or is it just easier to get an amino acid specific column? This is for amino acid profiling of ground insect samples to be used in fish feed -- amino acid profiles are vital to formulate nutritionally balanced feeds.
Dear Darja,
Well, why not thinking to analyse amino acids by Amino Acid Analyzer. If so please find my attachment.
Good luck
Aly R Abdel-Moemin
Article Analysis of phenolic acids and anthocyanins of pasta-like pr...
There are a bunch of methods out there that use a C18 column. It's been a long time, but I used one by Jones and Gilligan (J Chromatogram (1983) 266:471) and it was used at my old University Dept for years after I left. It uses OPA to make a fluorescent derivative of primary amino acids. Like most methods, however, if you want a complete profile you have to do more than one type of hydrolysis to get things like cysteine, methionine, tryptophan, glutamine and asparagine. For the work I was doing, for most amino acids hydrolysis with 6M HCl for 24 hr was pretty standard, followed by drying to get rid of the acid, and then pre-column derivatization.
What I'd suggest is maybe checking out the Jones and Gilligan method as a start and seeing what new hydrolysis procedures/derivatives might be available (i.e. so you can get Cys and Met at the same time). During one of my post docs I developed a method for hydroxyproline (2ndary amino acid) analysis using FMOC as a derivatizing reagent and capillary electrophoresis (J. Chromatography B, 744:195-199) so I'm sure there must be others out there. Good Luck!
If you're just starting with this type of analysis, make sure you start with a new column! When I first started I was given an old column to use, and I fooled around with it for a couple of months before I figured out it was screwed.
The column you are talking about is quite common, however the efficacy of the results depends upon the hydrolysis and derivatizing methods. Hydrochloric acid (6 molar) at 110 degree celsius for 24 hours gives standard hydrolysis, however this method could adversely affect the sensitive amino acids e.g. tryptophane and cysteine. You may add propionic acid and/or methansulfonic acid in hydrolysis system in order to improve the yield of sensitive amino acids and to perform the process quickly. Keep in mind that derivatization of the sample is responsible for modulation of chemical structures in a way that they could be easily separated inside the column, producing sharp peaks in recorded chromatogram. Moreover, derivatization is not a single method/procedure, and you can use any standard procedure of your choice. However, the final selection will be based upon the accuracy of the chromatogram peaks. When you will get a good derivatization of your sample, then HPLC operation would not be a serious problem. I shall suggest you to concentrate on hydrolysis method, derivatization process and the addition of reference/ standard chemical (if you require quantification of amino acids). Good Luck.
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