The Linear Dynamic Range and Limits of Detection of Fluorescein using the Agilent Cary Eclipse Fluorescence Spectrophotometer

Importance of the Topic

The accurate quantification of fluorescent probes such as fluorescein is central to many fields of analytical chemistry, including biological assays, environmental monitoring, and drug discovery. Establishing both a wide linear dynamic range and a low limit of detection (LOD) is critical to ensure reliable measurements across concentrations encountered in real‐world samples.

Objectives and Study Overview

This technical overview evaluates the performance of the Agilent Cary Eclipse Fluorescence Spectrophotometer for fluorescein in 0.01 M sodium hydroxide. Key goals include characterizing the instrument’s linear dynamic range and determining its LOD under defined measurement conditions.

Methodology and Instrumentation

• Instrument: Agilent Cary Eclipse Fluorescence Spectrophotometer with Cary WinFLR software.
• Sample matrix: Fluorescein solutions (0 pM to 1.06 µM) prepared in 0.01 M NaOH.
• Measurement settings: Excitation at 480 nm, emission at 520 nm, 10 nm slit widths, 1 s signal averaging time, PMT voltage adjusted as needed (up to 1000 V), total acquisition 60 s.
• Data treatment: Blank subtraction, log–log calibration plots, adjustment of detector voltage to extend linear response.

Main Results and Discussion

A calibration curve spanning five orders of magnitude (10 pM–1.06 µM) yielded an R2 of 0.99993, confirming linearity. Repeated measurements on five instruments produced an average LOD of 0.48 pM based on three times the blank standard deviation. An alternative LOD estimation using regression-derived equations gave a consistent value of approximately 0.45 pM.

Benefits and Practical Applications

• The wide dynamic range simplifies quantification across dilute and concentrated samples without frequent dilution or rerun.
• The sub‐picomolar LOD enables detection of trace levels of fluorescein in sensitive assays.
• Robust performance supports applications in biotechnology, environmental analysis, and quality control.

Future Trends and Possibilities

Advances in detector technology and optical filters may further lower detection limits. Integration with microfluidic platforms and high‐throughput screening systems could extend the method’s utility in real‐time monitoring and large‐scale studies.

Conclusion

The Agilent Cary Eclipse Fluorescence Spectrophotometer demonstrates exceptional sensitivity and a linear dynamic range over five orders of magnitude for fluorescein in alkaline solution. Its performance makes it well suited for demanding analytical workflows that require both precise quantification and trace‐level detection.

References

• ASTM E578-07: Standard Test Method for Linearity of Fluorescence Measuring Systems.
• ASTM E579-04: Standard Test Method for Limit of Detection of Fluorescence of Quinine Sulphate.