Electrophoresis in Forensic Science: High Voltage, Low Voltage and Immunoelectrophoresis

Electrophoresis exploits a simple fact: a charged particle in a uniform electric field experiences a force and moves. Anions (negatively charged) drift toward the anode (+), cations (positively charged) drift toward the cathode (−). Neutral molecules do not separate by this method.

The defining quantity is electrophoretic mobility:

μ = v / E = q / (6πηr)

where v is the migration velocity, E is the field strength (V/cm), q is the net charge on the particle, η is the viscosity of the medium and r is the Stokes radius. Three forensic implications follow.

1. Charge matters. At a given pH proteins carry a net charge that depends on their amino-acid composition. Change the buffer pH and you change the separation.
2. Size matters through the support medium. In free solution two proteins of similar charge density would migrate at the same speed. The gel (agarose or polyacrylamide) sieves them: smaller molecules thread through the matrix faster.
3. Heat is the enemy. I = V/R dissipates as Joule heat, distorts bands and can denature samples. This is why high-voltage runs use cooled plates and short distances, and why CE uses narrow capillaries (large surface-to-volume ratio for heat removal).

A common Indian textbook visual is the agarose submarine gel: a horizontal slab submerged in TBE or TAE buffer, samples loaded into wells at the cathode end, voltage applied for 30 to 60 minutes, bands visualised by ethidium bromide or a safer dye like SYBR Safe.

Low-voltage electrophoresis operates in the 50 to 500 V range with field strengths of roughly 1 to 10 V/cm. Runs are long (30 minutes to several hours) because the field is gentle and the heat load is small. The classical supports are paper, cellulose acetate and agarose.

Forensic uses of low-voltage runs:

• Blood group typing on dried stains. ABO antigens and Rh-related glycoproteins were typed routinely on cellulose acetate before DNA replaced them as the primary tool. Many SFSLs still keep the technique for confirmatory work on small or degraded stains, with method validation and SOP control sitting under the broader ISO 17025 and quality management in forensic laboratories framework.
• Isoenzyme polymorphisms. PGM (phosphoglucomutase), EAP (erythrocyte acid phosphatase), EsD (esterase D) and ADA (adenosine deaminase) were the pre-DNA backbone of Indian serology. Low-voltage agarose or starch gels resolve the isoforms.
• Haemoglobin variant screening. HbA, HbS and HbF separate cleanly on cellulose acetate at pH 8.6. Useful in mass-disaster identification where ancestry inference helps narrow candidates.
• Large protein separation and Western blot loading. SDS-PAGE for proteins above 30 kDa typically runs at constant voltage in this band.

The trade-off is resolution versus speed. Low voltage gives clean, sharp bands for big molecules but is impractical for small ions which would diffuse faster than they migrate.

High-voltage electrophoresis runs at 500 to 10,000 V with field strengths of 20 to 200 V/cm. Joule heating is the dominant problem, so the apparatus is built around it: thin paper or thin-layer plates sandwiched between cooled glass blocks, short separation distances (5 to 20 cm), and runs that finish in minutes rather than hours.

What it is used for in forensic and biochemical work:

• Amino acid and peptide mapping. Free amino acids carry small net charges and would never separate cleanly at low voltage; their slow migration is dominated by diffusion. High voltage outruns the diffusion.
• Small organic acids and nucleotides. Sugars phosphorylated to charged forms, nucleotides released by enzyme digestion, and metabolic intermediates separate well here.
• Inorganic ion screening. Forensic toxicology occasionally uses high-voltage paper electrophoresis as a quick orthogonal check on cation/anion identity in suspected poisoning cases (older Indian texts cite arsenite, antimonate and thiocyanate examples).
• Capillary electrophoresis. The modern incarnation of high-voltage electrophoresis. A 30 to 80 cm fused-silica capillary held at 10 to 30 kV, with sample volumes in the nanolitre range. CE inherits all the advantages of high voltage (speed, sharp peaks) and bypasses the heat problem because the capillary geometry sheds heat efficiently.

Immunoelectrophoresis, developed by Grabar and Williams in 1953, is the technique forensic serologists reach for when they need to know not just that a stain is blood, but whose species it came from. The method is a two-step procedure on an agarose plate.

Step 1: electrophoretic separation. Sample (serum, blood-stain extract, semen extract) is loaded into a well in an agarose layer. Low-voltage electrophoresis at pH 8.6 separates the constituent proteins by charge and size. Albumin runs fastest toward the anode; immunoglobulins migrate least.

Step 2: immunodiffusion. A trough is cut parallel to the run, filled with the antiserum of choice (for example anti-human serum for species identification). Antibodies diffuse outward from the trough, the separated antigens diffuse inward. Wherever an antigen meets its specific antibody at the equivalence ratio, a curved precipitin arc forms.

Forensic applications NTA tests:

• Species identification of bloodstains. A bloodstain extract run against anti-human serum produces species-specific arcs. Used routinely at CFSL/SFSL serology divisions to resolve "human or not human" before DNA work is committed.
• Bloodstain ABO and Gm typing on small or degraded stains where conventional agglutination is unreliable.
• Seminal stain identification. Anti-human-seminal-plasma antiserum confirms semen on a stained garment.
• Saliva and other body fluid identification via species-specific markers.

Two variants worth knowing by name:

• Counter-current immunoelectrophoresis (CIEP): antigen and antibody are driven toward each other by an applied field. Faster than passive diffusion; used in rapid species ID.
• Rocket immunoelectrophoresis (Laurell technique): antigen is electrophoresed into a gel containing antibody. Forms a rocket-shaped precipitin peak whose height is proportional to antigen concentration. Quantitative.

Capillary electrophoresis is the platform on which every modern Indian STR profile is read. After PCR amplification with a multiplex kit (Identifiler Plus, GlobalFiler, PowerPlex Fusion or the Indian-developed STR Indelplex by CDFD Hyderabad), the labelled fragments are injected into a narrow capillary filled with a sieving polymer (POP-4 or POP-7) and electrophoresed at roughly 15 kV. Fragments migrate by size; a laser excites the fluorophores; the detector captures four to six dye colours in parallel.

Three forensic implications you should be able to state in one line each.

1. Single-base resolution. CE distinguishes a 200-base from a 201-base STR allele, which is what makes accurate allele calling possible.
2. Multiplexing. Four to six colour channels let one run resolve 20 to 24 STR loci plus the amelogenin sex marker in a single injection.
3. Indian deployment. CDFD Hyderabad, the seven CFSLs (Hyderabad, Kolkata, Chandigarh, Pune, Guwahati, Bhopal, Delhi), NFSU Gandhinagar and most state DNA labs run Applied Biosystems 3500-series genetic analysers (an 8 or 24 capillary CE instrument). The DNA Data Bank framework under the DFSS rests on CE-generated profiles, with NABL accreditation under ISO 17025 and quality management in forensic laboratories governing instrument calibration, internal ladders and proficiency testing.

CE also underpins forensic toxicology screens for chiral drugs and explosive residue ion analysis, though those uses are not in the Unit II bullet.