Question about chemical compatibility of RP column resin

Record: 857

Category: Column Problem
Product: C18-4E
Sample:

Question
We have the C18-4E reversed phase column, and we are interested in doing analysis of free amino acid produced as a fermentation by-product.

There is a method to react primary amines with ninhydrin, which gives a compound that absorbs at 570nm. Our cells produce alanine as a fermentation product, and the amounts of alanine in the media should be much higher than any other amino acid. So we probably won’t need to get a lot of separation to see a clear alanine peak.

The ninhydrin reagent comes in DMSO. Do you have any knowledge about DMSO compatibility with the column packing? Will it be safe to load 5-10 ul of sample with about 50% DMSO in water?

Answer
The DMSO and reverse phase silica should not have compatibility issues; however, the DMSO may distort the peaks on your chromatogram blurring individual peaks together.

Ninhydrin is also sold as a solid and may be dissolved in methanol, water, and probably other HPLC compatible solvents. Just a question, but how will you differentiate the alanine-ninhydrin complexes from other amino acid-ninhydrin complexes? The other amino acids except for lysine will all have primary amine groups as well, right? Just wondering.

If you notice broad peaks that you cannot resolve the DMSO may be causing issues. If you have access to a lyophilizer, you may be able to remove some of the DMSO by diluting the sample with ACN: water, freezing the sample and lyophilizing it.

 
 
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