Using the Spectral Evaluation Function for Quantitative Analysis of Nucleotides and a Simultaneous Pass/Fail Judgment on Nucleotide Sample Purity

Significance of the Topic

UV-Vis spectrophotometry is widely used to quantify nucleic acids. Accurate measurement of nucleotide concentrations and sample purity is essential in molecular biology, diagnostics, and quality control. The spectral evaluation function adds an automated pass/fail criterion and flexible calibration options, improving analysis efficiency and data consistency.

Objectives and Study Overview

This work demonstrates the application of the Shimadzu UV-1900i Plus UV-Vis spectrophotometer and the LabSolutions UV-Vis spectral evaluation function to:

1. Quantitatively analyze oligonucleotides using calibration curves based on either fixed wavelengths or wavelength maxima.
2. Simultaneously assess sample purity via OD260/OD280 ratio with pass/fail judgment.

Methodology and Instrumentation

A series of M13 oligonucleotide dilutions (0.2–10 ng/µL) were prepared in phosphate buffer. Spectra were acquired over 220–400 nm at 0.5 nm intervals. Two calibration approaches were compared: fixed-point absorbance at 260 nm and peak absorbance at wavelength maxima. The spectral evaluation function was configured to process point pick and peak parameters and to apply a purity threshold of OD260/OD280 ≥ 1.8.

Instrumentation Used

• Shimadzu UV-1900i Plus UV-Vis spectrophotometer
• LabSolutions UV-Vis control software
• Super-micro cell holder with 10 mm path length micro black cell

Key Results and Discussion

Calibration curves based on fixed wavelength and wavelength maxima yielded correlation coefficients (r²) of 0.99969 and 0.99992, respectively, demonstrating improved accuracy when using peak absorbance. Purity assessment correctly flagged a contaminated sample (OD260/OD280 = 1.3) as fail against the threshold of 1.8, while pure samples passed.

Benefits and Practical Applications

• Simultaneous quantitation and purity evaluation reduces analysis time.
• Customizable evaluation items enable flexible analytical criteria.
• High accuracy in low-concentration measurements supports diverse research and QC needs.

Future Trends and Applications

Integrating automated spectral evaluation with emerging workflows, such as next-generation sequencing and thermodynamic stability studies, can further streamline nucleic acid analysis. Adoption of advanced software-driven criteria may expand applications to other biomolecules and enable real-time quality control in high-throughput environments.

Conclusion

The spectral evaluation function of the LabSolutions UV-Vis software, combined with the UV-1900i Plus system, provides robust tools for precise nucleotide quantification and automated purity judgments. This integrated approach enhances reliability and efficiency in nucleic acid analysis.

References

1. L. Braglia, S. Giani, D. Breviario, F. Gavazzi. Anal. Bioanal. Chem., 408, 8299 (2016).