Spectrophotometric Determination of Proline Concentration Using Ninhydrin Assay

Spectrophotometric Determination of Proline Concentration Using Ninhydrin Assay

This study describes a simple and sensitive method for proline quantification in samples using a spectrophotometric assay.

Materials and Methods:

1. Sample Preparation: 100 μL of the sample was mixed with 100 μL of 3% (w/v) sulfosalicylic acid, 200 μL of glacial acetic acid, and 200 μL of acidic ninhydrin solution (prepared by dissolving 1.25 g of ninhydrin in 30 mL of glacial acetic acid and 20 mL of 6 M orthophosphoric acid) in test tubes.2. Incubation: The test tubes were incubated at 96 °C for 60 min.3. Reaction Termination: The reaction was stopped by placing the test tubes in an ice bath. 4. Extraction: 1 mL of toluene was added to each test tube and vortexed for 20 s to extract the proline-ninhydrin complex into the organic phase.5. Measurement: The absorbance of the organic phase was measured at 520 nm using an Expectra Max Plus 384 spectrophotometer at room temperature.

Results:

The proline concentration in the samples can be determined by comparing the absorbance values with a standard curve generated using known concentrations of proline.

Conclusion:

This method offers a reliable and sensitive approach for proline quantification in various samples. The use of toluene extraction enhances the sensitivity and selectivity of the assay.