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.

The problem is how do I make sure that only caffein appears at the retention time from 7.3 minutes to and there are not more different chemicals?

My prof suggested me that I have to make sure that the addition is no more than double the size of the original peak - that means I need to calculate also the dilution of your reference with your sample (e.g. adding 1 mL reference with 1 mg/L to 1mL of your sample with potentially no caffeine will result in 2mL sample with 0.5 mg/L caffeine).

To be honest, I did not how to do as he said. What amount do I have to calculate? please help me to solve this problem.

Thank you for your help and have a good health.