Dear Aisha,

I'm not sure if you can properly measure amino acids with an DAD detector only in such a short runtime. We use precolumn OPA derivatisation and subsequent fluorescence detection and the gradient described below:

https://www.phenomenex.com/Application/Detail/15992

The order of elution from aspartate (1) to proline (32) is also shown.

Best

Michael

Dear Aisha,

I'm not sure if you can properly measure amino acids with an DAD detector only in such a short runtime. We use precolumn OPA derivatisation and subsequent fluorescence detection and the gradient described below:

https://www.phenomenex.com/Application/Detail/15992

The order of elution from aspartate (1) to proline (32) is also shown.

Best

Michael

You can check the elution order on this column in the literature. If the nimber of peaks correspond to the nimber of standards in your mixture the task is relatively simple.

Regards

G

As mentioned by others, without derivatization, analyzing amino acids might be challenging on reverse phase. We do Marfey’s derivatization as follows:

https://link.springer.com/article/10.1007%2Fs13361-018-2093-9

Dear Aisha

I agree with Michael re-derivatization/fluorescence detection. But leaving this aside and focusing on the identification of the different peaks in your chromatogram, I think the safest approach would be to use individual standards to confirm your retention times. I would not rely only on literature and absorption spectra, if available. I think it is imp to establish well how does the standard behaves in your system before you move into samples analysis.

Even when you use certified reference materials, it is important to ensure that you are using similar conditions to those used in the laboratory of reference, since changes in the solvents or column characteristics could lead to shifts in your retention times.

I use LCMS, which makes it easier to identify each amino acid using selective transitions. But even like this, we use different standard mixes and internal standards to confirm how my compounds of interest elute in my system.

Once you are sure of the order you get in your system, you can use your standard mix to make standard curves.

I hope it helps

Interesting! I am Following!!

Depdending on the available equipment in your lab, this recently published method without derivatization might be intersting:

Article A non-derivatized method for simultaneous quantitation of pr...