Chromatographic Techniques in Forensic Science: TLC, GLC, HPLC and HPTLC

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.

For UGC-NET, the highest-yield pairings are partition (GLC, RP-HPLC) and adsorption (TLC). Ion exchange and size exclusion are usually one-line distractors.

The other concept NTA likes is plate theory. A column behaves as if it were a stack of equilibrium plates; the more plates, the sharper the peaks. The Height Equivalent to a Theoretical Plate (HETP) is the column length divided by the plate count. Lower HETP equals higher efficiency. The van Deemter equation links HETP to flow rate through three terms (eddy diffusion, longitudinal diffusion, mass-transfer resistance), but for Paper 2 the formula itself rarely shows up; the conceptual link between plates, efficiency and peak sharpness does.

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.

R_f = (distance travelled by analyte) / (distance travelled by solvent front)

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
• Ink and dye comparison at GEQD Shimla: separating colourants in questioned ink samples and matching to a suspect pen
• Explosive residue screening: nitroaromatics and nitrate esters
• Pesticide residue work in toxicology casework on viscera samples

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.

For NET, three pairings carry the most marks: FID for hydrocarbons and fire debris, ECD for halogens and explosive nitro-compounds, and MSD (GC-MS) for confirmatory drug identification. The Bharatiya Sakshya Adhiniyam 2023 admissibility regime expects confirmed identifications, which in practice means GC-MS or LC-MS, not a single TLC spot.

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.

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.

Common HPLC detectors:

• UV-visible absorbance (single or variable wavelength), the workhorse for most drugs
• Diode Array Detector (DAD or PDA), gives a full UV spectrum at every time point and is used for peak-purity checks
• Fluorescence Detector (FLD), high sensitivity for fluorescent analytes like quinine, certain dyes and PAHs
• Electrochemical Detector (ECD, different from the GC ECD), for catecholamines and certain neurotransmitters
• Mass Selective Detector (LC-MS or LC-MS/MS), the modern courtroom standard for drug and toxin confirmation

UPLC / UHPLC is the high-pressure (over 1000 bar) sub-2-micrometre-particle variant of HPLC. It gives faster runs and sharper peaks; the principle is identical.

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.

Casework workflow you should be able to describe in one paragraph: seized suspected narcotic powder arrives at the SFSL drug section. The analyst runs a colour test (Marquis, Mecke or Mandelin) for a first indication, then a TLC plate against reference standards to narrow the identification, then HPTLC for semi-quantitative confirmation, and finally GC-MS or LC-MS for the courtroom-grade confirmed identification with quantitation. The same pipeline applies to toxicology (blood, urine, viscera) and to ink and dye comparisons at GEQD Shimla, with the chromatographic technique chosen for the matrix.