2.4. Coffee sample preparation
Raw coffee were ground and screened through 250 lm
sieve to get a uniform texture. An accurately weighed
amount of sieved coffee (approximately 50 mg) was dissolved
in 25 ml of distilled water. The solution was stirred
for one hour using magnetic stirrer and heated gently to
remove caffeine easily from the solution. In addition the
solution was filtered by a glass filter to get rid of particle
from solution.
2.5. Liquid–liquid extraction of caffeine
In this research for liquid–liquid extraction chloroform
and dichloromethane were used for comparison. However,
it had been observed that many interfering matrices were
extracted with chloroform than dichloromethane, due to
this dichloromethane was selected for the extraction. Literature
report also indicated that even if both solvents were
useful for decaffeinating caffeine from coffee beans according
to Clarke, (1980), Rofti (1971) the current most widely
used solvent for decaffeinating in coffee beans was dichlo-romethane. The efficiency of dichloromethane to extract
caffeine from coffee beans is 98–99%.
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.
2.6. Gaussian fit
In addition to caffeine spectra there are interfering
bands from other coffee components extracted by dichloromethane
and the peak of these bands was observed at
the wavelength of 308–310 nm. The compounds attributed
to this are known to be chlorogenic acid related
compounds (p-coumaroquinic acid). It is clear that this
interfering band has an effect on the maximum peak of
caffeine. Therefore, in this research matrices were eliminated
by Gaussian fit. The peak absorbance for calculating
the concentration of caffeine was obtained after
subtracting the Gaussian fit from the total caffeine
spectra.