I want to measure the concentrations of the free amino acids in a plant sample using HPLC can anyone guide me what is the best method to extract the free amino acids with reference if possible?
Hi Nader
By the nature, all free amino acid is amphoteric substance. The carboxilic group comes to be negatively charge at high pH, whereas the amine group is cationic species at low pH. Therefore, both at high or low pH the amino acid is water soluble. You can stir the suspension of powdered plant in HCl or NaOH solution to extract free amino acid. Separate the undissolved powder by centrifugation or filtration so you get amino acid solution in aqueous medium.
Since a plant contains numerous kind of amino acid, you must have the standard for all kind of amino acid (more than 20 items) for HPLC measurement. I will suggest you to start with top 5 of amino acid existed in your plant base on literature and optimize the HPLC system. Start the optimization using the C18 fixed phase and the gradient elution of methanol/ACN/TCA as mobile phase. Or get your idea of HPLC system from this paper.
Article Chromatographic method for determination of the free amino a...
Regards
Annas
Hi Nader,
I've found the following books. Maybe you can use them as a starting point:
https://www.hugendubel.de/de/buch/plant_analysis_procedures-3198944-produkt-details.html?gclid=EAIaIQobChMI5q3PuaXo3gIVC-R3Ch2ZygQPEAQYASABEgLULPD_BwE&adcode=832Q30Z22A41S
https://www.hugendubel.de/de/buch/analysis_of_plant_waste_materials-14517955-produkt-details.html?gclid=EAIaIQobChMI5q3PuaXo3gIVC-R3Ch2ZygQPEAQYBiABEgJPHfD_BwE&adcode=832Q30Z22A41S
https://www.springer.com/de/book/9789401176514?gclid=EAIaIQobChMI5q3PuaXo3gIVC-R3Ch2ZygQPEAQYCyABEgLgUPD_BwE#otherversion=9780792331827
https://www.hugendubel.de/de/buch/a_j_harborne-phytochemical_methods_a_guide_to_modern_techniques_of_plant_analysis-3680867-produkt-details.html?gclid=EAIaIQobChMI5q3PuaXo3gIVC-R3Ch2ZygQPEAQYDCABEgKYivD_BwE&adcode=832Q30Z22A41S
Regards Markus
Hi Nader
By the nature, all free amino acid is amphoteric substance. The carboxilic group comes to be negatively charge at high pH, whereas the amine group is cationic species at low pH. Therefore, both at high or low pH the amino acid is water soluble. You can stir the suspension of powdered plant in HCl or NaOH solution to extract free amino acid. Separate the undissolved powder by centrifugation or filtration so you get amino acid solution in aqueous medium.
Since a plant contains numerous kind of amino acid, you must have the standard for all kind of amino acid (more than 20 items) for HPLC measurement. I will suggest you to start with top 5 of amino acid existed in your plant base on literature and optimize the HPLC system. Start the optimization using the C18 fixed phase and the gradient elution of methanol/ACN/TCA as mobile phase. Or get your idea of HPLC system from this paper.
Article Chromatographic method for determination of the free amino a...
Regards
Annas
I suggest use of peptide column and reverse phase mobile phase for it....
I would do what Annas Binarjo have suggested, and would do it on ice because of the high heat generated by mixing with such stong acids, also I would suggest using Trichlouro acitic acid, which is more gentel than HCl on the hydrolysis front of the problem.
Anyhow, for the analysis it self i would pick an Ion-Exchange column with buffer gradient rathe than the C18 phase with methanol/ACN/TCA. The C18 works better on peptieds and proteins but the ion-exchange works better for free amino acids.
what kind amino acid you want it? if all of free amino acid, they are amphoteric substance, some of strong acid and some of strong basic or some of hydrophlic and some of hidrophobic. You could be extracted all of FAA with combine between aquades and methanol/ethanol. Remember dont do with HCL extracted because its not obtained the nature of FAA. Using HCL mean you break down of peptide/protein to be FFA.
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