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.

Basic Operation Procedure of Agilent 6890N-5975 MSD (2)

GC-MSD　Start & Turn Off（1, 2） Click the “BootP” icon on the desktop and CAG Bootp Server”(3, 4) Double click the “Instrument # 1” to access the GC-MS startup.　Now the MSD is not under Vacuum.(5) To view and go to Diagnostics/Vacuum Control ->Vacuum to select Pump Down up to 10-5 - 10-6 torr.　( will put another poster about how to check the leak in the GC-MSD next blog)( 6) ow to turn off the GC-MSD? Instrument Control -> View ->Diagnostics/Vaccum Control . Wait until Vent cycle，close the MSD Chemstation, CAG Bootp Server software. Thanks for the Agilent Technologies, Inc.

Note: In general, DO NOT need to turn off the GC-MSD unless you are sure will not use the instrument for several days. General, depends on the turbo pump tech, it takes 2-4 hours for theｔurn On the GC-MSD system, need 1 hour to turn off the instrument. Set up an “On-Hold” method to make sure instrument is standby to work next day. Call me if need some guild.

Basic Operation Procedure of Agilent 6890N-5975 MSD (1)

Lots of calling for asking some newbie questions “How to turn on GC-MS” or “Do I need to turn off at Friday afternoon?” “How to edit entire method” ect.

(1)Double click “Config” Icon to access the MSD System Configuration

(2, 3, 4) Select the “1” and click the “2” to enter the IP Address

(5) Always select the “Electron Ionization” as the Ionization Option, only select the “Chemical Ionization” when it is available

(6) Look up the Auto-tuning report or the Instruction manual of the MSD to secure the right DC Polarity

Now it is turn for GC system configuration

(7, 8) Select the “1” and click the “2” to enter the IP Address

(9) Select the “Enhanced Quantitation”(10, 11) Now, get a final review system configuration. Close the program, DONE!

(12) Then will two Icons on the computer desktop: “Instrument # 1” and “Instrument # 1 Data Analysis”PS:Thanks for the Agilent Technologies, Inc