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Tips & Tricks for Amino Acid Analysis – Part IV

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Ever wonder why chemical supplies are shipped or prepared a certain way?  Stability is often the answer.

Hi guys! We have a guest writer for today’s post, my colleague Bill from the small molecule columns team. He’s sharing some insight into why the extended amino acid standards from the AdvanceBio Amino Acid Analysis Kit are shipped separately and how to best prepare them. There’s even some electron pushing for anybody who secretly loves organic chemistry.

What is the proper sample preparation for the extended amino acids standards?

It has been noted that the procedures for preparation of Extended Amino Acid Standards have not been consistent. This application note describes making this solution just using de-ionized water. Another application note refers to making this solution using 0.01 N HCl and water. It does state however, “Solutions containing extended standards are unstable at room temperature. Keep them frozen and discard at first signs of reduced intensity.”

Some of the supplemental amino acid standards degrade in HCl, especially glutamine and asparagine. The correct answer is to dissolve the supplemental amino acids in de-ionized water, and if using them in a mixture with other amino acids, be aware that they are dissolved in dilute HCl, so they must be used quickly.

In addition, other amino acids are also noted to degrade under acid hydrolysis conditions. These include tryptophan and 4-hydroxyproline. However, the mechanism of their degradation is different.

Note the amide group on both amino acids. When exposed to acid, these groups would hydrolyze, releasing ammonia.

Mechanism illustration source

That wraps up our series on amino acid analysis. We’ll spend the next few posts on peptide mapping. Talk soon!

– Anne

Keywords: Bio columns, liquid chromatography, tips and tricks, amino acids, sample preparation, AdvanceBio blog

Parents

  • Thank you Anne and Bill for another great tips on Amino Acids Analysis. 

    I hope to see more tips and trick on sample preparation next time. 

    Working with animals feed myself required me to homogenize the samples with a blender and digest them in 6M HCl for 24 hours, and finally re-constituted the dry sample in 0.1M HCl prior to injection. 

     

    However, since I worked with Cysteine, I have been using DTDPA myself. As pointed out in application notes (both Zorbax Eclipse Plus and HPH C18), borate buffer (0.4N, pH 10.2) should be used to prevent solubility problem. I tried with both HCl and borate buffer, no crystallization/particulate formed after filtration with 0.2 um RC filter. However, letting samples stand at room temperature for a few hours, or thawing samples from -20'c freezer always see formation of precipitate. I have to filter them again before injection. I wonder what goes wrong. 

Comment

  • Thank you Anne and Bill for another great tips on Amino Acids Analysis. 

    I hope to see more tips and trick on sample preparation next time. 

    Working with animals feed myself required me to homogenize the samples with a blender and digest them in 6M HCl for 24 hours, and finally re-constituted the dry sample in 0.1M HCl prior to injection. 

     

    However, since I worked with Cysteine, I have been using DTDPA myself. As pointed out in application notes (both Zorbax Eclipse Plus and HPH C18), borate buffer (0.4N, pH 10.2) should be used to prevent solubility problem. I tried with both HCl and borate buffer, no crystallization/particulate formed after filtration with 0.2 um RC filter. However, letting samples stand at room temperature for a few hours, or thawing samples from -20'c freezer always see formation of precipitate. I have to filter them again before injection. I wonder what goes wrong. 

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