Hello. There is a LOT of information you need to provide for us to give you any meaningful suggestion. Such as what is your matrix (water, tablets, creams, tissue extracts, food, beverage, etc), which amino acids you need, desired limit of quantitation, degree of rigor for your method (validation, qualification in industry, GMP, R&D, academic, school work, etc).
As general suggestions go. Routine C18 column can work given that you only have a few amino acids to deal with (see below). If you need to analyze many amino acids at once, explore Waters AccQ•Tag Columns and derivatization procedure, FDNB dye for derivatization, and/or HILIC columns.
I have analyzed many natural and modified amino acids on regular C18 column with methanol or ACN + aqueous buffers (phosphate, ammonium acetate buffer, 0.1% formic acid/TFA for ion pairing). For most amino acids, you are looking to use 205-210 nm range (no methanol in your mobile phase in this case) and for aromatic ones you can use 280 nm (you can then use methanol). The problem arises if you have multiple amino acids that need to be resolved and analyzed, and when low quantitation limits are needed. Also when you have interfering components in the matrix.
Not sure why you see nothing since you didn't provide details about your method, gradient, mobile phase, injection/sample load, etc. They should be visible on UV even without derivatization. Some notes:
- Make sure you are not using solvents that have UV cutoff higher than your amino acids (for example, if you have TRIS buffer it will make it impossible to see analyte response at 205-210 nm).
- Same goes for your matrix.
- For UV detection without derivatization, load a minimum of 10 ug of amino acid per injection.
- In addition, check your mobile phase pH and your amino acids pKa values. If your amino acids are strongly charged at your mobile phase pH, they will not retain and will elute in the void volume along with your diluent.
- If you need to use C18 column, use not more than 2% organic phase at the beginning of your gradient since most amino acids will elute very early.
Thanks for your suggestion.
I am following the Agilent method.
I have not run samples yet.
I have Loaded only standard (1nmol/uL in 0.1M HCL).
I have used the online derivation method (File attached).
This is for research purposes (Academics).
Matrix is Food.
If you please send me your one of the papers which i can follow.
I want to analyse the essential amini acids.
I have purchased amino acid standard kit (Mix of 17 Amino acids), and other reagents which have mentioned in paper(Agilent paper attached) from agilent. But still getting only few peaks.
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