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 essential for accurate quantification and purity assessment of nucleic acids, critical for applications in genomics, diagnostics, and biomolecular research. The Spectral Evaluation Function enhances this capability by combining absorbance-based quantitation with real-time sample quality checks.

Objectives and Overview

This study demonstrates the use of the Shimadzu UV-1900i Plus spectrophotometer and LabSolutions UV-Vis software to:

• Perform quantitative analysis of oligonucleotides via calibration curves based on maximum-absorption wavelengths.
• Simultaneously execute pass/fail judgments on sample purity using the OD260/OD280 ratio.

Methodology and Instrumentation

The UV-1900i Plus instrument equipped with a Super-micro cell holder and 10 mm micro black cell recorded absorption spectra from 220 to 400 nm at 0.5 nm intervals. LabSolutions UV-Vis software’s “Spectrum” application and the Spectral Evaluation Function were configured to extract parameters such as point pick at fixed or variable wavelengths and calculate OD260/OD280. M13-F25mer oligonucleotide standards (0.2–10 ng/µL) were prepared in phosphate buffer for calibration.

Main Results and Discussion

Two calibration approaches were compared: fixed-wavelength analysis at 260 nm and variable-wavelength analysis at each sample’s maximum absorption peak. The respective correlation coefficients (r2) were 0.99969 and 0.99992, indicating superior accuracy when adapting to peak shifts. Purity assessment using an OD260/OD280 threshold of 1.8 effectively identified samples adulterated with bovine serum albumin.

Benefits and Practical Applications

The integrated workflow streamlines nucleic acid quality control by combining precise concentration measurements with automated purity screening. This dual evaluation is valuable for next-generation sequencing sample preparation, thermal stability (Tm) analysis, and thermodynamic parameter studies.

Future Trends and Applications

Emerging developments may include AI-driven spectral interpretation, real-time multivariate data analytics, broader application to diverse biomolecules, and seamless integration with automated laboratory systems. Enhanced spectral evaluation algorithms and colorimetric analyses are expected to expand utility further.

Conclusion

The Spectral Evaluation Function on the UV-1900i Plus UV-Vis spectrophotometer provides a robust, efficient solution for simultaneous nucleotide quantification and purity verification, enhancing reliability in analytical workflows.

References

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