Chromatographic Techniques in Forensic Science: TLC, GLC, HPLC and HPTLC
UGC-NET Paper 2 Unit II notes on chromatography. TLC, GLC, HPLC and HPTLC principles, detectors, Rf values and Indian forensic applications. With PYQ pointers and MCQ takeaways.
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Chromatography is the separation workhorse of every Indian forensic laboratory. Unit II of UGC-NET Forensic Science (subject code 82) groups four techniques under one syllabus bullet: thin layer chromatography (TLC), gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC) and high-performance thin layer chromatography (HPTLC). The bullet is consistently tested because each technique has a clean set of one-line answers (mobile phase, detector, typical sample) that suit MCQs.
Treat this topic as a comparison drill. NTA rarely asks you to derive plate theory from scratch; it asks you to match the technique to the sample, the detector to the analyte, or the Rf calculation to a TLC plate diagram. Learn the four-way comparison table first, then the detector-to-analyte pairings (FID for organics, ECD for halogens, MSD for confirmation), then the Indian CFSL application examples.
- Stationary phase
- The fixed phase across which the sample moves. Silica gel on TLC/HPTLC plates, a packed or capillary column coating in GLC, a bonded silica packing in HPLC.
- Mobile phase
- The phase that carries the analyte through the stationary phase. A solvent system in TLC and HPLC, an inert carrier gas (nitrogen, helium, hydrogen) in GLC.
- Rf value
- Retention factor in planar chromatography. Rf equals the distance travelled by the analyte divided by the distance travelled by the solvent front. Always between 0 and 1.
- Partition vs adsorption
- Partition chromatography separates by differential solubility between two liquid phases (GLC, reversed-phase HPLC). Adsorption chromatography separates by surface binding to a solid (classical TLC on silica).
- Isocratic vs gradient elution
- Isocratic uses a fixed mobile-phase composition throughout the run. Gradient progressively changes the composition (typically increasing organic strength) to elute strongly retained analytes.
- FID
- Flame Ionisation Detector. GLC detector for organic compounds with C-H bonds. High sensitivity, near-universal for hydrocarbons, destructive.
- ECD
- Electron Capture Detector. GLC detector selective for halogenated and other electron-affinic compounds. Workhorse for pesticide and explosive residue analysis.
- MSD
- Mass Selective Detector. GC-MS or LC-MS detector that identifies analytes by mass spectrum. The confirmation standard for drug and toxicology casework.
- Plate theory
- Model that treats a column as a series of theoretical plates. The Height Equivalent to a Theoretical Plate (HETP) measures column efficiency; lower HETP means better separation.
Principles of chromatography
Four separation mechanisms, one underlying idea.
Chromatography fundamentals all rest on the same idea: a mixture is separated by repeatedly distributing the analytes between two phases, a stationary phase that does not move, and a mobile phase that does. Analytes that interact more strongly with the stationary phase move slowly; analytes that prefer the mobile phase move quickly. The differential migration produces separation.
NTA expects you to recognise the four classical separation mechanisms.
| Mechanism | Basis of separation | Typical technique |
|---|---|---|
| Adsorption | Surface binding of analyte to a solid stationary phase (silica, alumina) | Classical TLC, normal-phase HPLC |
| Partition | Differential solubility between two liquid phases, or between a gas and a liquid film | GLC, reversed-phase HPLC, paper chromatography |
| Ion exchange | Electrostatic attraction between charged analytes and an oppositely charged resin | Amino-acid and inorganic-ion analysis |
| Size exclusion | Separation by molecular size through a porous gel; large molecules elute first | Gel permeation chromatography for polymers and proteins |
TLC and HPTLC
Cheap, fast, qualitative, and the first screen in every Indian drug case.
Thin Layer Chromatography uses a thin layer (typically 0.25 mm) of silica gel or alumina coated on a glass, aluminium or plastic plate. The sample is spotted near the bottom edge, the plate is placed in a developing chamber with the solvent system, and capillary action draws the solvent up through the layer. Components separate by adsorption (or partition, depending on the plate chemistry) and are visualised after drying.
The single most-tested formula in this section is the retention factor.
Rf is always between 0 and 1, dimensionless, and reproducible only when the plate, solvent system, chamber saturation and temperature are held constant.
Visualisation methods to memorise:
- UV fluorescence at 254 nm and 365 nm (most plates contain a fluorescent indicator that quenches under UV where the analyte sits)
- Iodine vapour (general-purpose, reversible staining for organics)
- Ninhydrin spray (amino acids, purple-pink spots)
- Dragendorff reagent (alkaloids, orange-brown spots, used for opium, cocaine, strychnine screening)
- Marquis, Mecke and Mandelin reagents (colour-test sprays for amphetamines, opiates and other controlled substances)
HPLC and HPTLC sit at the modern, instrumented end of this family. HPTLC is the instrumented version of TLC. The plates use finer particle silica (around 5 micrometres versus 10 to 12 for TLC), giving sharper spots and better resolution. Application is by automated band spotter, development happens in a controlled chamber, and detection uses a densitometer that scans the plate at chosen wavelengths to produce quantitative peaks. HPTLC is faster than HPLC for screening, runs many samples in parallel on one plate, and produces a permanent visual record, which is why CFSLs use it for drug screening and ink comparison.
Indian forensic uses to remember:
- Drug screening at CFSL chemistry divisions: cannabis (THC, CBN, CBD), opiates, amphetamines, benzodiazepines
GLC: gas-liquid chromatography
Carrier gas plus a liquid-coated column plus a detector tuned to the analyte.
Gas-Liquid Chromatography (GLC) and detectors is the older name UGC still uses; the modern term is simply gas chromatography (GC). The sample is vaporised in a heated injector, swept through a column by an inert carrier gas, and detected as it elutes. Only volatile, thermally stable analytes are suitable, which is the single biggest scope rule for MCQs: if the analyte degrades on heating (proteins, sugars, most pharmaceuticals as free bases), GLC is wrong unless the analyte is derivatised first.
Columns: modern forensic labs use fused-silica capillary columns (typically 15 to 60 metres long, 0.25 mm internal diameter) coated with a thin film of stationary phase (commonly 5 percent phenyl-methylpolysiloxane for general use, polyethylene glycol for polar analytes). Older packed columns (2 to 3 metres, glass or steel, with a liquid phase coated on a solid support) still appear in textbook diagrams.
Carrier gases: nitrogen (cheapest, slower), helium (standard for most labs), hydrogen (fastest, requires safety controls). NTA likes the order: helium is the most common choice in Indian forensic labs.
Detectors: this is the highest-yield sub-topic. Learn the pairings.
| Detector | Selectivity | Typical forensic analyte |
|---|---|---|
| FID (Flame Ionisation) | Organic compounds with C-H bonds; near-universal for hydrocarbons | Petrol and diesel in fire-debris, alcohols, general drug screening |
| ECD (Electron Capture) |
HPLC: high-performance liquid chromatography
High-pressure pump plus a packed column plus a selective detector.
HPLC pushes a liquid mobile phase at high pressure (typically 50 to 400 bar) through a stainless-steel column packed with small (3 to 5 micrometre) porous silica particles. The high pressure plus small particles is what gives HPLC its resolution. Unlike GLC, the analyte does not need to be volatile or thermally stable, which extends HPLC to most drugs, dyes, explosives and biomolecules.
Normal-phase vs reversed-phase. This pairing is the single most-tested HPLC distinction in Paper 2.
| Mode | Stationary phase | Mobile phase | Eluting order |
|---|---|---|---|
| Normal-phase HPLC | Polar (bare silica, cyano, amino) | Non-polar (hexane, dichloromethane) | Non-polar analytes elute first |
| Reversed-phase HPLC | Non-polar (C18, C8 bonded silica) | Polar (water, methanol, acetonitrile) | Polar analytes elute first |
Reversed-phase HPLC (often written RP-HPLC, C18) is the default in forensic casework because most analytes of interest (drugs, toxins, dyes) dissolve well in water-methanol or water-acetonitrile mobile phases.
Isocratic vs gradient elution. Isocratic holds the mobile phase composition fixed; gradient ramps the organic content (for RP-HPLC, typically from low to high methanol or acetonitrile percentage) during the run. Gradient elution is needed when analytes have a wide range of polarities, because isocratic conditions either elute the polar ones too quickly or trap the non-polar ones on the column.
Forensic applications and the four-way comparison
What gets used for what in an Indian CFSL workflow.
Each technique has a niche in the casework pipeline. Most Indian CFSLs and SFSLs run TLC or HPTLC as a fast screen, then move the positive samples to GC-MS or LC-MS for confirmation. The court wants the confirmed result; the screening result is investigative.
| Technique | Mobile phase | Sample type | Sensitivity | Typical Indian forensic application |
|---|---|---|---|---|
| TLC | Liquid solvent (ascending capillary action) | Qualitative screening of volatiles, non-volatiles, drugs, inks | Microgram | First-pass drug screening at SFSL chemistry; ink comparison at GEQD Shimla |
| HPTLC | Liquid solvent (controlled chamber, densitometer scan) | Semi-quantitative screening of drugs, dyes, herbal samples | Nanogram to high microgram | Cannabis profiling and explosive screen at CFSL chemistry divisions |
| GLC (GC / GC-MS) | Inert carrier gas (helium, nitrogen, hydrogen) | Volatile and semi-volatile organics (drugs after derivatisation, explosives, fire-debris hydrocarbons) |