Achieving High-Performance Absorbance Linearity in Affordable Spectrometers
Traditionally, high-performance absorbance linearity in spectrometers has been confined to complex and costly instruments. However, newer, more accessible spectrometers like the Ocean ST are now delivering similar measurement capabilities.
Evaluating the Ocean ST Microspectrometer
Ocean Optics tested the absorbance linearity of the Ocean ST microspectrometer, focusing on its application for protein and DNA measurements. This spectrometer stands out due to its exceptional UV response (185-650 nm), impressive signal-to-noise ratio, and ultra-compact design—smaller than a smartphone.
The Importance of Absorbance
Absorbance measurements are crucial for understanding the chemical makeup of materials. When light interacts with a sample, it can be transmitted, absorbed, or scattered. Transmitted light passes through the sample, while light that encounters molecules can be absorbed or scattered. Absorbance is used both qualitatively, to identify substances, and quantitatively, to determine the concentration of molecules in a solution.
Absorbance techniques are vital in various fields, particularly in biomedical and life sciences, where they help with tasks ranging from protein analysis to point-of-care diagnostics.
Understanding Absorbance Linearity
A spectrometer’s measurement accuracy is partly indicated by its absorbance linearity, as described by Beer’s Law. According to this law, the absorbance of a solution at a specific wavelength will show a peak whose intensity changes linearly with the solution concentration. For instance, doubling the concentration will double the absorbance peak height. High absorbance linearity up to 2 AU (absorbance units) means that beyond this point, the relationship between concentration and absorbance peak height becomes non-linear due to factors like detector noise and stray light.
Spectrometers with high absorbance linearity can accurately measure samples across a broad concentration range, eliminating the need for complex dilution steps that could introduce errors. This is especially critical for applications demanding high measurement accuracy, consistency across experiments, or limited sample quantities.
Assessing Protein and DNA Absorbance with Ocean ST
To test the Ocean ST’s absorbance linearity, measurements were conducted on bovine serum albumin (BSA) and DNA oligo. BSA is a widely used protein in biochemical applications, while oligos are synthetic DNA or RNA used in numerous applications, including genetic testing and diagnostics.
Note: Dr. Yvette Mattley conducted the measurements for this evaluation.
Equipment Setup
The equipment setup included:
- Ocean ST-UV (185-650 nm): Integration time set to 3.8 ms, 100 scans to average, boxcar width set to 3, dark spectrum stored, and nonlinearity correction enabled. The spectrometer and light source were pre-warmed for 30 minutes to ensure thermal stability.
- DH-2000-S-DUV-TTL Light Source: Only the deuterium lamp was used for these experiments.
- SQ1-ALL Cuvette Holder: Suitable for 1 cm cuvettes.
- CV-Q-10 Quartz Cuvette: Best for UV measurements.
- QP450-1-XSR Optical Fibers: Extreme solarization resistant fibers with stainless steel BX jacketing.
Sample Preparation and Measurements
For this evaluation, 30 BSA (Sigma A2153) concentrations ranging from 0.02 to 5 mg/mL and 35 DNA oligo (Sigma D-3159) concentrations ranging from 0.3 to 150 µg/mL were prepared. All dilutions were performed directly in the quartz cuvette by mixing 0.5 mL sample with 0.5 mL distilled water, ensuring minimal contamination and alignment issues by not removing the cuvette during measurements.
Results
The absorbance measurements for BSA demonstrated linearity up to 2 AU, surpassing expectations for the Ocean ST (Figure 1). This is significant because BSA is a common protein in various life science research and development applications.
This achievement highlights the potential of the Ocean ST spectrometer to provide high-performance absorbance linearity in a compact and cost-effective package, making advanced measurements more accessible to a broader range of users.
Figure 1. The Ocean ST microspectrometer demonstrates remarkable absorbance linearity over a wide range of protein concentration levels.
Similarly impressive absorbance linearity results were achieved with the Ocean ST measurements of DNA oligo (Figure 2). This highlights the value of the Ocean ST for researchers using oligos as a foundation for various molecular biology studies and for custom oligo production, where UV-visible spectroscopy is crucial for quantification during the manufacturing process.rs.
Figure 2 The Ocean ST microspectrometer demonstrates remarkable absorbance linearity over a wide range of protein concentration levels.
Extensive Configuration Options
Currently, Ocean Optics offers a wide range of configurations for their spectrometers. It is crucial to carefully select or consult with engineers to determine the most suitable model for specific applications. This ensures the best possible performance and accuracy for measurements.
