We tried a Luna C18 260 x 4.6 but analysis time is too long and separation is not good for 6 amino acids. We are looking for a column which a researcher has worked with and is satisfied and not only a column from an application brochure of a company.
See this discussion on Researchgate.
https://www.researchgate.net/post/Does_anyone_have_a_pre-column_derivatization_method_for_amino_acid_analysis_OPA_not_suitable_for_autosampler_FMOC_tried_but_has_problems-help_here#view=5883b97d93553b8fb1240f6a
Bruce they use a special derivatization agent which is quite expensive to purchase either alone or the entire kit. It is not PITC derivatization. I am looking for someone who has worked with PITC amino acids and used a column he/she can suggest. There are columns in the market in brochures but I trust more the experience of an independent researcher.
It would be helpful if you would provide your actual analysis conditions and column details (i.e. was this a 5u, 3u ... support?). There are multiple versions of this procedure. Most importantly, which AA's are you having problems with? Can you provide a chromatogram using your current method? I have used the older PITC pre-column derivatization technique with success hundreds of times over the past decades. As I recall, we usually used a high quality C18 column such as a Zorbax (stay away from the Phenomenex rebranded columns) Eclipse Plus C18 or equivalent columns. The key is of course sample prep and matching the correct column dimensions to the gradient conditions used (to the exact method/sample). Some of the AA's are always going to elute close together using this specific method, but again, which ones are you having issues with?
Dear Bill thank you for your answer and comments. We use a Luna C18 3μm 250 x 4.6mm. The mobile phase is A: buffer pH6.5 and B: ACN:H2O 4:1 under 0.3 ml/min flow and a gradient starting from 3% B. The amino acids which can not be separated are GABA and valine, glutamine and serine. We noticed that when the standards are dissolved in water the separation is better than when they are dissolved in 0.1 N HCl. But we will use 0.1 N HCl to extract the amino acids from our tissue. Do you have any other idea for the solvent used to extract the amino acids? Furthermore, cysteine is being broken to three peaks uder the conditions we work. I believe that if we change the pH value of the samples, chromatograph could be improved. Is that right? Have you any suggestions? I was thinking of getting a column for UPLC where in the company's brochure they show that it can separate 17 amino acids within 4 minutes, but as we all know these are sometimes too good to be true.
Thank you for adding some additional information. So here are a few quick comments.
"UPLC" is a trademark, not a column. Perhaps you were referring to an UHPLC method? It is all just 'HPLC'. Please do not worry about trying any columns with super low volumes in them unless you want to start over from scratch and also have fully optimized the flow path and dwell volume of your entire HPLC system. Stick with the simple, well proven methods for now (unless money and time are plentiful). PITC is not always the best AA approach, but it works for many samples.
YES, pH is very important. It is one variable of many in HPLC method development. Remember, change only one thing at a time! Try pH 5.5 (or even 5.2) with your method. Peaks may shift, but resolution will probably improve. Temperature (you did not provide us any info?) if too low, can be raised AFTER better resolution has been obtained to reduce total run time too.
Preparing samples in 0.1N HCL ! Wow, that is too strong. We often stay under 1mM or keep it closer to neutral. You are causing problems before you start.
BTW: Make sure you are properly cleaning and washing that column down after each run (Use high percentages of MeOH, ACN in addition to water).
OK, that is enough free info to get you started again. Good luck.
http://hplctips.blogspot.com/2015/08/terminiology-which-is-it-uplc-uhplc-or.html
Bill are you suggesting using 1mM HCl for extraction and pH 5.5 for mobile phase A? We are running the samples at 30 C. We will try 35 and 40 after having optimized the method as you said. Thank you for your support. We will keep on trying with Luna and let as last choice the column for UHPLC analysis, as the cost is quite high as you already know!
Yes. 1 mM ~ pH 3.0, but your samples were not my samples... After prep, try to dissolve in your mobile phase for better results.
Ok, thanks, I ll let you know our progress based on you suggestions Bill. Thank you very much, deeply appreciated!
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