Please let me know how I can detect ethanol in the fermentation media. Is there any specific methodology (injector, column temp, detector temp) for the detection of ethanol in the media? And do we have to separate the ethanol from the media by distillation or we can directly use the supernatant after centrifugation to the GC analysis. Is there any specific protocol for separation of ethanol from the fermentation media.
The conditions depends on the column type, length, etc., but in generally, if you inject pure ethanol onto the column you will see the retention time and that's changing with change the injection temp., heating rate, etc. Depending the other components in the fermentation media you have to solve the problem, that the ethanol peak will be separated from those. Number of peaks of components and their positions depends on the ferment liquor composition and GC conditions , so it is important, that you have a pure ethanol and some byproduct contg. media, or e.g. acetone-butanol-ethanol from ABE fermentation, or acetic acid contg. media, etc.
When you select a column it should be taken into consideration that the other components polarity and boiling point differs or not from the ethanol parameters to ensure the best separation. THe boiling point and the polarity can change the elution order on different columns.
IF you started from sugar and did fermentation, I strongly suggest distillation and collecting a distillate, because non-volatiles are present in the ferment liquorwhich will be contaminate your column soon.
Furthemore, the distillate will contains less components, the chromatogram will contains less peaks, so in this way will be easier to separate the peaks well.
In Randy Lewis's lab at BYU we detected ethanol using GC directly from the supernatant of a bacterial suspension. This was handy because we could then detect other products such as acetic acid as well. I admit I don't know the full protocol as I was not personally the one doing the GC, but if you get in contact with Dr. Lewis he might be able to give you more specifics or point you in the right direction. He is in the Chemical Engineering department.
Ethanol at reasonable concentrations is readily detectable by GC. Most of the manufacturer's catalogues, or the application notes on their websites, will give you an idea of phases and operating conditions to evaluate.
The sample preparation you need to do depends on the concentration of ethanol in the liquid and its other components. Distillation would probably work, but why not consider using a headspace autosampler, which will effectively distill and inject automatically?
If you wish to directly analyse the medium, filtration and/or centrifugation would be important to remove particulate material, which might otherwise jam or clog the injection syringe. And I'd strongly recommend you clean the injector liner and replace the septum regularly. Inorganic matter will cake it otherwise, as will involatile and pyrolysed organic material.
As an alternative route, why not sample the vapour above the medium in a closed vessel, perhaps using something like an alcohol "breath-test" device?
Greetings
I am not sure what equipment you have available but look at this paper were we describe a simple method that include organic extraction. You can find the paper here:
http://tiny.cc/elu2ax
Either way do not inject water directly to the GC as it could damage column and detectors.
Yoram
Small volumes of water won't cause damage to modern commercial GC stationary phases or detectors, and probably won't even give a significant detector response. Unless you are using on-column injections with a 0.53mm id column, all the water will be vaporised in the injector. Hence my suggestions about maintaining the liner, where dissolved material will be deposited.
Many thanks, but given that fermenting broth often contain high concentration of sugars, proteins etc, shouldn't one worry about clogging the column? A liner will obviously solve some of the problems by collecting these but at least one system I have used (SRI-GC 8610C) used a non-disposable liner. The use of an organic extraction step (we used Ethyl Acetate) to extract the ethanol from the water phase should reduce dramatically this issue.
Yoram
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