Details of the operation follow a step by step protocol follows the following preparations.
Details are given elsewhere: http://www.umich.edu/~techserv/massspec/lct.pdf
Preparing the Syringe and Syringe Pump Use a 250-µL Hamilton syringe, various syringe fittings, and the a standard or sample.
1. Flush the syringe three times with methanol or a volume-to-volume mixture of 70% methanol : 30% water. 2. Load the syringe with the setup solution, and connect the Rheodyne 9013 needle port fitting to the PEEK union, finger tightening it. 3. Clip the ground cable (with the plug-in clip), located on the lower-right side of the front panel, into the syringe needle. 4. Fit the syringe into the syringe pump, and set the syringe stop accordingly
Caution: To avoid electrical shock, always ground the needle. Caution: The syringe pump includes a positive syringe stop to prevent certain syringe types from breaking. Nevertheless, as added protection against syringe breakage, set the syringe stop adjustor. This prevents the syringe plunger from traveling its full stroke inside the syringe barrel, reducing the potential for breakage.
Tuning
Tuning involves adjusting source settings, analyzer settings, and gas flows to produce optimal peak intensities. First tune then always calibrate the instrument in electrospray (ESI) mode.
Opening MassLynx and Starting the Instrument
1. Double-click the MassLynx desktop icon to open the application. The MassLynx Login dialog box appears
2. Complete the Logon Name, Password, and Domain fields. Note: The Login dialog box appears only if MassLynx security is enabled. Otherwise, the MassLynx Main window appears after clicking the MassLynx desktop icon.
3. Click OK. The MassLynx Main window appears Note: After initiating, the Main window displays "Instrument Present" in the status bar. 4. The shortcut bar should appear in the MassLynx Main window, and "Instrument" should appear at its top. If it fails to appear, click Shortcut, in the toolbar, to open it. Then click Instrument, at the left edge of the shortcut bar. 5. Select MS Tune from the Main window shortcut bar to open the Tune Window. The following paragraph describes the Tune window's ES+ Source page parameters. Note: The voltage parameters shown optimize sensitivity and stability. The temperature and flow rate parameters control the extent of solvent evaporation and adduct formation.
Source and Desolvation Temperatures
The Source and Desolvation temperature settings control desolvation for a specified flow rate. The paragraph gives temperature ranges for specific flow rates. Specifying Parameter Settings on the ES+ Source Page 1. Click (API Gas) in the Tune window to toggle the nitrogen flow to On. 2. Make sure the instrument is operating, noting whether peak activity appears in the Tune window's Peak Display area. If the instrument is not operating (indicated by no peaks), click Press for Operate. Desolvation gas flow Optimizes gas flow depends on mobile phase composition and flow rate (>100 L/hr). Cone gas flow Helps reduce adduct ions and keep the sample cone clean (50 to150 L/hr).
Attention: Toggle API gas to Off before reopening the nitrogen supply once it has been shut off. Otherwise, the sudden in-rush of gas can damage the flow meter.
3. Click the ES+ Source tab, and specify these suggested starting parameters in the corresponding fields of the ES+ Source page. 4. Select all four Peak Editor fields, and enter these mass assignment values. 5. Select Options > Syringe Type. The Syringe Selection dialog box appears.
Attention: Monitor readbacks, letting the source temperature, desolvation, gas flow, desolvation temperature, and cone gas flow reach their setpoints before proceeding. 6. Ensure the drop-down list displays Hamilton 250uL, then click OK. 7. Set the syringe pump to deliver 10 µL/min by entering that rate in the Pump Flow field of the Analyser page. 8. Click (Syringe pump) to infuse the startup solution into the source. 9. Click the Analyser tab, and ensure the syringe pump flow rate is 10 µL/min. 10. Turn the probe adjustor (see Figure 2-8) in one direction or the other to optimize peak intensities. 11. Adjust the capillary, cone, extractor, and RF lens voltages to optimize peak intensities.
Specifying Parameter Settings on the Analyser Page Analyser settings optimize mass peak resolution. With span settings at 5, the bases of mass peaks as they appear on the Tune window Analyser page should measure 1 da.
Note: The parameters LM Resolution, HM Resolution, and Ion Energy optimize resolution. 1. Click the Analyzer tab. The Tune window Analyser page appears. Affect mass peak concentration. Usually, 15, an arbitrary unit, yields adequate mass resolution. Increasing the value lowers sensitivity; decreasing it enhances sensitivity. Ion Energy (V) Decreases resolution. Set between –1 and 3 V, as low as possible without reducing peak intensity. Multiplier (V) Modifies gain (attenuation). Settings between 400 and 450 yield a suitable signal-to-noise balance. Tuning in APCI Mode. 2. Specify these suggested starting parameters in the Analyser page fields. Note: Any change of HM and/or LM Resolution after calibrating the instrument requires recalibrate. Otherwise, any data the instrument acquires could fall outside the calibration mass range.
Tuning in APCI Mode After the tune and calibrate in ESI mode, then it is possible to tune for APCI operation. This entails preparing the source, installing a tee fitting upstream of the probe, and specifying APCI parameters.
Tuning in APCI Mode
Installing the Tee Fitting -- To optimize APCI peaks, temporarily install a tee fitting to merge the sample flow from the syringe pump with the solvent flow from an HPLC pump. The combined sample/solvent stream flows into the probe.
Combined Flow into the Tee 1. Connect the HPLC pump tubing to injector port 2. 2. Connect the capillary from the syringe pump to one of the tee's three ports. 3. Connect a 1/16-in. OD × 0.007 in. ID × 1 M tube between injector port 3 and a second tee port. 4. Connect another 1/16-in. OD × 0.007 in. ID × 1 M tube from APCI probe to the tee's third port.
