A visible difference at the end of the reaction
Two mirror-image forms of tryptophan produced clearly different gold nanoparticle suspensions in a laboratory test, giving researchers a one-step color-based approach for distinguishing d-tryptophan from l-tryptophan and estimating their proportions in defined binary samples. The method forms the particles in the reaction mixture itself.
Each assay mixed 1 mL of tryptophan solution with 1 mL of copper(II) sulfate and 1 mL of gold(III). The selected final recipe contained 0.16 mM gold(III) and 3 µM copper(II), with total tryptophan concentrations ranging from 104 µM to 3.3 mM. The reaction ran at 25 °C for 15 minutes.
At the endpoint, l-tryptophan produced a purple suspension that was aggregated and partly sedimenting, with a weaker spectral band near 570 nm. The d-tryptophan suspension was predominantly dispersed and light pink, with its band centered at about 590 nm.
Turning color into a composition estimate
To turn that visible difference into a composition estimate, the researchers held the total tryptophan concentration constant while changing the share of d-tryptophan. That share, called the d-tryptophan mole fraction, ran from 0 for pure l-tryptophan to 1 for pure d-tryptophan.
Absorbance at 570 nm, a measure of how much light the sample takes up, increased as the d-tryptophan share rose. The researchers used that A570 response to build the calibration relationships. They also examined the ratio of absorbance at 590 nm to absorbance at 570 nm, but did not use it because its linearity was inconsistent.
The calibration was evaluated at five fixed total tryptophan concentrations: 104 µM, 204 µM, 0.41 mM, 0.83 mM and 3.3 mM. Each level had its own linear regression, so the results did not represent one continuous concentration range.
The reported fitted-line slopes for the 570 nm response were 0.0759 at 104 µM, 0.2069 at 204 µM, 0.3151 at 0.41 mM, 0.3266 at 0.83 mM and 1.3707 at 3.3 mM. The response therefore depended on the total amount of tryptophan present.
The particles differed more in arrangement than size
Electron microscopy showed overlapping particle-size distributions, but different patterns of association. The mean primary-particle diameter was 42.9 ± 2.8 nm in the d-tryptophan system and 47.0 ± 7.4 nm in the l-tryptophan system. Particles in the d-tryptophan samples were predominantly discrete, while those in the l-tryptophan samples were extensively associated.
Before selecting the working conditions, the researchers varied gold(III) concentration, reaction time, temperature, pH, copper(II) concentration and tryptophan concentration one factor at a time. Fitted trendline slopes were used to choose the conditions for the assay.
A narrow validation produced repeatable results
Precision testing used three replicate measurements of a 0.83 mM total-tryptophan standard within one day, producing a relative standard deviation of 2.8%. Measuring the same standard over three consecutive days gave a relative standard deviation of 4.3%.
For the accuracy check, the researchers spiked d-tryptophan into l-tryptophan to create mixtures with d-tryptophan mole fractions of 0.25 and 0.65. The reported t-test values were 2.5 and 1.8, respectively. With three measurements at a 95% confidence level, the tests found no significant difference between the measured and theoretical mole fractions.
The measured value for the mixture expected to contain 0.250 d-tryptophan was 0.2240, with an 8.04% relative standard deviation and a relative error of −10.4%. For the mixture expected to contain 0.650 d-tryptophan, the measured value was 0.6374, with a 1.88% relative standard deviation and a relative error of −1.94%.
What the experiment does not establish
The validation was limited to composition-defined binary mixtures of d- and l-tryptophan, without added amino acids or other matrix components. The work did not establish selectivity in the presence of other amino acids, reducing agents, chelators or complex samples.
The experiment also leaves open why the two forms produced different endpoint behavior. Ultraviolet-visible measurements and electron microscopy demonstrated an enantiomer-dependent difference in nanoparticle formation and aggregation, but did not establish an intrinsic chiroptical origin for the particles. Direct chiroptical, structural and time-resolved studies would be needed to resolve that question.
Taken together, the results describe a laboratory method for estimating composition in defined d-tryptophan and l-tryptophan mixtures. They show that a color and absorbance signal can track composition under the tested conditions, while leaving broader chemical selectivity and the underlying mechanism unresolved.
Paper data and sources
Original title: In situ synthesis of gold nanoparticles using tryptophan enantiomers for determination of enantiomeric composition
Authors: Radmanesh R, Ahmadi M, Madrakian T, Soloki A
Journal/Repository: RSC advances
Status: Peer-reviewed
First online: 2026-10-08
DOI: Not available
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