UV/Visible Spectroscopy Essentials
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Questions and Answers

What phenomenon is primarily responsible for electronic transitions in UV/visible spectroscopy?

  • Ionic interactions
  • Electronic spin transitions
  • Allowed transitions in a molecule (correct)
  • Nuclear transitions

    • What effect do solvent collisions have on absorbance peaks?

  • They eliminate vibrational bands.
  • They broaden the absorbance peaks. (correct)
  • They create a discontinuous absorption spectrum.
  • They narrow the absorbance peaks.

    • Which of the following can occur when additional energy is provided during electronic transitions?

  • Loss of absorbance characteristics
  • Excitation to vibrational states (correct)
  • Destruction of electronic states
  • Formation of new electronic states

    • Which colored compound is represented by the spectrum showing absorbance near 580 nm?

    <p>Rhodamine 6B</p> Signup and view all the answers

    What is indicated by vibrionic bands in an absorbance spectrum?

    <p>Absorption at multiple distinct wavelengths</p> Signup and view all the answers

    Which factor does NOT directly contribute to the shape of the absorbance peak?

    <p>External temperature fluctuations</p> Signup and view all the answers

    Which spectral region is primarily investigated in UV/visible spectroscopy?

    <p>Ultraviolet and visible region</p> Signup and view all the answers

    What is a consequence of using a solvent in UV/visible spectroscopy?

    <p>Broadening of the absorption peak</p> Signup and view all the answers

    What does absorbance spectroscopy primarily measure?

    <p>The attenuation of light intensity after it passes through a sample</p> Signup and view all the answers

    What is the primary function of diffraction gratings in a monochromator?

    <p>To reduce stray light</p> Signup and view all the answers

    What optical spectrum range is most relevant in UV-visible spectroscopy?

    <p>200-750 nm</p> Signup and view all the answers

    How does light get diffracted by a diffraction grating?

    <p>When the distance traveled from successive ridges is an integer multiple of the wavelength</p> Signup and view all the answers

    What occurs when UV-visible light photons are absorbed by molecules?

    <p>Valence electrons in molecules are excited to higher energy states</p> Signup and view all the answers

    What is the result of a sample absorbing green light from white light?

    <p>The transmitted light appears red</p> Signup and view all the answers

    What is one disadvantage of photomultiplier tubes (PMTs)?

    <p>They require a stable high voltage power supply</p> Signup and view all the answers

    What phenomenon may occur as a result of absorption of UV-visible radiation?

    <p>Photochemistry leading to chemical changes</p> Signup and view all the answers

    Which characteristic makes photodiodes different from photomultiplier tubes?

    <p>Photodiodes are much cheaper and reproducible</p> Signup and view all the answers

    Why can predicting colors based on absorption be complex?

    <p>Many compounds have multiple absorption bands</p> Signup and view all the answers

    What is the main advantage of double-beam spectrophotometers over single-beam instruments?

    <p>They minimize drift by measuring simultaneously</p> Signup and view all the answers

    What happens when light strikes the p-layer of a photodiode?

    <p>It creates an electron-hole pair</p> Signup and view all the answers

    What does the relationship $c = l n$ describe in relation to spectroscopy?

    <p>The absorbance of light in a sample</p> Signup and view all the answers

    How does light energy transform when absorbed by molecules?

    <p>It is transformed into vibration/rotation/translation energy</p> Signup and view all the answers

    What is the typical amplification factor of photomultiplier tubes?

    <p>Around 10 million-fold</p> Signup and view all the answers

    Which property is NOT associated with photomultiplier tubes?

    <p>Reliance on low voltage operation</p> Signup and view all the answers

    What happens to electronic peaks when analytes are measured in a nonpolar solvent?

    <p>Only distinct electronic transitions are observed.</p> Signup and view all the answers

    Which light source produces a black-body spectrum at 2900K?

    <p>W-Halogen Lamp</p> Signup and view all the answers

    What is the primary disadvantage of using a deuterium arc lamp?

    <p>It has a limited lifetime.</p> Signup and view all the answers

    What is the role of a monochromator in a spectrophotometer?

    <p>To select a single wavelength of light from a spectrum.</p> Signup and view all the answers

    What type of light does the W-Halogen lamp produce?

    <p>Very low noise light.</p> Signup and view all the answers

    What occurs to analytes in aqueous buffers during spectrophotometric analysis?

    <p>They produce few broad peaks.</p> Signup and view all the answers

    What is termed 'stray light' in the context of spectrophotometry?

    <p>Light that exits the monochromator except the desired wavelength.</p> Signup and view all the answers

    What is a characteristic of the light produced by deuterium arc lamps?

    <p>It produces intense light across the UV continuum.</p> Signup and view all the answers

    Which features are primarily lost when analytes are examined in a nonpolar solvent?

    <p>Vibrational and rotational structure.</p> Signup and view all the answers

    What contributes to the broadening of electronic peaks in a polar solvent?

    <p>Strong intermolecular forces.</p> Signup and view all the answers

    What does the shift in the plasmon angle indicate in SPR?

    <p>Binding interactions occurring</p> Signup and view all the answers

    Which parameter is determined from the exponential curve during the association phase?

    <p>Binding constant, kon</p> Signup and view all the answers

    What occurs once binding of the analyte is complete in an SPR experiment?

    <p>Flow switches back to the running buffer</p> Signup and view all the answers

    How can the ligand be attached to the chip surface in SPR?

    <p>Using an affinity tag or chemical linkage</p> Signup and view all the answers

    What does the ratio kon/koff represent in SPR?

    <p>Dissociation constant, KD</p> Signup and view all the answers

    What is indicated by a signal $A > 2.0$ when measuring absorbance?

    <p>The incident light is insufficiently reaching the detector.</p> Signup and view all the answers

    What characteristic does a good optical spectrum possess at short wavelengths?

    <p>Black appearance</p> Signup and view all the answers

    What factor contributes to the total attenuation of the incident light in absorbance spectroscopy?

    <p>Scattering and absorbance by the sample.</p> Signup and view all the answers

    Which of the following can result in common artefacts during UV-vis spectrophotometry?

    <p>Presence of particulates or colloids.</p> Signup and view all the answers

    How should a cuvette be prepared to avoid measurement issues?

    <p>The exterior must be clean and dry.</p> Signup and view all the answers

    What is a common misconception regarding the peaks in absorbance spectra?

    <p>Samples with cloudy appearances are acceptable.</p> Signup and view all the answers

    What is advised for monitoring chromatography column eluates?

    <p>Single-read measurements can be used.</p> Signup and view all the answers

    What effect does scattering have on absorbance measurements?

    <p>It attenuates incident light.</p> Signup and view all the answers

    Study Notes

    Optical (UV-visible) absorbance spectroscopy

    • This technique measures the attenuation of light intensity as it passes through a sample.
    • The attenuation is dependent on wavelength.
    • The resulting UV-visible spectrum provides information about the chemical solutes present in the sample, their chemical state, and their concentration.

    Electromagnetic spectrum

    • The UV-visible range is approximately 200-750 nm.
    • This range corresponds to 600-1500 kJ/mol.
    • Typical covalent bond energies are 150-900 kJ/mol.
    • UV-visible light photons can excite valence electrons in molecules.
    • Molecules are raised to electronically excited quantum states.
    • Light energy converts to vibrational/rotational/ translational energy (heat).
    • Chemical changes (photochemistry) might occur.
    • Less light exits the sample compared to the initial amount.

    Optical absorbance and visible colour

    • If green light is absorbed from white light, the transmitted light appears red (complementary colour).
    • Compounds/mixtures often have multiple absorption bands.
    • The spectrum of "white" light varies with the light source.
    • Colour perception involves physics, physiology, and psychology.
    • Analyzing spectra is more objective.

    Absorption of UV-visible radiation

    • c=λ ν (wave equation)
    • E = hv (Planck equation)
    • UV-visible spectral range is about 200-750nm.
    • 600–150 kJ/mol. is a suitable value for the spectral range.
    • Typical covalent bond energies are within 150-900kJ/mol.

    Solvents collisions broaden absorbance peaks

    • UV absorption spectra for various molecules.
    • In the gas phase, electronic, vibrational, and rotational transitions are present
    • In a nonpolar solvent, electronic transitions are distinct but have no vibrational and rotational structure.
    • In a polar solvent, strong intermolecular forces cause broader electronic peaks, creating a smooth absorption band.
    • Conclusion: Sharp peaks for analytes are common, but aqueous buffers generate wider peaks.

    Spectrophotometers

    • Devices for measuring light absorbance.
    • Different types exist in various designs and capabilities.
    • Key components include light source, monochromator, sample holder, and detector.

    Single-beam spectrophotometer: Schematic

    • Light source: Device producing a suitable spectrum.
    • Monochromator: Isolates a single wavelength from the spectrum.
    • Detector: Quantifies the incoming light intensity.

    Light source 1: W-Halogen Lamp

    • W-halogen lamps produce a black-body spectrum at 2900 K.
    • Light has very low noise.
    • Lamps have a long lifetime.
    • Disadvantage: Low light output in the UV range.

    Light source 2: Deuterium (or H₂) Arc Lamp

    • Deuterium arc lamps use high voltage to excite deuterium for electron relaxation to emit light.
    • Produces intense light across the UV continuum (180 nm–370 nm).
    • Low noise light is produced.
    • Disadvantage: limited lifetime (2000–5000 hours).

    Monochromator

    • Devices use prisms or diffraction gratings to split incoming light.
    • A slit blocks all light except for the desired wavelength.
    • Any stray light adds noise to the signal, especially at high absorbance.
    • Using two monochromators reduces stray light.

    Diffraction gratings

    • Are formed from any periodic repeating pattern of holes or ridges.
    • CDs are an example.
    • Are built as a series of ridges with defined spacing and angle.
    • Light diffracts only when the distance light reflects matches a whole number of wavelengths.
    • The wavelength determines which wavelength is diffracted at a particular angle.
    • UV/visible light's d is typically between 1200 and 1400 nm.

    Detectors: Photomultiplier tube

    • Each successive dynode is about 200 V higher.
    • Each electron impact yields about 4 secondary electrons.
    • Typically ~10 stages, resulting in a 10⁶-fold amplification.
    • Photomultipliers have high sensitivity (detect single photons).
    • Disadvantages: Expensive and mechanically fragile, require a stable high-voltage power supply.

    Detectors: Photodiodes

    • Light-sensitive semiconductor devices (mini solar panels).
    • Light striking the p-layer creates electron-hole pairs.
    • Electron travel to the n layer allows current flow.
    • They are small, light, cheap, and reproducible.
    • Lower sensitivity compared to photomultipliers.

    Beer-Lambert Law

    • Absorbance is proportional to the number of molecules in the path.
    • Each successive 'layer' absorbs the same fraction of incident light.
    • Absorbance (A) is unitless.
    • Molar extinction coefficient (ε) has units of mol⁻¹L cm⁻¹.
    • ε is a characteristic property of a specific molecular species.

    Practicalities

    • For standard cuvettes (l = 1 cm) and a typical chromophore (ε = 2000), a suitable concentration for quantitative analysis is ~0.1 mM.
    • Molecules usually absorb at UV-Vis when they have several conjugated double bonds.
    • Peptide bonds absorb at 190 nm and (more weakly) at 210-220 nm.

    Cut-off wavelength

    • Below a certain wavelength, any solvent strongly absorbs UV light so that nearly no light is transmitted.
    • A cut-off's wavelength defines the shortest measurable wavelength in UV-Vis spectroscopy.
    • Some organic solvents absorb strongly at 260-280 nm, which can interfere with DNA or protein quantification.

    Optimal reading ranges

    • The instrument gives a reading, but not always a meaningful one.
    • The most accurate range for absorbance measurements is ~0.1-1.
    • If A > 2, the incident light is too small; the sample should be diluted.
    • If A < 0.01, the difference between I and I₀ is too small, suggesting you've reached the sensitivity limit.

    Characteristics of a good optical spectrum

    • "Black" at short wavelengths.
    • Smooth drop to A = 0 at long wavelengths.
    • Smooth, broad peaks (Gaussian-like).

    Scattering

    • Scattering is a process where objects absorb light energy and re-emit it.
    • Scattering allows the observation of light even if observers aren't in the light's path.
    • Wavelength changes but is unaffected (elastic scattering); direction changes.
    • Scattering physics is sensitive to the particle's concentration and size, and the wavelength of light.
    • Proteins (2–10 nm) are much smaller than visible light (400-700 nm).
    • Rayleigh scattering dominates for smaller particles. Larger particles (40–900 nm) exhibit stronger Tyndall scattering. Protein aggregates scatter more strongly, complicating optical measurements.

    Rayleigh scattering

    • Rayleigh scattering strongly depends on wavelength (proportional to λ⁻⁴).
    • Blue light scatters significantly more than red light.
    • This is the primary reason why the sky is blue. Blue wavelengths of sunlight are preferentially scattered toward observers by atmospheric nitrogen.
    • Rayleigh scattering is weak for small particles unless the path length is substantial (like in the atmosphere).
    • Scattering is linearly proportional to molecular weight; significant for larger proteins.

    Static light scattering

    • A biochemical application of scattering.
    • Scattering depends on particle size and molecular weight.
    • Provides accurate readings of molecular weight.
    • Scattering also depends linearly on concentration which means that using UV absorption or refractive index measurements gives accurate protein concentration.
    • For large particles, the scattering angle drops off at higher angles, which depends on 'r'.
    • Proteins tend to be too small for reliable measurement.

    SEC-MALS

    • SEC-MALS resolves oligomerization equilibrium of proteins.
    • Static light scattering gives a reliable molecular weight measurement if only one particle type is present.
    • SEC separates species before measuring multi-angle light scattering.
    • UV absorbance and refractive index measurements allow accurate calculation of protein and other solute concentrations.
    • Molecular weight can be calculated with ~5% accuracy.

    Surface Plasmon Resonance

    • Light propagates along the interface between two media.
    • The angle of light reflection is sensitive to the refractive index of the media.
    • Provides measurements of small changes in refraction index in a surface boundary layer.
    • Measurement with SPR can be a macromolecule's binding to a specifically prepared surface, even small molecule binding is measurable via changes in the refractive index.

    SPR gives binding constants

    • Precise estimates of Kď require ligand concentration range measurements then data fitting.
    • Measurement precision is approximately 1%.

    Typical applications of UV-visible absorbance spectroscopy

    • Directly measure the concentration of pure proteins, nucleic acids, small molecules.
    • Indirectly measuring concentrations via chromogenic assays.
    • Monitoring chemical changes (e.g., protonation equilibria, redox state changes in metals or cofactors).
    • Monitoring eluate from chromatography columns.
    • Characterizing protein prosthetic groups (e.g., heme, flavins).
    • Various enzyme assays that involve changes in cofactors, substrates, or coupled reactions (e.g., NADH depletion).

    UV-visible spectroscopy of biomolecules: overview

    • Nucleic Acids: Excellent.
    • Proteins: Good, but accurate quantitation depends on composition.
    • Prosthetic groups: Excellent if they absorb.
    • Carbohydrates: Usually poor.
    • Lipids: Unsaturated or conjugated only.
    • A variety of substances can be measured using chromogenic assays.

    UV spectra of protein and DNA

    • Proportional relationships in the absorbance between DNA, RNA, and protein at 260 nm and 280 nm.

    Unless they contain prosthetic groups, most proteins have similar UV spectra.

    • Proteins lacking prosthetic groups have similar UV spectra.

    Protein UV absorbance depends on Trp, Tyr, and Cystine

    • Trp strongly absorbs UV light.
    • Tyr absorbs UV light ~1/3 as strongly as Trp.
    • Cystine absorbs UV light at low amounts.
    • Formula: ε₂₈₀ = (#Trp × 5,500) + (#Tyr × 1,490) + (#Cystine × 125).

    Sample calculation of protein absorbance

    • Example calculation using the Beer-Lambert Law, to calculate protein absorbance from concentrations.

    UV spectroscopy can detect protein prosthetic groups

    • Use UV spectroscopy to detect protein prosthetic groups.

    Changes in some active sites can be monitored by changes in the absorbance spectrum

    • Changes in active sites can be monitored in the UV-Vis spectrum.
    • Examples include cytochrome c, and oxygen binding.

    Isosbestic points

    • A reaction that follows a simple stoichiometry (A → B) exhibits at least one (or more) isosbestic points.
    • These points show that the absorbance remains constant during the process.
    • This isn't necessarily true if A → B + C.

    Isosbestic points are often seen in redox changes, titrations, ligand binding, and other stoichiometrically simple reactions.

    • Examples show that isosbestic points are common in redox changes, titrations, ligand binding and other simple reactions.

    UV-visible spectroscopy: Summary

    • Molecules absorb photons, exciting electrons in molecular orbitals.
    • Excited states decay via collisions, releasing energy as heat.
    • Quantitative analysis of molecules with useful chromophores is possible.
    • Structural information (e.g., protonation or redox equilibria) can be obtained. -Strengths: Routine instrumentation, high-throughput mode (e.g., microplate readers), direct measurement of analyte concentration. -Weaknesses: Applicable only to molecules with good chromophores, susceptible to interferences, lower sensitivity than fluorescence spectroscopy.

    Practice question

    • Identify suitable wavelengths for monitoring an enzyme's activity that catalyzes the reduction of NAD+ to NADH. The key is identifying which wavelength shows the largest change in absorbance.

    Answer

    • 340 nm is the best wavelength to measure because it shows the largest difference in absorbance between NAD+ and NADH.

    Circular Dichroism Spectroscopy

    • UV light (<220 nm) is absorbed by peptide bonds.
    • Differential absorption of left and right-handed circularly polarized light, differing due to chiral environments within the protein.
    • Measured using an instrument similar to a spectrophotometer.
    • Differences in absorbance are small and necessitate extremely clean samples (free from scatter).
    • Provides insights into secondary structure by measuring changes in chromophore environment.
    • Useful for monitoring changes in secondary structure, such as temperature, mutations, ligand binding, etc.

    CD gives insight into secondary structure

    • a-helical proteins are characterized by specific peaks.
    • β-sheets have a more complex pattern.
    • Unfolded proteins have a different spectrum.
    • Software helps interpret these complex spectra based on fractional fitting.

    CD monitors protein structural transitions

    • Changes in protein structure can be monitored via changes in CD spectra at various pH ranges.

    Refractive index gives solute concentration

    • Medium's refractive index (RI) reflects light speed.
    • This bending of light can be precisely measured.
    • RI of solutions is linearly correlated to solute concentrations (including proteins).
    • Protein concentration can be accurately measured by RI measurements if other solutes are accounted for.

    Scattering

    • Objects absorb light energy and re-emit it in a different direction (scattering).
    • Scattering reveals light even if observers aren't in the light's direct path.
    • Direction changes but no wavelength changes (elastic scattering).
    • Scattering sensitivity is related to the particle's concentration, size, and wavelength of light.
    • Proteins (2–10 nm) are much smaller than visible light (400-700 nm). Rayleigh scattering dominates for smaller particles
    • Larger particles (40-900nm) show stronger Tyndall scattering, which is problematic for optical methods.

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