Extraction has been done according to the following
procedures. The coffee solution prepared above (under coffee
sample preparation) was mixed with dichloromethane
by volume ratio (25:25 ml) for the extraction of caffeine
from coffee. First, a mixture of the solution was stirred
for 10 min. Then, using separatory funnel caffeine was
extracted by dichloromethane from the solution. The
extraction of caffeine proceeded 4 times with 25 ml dichloromethane
at each round. The caffeine extracted by dichloromethane
at each round was stored in volumetric flasks.
Finally, the absorbance of the solution was measured by
UV/vis spectrophotometer in the range of 200–500 nm
against the corresponding reagent blank. All glassware
was thoroughly cleaned, rinsed with distilled water and
dried before use.