{ "id": "ep2dzrjhfv", "slug": "ultraviolet-spectrophotometry", "title": "Ultraviolet spectrophotometry", "updated": "2026-10-15", "topic_path": [ { "id": "physical", "label": "Physical world and mathematics", "api_url": "https://www.edgepedia.org/api/v1/topics/physical" }, { "id": "physical.chemistry", "label": "Chemistry", "api_url": "https://www.edgepedia.org/api/v1/topics/physical.chemistry" }, { "id": "physical.chemistry.methods", "label": "Chemical principles and methods", "api_url": "https://www.edgepedia.org/api/v1/topics/physical.chemistry.methods" }, { "id": "physical.chemistry.methods.analytical-chemistry", "label": "Analytical chemistry", "api_url": "https://www.edgepedia.org/api/v1/topics/physical.chemistry.methods.analytical-chemistry" }, { "id": "physical.chemistry.methods.analytical-chemistry.optical-spectrometry-and-photometry", "label": "Optical spectrometry and photometry", "api_url": "https://www.edgepedia.org/api/v1/topics/physical.chemistry.methods.analytical-chemistry.optical-spectrometry-and-photometry" } ], "geo": [ { "id": "geo.nongeo.t1800.physical.chemistry.methods.analytical-chemistry", "label": "Non-geographic · 1800 to 1945: Analytical chemistry", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1800.physical.chemistry.methods.analytical-chemistry", "path": [ { "id": "geo.nongeo", "label": "Non-geographic", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo" }, { "id": "geo.nongeo.t1800", "label": "Non-geographic · 1800 to 1945", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1800" }, { "id": "geo.nongeo.t1800.physical", "label": "Physical world and mathematics", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1800.physical" }, { "id": "geo.nongeo.t1800.physical.chemistry", "label": "Chemistry", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1800.physical.chemistry" }, { "id": "geo.nongeo.t1800.physical.chemistry.methods", "label": "Chemical principles and methods", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1800.physical.chemistry.methods" }, { "id": "geo.nongeo.t1800.physical.chemistry.methods.analytical-chemistry", "label": "Analytical chemistry", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1800.physical.chemistry.methods.analytical-chemistry" } ] }, { "id": "geo.nongeo.t1946.physical.chemistry.methods.analytical-chemistry.optical-spectrometry-and-photometry", "label": "Non-geographic · 1946 to 2000: Optical spectrometry and photometry", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.physical.chemistry.methods.analytical-chemistry.optical-spectrometry-and-photometry", "path": [ { "id": "geo.nongeo", "label": "Non-geographic", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo" }, { "id": "geo.nongeo.t1946", "label": "Non-geographic · 1946 to 2000", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946" }, { "id": "geo.nongeo.t1946.physical", "label": "Physical world and mathematics", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.physical" }, { "id": "geo.nongeo.t1946.physical.chemistry", "label": "Chemistry", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.physical.chemistry" }, { "id": "geo.nongeo.t1946.physical.chemistry.methods", "label": "Chemical principles and methods", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.physical.chemistry.methods" }, { "id": "geo.nongeo.t1946.physical.chemistry.methods.analytical-chemistry", "label": "Analytical chemistry", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.physical.chemistry.methods.analytical-chemistry" }, { "id": "geo.nongeo.t1946.physical.chemistry.methods.analytical-chemistry.optical-spectrometry-and-photometry", "label": "Optical spectrometry and photometry", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.physical.chemistry.methods.analytical-chemistry.optical-spectrometry-and-photometry" } ] } ], "excerpt": "Ultraviolet spectrophotometry is an absorption technique of analytical chemistry that measures how much ultraviolet light a dissolved substance absorbs, typically from 190 to 900 nm, and converts absorbance into concentration.", "snippet": "Ultraviolet spectrophotometry is an absorption technique of analytical chemistry that measures how much ultraviolet light a dissolved substance absorbs, typically from 190 to 900 nm, and converts absorbance into concentration.", "node": "physical.chemistry.methods.analytical-chemistry.optical-spectrometry-and-photometry", "markdown": "# Ultraviolet spectrophotometry\n\nUltraviolet spectrophotometry is an absorption technique that measures how much ultraviolet light a dissolved substance absorbs and converts that measurement into an absorbance spectrum and, through calibration, a concentration. Commercial instruments typically cover 190 to 900 nm, with some designs extending to 1100 nm or into the near-infrared.[1](https://www.agilent.com/cs/library/primers/public/primer-uv-vis-nir-basics-5994-7919en-agilent.pdf) • [2](https://media.iupac.org/reports/V/spectro/partVII.pdf) The readout is absorbance, \\( A = \\log_{10}(1/T) \\), where \\( T \\) is the fraction of light the sample transmits.[3](https://trungtamthuoc.com/usp-en/ultraviolet-visible-spectroscopy) UV spectra generally lack the specificity needed to identify an unknown on their own, but the method is highly suitable for quantitative assay.[4](http://uspbpep.com/usp29/v29240/usp29nf24s0_c851.html)\n\n| Key fact | Value |\n|---|---|\n| Typical wavelength range | 190–900 nm (IUPAC region: 190 to about 1000 nm) [1](https://www.agilent.com/cs/library/primers/public/primer-uv-vis-nir-basics-5994-7919en-agilent.pdf) • [2](https://media.iupac.org/reports/V/spectro/partVII.pdf) |\n| Working equation | \\( A = \\varepsilon \\cdot b \\cdot c \\) (Beer–Lambert law) [1](https://www.agilent.com/cs/library/primers/public/primer-uv-vis-nir-basics-5994-7919en-agilent.pdf) |\n| Absorbance from about 10 µg/mL in a 1 cm cell | 0.2–0.8 AU in the UV or visible [3](https://trungtamthuoc.com/usp-en/ultraviolet-visible-spectroscopy) |\n| Best-precision absorbance | ≈0.43 (photodiode/phototube) to ≈0.86 (photomultiplier); work within 0.1–1.0 [2](https://media.iupac.org/reports/V/spectro/partVII.pdf) |\n| Cuvette for UV work | Quartz or fused silica below 350 nm; 10 mm path length standard [5](https://nanoqam.ca/wiki/lib/exe/fetch.php?media=an_introduction_to_ultraviolet-visible_molecular_absorption_spectrometry.pdf) • [1](https://www.agilent.com/cs/library/primers/public/primer-uv-vis-nir-basics-5994-7919en-agilent.pdf) |\n| Microvolume dsDNA range | 2–27,500 ng/µL from 1–2 µL of sample [6](https://assets.thermofisher.com/TFS-Assets/CAD/Specification-Sheets/NanoDrop-One-Specifications.pdf) |\n| Landmark instrument | Beckman DU, produced from 1941; over 30,000 units by 1976 [7](http://pubsapp.acs.org/supplements/chemchronicles2/pdf/087.pdf) • [8](https://www.beckman-foundation.org/about-foundation/inventions/spectrophotometer/) |\n\n## How it works\n\nUV-visible absorption is an electronic transition technique: incident radiation promotes outer or bonding electrons from the ground state into higher-energy states, with the photon energy matching the gap, \\( h\\nu = E_{2} - E_{1} \\).[9](https://www.drugfuture.com/Pharmacopoeia/usp38/data/v38332/usp38nf33s2_c1857.html) • [10](https://polymerscience.physik.hu-berlin.de/docs/manuals/UV.pdf) The wavelength of maximum absorbance, \\( \\lambda_{\\max} \\), gives a rough estimate of the [HOMO–LUMO gap](https://www.edgepedia.org/homo-lumo-gap) and increases with the number of conjugated double bonds, which is why extended chromophores absorb at longer wavelengths.[10](https://polymerscience.physik.hu-berlin.de/docs/manuals/UV.pdf)\n\nQuantification rests on the Beer–Lambert–Bouguer law. IUPAC writes it as \\( -\\log_{10}(F_{t}/F_{0}) = \\varepsilon \\cdot c \\cdot b = A \\), where \\( F_{t} \\) and \\( F_{0} \\) are transmitted and incident radiant power, \\( \\varepsilon \\) the molar absorption coefficient, \\( c \\) the amount concentration, and \\( b \\) the path length.[2](https://media.iupac.org/reports/V/spectro/partVII.pdf) The law holds only if absorbing species behave independently, the medium is uniform, and the radiation is parallel and monochromatic.[2](https://media.iupac.org/reports/V/spectro/partVII.pdf) Deviations arise from chemical variables such as association, dissociation, and ionization, and from instrumental variables including polychromatic radiation and stray light.[4](http://uspbpep.com/usp29/v29240/usp29nf24s0_c851.html) At high concentrations, usually above 0.01 M, solute–solute and solute–solvent interactions change the absorptivity.[5](https://nanoqam.ca/wiki/lib/exe/fetch.php?media=an_introduction_to_ultraviolet-visible_molecular_absorption_spectrometry.pdf)\n\n## How it is done\n\nA spectrophotometer consists of a light source, a sample holder, a monochromator, and a detector.[11](https://www.mt.com/dam/MT-MX/UVVIS_Spectrophoto_Guide_EN_30256131C_V08.21_Original_69649%20%281%29.pdf) A measurement begins with blanking: the solvent-filled cuvette is measured so the instrument stores the reference intensity and solvent absorbance is removed from sample readings.[12](https://kh.aquaenergyexpo.com/wp-content/uploads/2024/02/Fundamentals-Of-UV-Visible-Spectroscopy.pdf) The spectral bandwidth should be about one-tenth of the analyte's natural bandwidth, whose peaks are typically 20 nm or wider.[1](https://www.agilent.com/cs/library/primers/public/primer-uv-vis-nir-basics-5994-7919en-agilent.pdf) Analytical wavelengths are chosen at absorption maxima to minimize wavelength error, and isoabsorptive (isosbestic) points are frequently useful.[13](https://ctech.repligen.com/wp-content/uploads/2021/03/ASTM-Std-Practices-for-General-UV-VIS-Quantitative-Analysis-1.pdf)\n\nCuvettes for work below 350 nm must be quartz or fused silica; glass and acrylic do not transmit UV light, and cheaper plastic cuvettes generally do not either.[5](https://nanoqam.ca/wiki/lib/exe/fetch.php?media=an_introduction_to_ultraviolet-visible_molecular_absorption_spectrometry.pdf) • [1](https://www.agilent.com/cs/library/primers/public/primer-uv-vis-nir-basics-5994-7919en-agilent.pdf) • [11](https://www.mt.com/dam/MT-MX/UVVIS_Spectrophoto_Guide_EN_30256131C_V08.21_Original_69649%20%281%29.pdf) Solvents should transmit at least 40% at the wavelength of interest.[3](https://trungtamthuoc.com/usp-en/ultraviolet-visible-spectroscopy) Pharmacopeial verification uses holmium perchlorate solution or deuterium and mercury lamp lines (tolerance ±1 nm in the UV) and potassium dichromate solutions for absorbance control; stray light is checked with a 12 g/L potassium chloride solution, whose absorbance must exceed 2.0 at 198 nm.[14](https://www.drugfuture.com/Pharmacopoeia/BP2012/data/1015.html) IUPAC recommends adjusting concentration or path length to keep absorbance within 0.1–1.0.[2](https://media.iupac.org/reports/V/spectro/partVII.pdf) USP describes detection-limit estimation as 3.3 times the standard deviation of at least six replicate blank measurements, with linearity shown using at least five standard solutions.[3](https://trungtamthuoc.com/usp-en/ultraviolet-visible-spectroscopy)\n\n## Origin\n\nQuantitative absorption measurement relates the attenuation of light to the properties of the absorbing medium.[15](https://link.springer.com/article/10.1007/s10698-024-09501-5) One of the first photoelectric spectrophotometers used cesium photocells, and such an instrument was a UV-visible self-recording spectrophotometer available commercially.[7](http://pubsapp.acs.org/supplements/chemchronicles2/pdf/087.pdf) • [15](https://link.springer.com/article/10.1007/s10698-024-09501-5)\n\nThe modern instrument lineage begins with prototype models A, B, and C before the DU.[8](https://www.beckman-foundation.org/about-foundation/inventions/spectrophotometer/) The first DU was built in 1940 with a glass prism, later models using quartz, and the Model DU launched in 1941.[16](https://digital.sciencehistory.org/works/6q182k88v) • [7](http://pubsapp.acs.org/supplements/chemchronicles2/pdf/087.pdf) The first publication using a DU appeared in 1942 for vitamin A analysis, reducing a three-week rat bioassay to a ten-minute absorption measurement, and the instrument was used in penicillin production, synthetic rubber research, and explosives work.[8](https://www.beckman-foundation.org/about-foundation/inventions/spectrophotometer/) • [16](https://digital.sciencehistory.org/works/6q182k88v) The Cary 10 and 11 were UV-Vis spectrophotometers with a double monochromator, affording much less stray light; the Cary 11 was a self-recording instrument resembling modern laboratory models.[7](http://pubsapp.acs.org/supplements/chemchronicles2/pdf/087.pdf) • [15](https://link.springer.com/article/10.1007/s10698-024-09501-5) Beckman produced over 30,000 DUs before discontinuing the model in 1976.[8](https://www.beckman-foundation.org/about-foundation/inventions/spectrophotometer/)\n\n## Variants\n\nSingle-beam instruments measure blank and sample sequentially in the same cuvette, which makes them susceptible to drift between the two readings; dual- or split-beam designs pass light through sample and reference cuvettes and measure the difference, giving flatter baselines.[12](https://kh.aquaenergyexpo.com/wp-content/uploads/2024/02/Fundamentals-Of-UV-Visible-Spectroscopy.pdf) • [1](https://www.agilent.com/cs/library/primers/public/primer-uv-vis-nir-basics-5994-7919en-agilent.pdf) Textbooks distinguish single-beam, double-beam-in-space, double-beam-in-time, and multichannel instruments.[5](https://nanoqam.ca/wiki/lib/exe/fetch.php?media=an_introduction_to_ultraviolet-visible_molecular_absorption_spectrometry.pdf) Diode-array instruments such as the Agilent 8453 (190–1100 nm) capture a full spectrum in seconds, enabling multicomponent analysis and multi-wavelength calculations.[17](https://www.agilent.com/cs/library/brochures/5989-8680EN.pdf) Microvolume instruments measure 1–2 µL droplets on auto-ranging pathlengths of 0.030–1.0 mm, covering 190–850 nm.[6](https://assets.thermofisher.com/TFS-Assets/CAD/Specification-Sheets/NanoDrop-One-Specifications.pdf)\n\nDerivative spectroscopy uses first or higher derivatives of absorbance with respect to wavelength, \\( dA/d\\lambda \\) and beyond, to resolve overlapping bands and discriminate against broad background absorption from turbidity or matrix.[18](https://www.whoi.edu/cms/files/derivative_spectroscopy_59633940_175744.pdf) • [19](https://www.sciencedirect.com/science/article/abs/pii/S000326700802196X) The Savitzky–Golay calculation underlies most commercial derivatization algorithms.[18](https://www.whoi.edu/cms/files/derivative_spectroscopy_59633940_175744.pdf) For mixtures, IUPAC's multicomponent analysis measures absorbance at \\( k \\) wavelengths with \\( k > n \\) analytes, using \\( A(\\lambda_{a}) = \\sum_{i} b \\cdot \\varepsilon_{i}(\\lambda_{a}) \\cdot c_{i} \\).[2](https://media.iupac.org/reports/V/spectro/partVII.pdf)\n\n## Applications\n\nNucleic acids absorb maximally at 260 nm and proteins at 280 nm, the basis of the Warburg–Christian method, in which the 280 nm reading compensates for protein contamination.[11](https://www.mt.com/dam/MT-MX/UVVIS_Spectrophoto_Guide_EN_30256131C_V08.21_Original_69649%20%281%29.pdf) Software converts measured \\( A_{260} \\) to concentration with the dsDNA mass extinction coefficient of 50 ng/µL·cm⁻¹ and Beer's law; microvolume instruments accurately measure dsDNA from about 2–3 to 27,500–28,000 ng/µL using 1–2 µL.[20](https://assets.thermofisher.com/TFS-Assets/MSD/Application-Notes/EB53212-acclaro-nucleic-acid-resource-guide.pdf) • [6](https://assets.thermofisher.com/TFS-Assets/CAD/Specification-Sheets/NanoDrop-One-Specifications.pdf) The 260/280 ratio serves as a purity check, but it has blind spots: phenol absorbs strongly at 270 nm and its purity ratios resemble those of pure DNA and RNA, so small phenol contamination can drastically overestimate concentration while escaping ratio-based detection.[20](https://assets.thermofisher.com/TFS-Assets/MSD/Application-Notes/EB53212-acclaro-nucleic-acid-resource-guide.pdf)\n\nProtein \\( A_{280} \\) depends on tryptophan and tyrosine content, producing high protein-to-protein variability, and is affected by folding, solvent, and pH.[21](https://link.springer.com/content/pdf/10.1007/s00216-022-03910-1.pdf) [Extinction](https://www.edgepedia.org/extinction) coefficients \\( \\varepsilon_{1\\%} \\) differ greatly between proteins (for example, BSA 6.3–6.8 versus lysozyme 24.7–27.2), so a known coefficient is required; Gill and von Hippel showed these can be calculated from amino acid sequence data.[22](https://patofyziologie.lf1.cuni.cz/file/917/protein-determination-methods.pdf) • [23](https://doi.org/10.1016/0003-2697%2889%2990602-7) The classic Layne equation, \\( [\\mathrm{protein}]\\ (\\mathrm{mg/ml}) = (1.55 \\times A_{280}) - (0.76 \\times A_{260}) \\), introduced by Ennis Layne in 1957 in [Methods in Enzymology](https://www.edgepedia.org/methods-in-enzymology) [24](https://doi.org/10.1016/s0076-6879%2857%2903413-8), is not sufficiently accurate for routine calculations.[22](https://patofyziologie.lf1.cuni.cz/file/917/protein-determination-methods.pdf) [Measurement](https://www.edgepedia.org/measurement) at 205 nm, described by R. K. Scopes in 1974 in Analytical Biochemistry, extends UV quantification to proteins and amino acids with less dependence on aromatic residues.[25](https://doi.org/10.1016/0003-2697%2874%2990034-7) Proteins lacking aromatic residues, such as collagen and gelatin, cannot be measured at 280 nm.[26](https://www.jasco-global.com/solutions/protein-quantitation-using-a-uv-visible-spectrophotometer-uv-absorption-method/)\n\nAgainst colorimetric assays, UV absorption is simplest but often less accurate; the Bradford dye-binding assay, reported by M. Bradford in 1976 in Analytical Biochemistry [27](https://doi.org/10.1006/abio.1976.9999), responds nonlinearly to protein concentration, and plotting \\( A_{595}/A_{465} \\) linearizes it and raises sensitivity about tenfold, as shown by Tsaffrir Zor and Zvi Selinger in 1996 in Analytical Biochemistry.[22](https://patofyziologie.lf1.cuni.cz/file/917/protein-determination-methods.pdf) • [28](https://doi.org/10.1006/abio.1996.0171) For complex samples, LC with UV detection at 220 nm (estimated LOD 10 mg/L) gave robust protein-content results where colorimetric assays can severely over- or underestimate in unknown matrices.[21](https://link.springer.com/content/pdf/10.1007/s00216-022-03910-1.pdf)\n\n## Limitations and alternatives\n\nStray light, radiation outside the nominal wavelength band, decreases absorbance readings, distorts peak shape, causes Beer–Lambert deviation, and sets the maximum absorbance an instrument can reliably measure.[1](https://www.agilent.com/cs/library/primers/public/primer-uv-vis-nir-basics-5994-7919en-agilent.pdf) • [5](https://nanoqam.ca/wiki/lib/exe/fetch.php?media=an_introduction_to_ultraviolet-visible_molecular_absorption_spectrometry.pdf) [Absorbance](https://www.edgepedia.org/absorbance) should not be determined on turbid or light-scattering samples, because scattering removes intensity increasingly as wavelength decreases.[13](https://ctech.repligen.com/wp-content/uploads/2021/03/ASTM-Std-Practices-for-General-UV-VIS-Quantitative-Analysis-1.pdf) For a 50 mg/mL monoclonal antibody, uncorrected light scattering overestimated protein concentration at 280 nm by 2.9%, and variable-pathlength spectroscopy reduced %RSD from 3.3 to about 1.1–1.2 over a 0.05–300 mg/mL range.[29](https://www.repligen.com/ctech-resources/pdf/Biogen-Inc.-Comparison-of-Protein-Concentration-Determination-of-a-mAb-Drug-Substance-Using-Variable-Pathlength-Spectroscopy-and-UV-VIS-Spectroscopy.pdf) Single-beam designs add drift between blank and sample readings [1](https://www.agilent.com/cs/library/primers/public/primer-uv-vis-nir-basics-5994-7919en-agilent.pdf), and overlapping spectra limit specificity, so mixtures require multicomponent or chemometric treatment.[4](http://uspbpep.com/usp29/v29240/usp29nf24s0_c851.html)\n\nSensitivity compares favorably with infrared spectroscopy, where 1–10 mg/mL may be needed to reach similar absorbances in 1 cm cells versus about 10 µg/mL in the UV or visible.[4](http://uspbpep.com/usp29/v29240/usp29nf24s0_c851.html)\n\n## References\n\n1. [Agilent UV-Vis-NIR Basics Primer](https://www.agilent.com/cs/library/primers/public/primer-uv-vis-nir-basics-5994-7919en-agilent.pdf)\n2. [IUPAC Nomenclature, Symbols, Units and their Usage in Spectrochemical Analysis Part VII: Molecular Absorption Spectroscopy (UV/VIS)](https://media.iupac.org/reports/V/spectro/partVII.pdf)\n3. [USP General Chapter ⟨857⟩ Ultraviolet-Visible Spectroscopy (USP 2025)](https://trungtamthuoc.com/usp-en/ultraviolet-visible-spectroscopy)\n4. [USP General Chapter <851> Spectrophotometry and Light-Scattering](http://uspbpep.com/usp29/v29240/usp29nf24s0_c851.html)\n5. [Introduction to Ultraviolet-Visible Molecular Absorption Spectrometry (textbook chapter, Skoog et al., hosted copy)](https://nanoqam.ca/wiki/lib/exe/fetch.php?media=an_introduction_to_ultraviolet-visible_molecular_absorption_spectrometry.pdf)\n6. [NanoDrop One Microvolume UV-Vis Spectrophotometers Product Specifications (Thermo Fisher Scientific)](https://assets.thermofisher.com/TFS-Assets/CAD/Specification-Sheets/NanoDrop-One-Specifications.pdf)\n7. [Instruments of war (ACS chemical chronicles)](http://pubsapp.acs.org/supplements/chemchronicles2/pdf/087.pdf)\n8. [Spectrophotometer | Beckman Foundation](https://www.beckman-foundation.org/about-foundation/inventions/spectrophotometer/)\n9. [USP 38-NF 33 <1857> Ultraviolet-Visible Spectroscopy](https://www.drugfuture.com/Pharmacopoeia/usp38/data/v38332/usp38nf33s2_c1857.html)\n10. [Humboldt-Universität zu Berlin UV-Vis lab manual](https://polymerscience.physik.hu-berlin.de/docs/manuals/UV.pdf)\n11. [UVVIS Spectrophoto Guide EN 30256131C V08.21 Original 69649 (1) (mt.com)](https://www.mt.com/dam/MT-MX/UVVIS_Spectrophoto_Guide_EN_30256131C_V08.21_Original_69649%20%281%29.pdf)\n12. [Fundamentals of UV-Visible Spectroscopy (Agilent primer, third-party hosted copy)](https://kh.aquaenergyexpo.com/wp-content/uploads/2024/02/Fundamentals-Of-UV-Visible-Spectroscopy.pdf)\n13. [ASTM Standard Practices for General UV-VIS Quantitative Analysis (E169)](https://ctech.repligen.com/wp-content/uploads/2021/03/ASTM-Std-Practices-for-General-UV-VIS-Quantitative-Analysis-1.pdf)\n14. [Ph. Eur. method 2.2.25 / BP Appendix II B: Ultraviolet and Visible Absorption Spectrophotometry](https://www.drugfuture.com/Pharmacopoeia/BP2012/data/1015.html)\n15. [Introducing UV–visible spectroscopy at high school level following the historical evolution of spectroscopic instruments](https://link.springer.com/article/10.1007/s10698-024-09501-5)\n16. [The Beckman Photoelectric Spectrophotometer, Bulletin 79 (1941)](https://digital.sciencehistory.org/works/6q182k88v)\n17. [Agilent 8453 UV-visible spectrophotometer brochure](https://www.agilent.com/cs/library/brochures/5989-8680EN.pdf)\n18. [Uses of Derivative Spectroscopy (Agilent application note)](https://www.whoi.edu/cms/files/derivative_spectroscopy_59633940_175744.pdf)\n19. [Recent development in derivative ultraviolet/visible absorption spectrophotometry: 2004–2008 (Analytica Chimica Acta review)](https://www.sciencedirect.com/science/article/abs/pii/S000326700802196X)\n20. [NanoDrop One/OneC dsDNA dynamic range and Acclaro contaminant identification technical notes (Thermo Fisher Scientific)](https://assets.thermofisher.com/TFS-Assets/MSD/Application-Notes/EB53212-acclaro-nucleic-acid-resource-guide.pdf)\n21. [Determination of the protein content of complex samples by aromatic amino acid analysis, liquid chromatography-UV absorbance, and colorimetry](https://link.springer.com/content/pdf/10.1007/s00216-022-03910-1.pdf)\n22. [Assays for Determination of Protein Concentration (Current Protocols in Protein Science, hosted copy)](https://patofyziologie.lf1.cuni.cz/file/917/protein-determination-methods.pdf)\n23. [Calculation of protein extinction coefficients from amino acid sequence data (Analytical Biochemistry, 1989)](https://doi.org/10.1016/0003-2697%2889%2990602-7)\n24. [(73) Spectrophotometric and turbidimetric methods for measuring proteins (Methods in enzymology on CD-ROM/Methods in enzymology, 1957)](https://doi.org/10.1016/s0076-6879%2857%2903413-8)\n25. [Measurement of protein by spectrophotometry at 205 nm (Analytical Biochemistry, 1974)](https://doi.org/10.1016/0003-2697%2874%2990034-7)\n26. [Protein Quantitation using a UV-Visible Spectrophotometer – UV Absorption Method (JASCO application note)](https://www.jasco-global.com/solutions/protein-quantitation-using-a-uv-visible-spectrophotometer-uv-absorption-method/)\n27. [M Bradford (1976). A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry.](https://doi.org/10.1006/abio.1976.9999)\n28. [Tsaffrir Zor, Zvi Selinger (1996). Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies. Analytical Biochemistry.](https://doi.org/10.1006/abio.1996.0171)\n29. [Biogen Inc.: Comparison of Protein Concentration Determination of a mAb Drug Substance Using Variable Pathlength Spectroscopy and UV-VIS Spectroscopy](https://www.repligen.com/ctech-resources/pdf/Biogen-Inc.-Comparison-of-Protein-Concentration-Determination-of-a-mAb-Drug-Substance-Using-Variable-Pathlength-Spectroscopy-and-UV-VIS-Spectroscopy.pdf)\n\n---\n*Topic: Encyclopedia › Physical world and mathematics › Chemistry › Chemical principles and methods › Analytical chemistry › Optical spectrometry and photometry*\n\n*Initially written Sep 29, 2026 · Reviewed: Sep 30, 2026 · Edited: Oct 15, 2026 · Last review: Sep 30, 2026*\n\n*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*\n\nLicense: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license\n", "same_as": [], "url": "https://www.edgepedia.org/ultraviolet-spectrophotometry", "markdown_url": "https://www.edgepedia.org/ultraviolet-spectrophotometry.md", "license": { "name": "Edgepedia Community License 1.0", "url": "https://www.edgepedia.org/edgepedia/license", "summary": "Free with credit, commercial use included. AI training is open to everyone. For other uses, organizations over USD 100M in revenue or 100M monthly users license separately.", "spdx": "LicenseRef-Edgepedia-Community-1.0" }, "credit": "\"Ultraviolet spectrophotometry\", Edgepedia (EdgeChat), https://www.edgepedia.org/ultraviolet-spectrophotometry. Edgepedia Community License 1.0.", "credit_md": "\"[Ultraviolet spectrophotometry](https://www.edgepedia.org/ultraviolet-spectrophotometry)\", Edgepedia (EdgeChat), [https://www.edgepedia.org/ultraviolet-spectrophotometry](https://www.edgepedia.org/ultraviolet-spectrophotometry). [Edgepedia Community License 1.0](https://www.edgepedia.org/edgepedia/license).", "credit_html": "\"Ultraviolet spectrophotometry\", Edgepedia (EdgeChat), https://www.edgepedia.org/ultraviolet-spectrophotometry. Edgepedia Community License 1.0.", "speakable": "Ultraviolet spectrophotometry is an absorption technique of analytical chemistry that measures how much ultraviolet light a dissolved substance absorbs, typically from 190 to 900 nm, and converts absorbance into concentration." }