Toxicological Laboratory Techniques: TLC, GLC, AAS, HPLC, and Breath Alcohol Analyzer

1. Thin-Layer Chromatography (TLC)

• A concentrated sample extract and known standard drug solutions are applied as small spots at the baseline (origin) at the bottom of the TLC plate.
• The plate is placed in a closed developing chamber where the solvent (mobile phase) travels upward by capillary action, carrying compounds with it.
• Compounds separate based on their different affinities for the stationary vs. mobile phases - those more attracted to the mobile phase travel further.
• After development, the plate is sprayed or dipped in specific reagents to produce visible colored spots.

Rf = Distance traveled by compound / Distance traveled by solvent front

• TLC is 10 to 100 times more sensitive than traditional chemical methods.
• Widely used to screen for alkaloids, barbiturates, opiates, and other drugs of abuse.
• It detects volatile poisons, medicines, pesticides, and metabolites.
• The toxicologist chooses reagents based on the chemical properties of the compound being tested.

2. Gas-Liquid Chromatography (GLC)

• Carrier gas supply - inert gas (N₂, He, Ar) as mobile phase
• Injector/injector oven - where the sample is vaporized and introduced
• Column oven containing a coiled column (packed or capillary) - where separation occurs
• Detector in its own oven - measures compounds as they elute
• Processing unit - records and displays the chromatogram (peaks)

• Particularly preferred for analysis of volatile and thermostable compounds: pesticides, organic solvents, alcohols, chlorinated hydrocarbons.
• Can screen a wide range of basic drugs including alkaloids.
• Quantitative analysis requires prior sample preparation to isolate the drug and avoid interference.
• Produces a characteristic chromatogram with peaks at specific retention times for identification and quantification.

3. Atomic Absorption Spectroscopy (AAS)

1. Hollow cathode lamp - emits characteristic radiation for the specific metal being analyzed
2. Atomizer - flame (flame AAS) or graphite furnace (flameless/FAAS) that converts the sample into free atoms
3. Monochromator - selects the specific absorption wavelength
4. Detector (photomultiplier tube) - measures the transmitted light and calculates absorbance

• Lead (Pb) - blood lead levels in occupational/environmental toxicology
• Arsenic, Mercury, Cadmium, Antimony, Barium, Copper
• In forensic firearm residue analysis: AAS detects antimony, barium, and lead from primer residues, and copper from cartridge cases/bullet jackets. Hand swabs from suspects are analyzed to determine whether a person has fired a gun.
• Can identify bullet holes in clothing and tissues, determine range of fire, and trace common origin of bullet fragments.

4. High-Performance Liquid Chromatography (HPLC)

1. Eluent reservoir - holds the solvent (mobile phase)
2. High-pressure pump - forces the liquid through at pressures up to 6,000-9,000 psi
3. Injector - introduces the sample into the mobile phase stream
4. Stainless-steel column - packed with small particles (usually ≤10 μm) for high resolution
5. Detector - UV/Vis absorbance, photodiode array (PDA), fluorescence, or mass spectrometry
6. Recorder/integrator - produces the chromatogram

• Screening, identification, and quantification of heroin, LSD, amphetamines, cannabis, tricyclic antidepressants, barbiturates
• Detection of pesticide residues in blood, tissues, and drinks (e.g., organophosphates in contaminated drinking water)
• Analysis of snake venom, plant poisons, herbicides, and insecticides
• Ideal for drugs that are thermally labile (cannot withstand GLC conditions)

5. Breath Alcohol Analyzer (Breathalyzer)

(a) Infrared (IR) Spectroscopy (most common evidentiary instruments)

• An infrared beam is projected through the captured breath sample in a chamber.
• Ethanol molecules absorb infrared radiation at characteristic wavelengths (primarily 3.4 μm, corresponding to the C-H bond stretch).
• The amount of IR absorbed is proportional to the ethanol concentration (Beer-Lambert Law).
• Modern devices (e.g., Intoxilyzer 5000/8000/9000) use multiple wavelength filters (3.36, 3.4, 3.47, 3.52, and 3.8 μm) to differentiate ethanol from interferents like acetone, acetaldehyde, and toluene in exhaled air.
• The 3.4 μm wavelength detects alcohol; the 3.47 μm wavelength identifies interfering substances; a reference wavelength (3.9 μm) ensures baseline correction.

(b) Electrochemical Fuel Cell

• Exhaled ethanol is oxidized to acetic acid at the anode of a fuel cell:

  CH₃CH₂OH + H₂O → CH₃COOH + 4H⁺ + 4e⁻

• The electric current generated is directly proportional to the amount of ethanol - measured as BAC.
• Fuel cell sensors are very stable, require calibration only every ~6 months, and are commonly used in roadside screening devices.

(c) Color-based (historical - Breathalyzer original)

• Based on oxidation of ethanol by potassium dichromate in acidic solution; the orange dichromate changes to green chromic ion in proportion to ethanol concentration. Largely obsolete.

Summary Table