How Important to Tune LC/MS system (Agilen* 6410)

At the first half of 2007, always complained on the poor / False Negative ESI mode on the Agilen* 6410.
At the on-site setup@Feb-2007, already learned how to tune up the 6410. At March 2007, Mr. B-Cousin** just occasional stopped by Atlanta: then asked him for an update of Mass-Hunter Software. Then I performed an auto-tune on the 6410. After that, the nightmare on the Negative ESI happened. -> Found poor selectivity on the Negative ESI mode ->However the Positive ESI mode worked perfect with a good pass results of tuning. Tried to cal Agilent Tech, but faile to find out a solution.
Lots of unknown biofuels in the lab need to identified by PosESI and NegESI, felt huge pressure from the Co-works. Every morning, almost want to smash on the Agilen* 6410 when entered into the lab.
When cool down, checked with the old tune reports with new tune reports: for old tuning solution b4 upgrade the 6410 Masshunter software, tuning peaks@ 112.99, 431.98, 601.98, 1033.99, 1633.95, 2233.91. However when upgrade the software, the tuning m/z is: 112.99, 302.00, 601.98, 1033.99, 1333.97, 1633.95.
Then found out problem: maybe did not use the new tuning solution after upgrade the 6410 Masshunter software. Changed the new tuning solution, OMG, perfec. Huge response value for the MET** in the biofules.
Used the old tuning solution after upgrade the 6410 Masshunter software.: expected 302.00m/z , but only found @ 290.26 m/z.
So when the m/z of the analyte is from 230- 350 at NegESI, almost got poor results, made the catalyst guys crazy and cry everyday.

See an good GLP on LC/MS tune report @ FDA :http://www.cfsan.fda.gov/~comm/fluoroqu.html#attachb

Why NOT Ethanol in Reverse Phase HPLC?

Mik***: You did have lots of questions that hard to answer in one word.
Why NOt use ethanol in reverse phase - HPLC?
Actually, if I could choose, i definitely would use Ethanol, rather than the Methanol ( MeOH), Acetonitrile (ACN), from the chemist's health concern.

Ethanol (EtOH) & Methanol (MeOH): they do have similarly polarity, cut-off wavelength, the flashing/ boiling points. But Why we use a lots of Methanol (meOH), rather than the Ethanol.

(1) the HUGE discrepancy on Viscosity: Methanol — Viscosity: 0.59 mPa·s at 20 °C; Ethanol - Viscosity: 1.200 mPa·s (CP) at 20.0 °C

the general ideal viscosity of mobile phases should NMT <1cp. style="font-weight: bold; color: rgb(102, 51, 255);">the HUGE HUGE discrepancy on elute ability: Acetonitrile (ACN) > Methanol (MeOH) > >Ethanol ( EtOH). Using Ethanol, always obtain poor peaks separation, lots of unknown peaks or ghost peaks in the chromatogram.

(3) the Acidity (pKa in water): CH3CH2OH (ethanol), pKa 15.9.

the pKa of CH3OH methanol in water is 15.5, while that of pure water is 15.74.

(Pls do not ask me: why pKa of water is not 7 ??)

this bring more more gaps on the application of Ethanol on Reverse-phase HPLC: more easily to react with the analytre or esterification.

Toxicity: Acetonitrile (ACN) >> Methanol (MeOH) >>>> >Ethanol ( EtOH)

However, I DID read lots of application of Ethanol on reverse phase HPLC in the literature. And alwasy wish we could find a non-toxic or low toxicity, non-volatile liquid for RP-HPLC someday.
That is my day dream....Wake up... Malcolm...

Undate@ Aug-22-2008: At Page 75 th of the book [The HPLC Solvent Guide 2nd Edition] by
Paul C. Sadek: Neat ethanol has found limited use, not because it does not offer interesting and useful chromatographic properties, but because of the artificially high cost due to strict government control over its use and dispensation. Denatured ethanol, commonly called reagent alcohol, is readily available in many forms. However, only those with either a hydrocarbon at ~1% levels or ones containing methanol/IPA mixes at the 1-5% level are compatible with UV work. Note that the potential variability in the level of added denaturant poses potential reproducibility problems for the chromatographer.

Column Bleeding or Leakage?

To: WENSWTo my best knowledge, this analyte (Dimethyl 1,3-acetonedicarboxylate, cas#1830-54-2) should not be a problem for you.
If the assay method is well developed and transferred, so my first comment to your question is the bleeding of the Capillary Columns. ( Column bleed is a result of stationary phase fragments releasing from the inside wall of the column. )
Here are some comments:
(1) Double check the leakage of the GC system.
(2) If NOT, change another polar capillary column: Polyethylene glycol (PEG) Polar Capillary GC Columns or high performance low bleed, highly inert, polar fused silica GC columns ->50% Cyanopropylphenyl Polysiloxane.
(3) Perform a Grob test on that previous by following the on my Tech-Notebook 2006. {(K. Grob Jr. G. Grob, and K. Grob, J. Chromatogr. 156 (1978) 517; K. Grob, G. Grob, and K. Grob Jr., J. Chromatogr. 219 (1981) 13.). } or check the weblink of Grob Test.

Does Electrospray Ionization Produce Gas-Phase or Liquid-Phase Structures?

Just read a recent paper on the "Mass Spec Ion Structure Controlled by Solvent Effects"
(Tian, Z.; Kass, S. R. J. Am. Chem. Soc.; 2008; 130(33); 10842-10843. DOI: 10.1021/ja802088u)

Everyone know that the Electrospray Ionization ( ESI) converts the molecules from liquid-phase to gas phase for MS analysis. However, the biomolecules could produce different ion fragments by ionized at different sites, and the molecular ions in the liquid and gas phase are not the same. It is fundamental importamt to know about the whether the Electrospray Ionization Produce Gas-Phase or Liquid-Phase Structures for those bioresearchers who are research on the biomolecule reactivity or identify the protein fragments by MS.

Their strategy is: Use the trimethylsiyl Azide to derivatize tyrosine carboxylate ( stable ion in liquid phase) and Tyrosine Phenoxide ( stable ion in gas phase). These two ions have the same mass.

Will update @ Monday.

Column types for LC-MS/MS?

To MiK***: It depends. -> need to order the column according the analyte in your Lab.

Here is the column info I sued @ 2005' Tech.

2: eclipse c18 150 mm * 4.6 mm ,5 um.
1: zorbax-aq 150 mm * 4.6 mm, 3.5 um.
2: zorbax-aq 250 mm * 4.6 mm, 5 um.
4: zorbax-sq 50 mm * 4.6 mm, 5 um.
3: YMC-am-c18 50 mm * 4.6 mm, 5 um.
4: Waters C18 50 mm *2.0mm ， 5 um.

LC-MS: Prefer use the zorbax-sq 50 mm *4.6 mm,5 um and Waters C18 50 mm *2.0 mm，5 um, HUGE time saving with Syringe pump.

LC only: eclipse c18 150 mm *4.6 mm and zorbax-sq 50 mm *4.6 mm.

DONE! Annual Calibration of Agilent 6890N GC/FID

Update @ Wed.
using the Solution of 0.03% C14, C15 and C16 normal alkanes in hexane.

Then the calibration for Varia** 240 FS Atomic Absorption Spectrometer.
hope get it done NLT this Friday.

Thanks..

Polydimethylsiloxane (PDMS) Fibers ( non-polar) should be OK for the Flavor Components (2)

To Je***: Based on the searching on my NIST/EPA/NIH 2005 Mass Library, I got some crabs :>) Peak @ 21.36 min -> 2，6-di-tert-butyl-4-sec-butylphenol

Peak @ 30.91 min -> 4-sec-butyl-2-tert-butylphenol

So please DO NOT trust this Mass Library Searching. will update when I find something new.
Update@Aug-12-2008
Maybe PDMS works. Also it maybe work on the acids, alcohol, aldehydes (ketone). However, that is not a good one. -> “the Supelco 57301 the fiber ( 100 um) PDMS” has better SPE affinity to the high molecular weight non-polar compounds, that maybe the reason you got so many unknown peak ( high molecular compounds).
So if your analyte is low molecular polar acid, alcohol, aldehydes (ketone), recommend the SPME fiber assembly, Carbpwax-Polyethyele (PEG), Supelco#57355-U, it should be good to go for the polar low molecular compounds.

Polydimethylsiloxane (PDMS) Fibers ( non-polar) should be OK for the Flavor Components (1)

To: Je***: Polydimethylsiloxane (PDMS) ( non-polar) should be Good. What is the thickness of matrix active group (polydimethylsiloxane coating)? using 100μm coating? that 100 μm coating thickness is recommended for low molecular weight or non-polar high molecular weight compounds. Or equivalent with the one in the Your Reference Literature?

If want to obtain a decent result, need to pay attention to the optimization of the SPME method with PDMS: Carry-over effect / Position of the fiber in the GC injector/ Extraction conditions, such as the duration of absorption and desorption, temperature of extraction, and the polarity and structure of the fiber. (Here is the basic SPE info:
http://www.sigmaaldrich.com/Brands/Supelco_Home/Spotlights/SPME_central.html)
If use the 30 μm or a 7 μm PDMS fiber, could extract the non-polar semivolatiles or large molecular weight compounds more effectively.

So my suggestion is: try to do more research on that papers -> find out the right PDMS fiber they used. (100 μm coating or 30 μm or a 7 μm)?

Reference from Varia**: https://www.varianinc.com/media/sci/apps/spme04.pdf

How to Calibrate the Uncoded Hollow Cathode Lamps for 240 FS Atomic Absorption Spectrometer

To HJH: Yes, I agreed with your Lab manager -> It is NOT necessary to buy Coded (Certified) Hollow Cathode Lamps for your 220 FS AAS, however, he might neglect to give you some basic explanations.For Varia** AAS, I am NOT a Technical Expert, but according my 3-years experiences of Varia*: Coded Hollow Cathode Lamps is only good for the automatic lamp recognition SpectrAA software. So I prefer the Lower priced uncoded Hollow Cathode Lamps.

But, always remember that you need to Calibrate the Uncoded Hollow Cathode Lamps before the 1st analysis. 1-3 step is OK.

Determination of Ractopamine in Feedstuffs

To Mik**, the reason why failed in the assay of Ractopamine lies on the sample pre-treatment. Here are some information from my note.

(1) Transfer 3.0 g of feedstuff sample into 25 mL solution of 2% Ammonia in MeOH and place onto Vortex 1 min, then sonicate and swirl 5 min. Pipet 10 mL of upper clear solution and transfer to a 1 ml flask, make the dryness @ 50 C by Rotatory Evaporator.
(2) Using 2 mL of MeOH and 2 mL of n-hexane to dissolve the residue, discard the upper solution, and repeat the above procedure twice. Collect the 10 mL of MeOH portion and blow with N2 to dryness @ 45℃. Dissolve the residue again with 5 mL of Ethyl Acetate, add some anhydrous sodium sulfate, and centrifuge the solution@5000rpm for 3 min.
(3) Apply Polystyrene SPE column purify the analyte: (a) Column solvation: apply 5 mL of Ethyl Acetate and 3 mL of Acetonitrile/Ethyl Acetate (1:1). (b) Interference elution: Elute Analyte by using 10 mL of MeOH/Ethyl Acetate (1:1), collect the elute. (c) Concentrate: Under 40 degree C, evaporate on a stream bath to dryness (or with Nitrogen blowing Instrument). Mixing with 0.1 mL BSTFA + 1%TMCS by using Vortex for 1 min, Heat in oven 80 C for 1 hr. Blow with N2 to dryness by Nitrogen blowing Instrument. Bring to the volume with 0.2 mL of toluene for GC-MS analysis.
GC Column: 5% Phenyl-Methylpolysiloxane 30 m×0.25 mm I.D.×0.25μm.
Inlet Temp: 300℃.
Oven: Initial 150℃ (3 min), 10℃/min to 230℃ (10min)， 20℃/min to 280℃ (10min).
Carrier Gas: Helium（99.999%）1.0mL/min
Injection: 1.0µL, pulsed splitless, 50 ng on-cloumn per Inj.

EI Temp: 230℃.
MSD Interface：280℃.
EM voltage: 1506V.
Scan Rage: 30～550U.
SIM (m/z): 267、250、502，Quantitative ion 250.
Solvent Delay：5 min. ( I can not forget the accident we made @ Tech Dec-2005. How about you?).
Please let me know if this sample pre-treatment method works.
BTW, How do you valuate the Beijing 2008 Olympic Game Opening ceremony? for me, feel humble in the front of 5000-years China History.