It's time to clean the Inlet....

Agilent 6890N ECD.
Temp Ramp: 80℃（1min)----20℃/min--- 260℃（15min)----5℃/min---300℃（3min),
Detector Temp: 300℃
Inlet: 250℃
GC Column:HP-5.

Baseline drift?

Maybe the collision energy (CE) is too LOW

(1) Maybe the collision energy is too LOW. The First MS is not full a fragment.
(2) Are you using ESI mode?
(3) If the Molecular weight is 237, for SIM -> Here are three characteristic ions (m/z) 164，149，122 I highly recommend them.
(4) MS-MS (MRM): Need to search online for the product ion and parent ions.
(5) If only need the full scan, why not adjust the concentration of analyte.

Hope it helps.

Agilent MassHunter could meet your request.

Go to MassHunter, File->TIC->.....

m/z (563.10761) is NOT the Molecular Ion? (II)

(1) Try to adjust the the capillary voltage: increase it a little bit each time.
If the the mass response of [M＋H2O －Ｈ]－ is decrease, the mass response of [M-H]- increase: then the m/z (563.10761) definitely is the molecular Ion.
If No such change happened, then the m/z (563.10761) definitely is NOT the Molecular Ion.

(2) From my view, the possibility of ［Ｍ＋Ｈ２Ｏ－Ｈ］( belongs to Molecular ion) is very low. Under ESI (-) negative mode, the trend of forming
the ［Ｍ＋ＨＣＯＯmore high.
Doubt the H2O have that much acidity for forming the ion peak.

m/z (563.10761) is NOT the Molecular Ion? (I)

What is the capillary voltage?
Did you try to increase a little bit?

Where M/Z 425 from? ( II )

Please click the elucidation Scheme for more details:
(Sorry for the confusion in the last post.)

From M/Z (471) to M/Z (425) , maybe belong the loss of Formic Acid (46) .

That is interesting Mass Spectrum, Good luck.

Where M/Z 425 from? ( I )

The molecular weight of your analyte is 548: 1st Mass is 549, 2nd Mass is 489, 3rd Mass are 471, 425.

Here is HUGE challenge for the structural elucidation of your analyte.
From M/Z (471) to M/Z (425) , maybe belong the loss of Acetoxyl M/Z.

But it is NOT right because there are two Acyloxy in your analyte.

Will Update you by Monday Night.

PPG Standard Prep for AB API4000

Come on, Man: Don't be silly to send us this Newbie question? Are you want to test our GC knowledge?
(1) Condition the GC Column according the manufacturer' recommend.
(2) Check the septum and the inlet, make sure NO cross-contamination -> inject the Blank.
(3) Optimize your method, change the Oven temp Ramp -> Higher the Initial temp, Low down the Temp Ramp.
(4) Low down the Split Ratio.
(5) Adjust the GC gas Flow Rate.

Sample Overload on Column? Example 2

That maybe Sample Overload: Low down the injection Concentration per injection or Injection volume per column. Or increase the Split Ratio that maybe help.

Sample Overload on Column?

From my view, maybe Sample Overload on the column.

Inlet Temp: 250， Initial Temp: 100; Hold 5 min，then 10 C/min to 230 C，Hold 5 min.
FID Temp: 280，Injection Vol. 0.2ul.

How do you define the Limit of Detection?

In figure 5b, [M+Na-H2O]+ ion peak at m/z 475 was observed. The peak is hidden by back ground. How do you define the limit of detection?

(1) From the reviewer's point, you should explain why the Low response in the Mass Spectrum?

(2) Optimize the Mass Spectrum of product ion: adjust the CID little by little, do not need to change the energy of cone voltage. low down the gas flow rate of CID, wait 10 min until equilibrium, increase the collision energy at Q2.

(3) If the Professor only care the Low response in the Mass Spectrum, here I have one "non-professional" shortcut: Inject twice in one time ( do not use the syringe pump), one could use low CID, other use high CID for about 2-5 min. Then use the second one as background to extract the First one. you definitely get high abundance on your mass spectrum. ( actually they are the same, but you could hide the response value in the Y axis, only put the abundance.)
Do not abuse the tip a lots, some reviewer hates that so much.

Good Luck...

Safety, Productivity, Accuracy, Credibility, Education

Hi, Guys, I need your help on the my current concern NOW.

How do you value the "Efficiency" in the lab?

How do you evaluate "SPACE" (safety, productivity, accuracy, credibility, education) system of laboratory management.

Could you call me at NOW?

Thanks,

the Different between ESI and APCI sources

Update @ Friday

(MRL) Maximum Residue Levels for Pesticides in or on food and feed of plant and animal

On the regulation of (MRL) Maximum Residue Levels for Pesticides in or on food and feed of plant and animal:

I think European Union and Japan really did a good job on that.

It is really dilemma to the using of the Pesticides: Some "Environmental-concern" people always want to Pesticides free in all everything. Some"Profit-oriented" always try to use all high efficient Pesticides without caring the impact.

As for me, European Union and Japan: from the regulation view, they are good example for USA and China.

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:058:0001:0398:EN:PDF

( LC-MS/MS) Contamination of Detergents/Surfactant

Which detergent used in the Lab now? Look like the contamination of the detergent/surfactant which was used for the glassware.

Did you notice the m/z different of all the peaks in the mass spectrum?

Tips:
Calculated the most dominant five peaks in the mass spectrum.
305,349,393,437,481,525,569 -> there is 44 different in m/z.
Also checked the less dominant peaks:
333, 377, 421, 465, 509,553 -> there is also 44 different in m/z.
The m/a difference between the first line and second line is 16, look like [M+Na]+, for the second like [M+K]+.

As the m/z = 44.
I think that may be the PEG （polyethylene glycol）:-[-CH2-CH2-O-]-n .
The monomer of PEG is C2H4O ( m/z is 44).
That are the evidences why I doubt that is from the contamination of detergents/surfactant.

Decent HPLC Assay Method for Melamine

(From FDA: Melamine found in baby formula made in China.)

I am really feel sorry for the innocent baby. Feel shame for the authority in SFDA in China. They are not doing any professional jobs.

Sample Pre-treatment:
Transfer an accurately weighted quantity (5 g) of Baby (Infant) formula or milk powder sample to 50-mL volumetric flask. Add some amount of grade MeOH and mix well, sonicate it 10 min. Bring to volume with MeoH. Filter through a membrane filter (0.22 um) before injection.

Standard Preparation:
Using an accurately weighted quantity (50 mg) of USP Melamine RS, prepare a solution in MeOH having a known concentration of about 1 mg/ml.

Column: C18 4.6×250mm，5um.
Buffer:10mM Citric Acid, 10mM 1-Heptanesulfonic acid. using 1M NaOH adjust pH to 3.0.
Mobile Phase: Buffer:MeCN=85:15.
Injection Volume: 10uL.
Flow rate: 1.0mL/min.
Column Temp: 30℃.
Detector Wavelength: 236nm or DAD.

two Papers of Neutral Loss Scan LC/MS/MS Method

To Erics**, Here are two good papers about the Neutral Loss Scan LC/MS/MS Method.
Good luck for your Qualify. (I think the Boss is nice for the Qualify: didn't require 1st year should pass, but NO ONE is failed for the qualify yet in the group, all you need is relax and 100% focus on that).

(1) Screening and Sequencing of Glycated Proteins by Neutral Loss Scan LC/MS/MS Method. Anal. Chem., 79 (15), 5991 -5999, 2007. 10.1021/ac070619k S0003-2700(07)00619-1.

http://pubs.acs.org/cgi-bin/abstract.cgi/ancham/2007/79/i15/abs/ac070619k.html

(2) Multiple Neutral Loss Monitoring (MNM): A Multiplexed Method for Post-Translational Modification Screening. Journal of the American Society for Mass Spectrometry:Volume 17, Issue 3, March 2006, Pages 307-317.

http://dx.doi.org/10.1016/j.jasms.2005.11.002

the Different among the Q1 Full Scan, Product Ion Scan, Precursor Ion Scan

Q1 Full Scan (Start – Stop) : Q1 always used as single MS analyzer. Used primarily for identification of precursor ions.

SIM - Selected Ion Monitoring: Used to optimize analyzer for specific ions. SIM used for quantitative analyses. Q1 SIM used to “optimize” precursor ion. Maximize signal in preparation for MS/MS.

Product Ion Scan: After identification, the precursor ion is sent into the collision cell and fragmented by CID. Q1 is fixed, Q3 sweeps a given mass range. Used for structural elucidation. First step to developing quantitative methods:
m3 + scanned or m1+ fixed.

Precursor Ion Scan: Q1 sweeps a given mass range, Q3 is fixed.
Used to determine the “origin” of particular product ion(s) created in the collision cell.
Frequently well used for drug metabolite identification (common product ion observed in the metabolites).

Why Q-TOF can not perform neutral loss scanning, however precursor ion scan is OK?

To Erics**, That is a "no ending" topic if you allow me to elucidate: once a man walk into bar. (Just my 2 cents)

Precursor Ion Scan: (select a specific m/z in Q3)
Scan Q1, obtain all the precursor ions which could product the specific m/z ion in Q3.
Q1 masses Q3masses
from to from to 300 (m/z) or 400 (m/z) to the same -> 191 (m/z).

Neutral Loss Scanning: perform Q1 and Q3 at the same time.
The only different is that always keep a specific mass different
(the m/z is neutral loss)
Could locate the precursor ions that could product ions of neutral loss.
Q1 masses Q3 masses: from to from to
200 (m/z) -> 182 (m/z): loss 18 (m/z)
420 (m/z) -> 402 (m/z): loss 18 (m/z)
is good for identification of some functional groups ( -OH, -NH2)

So for Q-TOF: it is MS-MS too, but different Q-Q. The 1st MS is a Quadruple, the 2nd mass is TOF.
As I said above, neutral loss scanning requires the 100% exact same scan mechanism happening in 2nd mass. Simultaneous scan at the same ion:
The 1st mass for M, the 2nd mass is for M-m.
Separately Quadruple and TOF could not do that at the same time.

For both of them, MS1 full scan is not necessary: Full scan is only basic scan process, is not a requisite for all the neutral loss scanning and precursor ion scan.

For example, for product ion scan process: Q1 would allow you choose the specific m/z precursor ions, (just change RF/DC is ok), then Q2 CID, Q3 is for product ions scanning.

Nice Picture and Vedio from Varia* 240 Atiomic Absorption Spectrometer

Very Interesting Experimental Phenomenon happened Varia* 240 FS AAS.

Will update the video later.

Infants Milk Powder Contaminated with Toxic Melamine

Updated FCC Developmental Melamine Quantitation (HPLC-UV) and GC-MS Screen for the Presence of Melamine. The sample pre-treatment really matters a lots on the assay result.

Call me if want the detail Method (Adapted from FDA/ORA Forensic Chemistry Center SOP T015) .

Molecular ion of CH3I ??

I am not sure what is your question? So sorry if my answer is wrong, the molecular ion should be 127. But I had no idea of the second Mass Spectrum. Do then have any relationship? or you sent me a wrong Mass Spectrum?

Thanks for your update...

Agilen* 1100: Pump Preventive Maintenance

Today, firstly finished a "Preventive Maintenance" on the pump @ Agilen* 1100 system at Sept-08-2008. ( the fourth PM on the Agilen* LC system since 2004)

The system is working well, except a high frequency noise come from the Quan-Pump -> when the pump delivery mobile phase, the Sapphire Plunger forward and back -> a scratching noise from the pump.

-> found lots of dark matter in the pump, use the MeOH and H2O to wash ->

-> use the IPA to prime the LC system for 30 min. done!!!

Next step: should order the Preventive Maintenance kit and finish the PM task on the autosampler and VWD.

Note: some catalog #01018-68722, # G1311-68710.

Poor Response of p-Chlorophenol in Tandem LC-MS?

That is really a good example for Triple Quadrupole LC-MS.
Poor response at Positive ESI (+) mode, however, acceptable response at Negative ESI (-) mode.

Here, let's us assume that Tandem LC-MS instrument is under 100% perfect tuning mode ( just want to exclude the uncertainty of the Tandem LC-MS, just want to discuss about the nature of analyte in LC-MS)

General the Mass Response depends on the stability of molecular ion. -> look at the structure of p-Chlorophenol -> Positive ESI (+) mode, would very unstable due to the -Cl element (Cl element is EWG, that is to say, -Cl belongs electron withdrawing group. However, under Negative ESI (-) mode, the molecular ion would be more stable due to the present of Cl element. ( When this center is an electron rich carbanion or an alkoxide anion the presence of the Cl has a stabilizing effect.) that is the delocalization effect in the physical chem.

That is the reason why I always ask for the pKa value of each analyte @ Tech. For me, I NEVER under estimate the importance of pKa value.

So we always could change pH value to alter the disassociation of analyte in ESI mode. ( With ESI, convert the molecular ion from liquid phase into gas phase, enable the analyte evaporate into ion status -> obtain a decent response).

From the pKa, we know about the nature of analyte: basic,acid or neutral.

Then for basic analyte -> add some Acetic Acid/ Formic Acid ( pH ≈ pKa－2 ) -> enable the analyte generate positive (+) ion in solution for Positive ESI (+) mode. or for acid analyte -> add some ammonium to increase the pH ≈ pKa＋2 -> analyte is molecular ion with negative (-) for Negative ESI (-) mode.

Next time, will update my Tech Notebook about how to trouble-shooting the Poor Response of the Analyte based on the API 3200 Tandem QQQ LC-MS . (that is a golden treasure I learnd from Tech, U definetely need to buy me a drink for that)

Normalized Peak Area

Talked about this issue so many time, need to assume that response factor of each component is identical. However, we always negelect that at the assay of total impurities in the samples were performed by normalization method.
So I only recommend to use this method to estimate the relative amounts of small impurities or degradation compounds in a purified component at the specific detector wavelength, that is chromatographic purity.
From my view, the Second maybe ensure a better Repeatability for your results.
or if I were you, I will use manual integrate to get a decent result with batch options.
At the same time, I always double that the column maybe over-load, that mass of analyte per injection is too high, don't you think so?

What is Batch Review in ChemStation?

Huge Time saving with this Batch Review option.
And I also need to learn how to write Excel Macros/Excel VBA, hope john** you could give me some basic knowledge, thanks a lots.

Assay of Vitamin e

H2O + Vitamin D3 (Internal Standards) + Vitamin C Solution (in MeOH) + Sodium Hydroxide. Water bath 30 min, extract with ethyl ester and evaporate to dry, and then dissolve in MeOH. The peak @ rt=2.8 is the problem?(1) Double check the contamination from the needles and HPLC volumetric flash, vials.

(2) If doesn't work, -> what is the concentration of the Sodium Hydroxide? 0.1 N? Do you make sure that the internal standard (Vitamin D3) would not discomposed with the present the anti-oxidant (Vitamin C) during the saponification process?
(3) The final method is that follow the USP method to troubleshooting.

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.

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.