UV-Vis Spectroscopy: An Introduction

UV-Vis Spectroscopy

• Used primarily for quantitative analysis of molecules

Regions

• UV (Ultraviolet): 190–400 nm
• Visible (Vis): 400–800 nm
  • Violet (400–450 nm)
  • Blue (450–490 nm)
  • Green (490–560 nm)
  • Yellow (560–590 nm)
  • Orange (590–630 nm)
  • Red (630–700 nm)
• Near-Infrared (NIR): 800–2500 nm

Type of Spectroscopy

• Absorption spectroscopy measures light absorbed by molecules
• Molecular spectroscopy analyzes molecules, including organic ions and metal complexes

Instrumentation Basic Setup

• Radiation Source
  • Incandescent bulb emits Visible range (320–2500 nm)
  • Deuterium lamp emits UV range (160–375 nm)
  • Xenon arc lamp emits a Broad range (190–1000 nm)
• Wavelength Selector
  • Photocolorimetry (Filter)
    • Bandwidth of ~10–100 nm
    • Made of colored glass, recording full spectra is not possible
  • Spectrophotometry (Monochromator)
    • Selection achievable of narrow bandwidths
    • Full spectrum scanning allowable due to usage of diffraction devices
• Sample Holder (Cuvette)
  • Materials
    • Quartz: Transparent above 190 nm (UV-Vis)
    • Glass: Transparent above 300 nm (Vis)
    • Plastic: Only in the visible range
  • Requirements include clean optical sides (no fingerprints, droplets, or particles)
• Detectors
  • A Phototube detects from 150–1000 nm
  • Photoelectron Multiplier detects from 150–1000 nm (high sensitivity)
  • Photodiode detects from 350–1100 nm using Si semiconductor
  • Diode Array uses multiple photodiodes for simultaneous detection

Beam Configurations

• Single-Beam Spectrophotometer
  • Measures reference intensity (I0) and sample intensity (I) separately
  • Zeroing required, with a blank solution (same components as sample, minus analyte)
• Double-Beam Spectrophotometer
  • Measures I0 and I simultaneously at each wavelength
  • Compensates for cuvette differences, which leads to more stable spectrum recording

Principles of UV-Vis Absorption Electronic Transitions

• Molecules absorb photons to become excited: M+hν→M*
• Key Transitions
  • Organic Compounds: π→π* (most important for UV-Vis)
    • Conjugation shifts absorption to longer wavelengths (e.g., more conjugated bonds = visible color)
  • Transition Metal Complexes
    • Excitement of dd and ff electrons
    • Charge-transfer transitions (electron moves between metal and ligand) produce intense absorption

Beer-Lambert Law

• Aλ=ελ⋅l⋅c Aλ is absorbance at wavelength λ ελ is the molar absorption coefficient (L/mol·cm) l is the cuvette path length (cm) c is the analyte concentration (mol/L)
• Absorbance Calculation: Aλ=log(I0/I)

Limitations of Beer’s Law

• Real Limitations
  • High concentrations (>0.01 M): Molecular interactions distort linearity
  • Chemical changes: pH, associations, or reactions (e.g., unbuffered indicators)
• Instrumental Limitations
  • Polychromatic radiation (non-ideal monochromator)
  • Stray light reduces accuracy at high absorbance
  • Mismatched cuvettes

Quantitative Analysis Key Considerations

• Linear Range is 0.02–1.2 AU, deviations are expected outside range
• Detection Limit is typically 10−3 to 10−5 M
• Calibration needs to be reliable due to minimal instrumental variability

Selecting Analytical Wavelength

• Higher wavelengths reduce interference (other compounds are less likely to absorb)

• Maximizing sensitivity necessitates using λmax (peak absorption) for highest ε

• Avoid low wavelengths ( Eexcited)

• Excitation spectra is measured by monitoring the fluorescence intensity at a fixed emission wavelength (or a sum of wavelengths) while varying the excitation wavelength

• Excitation spectrum shares similarities with the absorption spectrum of the molecule

• Emission (or fluorescence) spectra is measured by scanning the emission wavelength while keeping the excitation wavelength fixed

• The emission spectrum shows what wavelengths of light are emitted by the sample

• Excitation spectra are generally at lower wavelengths (higher energy) than the emission spectra due to the Stokes shift

• Spectral bands in fluorescence spectroscopy are relatively wide and not highly characteristic of specific structures

• The excitation spectrum expands to shorter wavelengths while the emission spectrum expands to longer wavelengths

• Using an excitation wavelength away from the maximum excitation wavelength changes the intensity of the emission, but not the shape of the emission band

• The wavelength of the excitation source does not have to correspond exactly to the excitation maximum to observe fluorescence

Spectrometers

• Two common spectrometer types include filter-fluorometers and spectrofluorometers
• High-end systems include laser fluorescence systems used in microscopy and LIF (Laser-Induced Fluorescence)
• Filter-fluorometers use two filters: one to select the appropriate excitation wavelength range, and another for the appropriate emission wavelength range
• These instruments are suitable for measurements with somewhat fixed parameters; the scheme includes a lamp, excitation filter, sample, emission filter, and detector
• Spectrofluorometers use two monochromators
  • one to select the excitation light
  • the other for selecting the fluorescence light
• This instrument can register both excitation and emission spectra, and provides flexibility and precision; a two-beam spectrofluorometer has lamp, excitation monochromator, sample, emission monochromator, a splitter, and two detectors (one for reference)

Compounds that Fluoresce

• For a molecule to fluoresce, it must absorb light first
• Fluorescence is particularly favored for molecules that are relatively rigid, as lack of rigidity enhances internal conversion (kic) and the likelihood of radiationless deactivation
• This is beneficial for fluorescence
• Most molecules do not fluoresce because other radiationless processes are much faster
• The difference in fluorescence between Fluorene (which fluoresces) and Biphenyl (which does not) illustrates the effect of structural rigidity

Limit of Detection

• Measuring a single low-intensity light beam (as in fluorescence emission) is more accurate than comparing two close intensity light beams (as in absorption in UV-Vis)
• Limit of detection (LoDs) in fluorescence spectroscopy are much lower than in UV-Vis, often in the ppb or ppt range
• Fluorescence is well-suited for the detection of small residues if these compounds fluoresce
• Derivatization with fluorescent reagents is a common technique to enable the detection of the non-fluorescent compounds

Quantitative Analysis

• Emitted light intensity (F) is proportional to the concentration (C) of the fluorescent analyte: F = k∙C, where k is constant
• A wide linear range is observed for quantitative analysis in fluorescence spectroscopy
• The linear range is typically 3 to 6 orders of magnitude
• Quantitative analysis is usually performed by relying on the calibration graph method due to the dependence of fluorescence intensity on factors which include the light source, optics, and environment

Applications

• Applications span a wide range:
  • determination of metals using fluorescence reagents
  • many sensor applications, e.g., determination of O₂
  • liquid chromatography (HPLC) with a detector
  • biological analysis (e.g. immune analysis and the imaging of processes in living organisms)
  • environmental residue monitoring
  • metal ion analysis with fluorescent reagents

Pros and Cons

• Pros
  • A low Limit of Detection (LoD)
  • Wide linear range
  • It can be miniaturized (HPLC detector)
  • Can achieve ultra-high sensitivity (immuno-analysis)
• Cons
  • Narrower application range than UV-Vis (fewer compounds naturally fluoresce)
  • Sometimes less robust

Summary

• Fluorescence is a dual-wavelength process involving excitation at one wavelength and emission at a longer wavelength due to the Stokes shift
• Excitation and Emission spectra provide valuable, but different info about the fluorescent molecule
• Instrumentation includes
  • filter fluorometers (fixed wavelengths)
  • spectrofluorometers with monochromators (scanning wavelengths)
  • laser-induced fluorescence systems
• Only molecules that undergo transitions are likely to fluoresce
• Fluorescence offers great sensitivity advantage and low limits of detection, making it ideal for trace analysis
• Limited compounds naturally fluoresce, which presents the main limitation though deritivization can overcome this
• Emission intensity is proportional to analyte concentration, enabling quantitative analysis using calibration graphs
• Applications span chemical sensing, environmental monitoring, biological imaging, HPLC detection, and metal ion analysis