Calibrating Thermo Exactive with the Direct Analysis in Real Time (DART) Ambient Ionization Source (Protocol Adapted from US FDA FCC)
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1 1.0 Equipment: Calibrating Thermo Exactive with the Direct Analysis in Real Time (DART) Ambient Ionization Source (Protocol Adapted from US FDA FCC) Thermo Exactive Orbitrap Mass Spectrometer Direct Analysis in Real Time (DART) SVP ion source Chemyx Fusion 100 syringe pump (or equivalent) 1.1 Reagents: HPLC grade methanol Caffeine, Sigma Chemical Co. (p/n C6035) or equivalent Diazinon, AccuStandard, (p/n P033S) or equivalent ESIL Low Concentration Tuning Mix, Agilent (p/n G ) o Store Refrigerated 1.2 Calibration Solution: 1. Caffeine stock solution Prepare a 1 mg/ml stock solution of caffeine as follows: a. Dissolve 10.0 mg of caffeine in 10 ml of methanol and label the vial 1 mg/ml caffeine in methanol. Store in a refrigerator until it is needed. 2. Diazinon stock solution Prepare a 1 mg/ml stock solution of diazinon as follows: a. Dissolve mg of diazinon in 10.0 ml of methanol and label the vial 1 mg/ml diazinon in methanol. Store in a refrigerator until it is needed. 3. Positive and Negative Mix Calibration Solution for DART Source Prepare 1.0 ml of the calibration solution, as follows: a. Pipette 20 µl of stock solution of caffeine into a clean, dry autosampler vial. b. Pipette 20 µl of stock solution of diazinon into the vial. c. Pipette 960 µl of the Agilent ESIL Low Concentration Tuning Mix into the vial. Mix the calibration solution thoroughly. Page 1
2 1.3 Generating a User Defined Mass Calibration List in Exactive Tune: Note: The user will need to create a user defined mass calibration list in order to calibration the Exactive using the custom calibration solution outlined in section 1.2. In order to generate a user defined mass calibration list, the Current User Role setting in the Exactive s Instrument Status window must be selected as Advanced. Page 2
3 To ensure that the instrument calibrates using the ions in the Agilent calibration mixture, go to the Instrument Status window, open Control Procedures and right click on the word User Mass List, and then select User from the dropdown menu. Page 3
4 Check also that under Calibrate that both the Pos ion mode and Neg ion mode mass calibration settings are set to User. Right click on the Mass calibration text to obtain the dropdown box to select User. Page 4
5 Confirm that the user defined mass list contains the ions from the Agilent Calibration mix (plus caffeine and diazinon) by opening the drop down menu in the Instrument Status window under Procedures, User Mass List, Positive Ions or Negative Ions. If the list still contains the ions from the Thermo Calibration mix, right click on Positive Ions (or Negative Ions ) and select Clear List. Enter the correct ions for the prepared calibration mix (listed on page 6) by right clicking on the blank space next to Peak Centroid m/z and typing the ions under Set m/z. The list is automatically saved and will not be changed unless a user changes these mass values by entering in different mass values. Page 5
6 List of ions for calibration mix used to calibrate the Exactive with DART source. Source of [MH] Positive Ion, Negative Ion, Ionic Formula m/z m/z Caffeine fragment C 6 H 8 N 3 O ND Caffeine C 8 H 11 N 4 O ND hexakis(2,2 difluoroethoxy)phosphazine C 12 H 19 O 6 N 3 P 3 F ND hexakis(1h, 1H, 3Htetrafluoropropoxy)phosphazine C 18 H 19 O 6 N 3 P 3 F ND hexakis(1h, 1H, 4Hhexafluorobutyloxy)phosphazine C 24 H 19 O 6 N 3 P 3 F ND hexakis(1h, 1H, 5Hoxtafluoropentoxy)phosphazine C 30 H 19 O 6 N 3 P 3 F ND Diazinon C 12 H 21 N 2 O 3 PS ND Trifluoroacetic acid ammonium salt C 2 O 2 F 3 ND Tris(trifluoromethyl)1, 3, 5 triazine C 6 HON 3 F 9 ND Tris(heptafluoropropyl)1,3,5 triazine C 12 HON 3 F 21 ND hexakis(1h, 1H, 3Htetrafluoropropoxy)phosphazine C 20 H 18 O 8 N 3 P 3 F 27 ND trifluoroacetate hexakis(1h, 1H, 4Hhexafluorobutyloxy)phosphazine trifluoroacetate C 26 H 18 O 8 N 3 P 3 F 39 ND Page 6
7 1.4 Exactive Calibration in Positive Mode Using the DART Ion Source Scan Range to 2,000.0 m/z Fragmentation In Source CID 30.0 ev; HCD gas on Resolution Ultra High Polarity Positive Microscans 1 Lock Masses Off ACG Target Ultimate Mass Accuracy Maximum Inject Time 1000 ms Sheath Gas Flow Rate 0 Aux Gas Flow Rate 0 Sweep Gas Flow Rate 0 Spray Voltage (kv) 0 Capillary Temperature ( o C) 300 Capillary Voltage (V) Tube Lens Voltage (V) Skimmer Voltage (V) Attach the DART source and linear rail to the front of the Exactive. Ensure the He, Vapur pump, and the DART controller box are turned on. Attach the DIPit or Tweezer module to the linear rail. Turn the DART from Off to Standby. Set the He temperature to 300ºC. See Figure 1 for the general setup. Use a 500 ul syringe and the syringe pump provided by Thermo to deliver the liquid flow to the DART source. Run the infusion line from the syringe to the holder in the DIPit or Tweezer module that is positioned in front of the DART gas beam. Position the end of the infusion line in the center of the horizontal center point, but slightly above the vertical center point and about 2 mm from the exit of the DART source. The liquid flow rate can be adjusted from 0.5 to 5.0 ul/minute through the Exactive Tune page (the flow rate may need to be varied to steady the signal; 3.0 ul/minute is a good flow to start with). The DART source is set so that there is approximately 810 mm between the DART source exit and the inlet tube in the Vapur interface. Note: The DART source position is approximately halfway between 3.5 and 4 cm on the ruler on the right side of the DART horizontal adjustment bar. Page 7
8 Figure 1. Calibration using DART ion source and DIPit module with infusion line. The Insource CID fragmentation (Xcalibur tune page) must be on in order to detect the caffeine fragment at m/z 138. A beginning value of 30 ev is recommended. The value may be varied from ev (to increase the signal). Turn the Exactive from Standby to On and turn the syringe pump on. In the DART Mozilla Firefox software, turn the DART from Standby to On (ensure that the DART is operating in the positive mode). Monitor the spectrum for the presence of the ions contained in the calibration fluid (See Table on page 6). The signal stability will not be as steady as that for the ESI source as long as all of the ions are present consistently, the positive mass calibration may be performed. If the signal is unstable (i.e. the ions are not continuously present or if the intensity is dramatically different from scan to scan), adjust the positioning of the PEEK tubing in front of the DART source exit. If this does not improve the signal quality, make sure that the entrance to the inlet is clean and unblocked and that there are no leaks or bubbles in the calibration fluid transfer line. If the variation is still too high, try adjusting the syringe pump flow rate. When the appropriate ions in the TIC are consistently present, click on the Calibrate tab in the Exactive Tune window. Check the box for Positive Mass and press Calibrate. It is typically difficult to tune with the DART, so the calibration may be completed without tuning. Page 8
9 1.5 Calibration in Negative Mode Using a DART Ion Source The negative mode only needs to be calibrated if an analysis is going to be run in negative mode. Create a new tune file by setting the values listed in the table below. Do this by clicking in each appropriate field in the Instrument tab in the Exactive Tune window, entering the value listed below, and then clicking the Apply button at the bottom of each section within the Instrument tab. Save the file as DART_Agilent_ESI_Mix_neg by clicking on the disk icon on the menu bar and selecting Save As. For all future mass calibrations in negative mode using the DART, open this tune file by clicking the folder icon on the menu bar and choosing this file. Scan Range to 2,000.0 m/z Fragmentation HCD gas on Resolution Ultra High Polarity Negative Microscans 1 Lock Masses Off ACG Target Ultimate Mass Accuracy Maximum Inject Time 1000 ms Sheath Gas Flow Rate 0 Aux Gas Flow Rate 0 Sweep Gas Flow Rate 0 Spray Voltage (kv) 0 Capillary Temperature ( o C) 300 Capillary Voltage (V) Tube Lens Voltage (V) Skimmer Voltage (V) Begin infusing the calibration solution and when the TIC variation has become stable and the desired ions are present, select the Calibrate tab in the Xcalibur Tune window and choose Negative Mass and press Calibrate. Page 9
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