Experiment: Analysis of Caffeine in Various Teas Using Chromatography Methods
The lab aims to measure the content of caffeine in different types of teas to determine which has the
highest and lowest content of caffeine.
Caffeine is an important ingredient, also used as a flavoring agent to make drinks addictive. It has the
effect of temporarily warding odd drowsiness and restoring alertness. Most experience no behavioral
effects with less than 300mg caffeine, sleep is sensitive and can be disrupted by 200mg caffeine. Caffeine
is a naturally occurring substance found in the leaves, seeds or fruits of various species. Caffeine is a
central nervous system stimulant.
Caffeine Content (per eight ounces serving)
Black Tea: 40-70 mg
Green Tea: 45-35 mg
White Tea: 15-30 mg
Oolong Tea: 37-55 mg
Herbal Tea: None (made from dried flowers, leaves, etc. typically that are caffeine-free)
Magnetic Hot Plate
Discovery RP-AmideC1615 cm x 4.6 mm I.D, 5 um particles
Pure Caffeine (1,3,7-trimethylxanthine) solid:
Standard solutions of 10, 20, 50, 70, 100 mg (1 mg/L = 1 ppm)
Standard methods for Concentration:
Goal is to use the calibration curve correlation of peak area to absorbance to evaluate the concentration
within the tested teas.
For each of the three tea samples, brew each sample tea bag in 250 mL of boiled water. This solution will
then be injected via syringe into the HPLC instrument.
Methanol: 25 mM
K2HPO4, pH 7.0
1 mL / min
UV, 254 nm
Discovery RP AmideC1615 cm
x 4.6 mm I.D, 5
1 mL / min
UV, 254 nm
Standard Operating procedure:
Turn power to HPLC by pressing the switch behind the main HPLC unit
If there are air bubbles… Turn the metal knob to the left on the bottom middle-left of the instrument then
press the purge button to purge air from mobile phase line [50% DI water, 50% Methanol]. After the
process was done, tighten the knob prior to advancing the system operation
Select composition of mobile phase by first pressing “%ABC” then using the cursor, increase, decrease,
and select keys modify the compositions of solutions A ( DI water: 75%) and B (Methanol: 25%) with a
flow rate of 1 mL/min.
Turn on the AD20 Absorbance detector, and set the conditions to “high”, ensure the wavelength is at 254
Turn the metal knob below the lever on the left side of the HPLC to the left. Then press the purge button
on the instrument to allow the solutions to be transferred through the absorbance detector.
Ensure the injection slot is in the load position and inject sample with the X syringe at capacity
Turn the injection valve to inject. The software should start the detection automatically. Wait 1 minute
then turn the injection valve to load again and remove the syringe.
The software should automatically save the data for each run. Remember to record the file name for the
data recording for each runs.
Purchase answer to see full