Spektrofotometrijska metoda određivanja triptofana iz hidrolizata proteina

A novel spectrophotometric method for determination of tryptophan content in protein hydrolysates has been developed. The reagent used is diphenylamine sulphonate which is oxidised to diphenylbenzidine sulphonic acid after reacting with sodium nitrite in the sulphuric acid medium. The unstable oxidation product reacts quickly with sodium nitrite to produce a diazotized intermediate. When the diazotized intermediate is coupled with tryptophan, a pink colour product is developed, which is stable for at least 1 h at the ambient temperature. This coloured product has the absorption maximum at 522 nm and the molar absorptivity is 8900 L/(mol·cm). Beer’s law is obeyed in the range of 0.30–12 mg/mL. The method is applied for the analysis of tryptophan content in grass carp protein hydrolysates. Moreover, it is compared with the reversed-phase high-performance liquid chromatography analysis. There is no significant (p<0.05) difference between the two results. The method is simple, rapid and accurate compared to the previous methods.Razvijena je nova metoda određivanja udjela triptofana u hidrolizatima proteina. Kao reagens upotrijebljen je difenilamin-sulfonat, koji reakcijom s natrijevim nitritom u mediju sa sulfatnom kiselinom oksidira u difenilbenzidin-sulfonsku kiselinu. Nestabilan proizvod oksidacije brzo reagira s natrijevim nitritom te nastaje diazotizirani intermedijer. Spajanjem diazotiziranog intermedijera s triptofanom nastaje produkt ružičaste boje koji je barem 1 sat stabilan na sobnoj temperaturi. Taj obojeni produkt ima apsorpcijski maksimum pri 522 nm i molarni apsorpcijski koeficijent od 8900 L/(mol·cm), te se ponaša u skladu s Beerovim zakonom u rasponu od 0,30 do 12 µg/mL. Ova se metoda primjenjuje za analizu udjela triptofana u hidrolizatima proteina amura, te je uspoređena s visokotlačnom tekućinskom kromatografijom obrnutih faza (RP-HPLC). Nije primijećena značajna razlika (p