Only three amino acids (Trp, Tyr, and Phe) have conjugate system so they absorb UV light. But Phe absorption is negligible. Therefore you can only measure Trp and Tyr. The absorption maximum for Tyr and Trp is about 280 nm. They also fluoresces. You can measure their concentration from the absorption data at a particular wavelength using their molar extinction coefficient at that wavelength (use this formula OD = e.c.l; where e is the molar extinction coefficient, c is the concentration, l is the pathlenght of the cuvette used).

Absorption and fluorescence behaviour of free Trp and Tyr in water:

http://omlc.ogi.edu/spectra/PhotochemCAD/html/091.html

http://omlc.ogi.edu/spectra/PhotochemCAD/html/092.html

Trp and Tyr are also there in the proteins present in the serum. You also need to get rid of all the proteins in order to get the free trp an tyr concentration in serum.

Only three amino acids (Trp, Tyr, and Phe) have conjugate system so they absorb UV light. But Phe absorption is negligible. Therefore you can only measure Trp and Tyr. The absorption maximum for Tyr and Trp is about 280 nm. They also fluoresces. You can measure their concentration from the absorption data at a particular wavelength using their molar extinction coefficient at that wavelength (use this formula OD = e.c.l; where e is the molar extinction coefficient, c is the concentration, l is the pathlenght of the cuvette used).

Absorption and fluorescence behaviour of free Trp and Tyr in water:

http://omlc.ogi.edu/spectra/PhotochemCAD/html/091.html

http://omlc.ogi.edu/spectra/PhotochemCAD/html/092.html

Trp and Tyr are also there in the proteins present in the serum. You also need to get rid of all the proteins in order to get the free trp an tyr concentration in serum.

I agree with Uttam. Very good explanation.

Another cheap method is to use TLC and revealing by a ninhydrin solution. It is ver sensitive for amino acids and cheaper because you don't need a HPLC system/solvents or UV reader.

Unfortunately, human serum is complex. With no reagent used, you can measure with UV-Vis at 260 280, but this will also pick up nucleic acids, and is known to be highly variable. This method is the Warburg/Christion method, and ranges between 200-2000 ug/ml. More specific methods would require a dye in serum, such as in the Lowry or Bradfod methods, but with a more sensitive but narrow concentration range. The W/C Mothod is best for purified protein. The Bradford assay is really inexpensive. Several different Lowry methods exist. But, BCA is by far the most dominant method to measure serum protein concentrations. Hope this helps.

they usually do not absorb well in the UV except for hystidine, tyrosine alanine and tryptophan. For them no single/common peak

Thanks Uttam, Lendro, Jonathan and Teodoro for your guidelines..... I also want to reveal that thing I did some experiment with mixture of acid and polar solvents and got some peak near to 390 nm in serum sample for cystine is it okk??? I tried to get linearity with standard addition method and i getting good result in it so is it okkk???

You can analyse amino acid by HPTLC method with solvent system n-butanol:acetic acid: water (12:3:5) and plate was developed with ninhydrin reagent. detection was carried out at 340 nm. if you want i can give detail procedure.

Thanks Washim Khan I need it if possible for u..........

For the estimation of amino acid, HPTLC was performed using a mixture of Butanol: Acetic acid: water in the ratio of 12:3:5 as mobile phase. Linear ascending development was carried out in 20×10 or 10×10 cm twin through-glass chambers (Camag, Muttenz, Switzerland). The optimized chamber saturation time for mobile phase was 20-30 min at room temperature with relative humidity of 60%. The chromatogram was developed until the solvent moves 75% of the plate. The plate was air dried and the ninhydrin solution (0.1% in n-Butanol) was sprayed on the plate. TLC plate was dried at 100 degree celcius in hot air oven for 10 min. Densitometric scanning was performed by a Camag TLC Scanner III in absorbance mode at 400nm and analysis was done by winCATS software (version 1.2.0).

Thanks Washim

Please check this link

http://www.biotek.com/resources/articles/peptides-amino-acids-fluorescence.html