Distillation, Microdiffusion and Dialysis Techniques

Simple distillation is the workhorse for volatiles whose boiling points sit below water: methanol at 64.7, acetone at 56, chloroform at 61.2 and ethanol at 78.4 degrees C. Macerate the sample with distilled water, acidify lightly with tartaric acid to fix free ammonia, place it in a round-bottom flask on a sand bath, fit a Liebig condenser and collect the first 30 to 50 mL of distillate in a receiver on ice. The volatile of interest concentrates in the first fraction.

For forensic ethanol the historical Indian benchwork was the Cavett apparatus, a compact distillation train with the distillate trapped into a tube of acidified potassium dichromate. Ethanol reduced Cr(VI) (orange) to Cr(III) (blue-green), and a photometric reading converted the colour to a blood ethanol value. Cavett dominated state FSL ethanol workups through the 1970s and 1980s. The modern replacement is headspace GC-FID, but the Cavett remains documented in bench manuals at several Indian forensic science institutions.

The limitations are well known. Anything that decomposes near 100 degrees C is destroyed. Anything that azeotropes with water (ethanol forms a 95.6 percent w/w azeotrope) cannot be obtained pure in a single pass. For forensic purposes pure transfer is not the goal, an aqueous distillate clean of viscera, proteins and fat is enough for the colour test or direct injection.

1. 1. Macerate and acidify
   Weigh 50 to 100 g of viscera or take 5 to 10 mL of blood. Macerate with an equal volume of distilled water, acidify lightly with tartaric acid to suppress ammonia carry-over and to keep volatile bases protonated and non-volatile.
2. 2. Assemble the train
   Transfer to a 250 mL round-bottom flask with anti-bumping granules. Fit a Claisen head, thermometer at the side-arm and a Liebig condenser. Receiver flask sits in an ice bath to prevent re-volatilisation of low-boiling solvents.
3. 3. Heat slowly
   Use a sand bath or electric mantle. Bring the flask contents to a gentle simmer. Stop the distillation when 30 to 50 percent of the original volume has been collected, or when the side-arm thermometer reads close to 100 degrees C.
4. 4. Test the distillate
   Run the appropriate colour test on aliquots of the distillate: dichromate for ethanol, Fujiwara for chloroform, chromotropic acid for methanol after oxidation to formaldehyde, sodium nitroprusside for acetone.
5. 5. Preserve a portion
   Seal a 5 mL aliquot of the distillate in a screw-cap vial with PTFE liner, label and refrigerate at 4 degrees C for confirmatory GC headspace and for the court exhibit chain.

Steam distillation extends the principle to compounds that decompose at their own boiling point. Phenol boils at 182, cresols at 191 to 203, nicotine at 247, aniline at 184 and camphor at 209 degrees C. By bubbling live steam through the macerated sample, each co-distils with water below 100 degrees C because the combined vapour pressure reaches atmospheric well before either component reaches its own boiling point.

The Indian bench setup is a three-piece train. A separate flask of water on a Bunsen burner generates steam. A delivery tube carries the steam to the bottom of the sample flask, which holds the viscera and an acidic or alkaline buffer. A wide condenser condenses the mixed vapour into a receiver. For acidic volatiles (phenol, cresols, salicylates) the sample flask is acidified with dilute sulphuric acid and the receiver may hold dilute sodium hydroxide as an alkaline trap. For basic volatiles (nicotine, aniline, ammonia) the sample flask is alkalinised and the trap holds dilute acid.

A forensic steam distillation runs for 30 to 90 minutes. The receiver fills with a milky distillate when phenol is present, a sharp tobacco odour signals nicotine, and an aniline-bearing distillate develops the bleach-purple colour with hypochlorite. CFSL Chandigarh still routes unknown organic poisonings through a steam distillation arm in parallel with a Stas-Otto extraction, particularly when the case history points to industrial or agricultural exposure.

The Conway cell, designed by E. J. Conway in 1933 and unchanged in geometry since, is a miniaturised gas-phase extraction device that has remained in continuous use because of its simplicity and sensitivity on microlitre volumes. The unit is a flat circular glass dish, roughly 60 mm across and 10 mm deep, with an outer ring and a small central well that share a single gas phase. A ground-glass lid sealed with petroleum jelly closes the dish, and over one to three hours at 37 degrees C any volatile released in the outer ring diffuses across and is captured in the central well.

The choice of reagents is what makes the cell selective. For hydrogen cyanide the outer ring receives blood or gastric contents with 10 percent sulphuric acid as the release agent, which protonates cyanide salts and liberates HCN. The central well holds 0.1 N sodium hydroxide and traps the HCN as sodium cyanide. After incubation the central well is tested with sodium picrate (red), pyridine-pyrazolone (deep blue, the Aldridge test), or Schoenbein guaiacum-copper sulphate paper. For ethanol the outer ring holds the sample with saturated potassium carbonate and the central well holds acidified potassium dichromate, which shifts from orange to blue-green Cr(III) (the Beckman variant of the Cavett chemistry). For ammonia and volatile amines the outer ring is alkalinised with potassium hydroxide and the central well holds dilute sulphuric acid, with Nessler reagent for the final colour.

The procedural details are critical to reproducible results. The petroleum-jelly seal must be continuous, the lid pressed down evenly, and the 37 degrees C incubation must match the timetable used for the standard curve. Standard cyanide solutions of 1, 5, 10 and 25 micrograms per cell are run in parallel from a fresh NaCN stock. The central well is read at 520 nm for picrate or 620 nm for pyridine-pyrazolone. The cell quantitates down to roughly half a microgram of HCN, comfortably below the lethal blood range of 1 to 3 micrograms per millilitre.

1. 1. Charge the central well
   Pipette 1 mL of 0.1 N NaOH (for HCN), or 1 mL of 0.25 percent K2Cr2O7 in concentrated H2SO4 (for ethanol), into the central well of a clean dry Conway cell.
2. 2. Charge the outer ring
   Pipette 1 mL of sample (gastric content, blood, or aqueous extract) into one half of the outer ring, then 1 mL of the release agent (10 percent H2SO4 for cyanide, saturated K2CO3 for ethanol) into the other half, kept apart by tilting the cell.
3. 3. Seal and mix
   Smear petroleum jelly on the rim, lower the ground-glass lid and press it down. Tilt the cell to allow the sample and release agent to mix in the outer ring while the central well stays undisturbed.
4. 4. Incubate
   Place flat in an incubator at 37 degrees C for 1 hour (cyanide) to 3 hours (ethanol). Run a blank cell and a standards series in parallel under identical conditions.
5. 5. Read and confirm
   Withdraw the central well solution, develop colour with picrate or pyridine-pyrazolone for HCN, or read directly for the Cr(VI) to Cr(III) shift for ethanol. Confirm a positive HCN screen by spectrophotometry against the standards curve and by a parallel ion chromatography or GC-MS run on the residual sample.

Dialysis differs from distillation and microdiffusion in direction of analyte movement: rather than lifting a volatile into a clean phase, it allows small molecules to cross a semipermeable cellulose membrane while the bulky biological background is retained. The membrane carries a nominal molecular weight cut-off, typically 12 to 14 kDa. Albumin (66 kDa), lipoproteins, blood cells and tissue debris stay inside the bag. Drugs (almost all under 1 kDa), small peptides, metal ions and inorganic anions cross into the buffer over 24 to 48 hours of slow rocking at 4 degrees C.

The forensic use case is clean-up. Blood or homogenised viscera contaminated with lipids and proteins fouls reverse-phase HPLC columns, depresses LC-MS/MS ionisation through matrix suppression and leaves carbon residue on AAS. Dialysis against phosphate-buffered saline gives an essentially protein-free dialysate, ready for direct injection or a quick SPE step. NIMHANS Bengaluru and the AIIMS toxicology service use it as a pre-LC-MS/MS option when the analyte is small, hydrophilic and poorly recovered by liquid-liquid extraction.

Equilibrium dialysis is the related variant. A drug-spiked plasma sample sits on one side of the membrane and pure buffer on the other. After 6 to 12 hours at 37 degrees C the buffer concentration equals the free concentration in plasma, and the ratio gives the unbound fraction. For a case involving a highly protein-bound drug such as warfarin (99 percent bound) or phenytoin (90 percent bound), equilibrium dialysis distinguishes a high total concentration sequestered on albumin from a high free concentration that is actually toxic.

In practice, dialysis in an Indian FSL is straightforward. Cellulose tubing is boiled in EDTA-bicarbonate to remove sulphur and heavy metals, rinsed in deionised water, tied at one end, charged with 5 to 10 mL of sample, tied at the other end, and suspended in 200 to 500 mL of buffer on a slow rocker. The dialysate is concentrated by lyophilisation or rotary evaporation and submitted to the instrument.

The three methods occupy non-overlapping niches. The three methods occupy non-overlapping niches. Distillation transfers and concentrates the analyte. Microdiffusion does the same on a microscale in a sealed gas phase, sacrificing volume for selectivity. Dialysis filters out the matrix while leaving the analyte in dilute solution. They are usually chained: a steam distillation followed by a Conway confirmation, or a dialysis clean-up followed by HPLC quantitation.

Isolation is half the workflow, identification is the other half. For hydrogen cyanide the classical bench tests are three. Sodium picrate paper turns from yellow to brick red in alkaline cyanide, as picric acid is reduced to isopurpurate. Pyridine-pyrazolone gives a deep blue colour via a cyanogen chloride intermediate and is more sensitive than picrate. The Schoenbein test uses guaiacum tincture and dilute copper sulphate, turning blue with HCN. All three are presumptive, and the confirmatory in a modern Indian FSL is ion chromatography or GC-MS with cyanide derivatised as the pentafluorobenzyl bromide adduct.

For ethanol the dichromate oxidation gives the colour shift used in Cavett and Beckman. The modern routine is ADH enzymatic assay for clinical purposes and headspace GC-FID for forensic blood ethanol with n-propanol as internal standard. Headspace GC remains the only method admitted as proof in most Indian Motor Vehicles Act prosecutions.

For ammonia the central well after a Conway run is read with Nessler reagent (alkaline mercuric potassium iodide), producing an orange-brown precipitate at 425 nm. The same chemistry handles blood urea estimation and is one of the oldest reagents on the Indian FSL bench.

The Conway cell remains standard on every state FSL cyanide protocol. Cyanide cases in India fall into three patterns. Industrial exposure in electroplating units, gold and silver refining, and small-scale jewellery workshops in Delhi, Mumbai, Kolkata and Rajkot produces occupational HCN poisoning that AIIMS, KEM Mumbai and SSKM Kolkata document each year. Suicidal cyanide ingestion from stolen industrial NaCN appears intermittently. Mass cyanide poisoning is rare, with the Sahjanand factory case in Saharanpur in the 1990s as a published example. In every pattern the Conway cell delivers a same-day qualitative answer.

Cavett-style distillation has largely been retired in favour of headspace GC, but the older apparatus still sits on the bench at FSL Sector 14 Madhuban and at several Maharashtra and Gujarat units, used for teaching and as backup. CFSL Chandigarh continues to run steam distillation for unknown-organic workups when the case history points to phenol, cresol or nicotine.

Dialysis is a quieter presence. NIMHANS Bengaluru uses dialysis tubing for pre-LC-MS/MS clean-up of plasma, and the AIIMS poison information centre cites equilibrium dialysis as the reference method when free-fraction questions arise in phenytoin or warfarin overdose. Most state FSL units have moved to SPE and protein precipitation, with dialysis reserved for small hydrophilic analytes where SPE recovery is poor.