CHM 2290

Determination of the Concentration of Caffeine Using UV-Vis Spectroscopy

A. Introduction:
O Caffeine (C8H10N4O2, 194.19 g/mol) is an alkaloid found in a variety of plants in
N N nature including the coffee bean, tea leaves, cocoa beans and cola nuts. It is mild
central nervous system stimulant commonly used in non-alcoholic beverages.
O N N
According to the FDA publication: “Medicines in my Home: Caffeine and Your
Body” (see: http://www.fda.gov/downloads/UCM200805.pdf ) approximately 90
percent of people in the world ingest caffeine in some form (beverages, pills or via consumption
of natural products). Ingestion of less than 200 mg of caffeine per day is considered to be
relatively harmless. Physicians suggest that daily consumption of 600 mg or more could lead to
harmful side effects. An overdose of caffeine can be fatal. Data published by the Center for
Science in the Public Interest (see https://cspinet.org/eating-healthy/ingredients-of-
concern/caffeine-chart ) provides detailed tables of the caffeine content of several common
beverages and foods. A subset of their website’s data is provided in Table 1.

Table 1: Caffeine content of several common beverages and foods (Source:

Center for Science in the Public Interest)

Item Serving Size Caffeine (mg)

Starbucks Coffee venti, 20 fl. oz. 415
Folgers Classic Roast instant 2 tsp., 12 fl. oz. 148
coffee
McDonald’s coffee large, 16 fl. oz. 133
Black tea, brewed for 3 8 fl. oz. 30 to 80
minutes
Green tea, brewed for 3 8 fl. oz. 35 to 60
minutes
Arizona Iced Tea, black 16 fl. oz. 30
Diet coke 12 fl. oz. 47
Pepsi 12 fl. oz. 38
Coca-cola, Coke Zero or Diet 12 fl. oz. 35
Pepsi
5-hour Energy 1.9 fl. oz. 208
Full Throttle (Monster) 16 fl. oz. 200
Red Bull 8.4 fl. oz. 80
Awake Caffeinated Chocolate 1.55 oz. 101
Bar
Hershey’s Special Dark 1.5 oz. 20
Chocolate Bar
Dove Dark Chocolate Silky 5 pieces, 1.4 oz. 4
Smooth Promises
Zantrex-3 weight-loss 2 capsules 300
supplement
NoDoz or Vivarin 1 caplet 200
Anacin 2 tablets 64
B. UV-Vis Spectrophotometry and Beer’s Law

UV-Vis spectrophotometry can be used to quantitate the amount of caffeine in an aqueous

solution. When a molecule is exposed to light, it can absorb a photon and move from the ground
state electronic state to an excited electronic state (see Harris, Quantitative Chemical Analysis,
Chapter 18, “Fundamentals of Spectrophotometry”). Belay et. al, (Belay, Ture, Redi, & Asfaw,
2008) found that, at room temperature, pure caffeine in water exhibited strong UV absorbance in
the region of 243 to 302 nm. The wavelength of maximum absorbance was approximately 272
nm. The amount of light absorbed by a substance is linearly related to its concentration in
solution and the pathlength through which the light travels.

𝐴 = 𝜀𝜀𝜀 (𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 1)

This relationship (Equation 1) is called the Beer-Lambert law or Beer’s law where A is the
absorbance of the solution at a given wavelength, ε is the molar absorptivity of the analyte at that
wavelength, b is the pathlength through which the light travels (e.g. cuvet length), and c is the
concentration of the analyte in the solution. Beer’s law works well for dilute solutions (<0.01 M)
when the analyte is not participating in a concentration-dependent equilibrium. For example, it
does not work well in very concentrated solutions or one in which a weak acid and its conjugate
base do not absorb light to the same extent at a given wavelength. Spectrophotometers are most
accurate when the absorbance of the solution is in the range of 0.3 to 2 absorbance units. In
some cases, it may be necessary to measure the absorbance of a diluted sample of an analyte in
order to improve accuracy. The concentration of the analyte can then be calculated by correcting
for dilution.

C. Chemical Reagents:
• Solid Caffeine standard, reagent grade
• Deionized water
• Solution containing an unknown amount of Caffeine

D. Glassware:
• Five 50 mL volumetric flasks
• One 250-mL volumetric flask
• Three 250 mL beakers
• 100 mL graduated cylinder
• 1-cm pathlength cuvettes
• 10-mL graduated pipette

E. Procedure:
I. Preparation of 100 ppm standard stock solution:

1. Weigh, by difference, 0.0250 g of the caffeine standard and place it into a 250-ml
volumetric flask. Record the actual mass of the caffeine in your notebook.
 2. Add 50 to 100 milliliters of deionized water to the flask. Swirl the solution until
the caffeine is completely dissolved.
3. Add additional deionized water to the volumetric flask until the fill-line is
reached.
4. Using a 10-mL graduated pipette, prepare working standards by pipetting 10, 7.5,
5, 2.5, and 1 ml of standard stock solution into five separate 50 ml volumetric
flasks respectively. Record the ACTUAL volumes of the stock solution used in
your notebook.
5. Fill each flask by adding deionized water to the fill-line.
6. Stopper the flasks and gently invert and shake the solutions.

II. Measurement of the absorbance values of the working standard solutions:

1. Take aliquots of each of the working standard solutions made, place them into
cuvettes and analyze them using the spectrophotometer in the instrument room.
2. Don’t forget to condition the cuvets between samples of different concentrations.

Absorbance measurements: Operating instructions for HP-8453

3. Turn on the HP-8453.
4. Wait until the lamp monitor on the HP-8453 turns yellow.
5. Open UV-Vis ChemStation software (no password required).
6. From the Mode pull down menu select Fixed Wavelength
7. Set the wavelength to 272 nm
8. Insert the blank solution into the spectrometer using a plastic cuvette. You can
use deionized water as a blank for this experiment.
9. Click “blank”
10. Place your cuvette containing sample into the spectrometer and click “sample” to
measure the absorbance.
11. Record the absorbance of each of the working samples at 272 nm.
12. Construct a graph of the absorbance versus the ACTUAL concentrations of
standard caffeine solutions and calculate the equation of the linear regression line
formed using Excel. Print the graph and turn it in with your lab report.

III. Measurement of the absorbance value of the unknown solution

Your teaching assistant will give you ~20 mL of liquid containing an unknown
concentration of caffeine. Record the absorbance of the sample following the procedure
described in part II, above. To be very accurate, the absorbance values of your sample
should be greater than 0.250 and less than 0.700 units. If the absorbance value of your
unknown is greater than 0.700 absorbance units, you must dilute your unknown and
obtain a second absorbance value with the diluted sample. The ratio method can be used
to determine the dilution needed to bring the absorbance of the unknown into the correct
range.

𝐴𝑡𝑡𝑡𝑡𝑡𝑡 𝑟𝑟𝑟𝑟𝑟 𝐶𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑢𝑢𝑢𝑢𝑢𝑢

=
𝐴𝑢𝑢𝑢𝑢𝑢𝑢𝑢 𝐶𝑢𝑢𝑢𝑢𝑢𝑢𝑢
 Record all data related to the process of dilution. You will need this information to
calculate the concentration of the caffeine in the unknown prior to dilution.

F. Calculations:
Use the linear regression line from your calibration curve to calculate the concentration
of caffeine, in ppm, in your diluted sample. Correct this value for dilution and report the
actual concentration of caffeine, in ppm, in the original (i.e. undiluted sample) in your lab
report.

G. Safety Hazards:
• Concentrated caffeine is harmful if swallowed or inhaled, and can cause eye and skin
irritation. Wear gloves, safety goggles and a lab coat when handling this solution.

H. Waste Disposal:
• Solutions containing caffeine should be placed into the non-halogenated waste
containers.
• Residual solid caffeine standard should be placed into the solid waste container.

REFERENCES
Belay, A., Ture, K., Redi, M., & Asfaw, A. (2008). Measurement of caffeine in coffee beans
with UV/vis spectrometer. Food Chemistry, 108(1), 310–315.
http://doi.org/10.1016/j.foodchem.2007.10.024