We would like to analyze the concentrations of amino acids in samples derived from a fermentation experiment using HPLC. Besides amino acids, these samples contain different salts required for growth of the bacterium we use.
So far, we have analyzed the filtered samples directly without any further purification but the resulting chromatogram is dominated by peaks from impurities and the baseline is shifted.
We have tried to solve the problem by diluting the sample (1:5, 1:10, 1:20, ect.) which improves the baseline and eliminates most of the impurities. Unfortunately, some of the peaks from the amino acids disappear, so this does not seem to be the most ideal strategy.
I have considered to remove the salts/other impurities by Solid Phase Extraction (SPE) prior to the analysis of the samples.
Does anyone have experience with using this approach for sample purification?
Which SPE column would you recommend for purifying this type of sample (mainly one that binds salts) to obtain a purer sample of amino acids?
I have found different SPE columns at Sigma (https://www.sigmaaldrich.com/DK/en/applications/analytical-chemistry/sample-preparation/solid-phase-extraction) but I am not sure which one will be the most suitable for my samples.
Thank you in advance.
Dear Freja
Why did you think the impurities were due to salt? It is not possible to give an answer to your question without knowing the details of the analysis method apllied.
BioSpin columns from BioRad, Amicon filter from Merck Millipore or HiPPR™ Detergent Removal Spin Column Kit
Also you can try liquid-liquid extraction technique to purify the sample.
Thaís Pedrete thanks for the recommendations.
I have looked at the different Bio-spin columns from Biorad and it seems there are several types. Which one can you specifically recommend for purification of amino acids prior to HPLC analysis?
Freja Karlsen BioRad spin columns are size exclusion, so your amino acids should be removed with salts, due to the size. Maybe you can consider by affinity. https://www.thermofisher.com/order/catalog/product/89879
Or you can ask directly to Amos Heckendorf [email protected] from NestGroup https://www.nestgrp.com/protocols/protocol.shtml#deterg
He is very helpful!
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