Hello, to know unknown concentration of known substances you need to have some reference material with known concentration for estimation.

There is no other reliable way.

Good luck !

You can prepare a standard curve of either absorbance or fluorescence using the fluorescent lysine. Both measurements are directly proportional to concentration, but on different scales of concentration. For fluorescence, you should use nanomolar to low-micromolar concentrations. For absorbance, you should use low-micromolar to high-micromolar concentrations.

There is a risk using fluorescence that the quantum yield of the fluorophore differs between the two compounds, which will introduce an error when using one compound as the standard for the other. This is less likely to be a problem using absorbance, but you need much more of the compounds to use the absorbance method.

Adam B Shapiro Thanks for the guidance, but does that mean I can use either fluorescence or absorbance standard curve of Lysine for any other amino acid as well? I mean whether its an amino acid as small as Glycine or any aromatic amino acid, all can be considered from the standard curve of Lys.

Surbhi

Yes for sure, use fluorescent tagged Lys (I guess it should be tagged by amide NH bond) as standard for fluorescent tagged amino acid(I guess it also should be tagged by amide NH bond), they are same type of conjugate. Better than the use of the free fluorescent dye (acid). You better check the "know" concentration of your fluorescent tagged Lys by the extinction coefficient of the fluorescent dye to be sure. If you use UV spectrometer, better to adjust your absorbance between 0.2 to 0.9 (need 1ml or 3ml sample). If you use nanodrop, it will only need 1 to 2ul sample.

Ideally, you would use the same amino acid as a standard as the one you want to measure, in case the side chain affects the fluorescence quantum yield or extinction coefficient of the fluorescent dye. If you decide to use the fluorescent lysine as the standard, keep in mind the possibility that it may not be accurate for all amino acids. If the fluorescent dye absorbs in the ultraviolet (260-280 nm), then the absorbance of aromatic amino acid side chains will contribute to the extinction coefficient. In addition, tyrosine and tryptophan have fluorescence in the UV range.

If you use the fluorescence/concentration calibration line of one compound to predict the concentration of a similar compound, you may make a mistake due to different fluorescent quantum yield. However, this error will affect all your measurements of the second compound in the same way, i.e., by a constant factor. You need to decide whether you can live with that.