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Related Concept Videos

Ultraviolet and Visible (UV–Vis) Spectroscopy: Overview01:02

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Ultraviolet–visible (UV–visible or UV–Vis) spectroscopy is an analytical technique that investigates the interaction between matter and UV–Vis light within the electromagnetic spectrum. This method is widely used for its versatility, simplicity, and relatively quick data acquisition, making it valuable for both qualitative and quantitative analysis. When UV–Vis radiation passes through a material,  molecules absorb light depending on the energy required for...
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The absorbance of UV and visible (UV–visible) radiations is measured using a UV–visible spectrophotometer. Deuterium lamps, which emit UV radiation, and tungsten lamps, which produce radiation in the visible region, are used as light sources in UV–visible spectrophotometers. A monochromator or prism is used for diffraction grating, i.e., to split the incoming radiation into different wavelengths. A system of slits is used to focus the desired wavelength on the sample cell.
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When light passes through a substance, a portion of the light is absorbed while the remaining light is reflected or transmitted. If the molecule absorbs light between the wavelengths of 180–400 nm range, the UV spectrum is obtained, and if it absorbs light in the 400–780 nm wavelength range, the visible spectrum is obtained.     
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UV–Visible absorption spectra of conjugated dienes arise from the lowest energy π → π* transitions. The light-absorbing part of the molecule is called the chromophore, and the substituents directly attached to the chromophore are called auxochromes. A strong correlation exists between the absorption maxima, λmax, and the structure of a conjugated π system. The Woodward–Fieser rules predict the value of λmax for a given...
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The Beer-Lambert law describes the relationship between absorbance and concentration, which combines the principles established by scientists Johann Heinrich Lambert and August Beer. Lambert's law states that when light passes through a medium, the loss in intensity is directly proportional to the original intensity and the path length of the light. Beer's law proposed that the transmittance of a solution remains constant if the product of concentration and path length is constant. The...
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Interference leads to systematic error in atomic absorption (AA) measurements by enhancing or diminishing the analytical signal or the background. These interferences can be grouped into three main categories: spectral interference, chemical interference, and physical interference.
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Correcting Ultraviolet-Visible Spectra for Baseline Artifacts.

Andrew J Basalla1, Brent S Kendrick2

  • 1KBI Biopharma, Inc., Louisville, CO, USA; First Principles Biopharma, LLC, Louisville, CO, USA.

Journal of Pharmaceutical Sciences
|August 24, 2023
PubMed
Summary
This summary is machine-generated.

Accurate protein concentration measurements are crucial. This study introduces a novel curve-fitting method to correct light scattering errors in UV spectroscopy, improving measurement reliability for various samples.

Keywords:
Biopharmaceutical characterizationCorrectionCurve fittingMie light scatteringRayleigh light scatteringUltraviolet (UV) spectroscopy

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Spectroscopy

Background:

  • Light scattering from particulates and protein aggregates interferes with UV spectroscopy concentration measurements.
  • Existing correction equations may fail with variable sample compositions or deviations from their development basis.

Purpose of the Study:

  • To develop and validate a robust method for correcting light scattering artifacts in UV spectroscopy.
  • To improve the accuracy of protein concentration determination in the presence of scattering agents.

Main Methods:

  • A curve-fitting baseline subtraction approach was developed, integrating fundamental Rayleigh and Mie scattering principles.
  • The method incorporates instrument baseline artifacts into the correction model.
  • Validation involved diverse controls: protein size standards, forced degradation aggregates, lentivirus, and polystyrene nanospheres.

Main Results:

  • The proposed Rayleigh-Mie correction method demonstrated effective accuracy across various scattering scenarios.
  • The approach successfully addressed errors caused by particulates, soluble protein aggregates, and large proteins.
  • Validation confirmed the method's reliability with diverse biological and synthetic scattering agents.

Conclusions:

  • The curve-fitting Rayleigh-Mie correction offers a more reliable alternative to existing methods for accurate UV spectroscopic concentration measurements.
  • This approach enhances the precision of quantifying proteins and similar macromolecules in complex samples.
  • The validated method provides a valuable tool for biopharmaceutical analysis and research.