I would like to know how I can prepare my sample of supercritical extract of green coffee beans if I want to analyze chlorogenic acid and I need to prepare the sample to analyze it by HPLC.
I would like it to be a quick method that does not require many separations, especially I would like to know how to remove the oil, waxes, etc. from the polar phase after a supercritical extraction with solvent (etOH)
This is a relatively easy HPLC method and separation, but please seek the assistance of someone with professional experience in HPLC analysis before starting on your own. It takes many years of full-time industrial experience to learn just the basics of chromatography (emphasis on "basics", as learning method development takes many more years) and as a student I am sure you understand how your time is best spent working with someone local and experienced to complete this project in a timely manner. *Ask your teacher for assistance. **BTW: HPLC analysis of caffeine and CGA are WELL documented online. Try a simple keyword search on Google to find several hundred free examples of methods, articles, white papers etc to review before you start. This is one of the easier HPLC applications because COFFEE is BIG business !
Basically, the extract can be centrifuged and filtered before dilution in mobile phase to make up fresh samples. Extracts are sometime made in pure ethanol or taken from the supercritical-fluid-extraction process, then dissolved in ethanol. The HPLC system you use should have a scanning diode-array-detector (aka "DAD" or "PDA") as this is needed to help qualitatively ID the different compounds for method development (you need to view each peaks "Spectra"). Use a high quality, NEW C18 column with a gradient of Methanol and Water to get started. Acquire professional standards for all compounds used and run standards at multiple calibrations "Levels" (= concentrations) to create formal Calibration Tables. Make sure you measure and understand what the column's void volume is and the K prime of any detected peaks are for any method you use. Make sure values obtained follow good chromatography principles and never assume that just because you followed someone's paper or article for the same, that they do (Review of most published scientific articles by me has found that 20-30% of them are invalid as presented, so be cautious). Be sure to work with someone trained in chromatography so you can complete this basic project.
Here is a link my HPLC training blog page with free authoritative training articles that may assist you and the person you work with to insure you stay on track (it includes info on Column Void Volume, K prime and many other basic fundamentals that you must know to use HPLC): https://hplctips.blogspot.com/
I have a question: should I rotary evaporate before or after centrifugation?
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