Concentration Analysis of Ultra-microvolume Samples by UV-Vis Spectroscopy

Significance of the Topic

Direct measurement of ultra-microvolume samples such as nucleic acids and proteins is essential for preserving valuable samples while ensuring accurate, reproducible results in modern laboratories. Avoiding dilution reduces sample handling errors and accelerates workflows. The use of spectrophotometric techniques with minimal sample volumes also supports sustainability initiatives by reducing waste and power consumption.

Objectives and Study Overview

This study demonstrates the performance of the Agilent Cary 60 UV-Vis spectrophotometer equipped with the TrayCell 2.0 Ultra-Microvolume Cell. The goals include evaluating sensitivity, dynamic range, photometric linearity, and reproducibility for protein and DNA samples across a wide concentration spectrum without requiring dilution steps.

Methodology and Instrumentation

Sample preparation involved BSA protein and herring sperm DNA dissolved in phosphate buffered saline and serially diluted to target concentrations. Ultra-microvolume aliquots (3 to 10 microliters) were measured directly on the TrayCell 2.0 window with interchangeable caps providing pathlengths of 2.0, 1.0, 0.2, and 0.1 millimeters. Absorbance was recorded at 260 nanometers for DNA and 280 nanometers for protein. Data acquisition and analysis used Agilent Cary WinUV software modules for wavelength scans and concentration measurements.

Main Results and Discussion

Repetitive wavelength scans of a 20 ng per microliter DNA sample showed excellent reproducibility at absorbance of 0.04 with negligible variability. Photometric linearity for DNA extended from 5 to 1500 ng per microliter using the 1.0 millimeter cap, with reliable detection down to 5 ng per microliter. For BSA protein, linear absorbance up to 2.4 Abs was achieved for 1 to 40 mg per milliliter with the 1.0 millimeter cap. Higher concentrations were measured by reducing pathlength: 0.2 millimeter cap enabled quantitation up to 200 mg per milliliter (2.3 Abs), and the 0.1 millimeter cap extended the range to 400 mg per milliliter (2.0 Abs), corresponding to equivalent 10 millimeter cuvette absorbances of 24, 115, and 200, respectively.

Benefits and Practical Applications

The combination of the Cary 60 UV-Vis and TrayCell 2.0 offers a fast, non-destructive workflow that minimizes sample consumption, eliminates the need for dilutions, and reduces handling errors. The room light immunity and xenon flash lamp ensure stable photometric performance, while easy cleaning of the TrayCell window accelerates sequential measurements.

Future Trends and Potential Applications

Further developments may include integration with automated cell changers, long pathlength accessories, and temperature-controlled holders for kinetic studies. The technology can be adapted for microfluidic devices and high-throughput screening, enhancing its application in proteomics, genomics, and quality control in biopharmaceutical research.

Conclusion

The Agilent Cary 60 UV-Vis spectrophotometer with the TrayCell 2.0 Ultra-Microvolume Cell provides a robust platform for direct analysis of nucleic acids and proteins across a broad concentration range. Its sensitivity, linearity, and ease of use deliver precise, reproducible data with minimal sample consumption, streamlining workflows and supporting sustainable laboratory practices.

References

• Weragoda G, Alwan W, Zieschang F Application Note Concentration Analysis of Ultra-microvolume Samples by UV-Vis Spectroscopy Agilent Technologies Inc 2022