I am determining butyric acid in plasma.
For this, I am developing a method. I will be giving a brief on how I prepare my highest concentrated calibration standard.
I prepare my internal standard (d7- butyric acid) in 100% acetonitrile having a concentration of 100 uM. From which, I transfer 10 uL to a cold glass vial along with 10 uL of 10 mM of Butyric acid as my highest concentrated calibration standard. I use 10 uL of 20% PFFBr in 100% acetonitrile to derivatise my standard and heat it for 1 hour at 60*C.
- The problem I am facing is low signals of internal standard to an extent that it cannot be even detected within the given retention time.
- Also, I am receiving signals for butyric acid in my blank and no peak for internal standards.
Could you please help me?
Hello Prapti Chakraborty
You may have some contamination in your system. Check whether your evaporator tube is clean or whether deposits are visible. If in doubt, replace the evaporator tube.
The head of the separating column may also be contaminated; shorten the start of the column by at least 10 cm or more.
Another possibility is a contaminated ion source.
Since you will also find butyric acid in the blank, you should also check the autosampler for contamination and adjust the rinsing steps so that the injection syringe is really clean.
I hope that you can make progress with these tips
Best regards and good luck with your work
Joachim Horst
Thank you Joachim for the support and advice. I’ll definitely keep this in mind. Thank you for your answer. I appreciate your help.
If you cannot derivatize the short-chain fatty acids, they can accumulate in the inlet/liner/column head, etc due to their low volatility...
Therefore it is very critical to get a satisfactory dervizatization yield...Probably the yield of the derivate was low and free fatty acid along with its deutereted form remained intact and stuck to the sample introducing parts of the GC before chromatographic separation...
To increase the efficiency, increase the reaction time, test the elevated temperatures, and, add more PFFBr and organic solvent to overcome any stochiometry-related issues...At this point, it may be better to choose another aprotic organic solvent as a reaction buffer like hexane, and observe the efficiency in comparison. Investigate if any co-solvent or agent is necessary or not to promote reaction...
Methylation of butyric acid in many cases also works well and would be sufficient for quantification. So, I mean other than PFFBR derivatization there are other ways to do that such as TMS or acid catalysis followed by methanol spiking...
Here is an example;
Samples can be derivatized neatly after dissolving in a solvent. If appropriate, dissolve the sample in a nonpolar solvent (such as hexane, heptane, or toluene). If the sample is in an aqueous solvent, first evaporate to dryness then use neat or dissolved in an organic, non-polar solvent.
Weigh 1-25 mg of sample into a 5-10 mL micro reaction vessel.
Add 2 mL BCl3-methanol, 12% w/w. A water scavenger (such as 2,2-dimethoxypropane) can be added at this point.
Heat at 60 °C for 5-10 minutes. Derivatization times may vary, depending on the specific compound(s) being derivatized.
Cool, then add 1 mL water and 1 mL hexane.
Shake the reaction vessel (it is critical to get the esters into the non-polar solvent).
Thank you Ismail, for taking the time to help and write in such details. I appreciate that. About the water scavenger part, I am not quite sure what you mean. İsmail Emir Akyildiz
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