Precision Microvolume Spectrophotometry of Lysosyme at 100mg/ml

Importance of the topic

Accurate absorbance measurement of proteins in highly concentrated solutions is crucial for biochemical research and quality control. Traditional spectrophotometry instruments require large volumes and struggle with high or low concentration limits. Microvolume spectrophotometry overcomes these barriers by combining ultralow sample volumes with high precision, enabling new insights into protein behavior and interactions.

Objectives and Study Overview

This study demonstrates the capability of a microvolume cuvette (Hellma Traycell) installed in a Cary UV/Vis spectrophotometer to measure lysozyme at 100 mg/mL. The aim was to assess photometric noise, stray light performance, and reproducibility at pathlengths down to 0.2 mm, using only 1 μL of sample.

Methodology

• Sample preparation: Lysozyme from Sigma-Aldrich at 100 mg/mL in distilled deionized water.
• Instrument settings: Wavelength range 200–380 nm, bandwidth 2 nm, scan speed 60 nm/min, data interval 2 nm, averaging time 2 s.
• Traycell alignment: Adjusted to 3.8 % transmittance using a precision cell holder.
• Beam attenuation: Rear beam attenuator set to offset internal Traycell absorbance, limiting readings to 1.3 Abs.

Used Instrumentation

• Agilent (formerly Varian) Cary 6000i UV/Vis spectrophotometer (Cary 4000 equivalent performance in the UV range).
• Hellma Traycell microvolume cuvette, 0.2 mm pathlength, precision holder.
• Automated and manual rear beam attenuators for high-absorbance samples.

Main Results and Discussion

The 100 mg/mL lysozyme exhibited a plateau at 280 nm with absorbance ~2 Abs (equivalent to ~40 Abs at 1 cm pathlength), rather than the expected peak of ~200 Abs by extrapolation. Baseline noise was measured at ±0.0006 Abs at 315 nm, and the system reproducibly captured spectra after a 12-minute delay. This unexpected absorbance behavior suggests molecular association or bond distortion phenomena in highly concentrated protein solutions, requiring further investigation with dilution series and varied pathlengths.

Benefits and Practical Applications

• Measurement of protein and nucleic acid concentrations from ~20 µg/mL to 100 mg/mL using only 1 µL sample.
• Low noise (0.0002 Abs) and high dynamic range (up to >7 Abs in Traycell configuration) support both quantitation and purity scans.
• Minimal sample consumption accelerates analysis in laboratories with limited material.

Future Trends and Possibilities

Microvolume spectrophotometry is poised to expand into high-throughput screening, on-chip diagnostics, and real-time monitoring of bioprocesses. Advances in pathlength optimization, integration with microfluidics, and deeper investigation of concentration-dependent phenomena will broaden its applications in pharmaceuticals, biochemistry, and materials science.

Conclusion

The combination of a Hellma Traycell with a Cary UV/Vis spectrophotometer enables precise, reproducible absorbance measurements of highly concentrated protein solutions in ultralow volumes. The observed deviations from expected absorbance behavior highlight new avenues for research into molecular interactions at high concentration.

Reference

R. A. Keighley and D. Scott. Precision Microvolume Spectrophotometry of Lysozyme at 100 mg/mL. Sample Analysis Report No. 50, Varian Inc. (2005).