Dear, 5g fresh leaves A were placed in 70% aqueous methanol (40 mL) at boiling temperature in for 15 min. The mixture was cooled to room temperature and homogenized for 15 minutes using Ultrasonic. The homogenate was centrifuged in 4000rmp for 15 minutes. The supernatant was filtered through a 0.45 µm pore filter membrane and 5 ml of extracts were analysed directly by HPLC under suitable conditions. Five calibration standards are 10ug/ml, 20ug/ml, 30ug/ml, 40ug/ml, 50ug/ml. I got the result that leaves A had caffein amount 35,11ug/ml at the retention time 7,4.
My prof suggested me that I have to calculate the amount of caffeine in the fresh plant samples, this make more sense than giving the concentration of the measurement solution only.
How can I solve this problem? Please help me.
This would be rather straightforward, though you might have to play around with the details. If you measured 35.11 ug/ml from 5 ml, then you had a total caffeine content of 175.55 ug in your 5 ml sample (35.11 x 5). Though you would have to measure the total volume collected (after filtration) you can then adjust for that total volume. So for example, if your total volume was 40 mL (it should be close to this plus whatever volume was released from the homogenization and not subsequently filtered) then you would multiply your total content by 8 (as 5 mL represents 1/8 of the total volume) to get your total Caffeine from the plant sample. This would give you 1404.4 ug from all the leaves you used. Taking it a step further, you could say that you had 1404.4 ug per 5g of leaves.... or 280.88 ug Caffeine/g leaves. Let me know if this makes sense, or if I missed a step or two!?