Basic Operation Procedure of Agilent 6890N-5975 MSD (6)

Quantitative Analysis

Just like the LC-MS ChemStation Software, also need establish a new calibration table:-> calibration /Setup calibration database. (Here are four Quantitative Methods : AREA%,NORM%, ESTD, ISTD.) The different betw GC and GC-MS is that: Need to Qualtilative before Quantitative Analysis.

Load the Method and signals, Select->the Calibrate/Set Up Quantitation

Quantitation Database Globals:Type the Calibration Title, Default Multiplier, Default Sample Concentration, Reference Window/Non-Reference Window, Reference window for Internal STD,Correlation Window(For Scan: 0.05, For SIM:0.025)

Double click the Right button, type the chemical name. If it's a internal STD, Select the Internal icon. Move the “+” to select the targeted ions,click the Left/Right button at the same time. DO NOt forget "Save".

All the internal STD Need to present before compound quantitative

Type the Compound's Cas#. At 2nd Page, there is EnviroQuant for Envi for MDL. If the analyte is water/soil, then don't forget the MDL.

Calibration PlotCalibrate/auto quant setup, -> AutoQuant Setup: Choose Compound Name,Update Calibration,Edit CompoundsQuantitate /Calculate, or data analysis ->Method /Edit Method -> Select Reports-> Quant Report saved with method.Manual Qedit/GraphicsChanging Data States

Why the Molecular Weight of the analyte is 335.31?

To Xue*, maybe It's right that your client claimed Molecular Weight is 335.31, NOT 339 that you obtained from NegESI.

C15H17O6N3-Calculated Exact Mass: 12.0000 (15C) + 1.0078 (17H) + 14.0031 (3N) + 15.9949 (6O) =335.30925.
According to the Nitrogen Rule, the chemical structure contains an odd number of nitrogen atoms (3N), indicating an odd nominal mass. So [M+H]+, [M-H]- the molecular ion peak should be an even number. Under negative ESI mode, 334 maybe the [M-H]-.Also under Positive ESI Mode, there are peaks of 336[M+H]+， 358[M+Na]+，371[M+K]+. So the conclusion is 339 could not be [M-H]-.In practical of NegESI, it is difficult to explain the molecualr ion is the peak of 339: How could it disassociate a Fragments ion of 5 to obtain a Fragments ion of 334. Also, please double check the Mass Spectrum of Positive ESI, there is not Fragments ion of 341. Also confirm that the irrational of molecular ion of 339.

As for the fragment peak of 339, that maybe is from the system contamination or the impurity in the analyte. That is the reason why I asked you obtain a positive ESI Mass@ Friday.

Negative ESI Mode should be OK for Flavonoid

Question from jumn*** Why ESI mode doesn't Work for Flavonoids?According my Tech lab Notebook(#2006-09), my answer to your question is: 100% Negative ESI Mode should be Good for Flavonoids.

Your analyte is belong to Flavonoid family. {Molecular structure of the flavone backbone (2-phenyl-1,4-benzopyrone)}. It contains lot of Phenols, so technically it should be good under Negative ESI mode.
(1) Try to use H2O-MeOH as mobile phase, the Injection Conc. is 1ug/ml.
If above method doesn't work,
(2) Method Development: under Negative ESI mode, direct injection sample through syringe pump. (Use syringe pump, easy tuning & time saving, but need to pay attention on the cleaning of capillary after tuning or could try the six-valve direct injection if feel confident, could obtain a good baseline with min background, time costing:> )
Add some Ammonia to enhance the ion compounds or 5mM of ammonium acetate should be fine. Optimize spray-voltage, voltage plate, capillary voltage/Temp, shealth gas, aux gas, flow rate.
The starting analyte Injection Conc. is from 1 ppm.

If you still have any problem, highly recommend you talk to your BOSS: Double check that is the right "Flavonoid" or the purity of Flavonoid.

Try to use LC to testify your suspect,
Column:2.1 X 50 mm, 5 µm , 100 Å , C18
Temp: 40℃
Mobile: A (H2O) with B (MeOH) 30-70% gradient elute in 5 min.
Flow rate: 1 ml/min.
Wavelength: 340 nm.

if there are multi-peaks in the chromatograms, then maybe explain the problems in NegESI mode. Always talk to your manager first, Don't let him to be the last ONE surprised.

Please update me if have something new, thanks.

Reference Paper: JOURNAL OF MASS SPECTROMETRY, 2004; 39: 312–321: LC-DAD-ESI Tandem MassSpect.

Correcting Peak Tailing Problems in Reversed Phase HPLC

Peak tailing in reversed phase HPLC continues to be a common complaint. It is particularly prevalent when separating basic compounds and, therefore, a source of constant problems to those analyzing pharmaceutical compounds by HPLC.

(1) Cause: Sample solvent stronger than the mobile phase.
(1) Cure: Dissolve sample in mobile phase or at least reduce the strength of the sample solvent as much as possible.

(2) Cause: Peak Tailing Caused by Sample Mass Overload.
(2) Cure: Reduce the amount (mass) of sample injected, choose injection size for different column configurations.

(3) Cause: Peak Tailing Caused by Stationary Phase Silanol Interactions with Amines.
(3) Cure: Reduce mobile phase pH to < 3.0.Increase mobile phase ionic strength. 25mM to 50mM recommended.Add a competing amine to the mobile phase.10 mM TEA is usually sufficient.Select a stationary phase with a lower silanol activity.

(4) Cause: Peak Tailing Caused by Adsorption of Acidic Compounds onto Silica.
(4) Cure: To correct peak tailing in these cases increase the salt concentration of the mobile phase to suppress secondary interactions, reduce the mobile phase pH to protonate silanols and solutes and, if necessary, add a competing acid to the mobile phase.

(5) Cause: Peak Tailing Caused by a Void in the Column's Packing Bed.
(5) Cure: The best cure is to replace it. This saves time, money, and frustration.or call me if DON'T have the Lexus.

The Final Cure to # 3: Add a competing amine to the mobile phase. Triethylamine (TEA) is commonly added to mobile phases for this purpose. TEA interacts strongly with silanols and inhibits them from interacting with amines in the sample. About 10mM TEA is sufficient for most applications.

Ace C18 <>
Thanks for MAC-MOD Ana Inc. Tech Report # 07031TR

Basic Operation Procedure of Agilent 6890N-5975 MSD (5)

View, Search and Qualitative Analysis

Instrument/view/data analysis offline Navigation ButtonMouse functionality: Double Right Click -> Select the Mass Spectra in the selected Chromatogram or Searching the Mass Spectra Library. Drag the Right button -> selecting the average Mass. Drag the Left button ->Zoom In. Double Click Left -> Zoom Out. Double click R/L -> Make a note. File/Subtract Background(BSB)Edit Integrate EventsExtract Ion ChronogramsSIM / Sacnview / review peak purity Search Mass SpectraSearch StrategyresultsView/parametric retrieval : structures/ select structures database -> molstruc
or DOSCAN -> Right click @ Molecular Ion -> Tools/process scan list
or tools/Signal to Noise checkNIST MS Search or Tool -> Mass Spectrum Interpreter, tools -> isotope calculate.
data analysis/spectrum/AMDIS: Automatic Mass spectral Deconvolution and Identification System

Basic Operation Procedure of Agilent 6890N-5975 MSD (4)

How to Set Up a Method
Click the “Instrument # 1” access “Instrument Control”

Edit the entire MethodInlet and Injection InfoAccording specific method set up all parameterGC Real Time Plot and MS Tune FileMSD Parameter

Input EM Voltage, Solvent Delay and select Acquisition Method: MS SIM / ScanEdit the Scanning Mass Range, Threshold Sampling Rates, Real Time PlottingEdit the SIM ParamsSelect Reports, Options, Library Search ParamsLoad the Method and Run the sample

Basic Operation Procedure of Agilent 6890N-5975 MSD (3)

TUNE
GC/MSD -> Instrument Control -> Qualify\Checkout Tune

Wait for several min for (Auto-Tune) Tune ReportSave or PrintHow to Performance the Manual Diagnostics the GC-MSD System?

After the pump down, it is difficult for make sure the GC-MSD system is ready to run sample. Only have two resources to check: Ino gauge (<5.0>
Here is a technical note how to use the Diagnostics to check the leak, contaminants in GC-MSD.

(a) Instrument Control -> Diagnostics/Vaccum Control, Diagnostics -> Diagnostics\ Edit MS Params (b) Edit Parameters -> MoreParams -> AcqParams(c) Acquisition & Display Param, check the Mass(1~3, maximum 3) &Scan Range(d) Mass Peaks of Common Contaminants(e) When need to check the leak in GC-MSD system, focus on the air-ion peaks. i.g. the N2 (28) & O2 (32) . Acquisition & Display Param -> Mass1 is 69、Mass2 is 28, Mass3 is 32 AND Scan Range IS From 10 To 70.Go back Edit Parameters -> Scan: when the Ion 28 / Ion 32 is 4:1, that is to say there is a leakage in the GC-MSD. (Why 4:1, recall what are composites in the AIR?)

Note: Look the Blog@July-23-2008 for the Contamination Peaks.