I've to estimate caffeine content in tea sample. I found a lot of protocol but none contain the formula how to calculate caffeine content from area. I need details calculation.
Muhammad Abid Hasan Chowdhury
quantitative analysis should be performed based on the calibration curve of the standard and not on the single measurement of its peak area. while comapring its peak area at one concentration only, you do not know, if you are in the linear area of the standars. too high quantitiy of an injected standard, or on the other hand, a not sufficient one, will affect your calculations.
I would recommend you to prepare several dilutions of standards, starting from 5 mg/mL and going down to well calculate the calibration curve and find the linearity range. Later, the content of a certain compound in the sample should be within this range, too.
based on a good curve, you should receive proper results and you will be able to well calculate the LOD and LOQ values of the method.
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The best way is to use external standard-based calculation. The measurement is straight forward. You calibrate your HPLC system and get a standard curve from a serious of dilutions of pure caffeine – This is your external standard. For accurate calculation, you need to make sure that:
1) Use area under curve values for each caffeine standard concentration and construct a standard curve (concentration on the x-axis – versus area under curve value on the y-axis).
2) A straight line should be obtained with equation y = mx + c
3) The straight line must have an r2 (statistical coefficient of determination) value close to one (over 0.9, e.g., 0.999)
4) Your extract is injected in the same HPLC system and an area under curve is obtained. This value is entered in the straight-line equation to calculate the corresponding concentration value. For accuracy, use the straight-line equation – never extrapolate directly from the graph.
5) For accuracy, you need to do a minimum of three experiments and use a mean value and standard deviation or standard error of mean in your report.
As you did not ask about the experimental, I am assuming that you know how to extract and obtain the HPLC data – Good luck.
Thank you Solomon Habtemariam for details explanation. It will be very helpful for me.
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