Agilent 6520 LC/MS Instrument Operation Guidelines
General Instrument Guidelines
1. This instrument was purchased with Taplin funds. Please acknowledge in all
publications using the following language: LC/MS data was acquired on an Agilent
6520 Q-TOF spectrophotometer supported by the Taplin Funds for Discovery
Program.
2. The lower limit of detection in high-resolution mode is 5 pg, with a signal-to-noise
ratio of 10:1. The dynamic range is typically ~5 orders of magnitude.
3. Sample preparation, instrument setup, data acquisition, and data analysis are all
performed by users.
4. Only trained users may operate the instrument. New users should fill out a new user
form detailing their information and requested training times should be e-mailed to
jsanta@fas.harvard.edu.
5. All questions pertaining to instrument cost and finances should be directed to
Mirela_Vaso@hms.harvard.edu.
Scheduling and Calendar Signup
1. Please sign up for times on the online calendar (http://os.med.harvard.edu/cgibin/microcalendars.cgi?CalendarName=WalkerQTOF). Try to be as accurate as
possible to avoid interfering with other users’ run times.
2. Do not sign up more than 2 weeks in advance. Please be courteous to other users
when signing up for multiple times and during peak hours.
3. Users will be charged for scheduled time, even if runs are not performed. Users may
agree with each other to substitute in for times, provided they share a billing code.
4. Scheduled runs can be canceled up to 24 hours in advance to avoid being charged.
Instrument Maintenance
1. Weekly maintenance is performed by the Walker Lab to maintain instrument
integrity and sensitivity.
2. All users should immediately e-mail jsanta@fas.harvard.edu with instrument
shutdown/critical errors.
3. All users will be notified immediately if the instrument requires servicing. Users will
not be charged for runs that could not be used due to instrument downtime.
4. An email will be sent when the instrument is operational again so that users may
reschedule on the online calendar.
5. A week-long annual maintenance is scheduled during the summer and will be
announced to users in advance.
Sample Guidelines
1. DO NOT RUN a sample containing large organic cations (e.g.
Tetrabutylammonium+). They are difficult to remove from the MSD and saturate
its detecting capabilities. DO NOT RUN samples in DMSO. DO NOT RUN
extremely non-polar samples, polyaromatic hydrocarbons (PAHs), or
polychlorinated biphenyls (PCBs).
Ensure that your sample is fully dissolved. If there are solids in your sample,
filter it with a 0.2μm Nalgene filters before injection. Alternately, one can
centrifuge the sample at 700g for 1-2 minutes and extract the supernatant
fluids.
3. Ensure that selected solvents are compatible with your HPLC column.
4. Set injection volume to 3 uL. Start with a very low concentration. If you don’t
see any ion intensity, gradually increase.
2.
Solvent Guidelines
1. Please only use high-quality, clean, new glass bottles (ideally Kimex). Used and
washed glassware may contain trace amount of detergents and salts that can
damage the instrument and reduce data quality.
2. Please only use HPLC-quality, degassed solvents as lower quality solvents may
damage the HPLC system and introduce contaminants into the mass
spectrometer.
3. Please refrain from using trifluoroacetic acid, triethanolamine, phosphates,
borates, and sulfates as carrier solvents, as these lead to ion suppression and
chronic loss of instrument sensitivity. Formate and acetate are ideal, as well as
ammonium hydroxide.
4. Positive-mode pH ranges 2-7, and negative-mode ranges 7-10.
5. Before you begin your run, ensure that the solvent reservoir settings are correct.
6. If the pumps have been idle, perform the following priming procedure –
Open the purge valve
Set the flow rate to 3 mL / min for 1 minute
Set the flow rate down to 0.2 mL / min –
(Note: that too high a flow rate during a mass spectrometry run will overwhelm
the mass spectrometer vacuum system and cause serious problems - don’t go
above 0.5mL/min!)
a.
b.
c.
d.
7.
Close the purge valve
Do not mix acidic and basic solvents!!
8. If you are switching solvent systems, ensure that your glassware is analytically
clean! Thoroughly wash the pumplines + pumpfilters with HPLC grade H2O
before immersion into new solvent reservoir.
9. If you are unsure what solvents were used in the previous run, or you are
switching between acidic to basic solvent system, flush the system with (30%
MeOH or ACN, 70% H2O) @ 1 mL/min for a time duration of at least 10 minutes
as a bridge to your solvent system.
10. Solvents containing dissolved solids should always be washed out before shut
down. A recommended washing solvent is 50% MeOH or 50% ACN in H O.
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11. If you have primed the system and wish to leave the solvent system overnight,
leave the pump running slowly @ 0.1mL/min. Ensure that the solvent reservoir
is sufficiently large to sustain the overnight run.
Column Switching Protocol
1. Ensure that the starting solvent is NEUTRAL. A recommended system is 40%
MeOH, 60% H O.
2. Column switch valve should be set to COLUMN 1. This may be done using the
<Column thermostat Control> in the Chemstation software.
3. All swage compression fittings should be hand-tightened. Overtightening
damages the screw tracks and results in a poorer fit over time.
4. Use gloves! Check that the flow rate is at 0.2 mL / min. Allow some solvent to
drip into the void volume of the LC/MS. Hand-tighten the swage screw
compression fitting into the column and allow the eluant to drip out of the
column on the other end.
5. Tighten the other end of the column.
Mass Calibration and Instrument Setup
1. Under instrument mode (Acquisition or Tune), select Tune.
2. Select the correction instrument polarity (Positive or Negative)
3. Select the correct MS setting (High Resolution, 4Hz, or Extended Dynamic Range, 2
GHz).
4. Select Calibrant Bottle B, and ensure LC Flow is set to Waste.
5. Under source parameters, make sure variables are set close to experimental
parameters. Calibration will not proceed until the gas temperature is within 5
degrees of the setting.
6. Under TOF Mass Calibration, click the calibrate button and wait 10-15 seconds. Press
Apply. Ensure that the error is ~ <2ppm.
7. After calibration please switch off Calibrant Bottle B, and switch back to Acqusition
mode. Save changes to the Tune file. Please consult with superusers before pursui
8. Feel free to permanently save methods and worklists in addition to temporary data
storage in your user folder.
Sample Removal, Instrument Cleanup, and Data Migration
1. Please include cleanup and/or standby scripts in your worklist for the end of your
run.
2. Please switch out your column (and insert an empty union), purge the lines, remove
your samples, update the paper logbook, and migrate your data using server file
transfer or a thumb drive. We will regularly purge excess data from the instrument
workstation, so immediate data migration is IMPORTANT.
3. MAKE SURE to log out after you finish, or you will be charged for additional time!
4. MAKE SURE to complete the logbook.
5. Mass Hunter software for data analysis is available on the following computers: one
computer in the microbiology computer lab (NRB963),
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