Spectrophotometry for Forensic Science: UV, Visible, IR, Raman, AAS and AES

UV-Visible spans roughly 200 to 800 nm: ultraviolet (200 to 400 nm) and visible (400 to 800 nm). Molecules with chromophores (C=C, C=O, aromatic systems) absorb in this region, and the absorbance follows the Beer-Lambert law:

A = ε c l, where A is absorbance (unitless), ε is molar absorptivity (L mol⁻¹ cm⁻¹), c is concentration (mol L⁻¹) and l is path length (cm, almost always 1 cm with a standard cuvette).

A typical instrument has two sources (a deuterium lamp for UV, a tungsten-halogen lamp for visible), a monochromator (grating or prism), a sample compartment with quartz cuvettes (glass cuvettes block UV), and a photodiode or PMT detector. Double-beam designs reference a blank in parallel to cancel lamp drift.

Forensic uses in Indian casework:

• Questioned-document inks screened by UV-Vis absorbance and reflectance, especially at GEQD Shimla.
• Drug screening: many controlled substances and their colour-test products absorb in UV (e.g. cocaine after derivatisation, LSD's indole ring around 320 nm).
• Bloodstain age estimation using the shift in haemoglobin / methaemoglobin / hemichrome bands.

IR spectroscopy probes molecular vibrations: stretches, bends, twists. A bond absorbs IR only if the vibration changes the molecular dipole moment (the selection rule that separates IR-active from Raman-active modes).

The mid-IR range, 4000 to 400 cm⁻¹, is the practical forensic window. It splits into two halves:

• Functional-group region (4000 to 1500 cm⁻¹): broad O-H around 3200 to 3600, sharp N-H around 3300, C-H around 2900, C=O around 1700, aromatic C=C around 1600. Tells you what groups are present.
• FTIR fingerprint region (1500 to 400 cm⁻¹): dense, sample-specific pattern. Used to identify a substance by library overlay, not by individual peak assignment.

Modern instruments are FTIR (Fourier-transform IR) with a Michelson interferometer, a Globar or Nernst glower source, a KBr beamsplitter and a DTGS or MCT detector. ATR-FTIR uses a diamond or ZnSe crystal as a total-internal-reflection element so you can press a tablet, fibre or paint chip directly on it with no sample preparation. ATR has effectively replaced KBr-pellet IR for routine casework.

Forensic uses in Indian casework:

• Drug identification at CFSL Hyderabad and state SFSLs. FTIR libraries identify methamphetamine, MDMA, ketamine, mephedrone hits in seconds.
• Polymer and fibre analysis for paint chips, hit-and-run cases, tape and adhesives.
• Counterfeit ink and toner comparison in questioned documents.

Raman works on inelastic scattering of monochromatic light, usually from a 532 nm, 633 nm, 785 nm or 1064 nm laser. Most photons scatter elastically (Rayleigh scattering, same energy). A tiny fraction (about one in 10⁷) exchange energy with a molecular vibration. The shifted photon at lower energy is the Stokes line; at higher energy, the anti-Stokes line. Both sit symmetric about the laser line, and the shift, measured in cm⁻¹, equals the vibrational frequency.

The Raman selection rule is the mirror image of IR: a vibration is Raman-active if it changes the molecular polarisability. Symmetric stretches (C=C, S-S, C-S, ring breathing modes) are strong in Raman and often weak in IR, which is why the two methods are presented as complementary.

Forensic advantages over IR:

• Water-compatible. Water is a very weak Raman scatterer but a very strong IR absorber, so Raman runs aqueous samples cleanly.
• Non-destructive and through-container. A handheld 785 nm Raman can identify a powder through a sealed glass vial or plastic bag, valuable for narcotic field screening at Indian airports and customs.
• Pigment and ink identification without removing material from a questioned document.

Limitations: fluorescence from the matrix can swamp the Raman signal (move to longer-wavelength lasers like 785 or 1064 nm to suppress it), and the signal is weak so integration times are longer.

AAS measures the absorbance of light by free ground-state atoms in the gas phase. The sample is atomised, an AAS hollow cathode lamp made of the analyte element emits its characteristic resonance lines, the free atoms absorb at exactly those lines, and the loss in intensity gives concentration via a Beer-Lambert-style calibration.

Two atomisation routes dominate:

• Flame AAS (FAAS): air-acetylene flame around 2300 °C for routine metals (Cu, Zn, Fe, Pb), nitrous oxide-acetylene around 2900 °C for refractory metals (Al, Si, V). ppm detection limits.
• Graphite Furnace AAS (GFAAS): electrothermal heating of a graphite tube up to 3000 °C. ppb to sub-ppb detection limits. Slower, more interference, but mandatory for trace toxicology.

Key components: hollow cathode lamp (one per element, or multi-element), monochromator (to isolate the resonance line), and a PMT detector. Background correction (deuterium lamp or Zeeman) corrects for non-atomic absorbance.

Forensic uses in Indian casework:

• Heavy-metal toxicology at CFSL chemistry divisions and state FSLs. Arsenic, mercury, lead, cadmium in viscera, blood, hair, well-water and post-mortem tissue.
• Gunshot residue (GSR) for Pb, Sb, Ba on swabs from suspects' hands, complementary to SEM-EDX.
• Soil and paint comparison when trace-metal profiles distinguish two visually similar samples.

AES inverts the AAS idea. Atomise the sample, push the atoms into excited electronic states using a high-temperature source, and measure the light they emit when they relax. Because each element has its own line set, you can analyse many elements simultaneously, and you do not need an element-specific lamp.

The excitation source defines the technique:

• Flame emission (FES): the old flame photometer. Suitable for easily excited alkali metals (Na, K, Li, Ca) at low temperatures. Still used in clinical-style labs.
• Arc and spark AES: classical solid-sampling for metal-fragment comparison.
• ICP-OES (Inductively Coupled Plasma Optical Emission): argon plasma at 6000 to 10000 K. Simultaneous multi-element analysis, ppb detection limits, wide linear range.
• ICP-MS (Inductively Coupled Plasma Mass Spectrometry): ICP for atomisation plus a mass analyser for detection. ppt detection limits, isotope ratios for sample matching.

Forensic uses in Indian casework:

• Glass fragment comparison by ICP-MS trace-element profile at CFSL Pune physics division. Two glass shards from the same windowpane share a Sr, Hf, Zr, Pb signature.
• Soil profiling to link a suspect's shoes to a scene.
• Counterfeit-currency ink and paper elemental fingerprinting.
• Arsenic and selenium speciation in environmental and toxicology samples when HPLC is coupled to ICP-MS.

A typical exhibit-handling pipeline at an Indian CFSL or SFSL uses these instruments in sequence rather than in isolation. A seized white powder, for example, first goes to ATR-FTIR for a non-destructive identification against the lab's drug library. If FTIR is ambiguous (mixture, low purity, novel psychoactive substance), the analyst escalates to Raman for an orthogonal vibrational confirmation, then to GC-MS or LC-MS for the molecular-weight evidence. UV-Visible sits in the chemistry workflow as a quantification tool once the identity is known.

For the trace and toxicology divisions, the split is cleaner. AAS runs metals one at a time on the chemistry division benches at CFSLs (Hyderabad, Chandigarh, Pune, Kolkata) and at every state FSL, because it is cheaper to own and operate than ICP. ICP-OES and ICP-MS sit in the higher-capacity labs and at NFSU Gandhinagar for trace-element profiling, glass and soil comparison, and isotope-ratio work that AAS cannot do.

The reason NTA bundles all six techniques into a single Unit II bullet is that an Indian forensic analyst is expected to choose between them quickly: what is the analyte, what is the matrix, how much sample do I have, and what is the courtroom evidentiary value of the result. That choice is the conceptual core of this syllabus point.