We tried FMOC but we do not seem to achieve desired results. High peak of FMOC molecule and low derivatization of amino acids. Can you describe the method and reagents used in FMOC? OPA has worked but we want to use the autosampler, so it is not suitable. Has anyone used PITC? Any reliable method for PITC derivatization?
Thank you all for your answers. We will try that of Li since we do not have the instrumentation required to perform the method Prof Hayakawa suggested and let you know.
Dear Professor thank you very much for your suggestion. I have seen this and I believe we can try it. Have you also tried it in your lab? Can I consult you in case we experience any problems?
Dear Professor thank you for your willing to help. us. We will try it next week and let you know. I hope you have a joyful retirement period. Thank you again.
Here is AOAC method using FMOC-succinimide for glucosamine deriv.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596946/
Passed collaborative study. Is quantitative conversion. Requires triethylamine base catalysis, then neutralize w/ HOAc. Worked for me and hope it might be helpful. Glucosamine is not amino acid, I realize. HPLC separates analyte from excess reagent. Best of luck.
Thank you Matthew, if the one we are trying now does not work for us we will try your way!! Thank again!
Dear all thank you for your help. We have tried PITC derivatization and we have good seperation of 20 amino acids, using the method Professor Hayakawa suggested.Thank you Professor! Have all a great New Years eve and a happy new year!
Well abour AccQ Tag we have not tried it, but for us the cost was too high. It was approximately 2500 euros for a full set just for 250 samples. If we were use it for commercial purposes, i guess it might worth it, but for research purposes under economic crisis this method is too expensive. We find PITC quite easy and affordable. It may not have the LOD of that of AccQ Tag but for now it suits us fine. The reagent AccQ Tag uses to be bought alone is also quite expensive, so we preferred the other way.
Dear all, is it necessary to deproteinise the extract (plant tissue extracted with 0,1N HCl) before derivatization? If yes what would be the best way to do so (Acetonitrile, sulfosalicylic acid, TCA and at which ration to the sample?). Could the protein in the sample if not deproteinise, block HPLC column?
Dear Professor thank you for your continous help. I have seen that some researchers do not deproteinise the sample and use it directly for derivatization and I was wondering if we could follow that approach. We used ACN at a ratio 4:1 ACN:SAMPLE but the analysis was somehow worse than before. We will try TCA tomorrow to see if it is better for our samples. I will also use guard column to preserve the analytical column as you proposed.
If you extract using 0,1N HCl you have deproteinized in part your sample, as going at pH 1 (or below) you're under the isoelectric point of most proteins. Of course this don't mean all the proteins are precipitated, I have no idea of what plant proteins or peptides might be there. If it essential to you, you might try another precipitation pass using a different method like solvent precipitation, I can't say exactly at which parameters as I don't know the sample you're working on.
However what Prof. Hayakawa said about guard columns is sensible when working with proteins, notwithstanding the deproteination state of your samples.
Dear Manlio thank you for your response. We are trying now acetonitrile as precipitation agent, using 4 volumes of ACN and 1 volume sample, but it is diluting it too much. We will use smaller volumes, hoping to get good results. Another question raised is if during the derivatization process with PITC, where all the derivatization reagents are diluted in ACN, with its final (ACN) ratio to sample being 1:1, the proteins precipate. The derivatization procedure lasts for 1h with a sample to ACN ratio 1:1; could it be possible under these conditions to precipate the proteins, so no prior precipitation is required?
Try adding 25μl/ml of final volume 5N NaOH during ACN precipitation.....it improves precipitation a lot.
Dear Dimitris thank you for your help, we will try it. Does anyone have a suggestion on HPLC column for the analysis of the PITC amino acids?
Dear
This paper may be help: J.W. Henderson, R.D. Richker, B.A. Bidlingmeyer (2000). Rapid, Accurate, Sensitive and Reproducible HPLC analysis of amino acids. Agilent Technologies Co. (USA) part no. 598 – 1193E.
http://www.shimadzu.com/an/hplc/prominence/xr-ods.html
Dear Marious thank you for your interest and help. Best regards, Peter
mass propagation and ex-situ cultivation/domestication of many land-ward mangroves may be the unique sources of raw materials for medicinal & nutritional uses.
Sir, can you please share the PITC derivatization method as we are facing problem in peak resolution.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction