What is a Conway microdiffusion cell used for in forensic toxicology?

It is a small two-well glass dish with a ground-glass lid. The sample plus a releasing reagent is placed in the outer well, and a trapping reagent is placed in the inner well. The lid is greased and sealed and the cell is left at room temperature. Any volatile poison diffuses across the air gap and is captured in the inner well for colour or titrimetric measurement. The classical analytes are carbon monoxide (released from blood by potassium ferricyanide, trapped in PdCl2), hydrogen cyanide (released by H2SO4, trapped in NaOH and developed by König or picrate reaction), and the lower alcohols. The big advantage over steam distillation is that only 0.5 to 1 mL of sample is needed.

Which poisons are recovered by steam distillation from viscera?

Volatile poisons that co-distil with water below their pure boiling points. The standard list is ethanol, methanol, phenol, chloroform, nitrobenzene and formaldehyde. With concentrated sulphuric acid added to the distillation flask to liberate HCN from cyanide salts, steam distillation also recovers cyanide. The distillate is condensed and collected for colour tests, GC-FID or GC-MS confirmation. Steam distillation is not used for non-volatile alkaloids (Stas-Otto for those) or for metals (dry ashing or wet digestion for those).

Extraction, Isolation and Clean-up of Poisons from Viscera

UGC-NET Paper 2 Unit IV notes on viscera workup. Stas-Otto, ammonium sulphate, steam distillation, Conway microdiffusion, modern SPE, SPME and QuEChERS, with the Indian SOP.

Last updated: 18 May 2026

Viscera workup closes UGC-NET Forensic Science Unit IV, and it is the bullet that binds every other toxicology question together. Whatever the suspected poison, the analyst still has to get it out of liver, kidney, stomach and blood before any colour test, GC-MS run or AAS quantification can fire. NTA tests two layers: the classical procedures (Stas-Otto, ammonium sulphate, steam distillation, Conway microdiffusion) and the modern sample-prep family (SPE, SPME, ASE, QuEChERS, SLE) that has displaced most of them.

Learn the standard Indian viscera kit, the four Stas-Otto fractions, the volatiles that go through steam distillation, what a Conway cell is for, and the SPE / QuEChERS sequence. The book chapters give the bench-level recipes; this NET topic gives the memorisable framework.

Key terms

Viscera: The internal organs collected at autopsy for chemical analysis in suspected poisoning. The Indian standard set is stomach with contents, small intestine, liver and one kidney, packed in two glass jars.
Stas-Otto procedure: Classical 1851/1856 acid-base liquid-liquid fractionation. Acidify with tartaric acid and extract acidic and neutral organics into ether, then alkalise and extract bases into chloroform. Yields four fractions used to separate barbiturates, phenols, alkaloids and volatiles.
Conway microdiffusion cell: Two-well glass dish. Sample plus releasing reagent in the outer well, trapping reagent in the inner well, sealed lid. Volatile poison (HCN, CO, alcohols) diffuses across the air gap and is captured for colorimetry.
Steam distillation: Co-distillation of volatile poisons with water vapour at temperatures below their pure boiling points. Used for ethanol, methanol, phenol, chloroform, nitrobenzene, formaldehyde and (with H2SO4) cyanide.
SPE: Solid-phase extraction. Sample is loaded onto a packed sorbent cartridge (C18, mixed-mode MCX/MAX, polymeric Oasis HLB), washed and selectively eluted. The dominant modern clean-up step in Indian SFSL toxicology.
SPME: Solid-phase microextraction. A polymer-coated fibre is exposed to headspace or sample, analytes adsorb, the fibre is then thermally desorbed in a GC injector. Ideal for volatiles, no solvent needed.
QuEChERS: Quick, Easy, Cheap, Effective, Rugged, Safe. Acetonitrile partitioning with MgSO4 and NaCl, followed by dispersive SPE clean-up using PSA and C18. Originally a pesticide-residue method, now used broadly in forensic toxicology.
ASE: Accelerated solvent extraction (Dionex ASE). Pressurised solvent at elevated temperature pushes the analyte off the tissue matrix in minutes, replacing overnight Soxhlet.
SLE: Supported liquid extraction. Diatomaceous earth columns absorb the aqueous sample as a thin film and a single pass of organic solvent elutes the analyte. Single-pass replacement for shake-flask liquid-liquid.

Why viscera matter and how they reach the lab

The post-mortem matrix where poisons end up, and the BNSS chain that delivers it.

In a suspected poisoning death, the autopsy surgeon collects viscera for chemical examination. Liver concentrates lipophilic drugs and metals, kidney holds water-soluble metabolites awaiting excretion, stomach and intestine retain unabsorbed parent compound, and blood gives the circulating concentration at the moment of death.

Section 176 of the Bharatiya Nagarik Suraksha Sanhita 2023 (BNSS) mandates an inquest in unnatural deaths and requires viscera preservation in suspected poisoning. The collection is logged on Form 21, sealed with the police seal in cloth and wax, and dispatched under the mortuary register and the chain of custody entry.

The standard kit: Jar 1 holds about 200 g of stomach with its contents, the upper small intestine and about 30 mL of gastric fluid. Jar 2 holds about half of the liver and one whole kidney. Preservative is saturated sodium chloride in both jars. Blood (about 10 mL) goes into a separate tube of sodium fluoride and potassium oxalate; NaF blocks bacterial breakdown of ethanol, critical for the analysis of ethyl alcohol in beverages, blood and breath.

Two exceptions trip MCQ candidates up. For suspected cyanide, NaCl interferes with the colour tests, so viscera go in preservative-free and refrigerated. For suspected ethanol, NaF alone is the preservative, since alcohol partitions out of saturated NaCl. The book chapter on post-mortem and viscera collection in poisoning covers the full Form 21 workflow.

Classical extraction: the Stas-Otto and ammonium sulphate methods

Two acid-base steps, four fractions, the framework every Indian forensic textbook still teaches.

The single most tested classical procedure is the Stas-Otto fractionation, published by Jean Servais Stas in 1851 (for the Bocarmé nicotine case) and refined by Friedrich Otto in 1856. The principle is acid-base liquid-liquid partition: weak acids and bases swap between the aqueous and an organic phase depending on pH, so adjusting pH pulls different classes into different fractions.

The viscera are minced, mixed with rectified spirit and a little tartaric acid, warmed to denature proteins, filtered, and the filtrate is evaporated to a syrup. The syrup is taken up in water, acidified with tartaric acid, and shaken with diethyl ether. The ether layer carries acidic and neutral organics (Fraction A: barbiturates, salicylates, phenols). The aqueous layer is then alkalised with sodium bicarbonate or sodium carbonate and shaken with chloroform (often chloroform plus isopropanol), which carries the bases (Fraction B: strychnine, morphine, atropine, nicotine, cocaine, quinine). Volatile poisons are recovered separately by distillation (Fraction C); the protein residue is digested for metals (Fraction D).

The ammonium sulphate precipitation method is the protein-stripping cousin. Saturated ammonium sulphate is added to the homogenate to salt out proteins; the supernatant is filtered, and the small-molecule poisons are extracted with an organic solvent at the appropriate pH. Less laborious than full Stas-Otto, useful when viscera are heavily decomposed.

Both methods are slow and solvent-heavy, which is why Indian SFSLs have moved most casework to SPE. The classical procedures still get tested because every Indian textbook (Modi, Parikh, Reddy, Nandy) leans on them, and the chemistry teaches the underlying acid-base reasoning. The book chapter on classical extraction: Stas-Otto and ammonium sulphate walks through the bench recipes.

Distillation, microdiffusion and dialysis

Three techniques tailored to volatile and small-molecule poisons.

When the suspected poison is volatile, Stas-Otto is the wrong tool. Three older techniques hold their place.

Steam distillation drives steam through the macerated viscera in an acidified flask; the volatile analyte co-distils with water below its pure boiling point. The distillate is condensed and analysed by colour test, GC-FID or GC-MS. It is the standard recovery method for ethanol, methanol, phenol, chloroform, nitrobenzene and formaldehyde, and (with concentrated H2SO4 added to liberate HCN from cyanide salts) for cyanide.

The Conway microdiffusion cell is a small two-well glass dish with a lid. Sample plus releasing reagent in the outer well, trapping reagent in the inner well, lid sealed, cell left at room temperature. The volatile diffuses across the air gap and is captured for colour or titrimetric measurement. Used routinely for carbon monoxide (released by K3Fe(CN)6, trapped in PdCl2), hydrogen cyanide (released by H2SO4, trapped in NaOH, developed by König or picrate), and alcohols (released by warming, trapped in K2Cr2O7). Big advantage over distillation: 0.5 to 1 mL of sample is enough.

Dialysis moves small-molecule poisons across a semipermeable membrane while keeping proteins behind. Slow but gives a clean aqueous extract; used today mainly when SPE cartridges clog on greasy tissue.

For metals the residue from any of these workups goes through dry ashing or wet digestion before AAS or ICP-MS quantification. The book chapter on distillation, microdiffusion and dialysis techniques covers the full chemistry.

Modern clean-up: SPE, SPME, ASE, QuEChERS and SLE

The five-letter acronyms that have displaced most liquid-liquid work in Indian forensic toxicology.

The modern Indian SFSL toxicology bench runs on cartridges and dispersive sorbents, not separating funnels. Five techniques carry almost all current casework.

Solid-phase extraction (SPE) is the workhorse. The aqueous extract is loaded onto a packed sorbent cartridge, the matrix is washed off, and the analyte is eluted with a small volume of selective solvent. Dominant sorbents are C18 (reversed-phase, general drugs), mixed-mode MCX (basic drugs: amphetamines, opiates, benzodiazepines), mixed-mode MAX (acidic drugs: barbiturates) and polymeric Oasis HLB (broad-spectrum default for unknown screens).

Solid-phase microextraction (SPME) exposes a polymer-coated fused-silica fibre to the sample or its headspace; analytes adsorb, the fibre is then thermally desorbed in a GC injector. Solvent-free, ideal for volatiles (alcohols, accelerants) off blood or tissue homogenates.

Accelerated solvent extraction (ASE / Dionex) pressurises a solvent above its atmospheric boiling point at 80 to 200 degrees Celsius, pushing the analyte off the matrix in minutes instead of overnight Soxhlet. Used for pesticide residues in fatty tissue.

QuEChERS ("quick, easy, cheap, effective, rugged, safe") is the pesticide-residue method that has spread into wider toxicology. Shake tissue with acetonitrile, salt out with anhydrous MgSO4 and NaCl to drive analytes into the organic layer, then clean with dispersive SPE using PSA (removes acidic interferences and fatty acids) and C18 (removes lipids). Single-tube, minimal-solvent.

Supported liquid extraction (SLE) uses diatomaceous earth columns that hold the aqueous sample as a thin film while organic solvent flows through. Single-pass replacement for shake-flask liquid-liquid, no emulsions. The book chapter on

Bench workflow and quality control at the Indian SFSL

From visual inspection to confirmation, with the controls a court will ask about.

A realistic SFSL viscera workup runs five steps in order. Visual and olfactory exam first: kerosene smell, garlic smell of phosphide, bitter-almond smell of cyanide, and the colour of stomach contents (yellow for picric acid, blue-green for copper sulphate, red for nitric acid) are recorded before any reagent touches the sample.

Second, colour and spot screens narrow the field (Reinsch for heavy metals, Marquis and Mecke for alkaloids, Trinder for salicylates), and an immunoassay panel (ELISA, lateral-flow) gives a rapid yes/no across opiates, amphetamines, benzodiazepines, cannabinoids and cocaine. Third, if any screen fires, the sample is extracted using the appropriate method: Stas-Otto for an unknown alkaloid, steam distillation for a suspected volatile, SPE for a known drug class, QuEChERS for pesticides, dry ashing for metals.

Fourth, the extract goes on a confirmatory instrument: GC-MS for general drugs and pesticides, LC-MS-MS for thermally labile and polar drugs, AAS or ICP-MS for metals. Fifth, the analyst signs and seals the report.

Quality control is what wins the case in court. Every run carries a blank viscera control (extracted from known clean tissue) to flag contamination, spiked recovery controls to prove extraction efficiency, and deuterated or 13C internal standards added at the extraction step (not at injection) to correct for losses. The Indian institutional anchors NTA likes to test are the DFSS Forensic Toxicology SOP (2020 / 2022), CFSL Hyderabad and CFSL Chandigarh, AIIMS Forensic Medicine and Toxicology, and the state SFSL toxicology divisions. The book chapter on biological, non-biological and viscera matrices

MCQ takeaways

Stas-Otto = acidify with tartaric acid, extract acidic/neutral into ether, alkalise with NaHCO3/Na2CO3, extract bases into chloroform. Four fractions.Preservative for viscera is saturated NaCl, except preservative-free for cyanide and NaF for ethanol (blood: NaF + potassium oxalate, about 10 mL).Conway microdiffusion cell = two-well glass dish for CO, HCN and alcohols. Outer well: sample + releasing reagent. Inner well: trapping reagent.Steam distillation recovers ethanol, methanol, phenol, chloroform, nitrobenzene, formaldehyde, and with H2SO4, cyanide.QuEChERS = Quick, Easy, Cheap, Effective, Rugged, Safe. Acetonitrile + MgSO4 + NaCl, then dispersive SPE with PSA and C18.SPE sorbents: C18 = general reversed-phase. MCX = basic drugs. MAX = acidic drugs. Oasis HLB = broad-spectrum starting cartridge.SPME = polymer-coated fibre, headspace or direct, thermal desorption in GC injector. Solvent-free.Viscera kit: Jar 1 = stomach + small intestine + ~30 mL gastric contents (~200 g). Jar 2 = half-liver + one kidney.BNSS Section 176 + Form 21 govern viscera collection in suspected poisoning in India.Internal standard is added at the extraction step (not the injection step) so it corrects for extraction losses, not just injection variance.

What is the standard Indian viscera kit for a suspected poisoning autopsy?

Two glass jars. Jar 1 holds about 200 g of stomach with its contents, the upper small intestine, and roughly 30 mL of gastric fluid. Jar 2 holds about half of the liver and one whole kidney. Both jars are topped up with saturated sodium chloride solution as preservative, sealed with the police seal in cloth and wax, and dispatched under Form 21 of the medico-legal forms to the SFSL. Blood (about 10 mL) goes separately into a sodium fluoride and potassium oxalate tube. For suspected cyanide the jars are preservative-free; for suspected ethanol the blood preservative is NaF alone.

Why is the Stas-Otto procedure still taught when most Indian SFSLs run SPE now?

Two reasons. First, the chemistry teaches acid-base partitioning of weak acids and weak bases between aqueous and organic phases, which is the same principle underlying mixed-mode SPE (MCX and MAX cartridges). If you understand Stas-Otto you understand why an opiate sticks to MCX at pH 3 and elutes in methanolic ammonia. Second, NTA tests it explicitly: the four fractions, the reagents and the historical attribution (Stas 1851, Otto 1856) are common MCQ stems. The bench procedure has been displaced; the conceptual framework has not.

What does QuEChERS stand for and what is its workflow?

QuEChERS stands for Quick, Easy, Cheap, Effective, Rugged, Safe. The workflow is a single-tube method: the homogenised tissue or food sample is shaken with acetonitrile, then anhydrous MgSO4 and NaCl are added to drive the analytes into the organic layer (salt-out). The supernatant is cleaned with dispersive SPE using PSA (primary secondary amine, removes acidic interferences and fatty acids) and C18 (removes lipids). The cleaned extract is injected on GC-MS or LC-MS-MS. Originally developed for pesticide residues in fruit and vegetables, QuEChERS is now used for many forensic toxicology applications including viscera workup.

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