Chromatography Forum

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What are your three pesticide compounds? At 210nm you can detect most everything, not very specific or selective, you may be seeing a trace impurity in the methanol. Most pesticides absorb UV light at higher wavelengths, have you tried that?

We use higher UV wavelengths here when assaying our own pesticide products.

Can you give us a link to a chromatogram of a methanol blank? Do you see the same peak there? If not, then the peak is *not* coming from the methanol. You have some other source of contamination which you will have to track down (exposure to other solvent vapors, perhaps??).

If you *do* see the peak with a methanol blank, try injecting a "blank" of 50/50 methanol/water. Assuming you use the same bottle of methanol to prepare the mobile phase and the blank, you should then see a negative peak at that same retention time. If you *don't* see a negative peak, then, again, your contaminant is *not* coming from the methanol.

If you *do* get a negative peak, try injecting degassed methanol. Dissolved O2 in methanol does have some absorbance at 210nm.

"i went to my friends hplc system ( 2 different hplc system) with same sample and syring but i found the peak of methanol contaminant is very very small and comes before all the compounds and i used the same condition like in my system !!! . i returned to my system the proplem is still untill now and it was decided to get rid of compound number 1 because if i leave this big contaminant , i have to move it before all the compounds and this happen at 55% methanol water but my compounds number 3 eluted after 41 min. "

regarding this long analysis time - gradient elution is the solution

regarding "2 different hplc system" - i would check degaser first, maybe that's your problem, as your peaks are small this can be dissolved oxygen

two excellent responses so far - track the peak as tom said or go back to method development as proposed in second response (changing wavelength)

sorry for the late in reply .Me and my supervisor decided to work on this wavelength because our interest analyte has maximum absorbance only on 210nm .please forget now any thing about the compounds and focus only in pure methanol . I inject as a new injection only methanol ( blank) and i found in my system the same peak area which is very very big .I am difinetly sure that the contaminant was not from the syring or flask etc because my supervisor also test this by him self and he said "every thing clean where does this contaminant come from " surely comes from methanol but why it is too big .

when i prepared my compounds at different conc. the peak area still the same in all conc. This mean that the contaminant is from the methanol solvent .

i went to my friend system with my method , syring,solvent column all the same but my friend's system is not SHIMADZU then i inject methanol ( blank) but here the peak were very very small.

please note : at 210 in my system the peak is very huge but at different hplc system( my friends hplc system ) the contaminant peak is very samll ( negiligable) why ??

i am sure the proplem is inside the instrument but i don't know what is it ?

Could it be that your friends setup is much less sensitive? If it is not a gas problem it could be just the absorption of MeOH itself. Did you ever try what Tom mentioned, or just inject H2O?

Can you give us a link to a chromatogram of a methanol blank? Do you see the same peak there? If not, then the peak is *not* coming from the methanol. You have some other source of contamination which you will have to track down (exposure to other solvent vapors, perhaps??).

[size=12]please see the word file i attached now

http://www.2shared.com/file/7421405/c0c ... vent_.html

,the methanol blank contains the contaminant peak [/size]

If you *do* see the peak with a methanol blank, try injecting a "blank" of 50/50 methanol/water. Assuming you use the same bottle of methanol to prepare the mobile phase and the blank, you should then see a negative peak at that same retention time. If you *don't* see a negative peak, then, again, your contaminant is *not* coming from the methanol.

i do inject 50 50 % methanol water i saw a negative peak ( the peak down )

If you *do* get a negative peak, try injecting degassed methanol. Dissolved O2 in methanol does have some absorbance at 210nm.

i injected also pure acetonitril ( only acetonitril 100% ) i do see the same peak and if you see the word file you will see that the peak appears ,,, i am really frustrated for more than six months now with this proplem !!

download file
http://www.2shared.com/file/7421405/c0c ... vent_.html

McAfee advises against the use of this site!!!

i am really sorry i will try to upload it in another site