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Phosphoric acid in lcms
Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.
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I am developing a method for vitamin b1 and b6 that contain phosphate groups. The peaks show bad tailing. One way of geting the tailing out is ad phosphoric acid to the elluens to compete with the phosphate in my compound in adsorption. I know phosphate-bufers are not recomended om lcms but what about pure phosphoric acid?
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In general non-volatile buffers are not OK. Boiling point of H3PO4 is about 200 °C but I am not sure…
Best regards,
Dmitriy A. Perlow
Dmitriy A. Perlow
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Bp of phosphoric acid is 158 celsius, acatic acid ks 118.
i know about the buffer,, that is why i asked about phosphoric acid. by the way, my highest calibator is 0,5 umol/l so 0,5 mmol/l phosphoric acid will give me a ratio of 1000 in phosphoric acid vs analyzed compound. i think that would be enough for the compettion in adsorbtion.
i know about the buffer,, that is why i asked about phosphoric acid. by the way, my highest calibator is 0,5 umol/l so 0,5 mmol/l phosphoric acid will give me a ratio of 1000 in phosphoric acid vs analyzed compound. i think that would be enough for the compettion in adsorbtion.
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From the literature it is usually recommended to avoid phosphate buffers and phosphoric acid because of their low volatility. I am not a good chemist but it seems to me that boiling point and volatility (or vapor pressure) are two different things. Otherwise fog would not bother me in the winter. There are some good chemists on this forum who will certainly be able to expand on this.
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You can use low levels of phosphoric acid but the ion source must be cleaned more frequently (daily basis etc.). Have you tried TFA etc.?Aldijkhuizen wrote:
I know phosphate-bufers are not recomended om lcms but what about pure phosphoric acid?
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Vlad,
i am a litlebit scared to use TFA. the lcms system is used for 6 different aplications and even more in the near future. i hear scary stories from people not geting TFA out of the system once the used it.
i am a litlebit scared to use TFA. the lcms system is used for 6 different aplications and even more in the near future. i hear scary stories from people not geting TFA out of the system once the used it.
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Instead of phosphoric acid for protonation you can use 0.1% formic acid on your LC/MS since it is volatile.
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I tried formic acid but it gave very bad peaksbape die ons compound
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The phosphoric acid is much more "scary" than TFA I never had troubles with TFA (0.01 - 0.10 %v; of course some trace will remain, but i never had issues with that). On the other hand what choice do you have in this situation? IP-RP-HPLC? HILIC or Mixed-mode HPLC (may be, it's best choice)?Aldijkhuizen wrote:
i am a litlebit scared to use TFA. the lcms system is used for 6 different aplications and even more in the near future. i hear scary stories from people not geting TFA out of the system once the used it.
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I ame doing mixed mode. That is the only way i ame geting enough retention. The peak broadening is there in every mode even when i ame bypassing the column. Accept when the ph is 9.5 or higher but that is above limit of column and ionexchange probably wont work
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So tfa is mostly scrarry if you do full scan ms and not really a problem if you do mrm?
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In that case you will probably get bad peak shapes with TFA too. You will have to determine the pKa of these molecules so only 1 species is present during HPLC (protonated versus ionized).
This is the reason behind protonation via formic acid, phosphoric acid, TFA.
This is the reason behind protonation via formic acid, phosphoric acid, TFA.
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Suppression of ionization is main issue with TFA.Aldijkhuizen wrote:
So tfa is mostly scrarry if you do full scan ms and not really a problem if you do mrm?
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May be pH > 9 is only way to go (a lot of sorbents works fine in this range).Aldijkhuizen wrote:
The peak broadening is there in every mode even when i ame bypassing the column. Accept when the ph is 9.5 or higher...
Also try to search some info:
https://www.agilent.com/cs/library/post ... 202015.pdf
http://www.sielc.com/wp-content/uploads ... 2017-1.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489891/
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Sav,
The firts link is about thiamine, not thiamine pyrophosphate. I had seen the third link. I think the first peak comes to early so i like to avoid this if posible. The second link is the aplication vlad from sielc developed for me. I ame trying to reproduce this but get horible results with b1 on my agilent 1290.
The firts link is about thiamine, not thiamine pyrophosphate. I had seen the third link. I think the first peak comes to early so i like to avoid this if posible. The second link is the aplication vlad from sielc developed for me. I ame trying to reproduce this but get horible results with b1 on my agilent 1290.
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