I need to prepare ethanol standards for HPLC to quantify the bio-ethanol in my sample of interest. What range of known concentrations would I have to prepare for estimating ethanol concentration and how do I do it? Brief explanation is welcome.
Prepare a series of a standard solution of Ethanol of different concentration in the same diluent you have prepared your sample solution. Use diluent as blank.
Inject blank, standard solutions and sample solution. Plot the graph of peak area against concentration of ethanol in your standard solution. Determine the peak area of ethanol in your sample and find corresponding concentration from calibration curve, this is your unknown ethanol content in the sample. (Note your concentration should fall within the calibration cure obtained from standard solutions)
Dear Tabrez,
Thank you for your response. It is informative. I wanted to know what concentrations (ppm, molarity) we should be considering for preparing the standards?
Hello Navnit,
why would you like to determine the ethanol content by HPLC? When determining ethanol, I immediately think of GC. Which HPLC detector would you like to use?
Regards
Markus
Dear Markus,
Yes, your views are accurate. I am using the HPLC (RID) because, I would get an idea of the amount of sugars consumed and the ethanol accumulated in the sample. However, as a confirmatory quantification I do use the GC (FID) as well. For GC, would you please tell me what standard concentrations would be better?
Hello Navnit,
what alcohol concentrations do you expect? I would proceed (as an example) with an expected alcohol concentration of 40 % as follows: 500 µL sample in a 50 mL volumetric flask + 200 µL internal standard (1-butanol). Make up to 50 mL with water. For the calibration I would do a 5-point calibration. I would prepare ethanol solutions with different contents (30, 35, 40, 45, 50 %) and use them for calibration. If your ethanol content is higher or lower, you have to adjust your calibration accordingly.
Regards Markus
Dear Prof Markus,
Thank you for that explanation. This is what I was expecting.
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