Constitutional Isomers (Structural isomer) by HPLC

To Ren***, I am a newbie on the separation on the Constitutional Isomers (Structural isomer). I 100% knew that you might try all the combination of mobile phases and C18 Column. Here are my suggestions:
(1) Try to call the Column Supplier for some info of Chiral column. Maybe they have more information for your analyte. Or if you are lucky, ask them give you a favor -> provide a free chiral column for trial analysis.
(2) If the chiral column is not available, try to put some Quatemary Ammonium Salt in the mobile phase, it maybe obtains a better separation.

Good Luck, also thanks for your question and any update of your results in advance.

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.