UV/Vis Spectroscopy
UV/Vis spectroscopy for protein and DNA quantification
Low volume, high accuracy UV/Vis spectrometers
Proteins, DNA and RNA absorb light in the ultraviolet range in solution, which means you can apply UV/Vis spectroscopy to quantify their concentrations (Figure 1). Quantification with UV/Vis spectroscopy uses Beer’s law, sometimes called Beer-Lambert law, to determine a molecule’s concentration from its absorbance spectrum.
Figure 1. Protein dilution spectra and DNA dilution spectra.
What is UV/Vis Spectroscopy and How Does It Work?
UV/Vis spectroscopy is an analytical technique that measures how much ultraviolet or visible light a sample absorbs. The amount of light a protein, DNA or RNA sample absorbs is directly proportional to the concentration. Proteins absorb most at 280 nm while DNA and RNA peak at 260 nm. With Beer’s law you determine sample concentration from the absorbance at 280 nm or 260 nm (called A280 or A260), the known pathlength and the sample’s extinction coefficient or concentration factor. UV/Vis spectrophotometers perform these measurements and calculations automatically and without any additional reagents. The best spectrometers can measure at high-throughput and with low sample volumes.
Lunatic and Stunner spectrometers perform high-speed UV/Vis spectral analysis from 230–750 nm and 0.03–275 OD with just 2 μL of sample in fixed pathlength microcuvettes (Figure 2). The microcuvettes are part of unique microfluidic circuits that prevent any cross-contamination or evaporation while delivering highly accurate absorbance spectra. Having pathlengths that are fixed enables accuracy on Lunatic and Stunner to within 2% and precision within 1%. For more info on UV/Vis spectroscopy and Lunatic’s innovative technology, check out this tech note.
High Lunatic and Stunner plates have 2 microcuvettes in eac h circuit, with pathlengths measuring 0.1 and 0.7 mm. The 0.1 mm microcuvette detects a high maximum absorbance while the larger microcuvette is more accurate for lower absorbances.
These 2 different sized microcuvettes make the wide dynamic range possible. Lunatic or Stunner read 96 of these microfluidic circuits on their SBS-compatible plates in only 10 minutes. This wide dynamic range and high-throughput capability make Lunatic and Stunner ideal for protein quantification across a variety of sample types and concentrations.
Looking for accuracy? Lunatic and Stunner quality has been proven with Reference Materials (RMs) from the National Institute for Standards and Technology (NIST). NIST has 3 RMs for testing UV/Vis spectrophotometers: a monoclonal antibody (NISTmAb), tryptophan and uracil – all at certified concentrations in defined buffers. Lunatic and Stunner measure these samples within 2% of the certified value (Figure 3), easily meeting both USP and Ph. Eur. guidelines.
UV/Vis spectroscopy is one of the oldest and most well-established methods of directly quantifying proteins, DNA and RNA. Unchained Labs pushes UV/Vis quantification to the next level with innovative optics, well-controlled consumables and honed analysis techniques.
Figure 2. High Lunatic plate circuit.
Figure 3. Lunatic and Stunner accuracy of NIST RMs.
All Unchained Labs UV/Vis Spectroscopy instruments deliver:
- Accurate and precise full-spectrum UV/Vis absorbance results from 230–750 nm
- Results from tiny 2 µL sample volumes
- Wicked fast reads
- Wide dynamic range from 0.03 to 275 OD
- IgG quantification from 0.02 – 200 mg/mL
- Nucleic acid quantification from 1.5 - 13750 ng/µL
- Microfluidic circuits to protect samples from evaporation and cross-contamination
- Fixed pathlength microcuvettes for maximum sensitivity and reproducibility
- Purpose-built applications for quantification of purified or non-purified samples
- Low maintenance and no clean-up
Check out our UV/Vis Spectroscopy instrument line-up below, with two solutions designed to solve protein and DNA quantification problems.
Lunatic
Lunatic makes batch quantification of biologics, DNA and RNA a no-brainer. All you need is 2 µL and 10 minutes to measure up to 96 samples. Run them straight-up, even at high concentrations, without ever diluting. Measurements are spot-on with accuracy within 2% and precision within 1% and are fully compliant with USP and Ph. Eur. guidelines. The SBS plate-based format means integrating with lab automation and liquid handlers is a snap. Lunatic gets UV/Vis quantification on the money every time. Just drop, load and read.
Stunner
Stunner combines Lunatic’s plate-based, high-throughput UV/Vis accuracy and precision with dynamic light scattering for a one-of-a-kind combination. Pairing up these techniques checks concentration, hydrodynamic size, polydispersity and detection of aggregates off your to-do list in one shot. Sync up sample quantity and quality your way.
FAQs
UV-Vis spectroscopy is an analytical technique that measures the absorbance of ultraviolet and visible light by a sample. It is commonly used to determine the concentration of analytes, assess purity, and analyze molecular structures in various fields, including chemistry, biology, and materials science.
UV-Vis spectroscopy is utilized in numerous applications, including quantifying nucleic acids and proteins, contaminant detection and identification, monitoring chemical reactions, assessing water quality, and analyzing pharmaceuticals. Its versatility makes it a valuable tool in research, quality control, and clinical laboratories.
Unchained Labs' UV-Vis technology offers high-throughput capabilities with minimal sample volumes, enabling rapid and accurate measurements. Their proprietary plate design allows for quantification over a large dynamic range eliminating the need for sample dilution before analysis. Their instruments are designed for ease of use, with intuitive software and robust hardware that streamline workflows in various laboratory settings.
UV-Vis spectroscopy offers rapid, non-destructive analysis with relatively simple sample preparation compared to techniques like HPLC or mass spectrometry.
Regular maintenance of Unchained Labs' UV-Vis spectrophotometers includes periodic calibration checks, and software updates. Following the manufacturer's guidelines ensures optimal performance and longevity of the instrument.
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