1

A COMPARATIVE STUDY

OF THE CAFFEINE

CONCENTRATIONS IN

VARIOUS CAFFEINATED

AND DECAFFEINATED

BEVERAGES

CH4721

Andrew LeSage, Christina Welch and Ford Guo

Due: May 1st, 2015

Individual Project Lab Report

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Abstract

Caffeine is a central nervous stimulant that provides the body with extra energy by inhibiting the

adenosine receptors. Consistent caffeine consumption can lead to various side effects such as

nausea and anxiety. The goal of this experiment was to perform a comparative analysis of the

concentration of caffeine in caffeinated versus decaffeinated beverages. These caffeine

concentrations were quantified by UV-vis spectrophotometry and high pressure liquid

chromatography. A difference between the caffeine concentrations reported by companies and

experimental data was observed. Experimental results also indicated many decaffeinated

beverages contained caffeine concentrations that exceed the FDA allotted concentration to be

considered decaffeinated.

Introduction

Caffeine is a central nervous system stimulant that increases alertness, relaxes smooth

muscles, stimulates cardiac muscles, and causes excess urination. In medicine, caffeine can be

used to treatment migraines, relieve pain and alleviate drowsiness [1].

Caffeine is an inhibitor of adenosine, which is a central nervous system neuromodulator

that binds to A1 and A2A receptors on the cerebrum [2]. When adenosine binds to these receptors,

neural activity is slowed, causing the body to feel tired. While the body is resting, adenosine

facilitates sleep and dilates the blood vessels to ensure adequate oxygenation. Structural

similarities between caffeine and adenosine (Figure 1) allows caffeine to act as an adenosine-

receptor antagonist. Caffeine inhibits adenosine by binding to its receptors without reducing

neural activity, which causes an overall decrease in adenosine receptor availability and an

increase in neural activity. This increase in neural activity forces the pituitary gland to secrete

hormones that cause the adrenal gland to produce adrenalin—the hormone that increases

attention levels and provides the body with extra energy [3].

Caffeine Adenosine

Figure 1. Structure of Caffeine versus the structure of adenosine [1].

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Regular consumption of caffeine can lead to a mild physical dependence. Symptoms of

caffeine withdrawal include headaches, fatigue, anxiety, irritability, depressed moods, and/or

difficulty concentrating. For a variety of reasons, some people prefer to drink decaffeinated

beverages and herbal teas instead of caffeine containing beverages [4].

In 1903, Ludwig Roselius became the first scientist to successfully decaffeinate coffee

beans. His decaffeination process, the Roselius Process, involved treating green coffee beans

with chlorinated hydrocarbon solvents to extract the caffeine and then using a roasting process to

remove any solvent from the beans. In the 1970’s, Roselius patented a new process developed by

the Max Planck Institute that uses carbon dioxide to eliminate caffeine from coffee beans; this is

the primary method manufacturers utilize in the production of decaffeinated coffee beans [5].

According to Commercial Item Description (CID) A-A-20213B mandated by the United States

Department of Agriculture, decaffeinated coffee shall not exceed more than 0.10% of its original

caffeine content in dry, packaged coffee[6].

In this experiment, the amount of caffeine in a variety of beverages were quantified using

two different analytical techniques. For the first method, the caffeine extraction was performed

using dichloromethane (DCM). The solubility of caffeine in water at 25oC is 2.2g/L and 10.2g/L

in DCM [7]. Since water and DCM are immiscible in each other and have a partition coefficient

of 4.63, which makes DCM a good organic solvent for caffeine extraction [8].

The caffeine was extracted from the samples using DCM. A Rotovap was used to

evaporate the DCM and crystalize the caffeine. The crystallized caffeine was dissolved in water

and tested with a UV-Vis Spectrophotometer (according to the literature, caffeine should be

observed at 272.8nm in water [9]. After all of the spectrums from the various caffeine samples

were observed, they were quantified with Beer’s law.

Molecules containing π-electrons and non-bonding electrons were considered to be UV

active for their ability to be excited to a higher anti-bonding energy state [10].

The less energy the electron needs for transition, the higher the maximum absorption

peak will be observed at a longer wavelength, which can be understood with the following

formula,

(1)

𝐸 = ℎ𝑐/𝜆 [11]

where h is Planck's constant and c is the speed of light. As figure 1 demonstrates, the structure of

caffeine has multiple double bonds and carbonyl carbons, so it is should be UV active. Beer’s

Law demonstrates the relationship between the attenuation of light and the properties of material

which the light is traveling through. The formula of Beer’s Law is shown below,

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(2)

𝐴 = 𝜀𝑙𝑐

where the A is the absorbance number, 𝜀 is the molar absorptivity, l is the path length, which is

generally the width of the cuvette used in the spectroscopy, and c is the concentration of the

solution [12]. In order to apply the Beer’s Law more directly, it can be simplified as,

(3)

𝐴 ∝ 𝑐

High performance liquid chromatography (HPLC) was the second method used to

quantify the amount of caffeine in each sample. HPLC is an analytical technique that is used to

separate, identify, and quantify various components of a mixture. To perform HPLC, the sample

of interest is injected into a stream of a highly pressurized mobile phase. The pressurized stream

continues to the HPLC column that is filled with an absorbent solid. Each component of the

sample interacts differently with the solid inside the column, causing each to have a different

retention time. As a result, the various components of the sample of interest exit the column at

different times. After exiting the column, the components continue to the detector of the

machine. The detector sends the information to the processing unit, which in turn prints out a

chromatograph [13]. Figure 2 illustrates this process.

Figure 2. Diagram of typical HPLC set-up [14]

There are a variety of parameters that must be optimized to gather useable data including the

mobile phase, flow rate, attenuation, column material, and the type of detector [13]. The C18

column was selected to perform HPLC because the column material is very nonpolar and are

capable of interacting with polar molecules, like caffeine. C18 columns are extremely common

in HPLC because the packing material can provide a variety of different pore sizes, has an ability

to interact with a large range of molecules, and is relatively inexpensive to manufacture.

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Materials and Methods

Materials

sodium carbonate, dichloromethane, sodium chloride, methanol, acetonitrile, Sigma pure

caffeine, Pepsi, Decaffeinated Pepsi®, Mountain Dew® Kick Start Energizing Drink, Red Bull®

Energy Drink, Monster® Energy Drink, Monster Unleaded® Energy Drink, Great Value®

Decaffeinated Black Tea, Twining’s® Irish Black Tea, Folger’s® Half Caff Coffee, Folger’s®

Decaffeinated Coffee, Folger’s® Columbian Coffee.

Instruments

Shimadzu 2450 UV-Vis Spectrophotometer, Shimadzu SPD20A/LC20AD Prominence UV-Vis

Detector HPLC, Heidolph Laboratories Laborota 4000 Efficient Rotovap.

Methods

Method I: Caffeine Extraction

Sample Preparation

Caffeine Standards

Four pure caffeine standards (ranging from 1g to 2.9grams) were prepared and suspended in

100mL of deionized water.

Soda and Energy Drinks

To prepare the beverages and energy drinks, the carbonation in 100mL of each sample was

removed by boiling it on a hot plate for 10 minutes. To make sure the carbonation was

completely removed from the samples, the beaker containing the sample was spun for a few

minutes. If any bubbles began to form, the sample was placed back on the heater for 5 additional

minutes.

Tea

For each tea sample, three tea bags were weighed and boiled in 120 mL of water mixed with 3 g

sodium carbonate for 10 minutes.

Coffee

For each coffee sample, 5 g of coffee grinds were weighed out and added to 100 mL of boiling

water mixed with 2 g sodium carbonate. The solution was stirred for 5 minutes and filtrated to

remove any coffee grinds.

Extraction

For soft drinks and energy drinks, 75 mL of each beverage was measured and transferred into a

separatory funnel. Next, 1.5 g of sodium carbonate was added to the separatory funnel and mixed

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lightly until it was completely dissolved. After, 20 mL of dichloromethane was added and shaken

lightly. To make sure most of the caffeine went into the organic (DCM) layer, the solution was

left to separate for approximately 10 minutes. If an emulsion formed, 1 g of sodium chloride was

added and the separation time was increased to 15 minutes. Repetition of this step was performed

until the emulsion disappeared. The organic layer in the separatory funnel was transferred to a

vile and stored at 4°C.

For coffee and tea, 25 mL of each sample was measured and transferred into separatory funnel.

Next, 25 mL of dichloromethane (DCM) was added into the funnel and shaken lightly for 3

minutes to prevent the formation of an emulsion. To make sure most of the caffeine was

transferred from the drink to the organic layer, it was left to separate for 10 minutes. If an

emulsion formed, 1.5 g of sodium chloride was added into the mixture and shaken lightly for

another 2 minutes. The solution was left to separate for another 5 minutes. Repetition of this step

was performed until the emulsion disappeared. The organic layer in the separatory funnel was

transferred to a vile and stored at 4°C.

Crystallization and sample solution

The vile was connected to the rotary evaporation apparatus with an adapter to remove the

organic solvent. The temperature was set to 40oC and the sample was spun at an appropriate

speed. Once the DCM was completely evaporated, the purified caffeine crystals were suspended

in 5 mL of deionized water.

UV absorption

The UV-Vis Spectrophotometer was used to generate the standard curve of pure caffeine at 273

nm and observe the spectrum at that same wavelength. Every sample was scanned from 190-350

nm using quartz cuvettes to make sure there weren’t any visible impurity peaks. The maximum

absorption of caffeine in each sample appeared at 273 nm.

Method II: High Performance Liquid Chromatography

Preparation

Caffeine standards containing 100, 150, 200, and 250 ppm caffeine in distilled water were

prepared. The mobile phase, 60/40 (V/V) MeOH/H2O was prepared using reaction grade

methanol and distilled water. The mobile phase was then degassed and filtered.

To prepare the HPLC in Chemical Sciences 404, the machine was allowed to warm up for three

minutes. After, the lines inside the machine were purged with mobile phase for three minutes.

After the lines were purged, the pump was turned on. Mobile phase was pumped through the

lines for 20 minutes, allowing the equipment to equilibrate. Next, the detector was calibrated

using the auto-zero function. Between each injection, the lines of the machine were rinsed for 20

minutes using mobile phase.

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Isocratic Flow

The initial parameters used for each run trial used an isocratic flow of 60/40 MeOH/water at a

flow rate of 1.00 mL/min, C18 column, UV/Vis detection at 253 nm, and an attenuation of 6.

Useable data was not obtained from this study. The following documents the steps taken to

optimize the HPLC conditions in an attempt to collect useable data.

First, the attenuation on the machine was turned down because the differences in the peak height

of each standard were minimal. Adjusting the attenuation of the machine turns down the

sensitivity of the detector. Turning down the attenuation should have lowered the peak height of

the caffeine standard and provided data that showed a correlation between caffeine concentration

and peak height [15]. Adjusting the attenuation failed to lower the height of the peaks and

minimal differences in the peak heights of each standard were still observed.

The flat tops on that were observed on top of each peak also suggests that the samples are too

concentrated for the detector sensitivity. Additionally, a decrease in peak height was observed as

the concentration of the standard increased, suggesting that the caffeine standard was

aggregating in the column. The next step that was taken to optimize HPLC conditions was

sample dilution. Each sample was diluted 10x using distilled water [15]. After the samples were

diluted 10x the flat tops on the peaks were still present, an inverse correlation between

concentration and peak height was still observed, and no useable data was obtained. Lastly,

gradient flow HPLC was attempted to help minimize caffeine aggregation in the column.

Gradient Flow

Gradient HPLC was conducted using water and methanol under the following conditions: 95%

H2O/5% MeOH to 40% H2O /60%MeOH over 20 minutes and back to 95% H2O /5% MeOH

over an additional 20 minutes.

This method also failed to provide useable data. The following measures were taken to try

optimizing HPLC conditions for gradient flow.

Many peaks were present on the chromatograph, suggesting that the column was dirty. To try

cleaning the C18 column, acetonitrile was used to flush the column for 45 minutes. Acetonitrile

is known to help regenerate the C18 inside the column, by pulling off excess molecules off the

column. Acetonitrile is extremely polar and does an excellent job cleaning the column [16].

After running the acetonitrile through the column, the initial gradient that was set up was run

twice to help prepare the column for the caffeine standard. Cleaning the column with acetonitrile

should have cleaned the column and eliminated many of the impurities that were observed in the

original gradient flow chromatograph. This method actually increased the amount of impurities

that were detected, yielding more data that was not useable.

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Lastly, the column on the machine was switched out with a newer column. After preparing the

column, unusable results were obtained again.

Chromatographs from the initial isocratic flow, the initial gradient flow, and the gradient flow

after the machine was cleaned with acetonitrile can be found in the appendix.

Results

Method I: Caffeine Extraction

Figure 3. UV Spetrum for 1 mg/L, 1.45 mg/L, 2.2 mg/L and 2.9 mg/L caffeine standards at 253

nm.

The line of best fist from figure 3 was calculated using Microsoft Excel. Equation 4 shows the

formula for the standard curve.

(4)

𝐴 = 0.5142 ∗ 𝑐 − 0.0109

where A is the absorbance of the sample and c is the caffeine concentration in mg/L.

Table 1. The measured UV absorbance of each sample at 273 nm.

Sample 1/40 dilution 1/200 dilution

Pepsi 0.438

Decaffeinated Pepsi 0.108

Mtn. Dew Energy Drink 0.415

Red Bull 0.501

Monster Energy 0.532

Monster Energy Unleaded 0.823

Great Value Black Tea Decaf 0.203

Irish Black Tea 3.418

half calf 1.66

Columbian coffee 2.578

Decaffeinated coffee 0.472

0.478 0.7041.253 1.404

y = 0.5142x - 0.0109

R² = 0.957

0

5

0 1 2 3 4 5

Ab

sorp

tio

n

mg/L water

Caffeine Standard Concentration vs

Absorbance

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Equation 4 was used to calculate the concentration of caffeine in each sample, using the

absorbance values listed in table 1. The results of these calculations are displayed in table 2.

An overall scan (from 190nm to 350nm) was performed to make sure there weren’t any

impurities. All of the peaks are identified by the software and the peaks labeled “1” are the ones

observed at 273nm.

Figure 4 Overall scan for the samples ranging from 190nm to 350nm. (From highest to lowest

peak at 273nm) the red peak represents the irish black tea, the second red peak represents the

Columbian Coffee, the black peak represents the Half Caff, green peak represents standard

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curve, blue peak represents decaffeinated coffee, and the second black peak represents

decaffeinated Great Value black tea.

Table 2. The calculated concentration of caffeine in each sample.

Sample [Caffeine](mg/L)

Pepsi 4.21

Decaffeinated Pepsi 1.12

Mtn. Dew Energy Drink 19.98

Red Bull 24.01

Monster Energy 25.46

Monster Energy Unleaded 7.82

Great Value Black Tea Decaf 1.83

Irish Black Tea 29.29

half calf 14.27

Columbian coffee 22.11

Decaffeinated coffee 4.12

Method II: HPLC

Discussion

Due to time constraints, no further attempts were made to obtain a useable chromatograph from

HPLC. If more time were allotted, the HPLC machine would have been rinsed several additional

times using acetonitrile. If these attempts failed, the machine would then be professionally

serviced. According to the service-date sticker, the HPLC machine is in need of being

professionally serviced. The presence of flat peaks after steps were taken to optimize HPLC

peaks suggests that the bulb inside the machine is ready to be replaced. After the machine is

professionally serviced, the next step would be determination of the optimal flow rate.

Table 3. The comparison of caffeine concentration taken from the samples in the lab data versus

the concentration reported by each company.

Company reported data experimental data

[caffeine]

g/L

amount of

caffeine

mg

[caffeine]

g/L

amount of

caffeine

mg

Pepsi 0.11 38 0.42 149.438872

Pepsi caffeine free 0.00 0 0.11 39.5764802

Mountain Dew

Kickstart

0.16 92 2.00 1122.481928

Red Bull 0.32 80 2.40 600.6890605

Monster 0.34 160 2.55 1204.868737

Monster Unleaded 0.00 0 0.78 370.1336675

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When comparing the lab data with company reported data, the caffeine concentration in each

beverage seems to be significantly higher than what the company reported.

Figure 5. The concentration of caffeine reported by the company versus the concentration

gathered from the experimental data.

The serving size for each sample is shown in the table and figure below,

Table 4. Sample serving size and caffeine amount.

serving

size

(ml)

caffeine

(mg)

Pepsi 355 149.44

Pepsi Caffeine-Free 355 39.58

Mountain Dew

Kickstart

562 1122.48

Red Bull 250 600.69

Monster 473 1204.87

Monster Unleaded 473 370.13

Great Value Black Tea

Decaf

100 7.31

Irish Black Tea 100 117.15

half calf 100 57.09

Columbia coffee 100 88.45

decaff coffee 100 16.50

0.11 0.00 0.16 0.32 0.340.00

0.420.11

2.002.40 2.55

0.78

0.00

0.50

1.00

1.50

2.00

2.50

3.00

conce

ntr

atio

n(g

/L)

Comparison of labeled data and experimental data

(concentration)

company reported

lab

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The amount of serving size in the soft drinks and energy drinks are determined by each 12 oz.

can. For tea, the serving size is measured as one tea bag in 100 mL of water and for coffee, it is

2.5 g of coffee in 100 mL of water.

Figure 6. The amount of caffeine for each serving size gathered from the experimental data.

According to figure 6, the monster energy drink has the greatest amount of caffeine per one

serving among all of the other beverages, and great value decaffeinated black tea appears to be in

correlation with the federally regulated amount of caffeine present to be considered

decaffeinated, however, monster unleaded, which is advertised as caffeine free shows an

adequate amount of caffeine in the experiment.

Conclusion

Caffeine is a central nervous system stimulated that provides an increase in energy by inhibiting

the adenosine receptors. A consistent intake of caffeine can lead to various side effects such as

irritability or depression; for these reasons and others, people prefer to drink decaffeinated

beverages and teas. From our experiment, we were able to determine that the only decaffeinated

beverage that correlates with the federal regulation (caffeine content must not exceed .10%) is

Great Value black tea.

0

200

400

600

800

1000

1200

1400

pepsi pepsi

caffeine

free

mountain

dew kick

start

red bull monster

energy

monster

unlead

Great

Value

Black

Tea

Decaf

Irish

Black

Tea

half calf columbia

coffee

decaff

coffee

amo

unt

of

caff

eine(

mg)

sample

Amount of caffeine for each serve of the drinks

13

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Appendix

Figure A1. Chromatograph of 100 ppm caffeine in distilled water from isocratic flow of 60/40

MeOH/water at a flow rate of 1.00 mL/min, C18 column, UV/Vis detection at 253 nm, and an

attenuation of 6.

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Figure A2. Chromatograph of 100 ppm caffeine in distilled water from gradient flow of 95%

H2O/5% MeOH to 40% H2O /60%MeOH over 20 minutes and back to 95% H2O /5% MeOH

over an additional 20 minutes.

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Figure A3. Chromatograph of 100 ppm caffeine in distilled water from gradient flow of 95%

H2O/5% MeOH to 40% H2O /60%MeOH over 20 minutes and back to 95% H2O /5% MeOH

over an additional 20 minutes, after the machine was cleaned using acetonitrile.