UV/Vis Spectroscopy

UV/VIS Spectroscopy

Please acknowledge the SIP core facility ( RRID: SCR_018986) in publications, on posters, or in talks if you use any instruments in the SIP core facility. Please include SIP's RRID (RRID: SCR_018986) and the grant numbers for instruments funded through instrumentation grants in your acknowledgements. This is a requirement from the funding agencies and is crucial for future funding. Find example text on theÌýAcknowledgement PageÌýor the individual instrument pages.

UV/VIS Spectroscopy in Biochemistry, Biophysics and Structural Biology

UV/Vis spectroscopy is a widely used analytical technique across chemistry, biological sciences, biochemistry, and biophysics. It involves measuring the absorption of light by molecules across a wavelength range from the mid and near UV (200 nm–400 nm) to visible light (400 nm–900 nm).

The shape of absorption spectra, including absorption maxima and strength, is characteristic of specific chromophores. These features depend on the chromophore’s electronic properties and its chemical environment. UV/Vis spectroscopy is a powerful tool for identifying and quantifying chromophores and monitoring reaction kinetics, UV/Vis spectroscopy can detect intrinsic chromophores in proteins, RNA, and DNA. It is also used in colourimetric assays to observe reaction partners that exhibit measurable colour changes due to complex formation with biological macromolecules or chemical reactions catalysed by them.

UV/VIS RNA spectra at increasing temperatures, 10 C steps 15 to 95 C)

Key Highlights of UV/VIS Spectroscopy in Biology, Biochemistry and Biophysics

  • Protein, RNA, DNA quantification
  • Protein , RNA, DNA purity
  • RNA/DNA stability
  • Reaction kinetics
  • Binding curves
  • Bacterial growth rates
  • Cell viability and proliferation assay
  • And a lot more ...
Carry 300 Bio UV/Vis Spectrometer

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Beckman DU 640 Spectrometer

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Tecan Spark Plate Reader

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Instruments and Accesories

Agilent Cary 300 ÌýUV-Vis Spectrometer

We have an Agilent Cary 300 double beam UV-Vis spectrometer, with a working range of up to 6.0 Abs and a resolution of 0.24 nm. It is equipped with a single-cell Peltier temperature control. It has a high-performance R928 photomultiplier tube and two light sources, a deuterium arc ultraviolet source lamp (190-350 nm) and a tungsten-halogen visible source lamp (350-900 nm). Data acquisition and analysis is controlled by the Cary WinUV software.

Beckman UV/Vis DU 640 Spectrometer

The Beckman DU640 UV/VIS spectrometer is an older, yet robust and user-friendly UV/VIS instrument. It has a spectral range from 190 to 1100 nm, with a working range of up to 3.0 Abs. It is equipped with an automatic 6-cuvette holder, and temperature control is maintained via a circulating water bath.

Applied Photophysics Chirascan Plus CD Spectrometer

The Applied Photophysics Chirascan Plus CD can also function as a UV/VIS spectrometer. It can simultaneously measure both UV/VIS and CD. It has a wavelength range from 180 to 1100 nm. The instrument is equipped with a Peltier-controlled single-cell holder, as well as a Peltier-controlled automated 4-cuvette turret. In-cell temperature sensors ensure precise monitoring of the sample's temperature. Additionally, a stopped-flow module and an automatic titration unit are available. For more detailed information see the CD spectroscopy page.

Applied Photophysics SX 20 Stopped-Flow Spectrometer

The Applied Photophysics SX 20 Stopped-Flow Spectrometer can be used to capture rapid kinetics using fluorescence and absorption as detection modes. It has a wavelength range from 200 to 800 nm. The instrument can operate in single 1:1 mixing, ratio mixing, or sequential mixing modes. For more detailed information, refer to "Fast Kinetics of Reactions and Conformational Changes".

Tecan Spark Plate Reader

We have a multimode plate reader for high-throughput measurements. The Spark plate reader supports absorbance, fluorescence, and luminescence modes. It is equipped with a xenon flash lamp light source, with a spectral range of 200–1000 nm for absorbance and an optical density (OD) range of 0–4 OD. It accommodates 1 to 384-well plates and includes a reusable NanoQuant Plate for DNA or RNA quantification. The system features Peltier temperature control, and a humidity cassette is available if required.

Essential Information for Using SIP's UV/VIS instruments

Acknowledgements Protocols Training Scheduling Cost Locations
Acknowledgements

Why?

Acknowledgements are essential for ensuring the continued success of the Shared Instruments Pool (SIP). They enable us to secure the necessary funding to sustain and expand the SIP, ensuring that our instruments are in optimal working condition and that the methods we offer are at the forefront of biochemical and biophysical research.

Please include SIP's RRID number (RRID: SCR_018986) in your acknowledgements. This allows funding organisations and potential grant reviewers to easily locate publications supported by SIP, helping to evaluate the impact of SIP on our research community.

If facility staff have provided substantial assistance, please consider acknowledging them. If they contributed significantly to the intellectual aspects or conducted important experiments, co-authorship may also be appropriate.

Example text:

  • We thank the Shared Instruments Pool (RRID: SCR_018986), Department of Biochemistry, University of Colorado Boulder for the use of the Agilent Cary 300 spectrometer.Ìý
  • We thank the Shared Instruments Pool (RRID: SCR_018986), Department of Biochemistry, University of Colorado Boulder for the use of the Beckman DU640 UV/VIS spectrometer.Ìý
  • We thank the Shared Instruments Pool (RRID: SCR_018986), Department of Biochemistry, University of Colorado Boulder for the use of the Tecan Spark Plate Reader spectrometer.Ìý
  • We thank the Shared Instruments Pool (RRID: SCR_018986), Department of Biochemistry, University of Colorado Boulder for the use of the Applied Photophysics SX20 Stopped-Flow spectrometer. We also thank [Name and title of the facility member providing significant help] for their invaluable assistance with data collection and evaluation.

Please contact Dr Erbse to obtain detailed protocols and arrange an initial project consultation and personalised training sessions. Protocols are available as PDF files on the instrument computer, with printed copies stored alongside the instruments.

Interested users can contact Dr Erbse to discuss planned experiments and arrange training sessions. These sessions will involve the use of actual user samples alongside standards, enabling users to collect preliminary data during the training and receive help from core staff right away if Ìýtroubleshooting or optimisation is needed. Users are welcome to request additional training or support sessions at any time. We are always happy to provide a refresher if it has been a while.

After your training is completed, you will be invited to join the CD, the Spark plate reader, or the Stopped-Flow Spectrometer Google calendar.

Sign Up Rules:Ìý
Up to Friday the week before the planned experiment users can sign up for a maximum of 2 days. In the week of the experiment users can sign up for additional time if available.

  • Initial consultation is free. SIP staff are happy to assist with a short pilot experiment if it can be accommodated within SIP's resources.
  • Regular user groups are expected to buy into SIP with a monthly flat fee according to their SIP usage level. For detailed information, please contact Dr Erbse.
  • Users are required to provide all consumables specific to their experiments. This includes the syringes needed for fast kinetic measurements, cuvettes and multi well plates.
  • Costs for necessary repairs, services, or replacement parts due to normal wear and tear will be shared among all user groups, based on the time used over the past two years. Please note that assuming the instrument is handled properly, such repairs or replacements are infrequent, and costs may arise after a user’s period of use has ended.
  • Users are responsible for covering the costs of repairs or replacement parts needed due to damage caused by carelessness or neglect.
  • Prospective Industry users should contact Dr Erbse for rates and requirements.Ìý

The Carry 300 spectrometer, the Beckman DU 640 spectrometr and the Applied Photophysics SX 20 Stopped-Flow instrument are located on the third floor of JSCBB in the D-Wing, room D381, on East Campus. Proxcard access is required at all times.