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.

Use two p200 micropipettes set to 50 microliters, and aspirate the protease cocktail solution into one, and water into the second. Then, quickly place 10 embryos in a 1.5-milliliter microcentrifuge tube using a glass pipette and close the cap. Repeat this process for all samples to be tested, controls, and ethanol-treated embryos.

Working with one sample at a time, quickly open the cap, and aspirate all residual embryo media with a p200 micropipette set to 200 microliters. Rapidly, but gently, add and remove the 50 microliters of water, then, add the 50 microliters of protease cocktail, and close the tube cap.

Let the sample sit for 10 minutes at room temperature, then, quickly add 450 microliters of 5 Molar sodium chloride, and close the tube cap. Homogenize the sample by vortexing for 10 minutes, and transfer 2 microliters of supernatant to a gas chromatography vial. Seal the vial with a polytetrafluoroethylene cap.

After completing the startup method, load 436 Current spme ethanol 2013 3-minute absorb 2_5min rg run.METH from the Methods menu in the analysis software for each sample on the sample list. Fill out the sample list, starting with the air, water, and 5 Molar sodium chloride protease cocktail blanks. Then, enter in the ethanol standards in order from 0.3125 to 40 millimolar.

Follow the standards with the second round of blanks. Enter all homogenized embryo supernatant samples to be tested, from lowest to highest predicted ethanol concentration, and finish by entering a third round of blanks.

Add the gas chromatography vials to the autosampler in the order in which the samples were entered, and allow them to warm for 10 to 15 minutes. Then, start the sample runs using the software. After all runs are completed, activate the shutdown method by adding a final sample in the sample list and running standby.METH. Back up all data acquired during the sample run.

Once the shutdown method is complete, click on Open Chromatogram and open the folder with the results. Samples are automatically integrated in this program. In the results, make sure the correct peaks have been integrated. Once all samples have been confirmed, print or export the results.

Plot the peak height of the ethanol standards on a graph. Then, calculate the slope, Y-intercept, and R-squared values. Obtain the ethanol value for each sample by subtracting the peak height of the sample from the Y-intercept of the standards, and dividing this value by the slope of the standards.

To calculate the ethanol concentration in the embryos, calculate the dilution factor for each sample, and multiply the sample ethanol value by this dilution factor. Finally, calculate media reference samples by multiplying the media ethanol value by a dilution factor of 10.