Accuracy in Microvolume Measurements. Investigating the Precision and Reproducibility of Cary 50, 300 and 4000 series Instruments for Microlitre samples

Importance of Microvolume UV-Vis Measurements

The quantitation of DNA and other biomolecules in microvolumes is vital for molecular biology, clinical diagnostics and biochemical research. High precision and minimal sample consumption enable more efficient workflows and preserve rare or precious samples.

Study Objectives and Overview

This work evaluates the precision, detection limits and reproducibility of three spectrophotometers (Cary 50, Cary 300, Cary 4000) equipped with a Hellma Traycell microvolume accessory. The goal is to define operating ranges, noise performance and practical thresholds for DNA quantitation and kinetic assays.

Methods and Instrumentation

Three Agilent (formerly Varian) UV-Vis instruments were tested with a fiber-optic Hellma Traycell microvolume cell. Alignment used built-in software modules and mechanical adjustments to optimize beam throughput. Key settings:

• Cary 50: Xenon flash source, 1.5 nm bandwidth, single monochromator, sub-microsecond flash, no warm-up required; pathlength options down to 0.2 mm; dual-beam transmission ~25%.
• Cary 300: High-precision Czerny–Turner monochromator, absorbance range 0.0002–5 Abs; pathlength down to 1 mm; transmission with Traycell ~6%.
• Cary 4000: Dual Littrow monochromators, dynamic range to >9 Abs, baseline noise ~0.0001 Abs; pathlength down to 1 mm; transmission ~5%.

Main Results and Discussion

• Cary 50 reliably quantifies ssDNA at 2–5 ng/µL with ~3–5% error; below 2 ng/µL, noise approaches signal levels.
• Cary 300 extends linearity to absorbances up to 4 Abs in micro-pathlength cells, reproducing DNA quantitation to <1% variance despite lower percent transmission.
• Cary 4000 achieves sub-nanogram sensitivity (≈0.5 ng/µL) at ~10% quantitation error, with superior baseline stability enabling repeat scans and kinetic measurements over >30 minutes with minimal evaporation.
• Repeatability studies show variance <1% across replicate reads for DNA concentrations ranging from ~9 to 52 µg/mL.
• Traycell design prevents sample evaporation, supporting enzyme kinetics and time-course assays in volumes as low as 1–4 µL.

Benefits and Practical Applications

Microvolume cuvette integration offers rapid, low-consumption analysis with room-light immunity, easy swap to standard cuvettes and no lamp warm-up. The system suits DNA/RNA quantitation, protein absorbance assays and micro-volume kinetic experiments in research, QA/QC and clinical labs.

Future Trends and Opportunities

Further miniaturization, integration with automated microfluidics, improved detection algorithms, and coupling with complementary techniques (e.g., fluorimetry, mass spectrometry) will expand applications. Real-time kinetics in sub-microlitre volumes and single-cell analyses represent emerging frontiers.

Conclusion

The Cary 50 with Hellma Traycell provides a cost-effective solution for routine microvolume DNA quantitation down to ~2 ng/µL. The Cary 300 offers extended dynamic range with similar precision. The Cary 4000 optimizes sensitivity and stability, enabling sub-nanogram assays and kinetic studies. Choice of instrument should balance sensitivity needs, dynamic range and sample throughput.

References

• Agilent Technologies. Varian, Inc. now part of Agilent Technologies. www.agilent.com/chem