Headspace Gas Chromatography: A Technique to Assess Ethanol Concentration in Zebrafish Embryos

To analyze the concentration of ethanol - a volatile organic compound, begin by taking the homogenized ethanol-treated zebrafish embryo culture supernatant in a chromatography vial. The supernatant contains other non-volatile organic compounds in addition to ethanol. Place the sealed vial in a sampler unit of chromatography assembly and heat it at an appropriate temperature.

During heating, ethanol vaporizes and migrates into the gas phase or headspace, leaving behind the non-volatile residues. This allows the sampler to introduce gaseous ethanol into a continuous stream of carrier gas. This acts as a mobile phase.

Ethanol passes through the pre-heated coiled chromatography column, which contains a packed bed of the liquid stationary phase. Owing to their volatility and polarity, the moving ethanol molecules start adsorbing over stationary phase particles, partitioning themselves into the stationary phase.

Due to the high column temperature, the adsorption of ethanol molecules is unstable and temporary, allowing these molecules to gradually desorb and migrate along the carrier gas flow. These ethanol molecules undergo a series of adsorption-desorption events throughout the column length.

Subsequently, ethanol enters a detection chamber that helps determine its concentration in the sample corresponding to the area under the curve in the resulting chromatogram.