While studying bile acids from living organisms, Pregl was constantly limited by tiny sample sizes. He established ‘micro-elemental analysis’, allowing measurement of carbon, hydrogen, nitrogen and other elements in samples as small as one milligram. Using a microbalance with 0.01 mg sensitivity, he optimized the length of combustion tubes and oxygen flow to achieve complete burning, then trapped the minute gases with chemical absorbents. The technique shortened analysis time, saved costs and soon became indispensable in drug synthesis, food inspection and more. Microanalysis was later applied to trace environmental pollutants and stimulated the development of today’s high-sensitivity instruments such as mass spectrometers.

Pregl’s microanalysis miniaturized the classical Liebig combustion method and combined manometric and gravimetric measurements. By adding platinum catalyst he achieved complete oxidation at lower temperatures and quantitatively absorbed CO₂ and H₂O with soda-lime and phosphates, respectively. Nitrogen was determined from the volume of N₂ after denitrification, while sulfur was oxidized to SO₂ and precipitated as barium sulfate. Detection limits were reduced to less than 1/50 of previous methods, enabling composition determination of natural products and pharmaceutical compounds available only in microgram quantities. This achievement opened a new branch of analytical chemistry called ‘micro-chemistry’ and inspired the development of modern automatic elemental analyzers (CHN analyzers).

Pregl scaled the Liebig method down from 100 mg to 1 mg by overcoming thermal loss and reproducibility issues with a two-stage combustion tube containing a platinized platinum catalyst layer and a glass micro-column. CO₂ was quantified to the 10⁻⁴ level by mass difference after absorption in potassium hydroxide solution; H₂O was trapped in phosphorus pentoxide tubes, and residual O₂ reduced on Cu filament to stabilize internal pressure. For nitrogen he applied a modified Dumas/Kjeldahl approach: gases were separated on a vacuum line and their volume read on a recording manometer. The protocol achieved ±0.2 % absolute accuracy, allowing reliable elemental composition of protein degradation products, bile-acid derivatives and other complex matrices. Pregl’s schematics are essentially identical to the flow diagrams of modern CHNS/O analyzers, showing that the integration of microbalance, high-temperature catalytic combustion and selective absorption formed the technological foundation for later automation and high-throughput multi-sample analysis.