Chromatography Forum

Hello every one,

I need help, I want to determine the retention time of propionic acid,suberic acid and butyric acid which are present in urine.For that i prepared standards of different concentration and extracted them by solvent extraction using ethyl acetate as the solvent.And injected 1ul sample into the GCMS after derivatizing it.I failed to get the peaks of both the compounds(using NIST library for matching compounds).Even no peak is increasing its height in the chromatogram with the increasing concentration of the standards.

Where i am lacking i don't know please help me.


Thanks

Chandra

Try diluting some of each acid in a solvent, derivatize and inject. This will remove any questions of recovery in the extraction from the question. In this way you can be sure there is enough material to see. (The reccomendation of dilution is just to get a sufficently small quantity in the GC vial that you do not overload the GC column.)

Thanks Don for your valuable advice.I tried it and got the peak that increased with the concentration, but again arises a problem. When i injected the dilution containing propionic acid,i got a peak that increases with the increasing concentration.When i match it with NIST mass spectrum library , it indicate that it is the peak of butanoic acid,3-methyl trimethylsilyl ester.I repeated whole procedure thrice but it gives the same result.

What should i do now


Thanks

Do yoy have propionic acid-TMS ester in the list of alternative mass spectral hits? If you look at the spectrum of propionic acid-TMS ester http://webbook.nist.gov/cgi/cbook.cgi?I ... #Mass-Spec, you do not see a molecuar ion -- just fragments. The spectrum for butanoic acid,3-methyl trimethylsilyl ester looks similar http://webbook.nist.gov/cgi/cbook.cgi?I ... #Mass-Spec, with a strong cluster of ions between 70 and 80 mass units. The larger fragments should help you to tell the difference.

The library search routinc anc be confused by background noise or an incorrect setting for the search.

If you have only propionic acid, solvent, and silylating reagent present - you should only see propionic acid -TMS ester. If you think something else may be present, add a bit of methanol rahter than propionc acid and run that sample. All of the side products will be the same (except the methyl alcohol-TMS ether), which would confirm that you do not have anything else present in your sample.

Thanks for your valuable suggestions , i got paeks of all the compounds by diluting the sample compound directly into the solvent.
Now i want to make a standard calibration curve so that i can quantify the compounds.Most of the compounds which i have are in liquid state, so could you please tell me what concentration should i take and how to calculate it.I am very poor in calculations.


Thanks

chandra

Concentrations and calculations depend on what you are doing. If you know the expected conentrations of compunds in your samles you would make a series of standards to bracket the expected concentrations.

Also are you going to use an internal standard?

How you make up the calibration may depend somewhat on how you will prepare samples.