 3 Verify that the scan parameters are defined to acquire SIM data for reserpine or your analyte of interest as follows a Click the Define Scan button to open the Define Scan dialog box See Figure 45 b Compare the values in your dialog box to those in Figure 45 and cl...

Page 107: ... c Type a comment about your experiment For example describe the scan mode scan type ionization mode sample amount or method of sample introduction The Xcalibur data system includes the comment on hard copies of your data d In the Acquire Time box select the Continuously option button to acquire data continuously until you stop the acquisition 7 Leave the Acquire Data dialog box open during data a...

Page 108: ... μL injector loop with the 125 fg μL solution of reserpine or a solution of your analyte of interest 11 Select the Inject option button to inject the reserpine solution into the ESI source Leave the Divert Inject Valve dialog box open 12 Observe the reserpine peak m z 609 2 or that of your analyte of interest in the Spectrum view See Figure 48 Wait about 1 min before you inject again step 13 b bel...

Page 109: ...rpine b Select the Inject option button to inject the reserpine solution into the ESI source and then observe the Spectrum view c Wait 1 min before performing the next injection d Perform step 13 a through step 13 c three more times 14 Click Close in the Divert Inject Valve dialog box to return to the Tune Plus window 15 Click Stop in the Acquire Data dialog box to end the data acquisition Click C...

Page 110: ...lus window showing the SIM spectrum of reserpine during analysis by loop injection Review the mass spectrum and chromatogram in the Xcalibur Qual Browser window See Figure 49 For more information about reviewing the data you acquired using the LTQ XL MS detector with the Xcalibur data system refer to the Xcalibur Getting Productive Qualitative Analysis manual ...

Page 111: ...in the SIM Scan Thermo Electron Corporation LTQ XL Getting Started 99 Figure 49 Qual Browser window showing loop injections of reserpine in the Chromatogram view left and showing m z 609 in the Spectrum view Note that the injections occur at intervals of approximately 1 min ...

Page 112: ...5 Acquiring ESI Sample Data Using the Tune Plus Window Acquiring MS Data in the SIM Scan 100 LTQ XL Getting Started Thermo Electron Corporation ...

Page 113: ...pter provides information on setting up the ion source for acquiring data in the APCI MS MS mode This chapter contains the following sections Removing the ESI Probe Removing the Ion Max Ion Source Housing Removing the Ion Sweep Cone optional Installing the Corona Needle Installing the Ion Max Ion Source Housing Installing the APCI Probe ...

Page 114: ...ct Control dialog box ii In the Direct Control Panel group box click the Pump Off button to stop the flow of solvent b Click On Standby on the Control Scan Mode toolbar to place the MS detector in Standby 2 Disconnect the sample transfer tubing from the stainless steel ZDV fitting grounding union See Figure 50 3 Remove the 8 kV cable from the ESI needle high voltage receptacle shown in Figure 50 a...

Page 115: ...from the grounding bar on the ion source housing 7 Unlock the probe locking lever by rotating the lever open to its widest position 8 Carefully pull the probe straight back in the port in the housing until it meets with the slot in the API interlock block The guide pin on the probe manifold will prevent you from rotating the probe until the pin is aligned with the slot in the API interlock block O...

Page 116: ...ation counter clockwise to free the probe from the alignment notch Be careful not to break the fused silica sample tube or PEEK safety sleeve 9 Pull the probe straight out to remove it from the ion source housing 10 Store the ESI probe in its original shipping container The ESI probe is properly removed from the Ion Max ion source housing ...

Page 117: ...source housing from the ion source mount assembly 3 Remove the ion source housing by pulling the housing straight off of the ion source mount assembly 4 Place the housing in a safe location for temporary storage The Ion Max ion source housing is now properly removed If you want to remove the ion sweep cone go to the next section Removing the Ion Sweep Cone Otherwise go to the topic Installing the ...

Page 118: ...t off of the API cone seal 3 Store the ion sweep cone in its original shipping container The ion sweep cone is now properly removed If you need to install the corona needle go to the next section Installing the Corona Needle Otherwise go to the section Installing the Ion Max Ion Source Housing on page 108 CAUTION AVOID BURNS At operating temperatures the ion transfer capillary can severely burn yo...

Page 119: ...o the needle contact in the Ion Max ion source housing Be careful not to scratch the needle See Figure 52 Figure 52 Corona needle view from inside of the Ion Max housing 2 Make sure the tip of the needle is aligned with the path of travel between the APCI probe and the ion source interface on the instrument The corona needle is now properly installed CAUTION AVOID INJURY The corona discharge needl...

Page 120: ... Reinstall the Ion Max ion source housing 1 Carefully align the two guide pin holes on the rear of the ion source housing with the ion source housing guide pins on the MS detector and carefully press the ion source housing onto the ion source mount See Figure 53 and Figure 54 Figure 53 Rear view of the Ion Max ion source housing Guide Pin Holes Ion Source Housing Drain Ion Source Housing Locking L...

Page 121: ...uid in the drain tube is able to drain to a waste container and that the outlet of the drain tube is above the level of liquid in the waste container CAUTION Do not vent the API source drain tube or any vent tubing connected to the waste container to the same fume exhaust system to which you have connected the forepumps The analyzer optics can become contaminated if the API source drain tube and t...

Page 122: ... Getting Started Thermo Electron Corporation a Connect the 1 in ID Tygon tubing to the ion source housing drain fitting b Attach the free end of the hose to a waste container Ideally the waste container should be vented to a fume exhaust system The Ion Max ion source housing is now properly installed on the MS detector ...

Page 123: ...nstall the probe 3 Insert the APCI probe into the port in the ion source housing align the guide pin on the probe body at a 45 degree angle from the API interlock block See Figure 55 4 Push the probe into the port until the guide pin meets with the probe collar on the ion source housing 5 Turn the probe 45 degrees clockwise and align the guide pin with the slot in the API interlock block you might...

Page 124: ...porizer heater cable from the ESI interlock plug on the ion source housing 9 Connect the vaporizer heater cable to the vaporizer heater cable socket on the APCI probe 10 Connect the sheath gas fitting blue to the sheath gas inlet S on the probe See Figure 55 11 Connect the auxiliary gas fitting green to the auxiliary gas inlet A on the probe Figure 55 12 Connect the sample transfer line to the APC...

Page 125: ...LC MS system in Standby and go to Chapter 7 Optimizing the MS Detector with Your Analyte in APCI MS Mode CAUTION Prevent solvent waste from backing up into the ion source and MS detector Always ensure that liquid in the drain tube is able to drain to a waste container and that the outlet of the drain tube is above the level of liquid in the waste container ...

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Page 127: ...be lens voltage for your particular analyte The capillary voltage and ion transfer capillary temperature may then be optimized manually to enhance ion transmission This chapter includes the following sections Setting Up the Inlet for Tuning Using High Flow Infusion Setting Up the MS Detector for APCI MS Operation Optimizing the Tune of the MS Detector Automatically in APCI MS Mode Saving the APCI ...

Page 128: ... with 450 μL of a 125 fg μL solution of reserpine or your analyte of interest See Appendix A Sample Formulations for a procedure for making the reserpine tuning solution 3 Insert the needle of a syringe into the segment of Teflon tubing and place the syringe in the syringe holder of the syringe pump 4 Connect a fused silica infusion line from the black LC union to the black LC Tee union as follows...

Page 129: ... on the APCI probe Grounding Union P N 00101 18182 LC Tee Union P N 00101 18204 From LC Union Fingertight Fittings P N 00101 18081 Stainless Steel Ferrule Infusion Line Fused Silica Capillary P N 00106 10504 PEEK Tubing P N 00301 22912 From Divert Inject Valve Ferrules P N 00101 18196 Ferrules P N 00101 18120 Stainless Steel Nut Fingertight Fitting P N 00101 18195 PEEK Tubing P N 00301 22912 Note ...

Page 130: ...Connect an appropriate length of PEEK tubing transfer line from the LC from the divert inject valve to the LC as follows a Connect a length of PEEK tubing with a stainless steel nut and a stainless steel ferrule to port 2 of the divert inject valve b Connect the other end of the PEEK tubing with a proper fitting and a ferrule to the outlet of the LC 8 Connect an appropriate length of PEEK tubing w...

Page 131: ...eal time display in the Spectrum view 2 Open the APCIhighflow LTQTune Tune Method the Tune Method for high flow APCI operation as follows a Choose File Open to display the Open dialog box b Scroll down until you see the folder C Xcalibur methods Then select the file APCIhighflow LTQTune c Click OK to open the file LTQ XL MS detector downloads the Tune Method parameters to the MS detector 3 Verify ...

Page 132: ... from the one shown in the figure it is probably because the advanced settings are not displayed You can turn on the advanced settings as follows In Tune Plus choose ScanMode and then click Advanced Scan Features to select the option b In the Scan Description group box in the Mass Range list box select Normal to allow for a selection of mass ranges between m z 150 to 2000 c In the Scan Rate list b...

Page 133: ...th to make available the Center Mass and Width text boxes in the Scan Ranges table i In the Scan Ranges group box in the Scan Ranges table in the Center Mass text box type 609 20 to set the center mass for the scan range to m z 609 20 j In the Width text box type 2 00 to set the width of the scan range to m z 2 00 k Verify that the settings in your Define Scan dialog box are the same as those show...

Page 134: ...or APCI MS Operation 122 LTQ XL Getting Started Thermo Electron Corporation 6 Click the Positive Negative button to toggle the ion polarity mode to positive The picture on the button should be the same as that shown here You have now completed setting up your MS detector for APCI MS operation ...

Page 135: ... rate for example 400 μL min See Table 3 on page 12 for guidelines about flow rates and temperatures Automatically optimize the MS detector 1 On the Control Scan Mode toolbar click Tune to display the Automatic tuning page See Figure 60 2 In the What to Optimize On group box select the Mass option button to make active the Mass spin box 3 In the Mass spin box type 609 2 to specify that you want to...

Page 136: ...omatic tuning is in progress the LTQ XL MS detector displays various tests in the Spectrum and Graph views in the Tune Plus window and displays various messages in the Status group box in the Tune dialog box Your Tune Plus window should now look similar to the one shown in Figure 61 You have now successfully tuned the MS detector in APCI MS mode for the compound reserpine or your analyte of intere...

Page 137: ...our Analyte in APCI MS Mode Optimizing the Tune of the MS Detector Automatically in APCI MS Mode Thermo Electron Corporation LTQ XL Getting Started 125 Figure 61 Tune Plus window with the Tune dialog box showing the Automatic tuning page ...

Page 138: ...obtained using reserpine You can recall the Tune Method and use it as a starting point for optimizing the MS detector on reserpine at a different flow rate Save the APCI MS Tune Method 1 Choose File Save As to display the Save As dialog box See Figure 62 Select the C Xcalibur methods folder 2 Click the File Name text box and then enter APCImyTune to name the Tune Method APCImyTune LTQTune Figure 6...

Page 139: ...ne Method Thermo Electron Corporation LTQ XL Getting Started 127 3 Click Save to save the Tune Method and close the Save As dialog box Note that the Tune Method is named APCImyTune LTQTune Before you acquire data go to the next section Cleaning the MS Detector after Tuning in APCI Mode ...

Page 140: ...from the Teflon tubing 3 Clean the syringe thoroughly as follows a Clean the syringe with a solution of 5 formic acid in water b Rinse the syringe with a solution of 50 50 methanol water c Use acetone to rinse the syringe Repeat this step several times 4 Remove the Ion Max ion source housing as described in the topic Removing the Ion Max Ion Source Housing on page 33 On Standby CAUTION Always plac...

Page 141: ...ing the syringe plunger Visually check that the solution is exiting the tip of the APCI probe on the inside of the probe assembly Use a lint free tissue to gently remove the excess solution as it exits the probe d Remove the needle of the syringe from the Teflon tube 6 Repeat step 5 with a solution of 50 50 methanol water 7 Reinstall the Ion Max ion source housing as described in Installing the Io...

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Page 143: ...lowing topics Setting Up to Introduce Sample by Loop Injection into Solvent Flow from an LC Acquiring APCI Data in the SIM Scan Mode Note The following procedures assume that you are familiar with your LTQ XL instrument and the Tune Plus window If you need information refer to the LTQ XL online Help LTQ XL Getting Connected and or LTQ XL Hardware Manual Verify that the procedures in Chapter 3 Tuni...

Page 144: ... Figure 64 2 Connect a 5 μL sample loop with stainless steel set nuts and stainless steel ferrules to ports 1 and 4 of the divert inject valve 3 Connect an appropriate length of PEEK tubing transfer line from the LC from the divert inject valve to the LC as follows a Connect a length of the PEEK tubing with a stainless steel nut and stainless steel ferrule to port 2 of the divert inject valve b Co...

Page 145: ... line from the divert inject valve to a waste container as follows a Connect a length of PEEK tubing with a stainless steel nut and stainless steel ferrule to port 6 of the divert inject valve b Insert the other end of the PEEK tubing into a suitable waste container The MS detector is now set up to introduce sample by loop injection into solvent flow from an LC Vaporizer Heater Cable Socket Guide ...

Page 146: ...Spectrum view 2 Ensure that the Centroid data type is selected The picture on the button should be the same as that shown here 3 Ensure that the scan parameters are defined to acquire SIM data for reserpine or your analyte of interest as follows a Click the Define Scan button to open the Define Scan dialog box Figure 65 b Verify that the values in your dialog box are the same as those in Figure 8 ...

Page 147: ...our experiment For example enter SIM APCI 50 pg loop to specify the scan mode ionization mode sample amount and or method of sample introduction The Xcalibur data system includes the comment on hard copies of your data d In the Acquire Time box select the Continuously option button to specify the continuous acquisition of data until you stop the acquisition 7 Leave the Acquire Data dialog box open...

Page 148: ...tarted Thermo Electron Corporation Figure 67 Tune Plus window showing the SIM spectrum of reserpine during analysis by loop injection 9 Inject the reserpine solution into the APCI source from the Instrument Setup toolbar as follows a Click Divert Inject Valve to display the Divert Inject Valve dialog box See Figure 68 ...

Page 149: ...SIM scan mode Wait about 1 min between injections a Select the Load option button to put the divert inject valve in the Load position Overfill the injector loop with the 125 fg μL solution of reserpine b Select the Inject option button to inject the reserpine solution into the APCI source Then observe the Spectrum view c Wait 1 min before the next injection d Repeat step 11 a through step 11 c thr...

Page 150: ...ctron Corporation Figure 69 Qual Browser window showing loop injections of reserpine in the Chromatogram view left and m z 609 in the Spectrum view Note If you want to acquire MS MS Full scan data in APCI mode see Setting Up to Acquire MS MS Data in the Full Scan Type on page 84 for information about setting up the LTQ XL MS detector ...

Page 151: ...s used in procedures throughout this manual include Acetic acid Acetonitrile Methanol Reserpine Formic Acid CAUTION Store and handle all chemicals in accordance with standard safety procedures The Material Safety Data Sheet MSDS describing the chemicals being used are to be freely available to lab personnel for them to examine at any time Material Safety Data Sheets MSDSs provide summarized inform...

Page 152: ...solution MRFA stock solution Ultramark 1621 stock solution Note Vials of caffeine MRFA and Ultramark 1621 are included in the API accessory kit These compounds are available from Sigma Chemical Co The Sigma caffeine Product Number is C6035 Caffeine Solution drug standard 1mg mL 5 in methanol The Sigma MRFA Product Number is M1170 MRFA Met Arg Phe Ala acetate salt To order more of these compounds f...

Page 153: ...esis write or call Lancaster Synthesis Inc P O Box 1000 Windham NH USA 03087 9977 603 889 3306 800 238 2324 in the USA Canada 44 0 1524 36101 UK International www lancastersynthesis com N P P N N P O O O O O O H CF2CF2 x H2C H CF2CF2 x H2C H CF2CF2 x H2C CH2 CF2CF2 xH CH2 CF2CF2 xH CH2 CF2CF2 xH CAUTION AVOID EXPOSURE TO POTENTIALLY HARMFUL MATERIALS Always wear protective gloves when you use solv...

Page 154: ...3 Mix the solution 5 0 nmol μL thoroughly 4 Transfer 50 μL of the 5 nmol μL solution into a clean polypropylene tube 5 Add 1 45 mL of 50 50 methanol water to the tube 6 Mix this solution 166 7 pmol μL thoroughly 7 Label the tube MRFA stock solution Stock Solution Ultramark 1621 Prepare a 10 mL stock solution of 0 1 Ultramark 1621 in acetonitrile 1 Obtain the vial of Ultramark 1621 in your accessor...

Page 155: ...1621 into the flask 4 Pipet 100 μL of glacial acetic acid into the flask 5 Pipet 5mL of acetonitrile into the flask 6 Bring the volume of the solution up to the 10 mL mark on the flask with 50 50 methanol water 7 Mix the calibration solution thoroughly 8 Transfer the solution to a clean dry vial 9 Label the vial ESI Calibration Solution and store it in a refrigerator until it is needed Note Use on...

Page 156: ...to use the Reserpine Stock solution as soon as it is made If it is necessary to store the solution keep it in a refrigerator until it is needed Reserpine Tuning Solution and Reserpine APCI Sample Solution Prepare 1 mL of either the Reserpine Tuning Solution of 10 pg μL 16 4 fmol μL step 9 or the Reserpine APCI Sample Solution of 250 fg μL 411 amol μL step 16 in 1 acetic acid 50 50 methanol water P...

Page 157: ...APCI Sample Solution continue with step 10 10 Transfer 100 μL of the 10 pg μL solution into a clean polypropylene tube 11 Add 900 μL of 1 acetic acid in 50 50 methanol water to the tube 12 Mix this solution 1 pg μL 1 64 fmol μL thoroughly 13 Transfer 100 μL of the 1 pg μL solution into a clean polypropylene tube 14 Add 300 μL of 1 acetic acid in 50 50 methanol water to the tube 15 Mix this solutio...

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Page 159: ...ns for the high mass range calibration of the LTQ XL The presentation in this Appendix assumes that you are familiar with the contents of Chapter 3 Tuning and Calibrating Automatically in the ESI MS Mode and Appendix A Sample Formulations This Appendix Contains the following topics High Mass Range Calibration Solution Normal Mass Range Calibration Enter Normal Mass Range Data into Tune Plus Tune o...

Page 160: ...call Sigma Chemical Company P O Box 14508 St Louis Missouri USA 63178 9916 800 325 3010 in the USA 905 829 9500 in Canada 314 771 3750 outside the USA or Canada www sigmaaldrich com CAUTION Store and handle all chemicals in accordance with standard safety procedures The Material Safety Data Sheets MSDS describing the chemicals being used are to be freely available to lab personnel for them to exam...

Page 161: ...SI MS Mode to perform the normal mass range calibration The calibration solution contains caffeine as the low molecular mass component MRFA as the mid molecular mass component and Ultramark 1621 as the high molecular mass component Four pieces of information from this calibration are needed for the high mass range calibration 1 The expected low mass caffeine component 195 10 2 The expected high ma...

Page 162: ... Plus window open the Diagnostics dialog box Diagnostics Diagnostics shown in Figure 70 Figure 70 Tune Plus Diagnostics dialog box normal mass range data 3 Click Tools in the upper left of the window Click on Mass Calibration in the pane below the Tools button as shown in Figure 70 4 Items mentioned in step 5 through step 7 below are inside of the Manual Coarse Calibration group box 5 In the Calib...

Page 163: ... respective text boxes These latter values are obtained as discussed in the preceeding section Normal Mass Range Calibration After entering the observed mass values click the Execute button 6 In the Estimate Other Calibration Modes Using Current Full Scan box click Execute 7 Click Save at the bottom right of the Manual Coarse Calibration window 8 Click OK at the bottom of the Diagnostics dialog bo...

Page 164: ... necessary click on the Automatic tab to display the Automatic tuning page See Figure 71 3 In the What to Optimize On group box select the Mass option button to make active the Mass spin box 4 In the Mass spin box type 524 3 to specify that the LXQ MS detector uses the peak at m z 524 3 to optimize your tune Figure 71 Tune window showing the MS detector Tune on m z 524 3u 5 Click Start A message b...

Page 165: ...ge Calibration on page 149 6 Click OK to close the message box and return to the Tune Plus window 7 In the File Display toolbar click the Graph View button to display the Graph view 8 Observe the Tune Plus window and the Tune dialog box While automatic tuning is in progress the LXQ MS detector displays various tests in the Spectrum and Graph views in the Tune Plus window and displays various messa...

Page 166: ...te spin box and c choose Full in the Scan Type spin box In the Scan Ranges box type 2000 for the First Mass and 4000 for the Last Mass Click Apply The spectrum should look like that shown in Figure 72 3 Do a two point mass calibration Use the Tune Plus Diagnostics dialog box Figure 73 Access the Diagnostics dialog box as instructed in Enter Normal Mass Range Data into Tune Plus on page 150 Type th...

Page 167: ...B High Mass Range Calibration Procedure Thermo Electron Corporation LTQ XL Getting Started 155 Figure 73 Tune Plus Diagnostics dialog box two point high mass calibration ...

Page 168: ... dialog box 5 For Mass Range select the High button upper right of the Calibrate dialog box The High Mass Range Calibration tabbed page appears as shown in Figure 74 6 In the What To Do box select the Calibrate button Type a calibration mass list name in the Name list box inside the Calibration Mass List group box ...

Page 169: ...ult PPG2700 name that is in the Name list box or you may assign a new name for this mass list b To assign a new name click on the Save As button The Unique Mass List Name dialog box appears c Type the new name in the Unique Mass List Name dialog box and click OK to accept the name The dialog box closes and the new name appears in the calibration mass list Name list box d Do not change the masses i...

Page 170: ...gress the Spectrum and Graph views display a variety of test results and the Status pane in the Calibrate dialog box displays status messages The system will automatically use the average mass values for the Turbo Scan calibration Monoisotopic masses will automatically be used for the normal Zoom Scan and isolation waveform calibrations 13 Observe the PPG2700 calibrations are complete it may be ne...

Page 171: ... Scan Enhanced Scan Isolation Waveform and High Mass Isolation Waveform calibrations This button changes when the High Mass Scan mode is selected In this case clicking on the Execute button causes the High Mass Isolation to be estimated from the Current Normal Mass Range Isolation calibration assuming that it has been calibrated This feature has been added to extrapolate the High Mass Isolation ca...

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Page 173: ...displaying 62 figure 64 messages in Status box 63 calibration discussion 13 ions 53 LTQ XL setup 47 procedure 62 solutions caution 74 preparing 143 syringe pump setup for 45 calibration solutions preparation 143 CAUTIONS avoiding burns from APCI vaporizer 29 30 66 128 from ion sweep cone 106 106 from ion transfer capillary 35 66 avoiding injury from corona discharge needle 31 107 Cautions avoiding...

Page 174: ...on Max ion source discussion 4 installing 108 removing 33 105 Ion Optics dialog box displaying 57 ion sweep cone avoiding burns from CAUTION 106 106 description 34 installing 34 removing 106 ion transfer capillary avoiding burns from CAUTION 35 66 106 cleaning 66 discussion 11 70 flushing 67 gaining access to 65 optimizing temperature of discussion 115 123 note 53 79 parameters affecting signal qu...

Page 175: ...17 placing in Standby 28 procedures acquiring data using Tune Plus 83 131 calibrating LTQ XL automatically 62 cleaning Finnigan LTQ after tuning and calibrating 128 cleaning LTQ XL after tuning and calibrating 65 installing APCI probe 111 corona discharge needle 107 ESI probe 39 Ion Max ion source 108 ion sweep cone 34 optimizing APCI tune 123 collision energy 89 ESI tune 77 isolation width 84 pre...

Page 176: ...thods opening predefined 49 74 119 Tune Plus window opening 28 47 74 placing Finnigan LTQ in Standby from 28 tuning results figure 124 using to acquire data 83 131 tuning automatically 56 discussion 13 ESI tune files note 49 LTQ XL for LC ESI MS operation 69 setup for 47 74 LTQ XL note 57 syringe pump setup for 45 voltages 56 U Ultramark 1621 cleaning spray shield of 67 contamination from in cauti...

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