Joshua S.

De Guzman BSChE-IV
Rizella C. Galban September 16, 2021

DETERMINATION OF CAFFEINE IN SODA USING HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)
Abstract

This experiment aims to determine the levels of caffeine concentration in soft drinks.
Quantitative estimation of caffeine concentration in drinks was performed by a High
performance liquid chromatography (HPLC). The experiment resulted with the highest suggested
caffeine content of 375 mg/l for energy drink. It was found out that the energy drink Redbull had
the highest concentration of caffeine. With diet soda and regular soda, as shown in Figure 3 and
4, that they are only slightly higher as compared to pure caffeine.

Introduction

The presence and amount of caffeine found in common beverages can be measured using
HPLC. To determine the actual quantity of caffeine in a beverage requires generating a
calibration curve from known concentrations of caffeine. Using the calibration curve for pure
caffeine, the concentration of caffeine in a caffeinated beverage, such as soda, coffee, tea, or any
other “modern” caffeinated beverage, can be determined.

Caffeine is a stimulant that is commonly found in many foods and drinks that we
consume. Concerns exist about the potential adverse health effects of high consumption of
dietary caffeine, especially in children and pregnant women. Recommended caffeine intakes
corresponding to no adverse health effects have been suggested recently for healthy adults (400 –
450 mg/day), for women contemplating pregnancy (300 mg/day), and for young children age 4 –
6 years (45 mg/day).
 Figure 1

High performance liquid chromatography (HPLC) is a type of chromatography which is

very similar to liquid chromatography. In HPLC, however, the stationary phase column is more
tightly packed than in other types of liquid chromatography. In this lab, the column is packed
with C18 particles that are less than 10µm in diameter. The small diameter of the particles allows
unprecedented resolution and high efficiency. Since the particles in the column are so small, it is
necessary to pump the mobile phase through the column at a very high pressure. The pump keeps
a precise flow rate so that the positions of the peaks in time can be used to identify the species in
a sample. This is done by comparing the chromatographs of prepared standards of the particular
species to be determined. The common peak is an indication of the standard. A small sample
(100Lx2) is injected into the injector port where the mobile phase moves it through the
column. Each component, being different in physical composition, will move at different rate
through the C18 column. Thus, the components will be separated according to the size and shape
of the molecules. The smallest and least hindered molecules will be eluted first since it is easiest
for them to pass through the finely packed column. As each set of molecules elutes from the
column, a detector (most often UV) recognizes it and records a peak. The area of this peak (in
relation to the area of other peaks) is proportional to the concentration of that particular species
in the sample. The identity can also be found by comparing the sample peaks to
standards. Identical substances (peaks) will have identical retention times.

Related literature

Nishitani and Sagesaka (2004) developed an improved HPLC analytical method for
simultaneously determining caffeine and the eight catechins as well as other phenolic compounds
in tea. The proposed method provided additional ability to analyze phenolic compounds when
compared with former HPLC methods. This procedure was based on an improved reverse-phase
ODS column operated at 4°C, a binary gradient elution system of water-methanol-ethylacetate-
phosphoric acid, and a photodiode array detector. The quantitative measurement of eight
catechins and caffeine confirmed the validity of this proposed method. The detection limits of
these analytes ranged from 1.4-3.5 ng per injection volume. The recovery rates of the analyses
were in the range of 96-103%. The caffeine contents of Sencha, Matcha, Gunpowder, Tie Kuan
yin, and Darjeeling determined in this study were 2.94±0.007, 3.62±0.005, 2.61±0.059,
2.51±0.019, and 3.24±0.016% (dry weight), respectively (Nishitani and Sagesaka 2004).

Pura (2001) modified a HPLC method for determining caffeine and theobromine contents
in aqueous cocoa extracts. Instead of directly injecting the extracts on the column, the improved
method can successfully remove the interfering cocoa pigments by passing them through a Sep-
pak C18 cartridge which was also used to separate the theobromine and caffeine. This method
enhanced the efficiency of the column and prolonged its life. After this treatment, the recoveries
of caffeine and theobromine were 98.0-100.1 and 97.8-100%, respectively. The modified method
displayed good resolution and sharp peaks on chromatograms that favoured correct
determination of theobromine and caffeine.

HPLC method with diode array detection developed in our laboratory for the quantitative
determination of caffeine in carbonated beverages provides stable retention times and a detection
limit of 0.01 mg/L for a signal-to-noise ratio of 3. The mean recovery of caffeine was
99.25%.Wanyika discussed the levels of caffeine in certain coffee (Nescafe, africafe, Dorman's)
and tea (chai maramoja, kericho gold, sasini, final's premium) brands found in the Kenyan
market were determined using High performance liquid chromatography (HPLC) and UV/ Vis
spectrophotometer (Shimadzu) at 274 nm which gave a concentration of 471 .73 ± 1 96.92 ppm
(Nour, et. al)

Farah (2006) investigated the relationship between the Arabica coffee cup quality and the
contents of sucrose, caffeine, trigonelline, and chlorogenic acids. The researchers applied
reverse-phase HPLC analysis to determine each compound. Sucrose was analyzed by using 80%
acetonitrile and 20% water as the mobile phase and a refractive index detector. For analyzing
caffeine, the UV detector was set at 272 nm. The mobile phase was composed of 60% water and
40% methanol. The results demonstrated that the caffeine content was the highest in the highest
quality sample and the lowest content was found in the poorest quality sample. However,
trigonelline and 3,4dicaffeoylquinic acid gave a better indication of high quality coffee.

Chen and Wang (2001) analyzed the level of artificial sweeteners (sodium saccharin,
aspartame, acesulfame-K), preservatives (benzoic acid, sorbic acid), caffeine, theobromine, and
theophylline in carbonated cola drinks, fruit juice drink, fermented milk drink, preserved fruit,
and one pharmaceutical preparation by an ion exchange chromatography method. Analytes were
separated using an anion-exchange analytical column maintained at 40°C and detected by
wavelength-switching ultraviolet absorption. The detection limits ranged from 4-30 ng/mL for all
analytes. The average recoveries for samples ranged from 85 to 104%. In addition, the data
obtained from this method were in good agreement with those determined by reference HPLC
procedures.
Objectives

 The purpose of this lab is to determine the amount of caffeine in a sample of soda using
HPLC.
 To demonstrate High Performance Liquid Chromatography.

Experimental
Energy Drink (Redbull)
Materials
 100ml Beaker x2 Spatula
 100ml Graduated Cylinder Syringe w/45um Filter x6
 Delivery Funnel 100ml Volumetric Flask x5
 HPLC Vial x7 500 ml Volumetric Flask
 10ml Pipette Weigh Boat
 Pipette Pump Acetonitrile
 Reversed Phase Column Red Bull
 Solvent Filter Ultra-Pure Water
 500ml Solvent Reservoir

Procedure:
 Measure com Acetonitrile and transfer it to a 500mL Volumetric Flask.
 Fill flask with Ultra-Pure Water and check the meniscus at eye-level.
 Insert the flask to mix.
 Set up the vacuum filter and switch it on.

1. 300ml filtering cup

2. Filtering head
3. Filtering membrane (60mm)
4. Clamp
5. 1000ml conical flask
 Degas the Mobile Phase.
 Transfer the Mobile Phase into a 500ml Reagent Bottle.
 Invert the flask to mix.
 Put this into the mobile phase reservoir and allow it to flush through the HPLC for 45
minutes.
 Open the purge valve.
 Prepare an extra batch of Mobile Phase to be used during the preparation of the Caffeine
Standards.

Preparation of Caffeine Standards

 Weight exactly 0.5g of Pure Caffeine.
 Dissolve and Transfer it in a 500ml Volumetric Flask.
 Fill this to the mark with Ultra-Pure Water and check the meniscus at eye-level.
 Invert the flask to mix it
 If the Caffeine doesn't easily dissolve, at a magnetic stir-bar, stir it with a magnetic
stirrer.
 This makes a 1000ppm Caffeine standard solution.
 Filter each of the standards into HPLC vials using a Syringe with 45pm Filter.
 Pour the RedBull into a beaker.
 Degas it using a Sonicator for 10 minutes.
 Transfer the RedBull sample into a HPLC Vial, filtering it using a syringe w/ filter.
 Fill a HPLC Vial with Mobile Phase and label it "Blank".
 Place each of the vials inside the sample holder and run the analysis.

Regular soda and Diet soda

Sample preparation
 In order to identify that there is indeed caffeine in the beverage we need to run a pure
sample of caffeine to use pure sample caffeine took a small of it and put it on motor and
pestle grind up so it will dissolve faster.
 Put it on about 3 mL of mobile phase which is used in instrument using a pipette, mix it
up so that it was dissolve fully.
 To ensure its high quality enough to run through the instrument, filter it.
 Use a 3 ml syringe and filter patch fill the syringe with beverage sample (regular soda,
diet soda) and mobile phase mixture.
 In HPLC it need a small about so get about 1 to 2 ½ ml and attach the filter patch into the
syringe sample and transfer it into a new test tube.
  Put it in the flask and shake it to combine it and transfer to a new test tube and repeat the
procedure for filtering and through the filter pad and covering it.

Set-up

 Inject the pure caffeine into the system make sure that you don't capture bubble in the
syringe.
 Inject it up to three times to run it through the system press auto zero in the first box then
press auto zero to the second box.
 Switch the load position to the injection position go over to the computer.
 The scan takes between 2 to 5 minutes during this time take the syringe used to inject the
pure caffeine into the sample and rinse it out with the motor phase.
 After about 5 minutes switch off the load position so you're ready for the next sample
repeat the set up for the next sample beverage.
 Each of the defectives has returned to the baseline and no longer rising go to the
acquisition and stop.
 Stop the instrument and complete the scan.
 After the run of the caffeine the beverage sample such as regular soda and diet soda was
continued to run.
 After each sample was analyzed printed the graphs so it can analyze to see if there is
indeed caffeine its sample.

To shut down HPLC

 Clean syringe with methanol (HPLC grade).

 Run methanol as a sample a few times.
 Run 100% methanol (HPLC grade) as a solvent for 5-10 minutes.
 Turn off pump, detector, and computer.

Results and Discussion

Energy Drink ( Redbull)
The caffeine content was found to be 375 mg/ L which is higher than the 320 mg /L as
stated on the can.

Regular Soda & Diet

 Fig.2 (Pure Caffeine) Fig.3 (Regular Soda)

Fig. 4(Diet Soda)

In the figure 3 and 4, it shows there that the Regular Soda there was a peak occurred at
3.13 and the Diet Soda had a peak of at 3.13. The sample such as Regular Soda and Diet Soda
are exactly similar to identify the caffeine. A pure sample shows in figure 2 that the Pure
Caffeine was run the peak at 3.123 ,therefore the regular soda and diet soda are successfully
identify that there was a caffeine in there.

Hazards
Chemical Hazards
Chemical hazard using chemical in lab is inevitable, but so can be injured or harm if the
chemicals are misused or mishandled.
 Caffeine -harmful if swallowed
 Methanol -highly flammable liquid and vapour
-Toxic if swallowed toxic in contact with skin
-Toxic if inhaled
-Cause damage to organs.
 Theobromine - toxic if swallowed
-Avoid contamination.
Physical Hazards
Physical hazard such as electrical safety hazard ergonomic hazard associated with manual
material handling and equipment use handling sharp objects and basic housekeeping issues.
 Radiation
 Exposure to hazardous chemicals.
 Accidental Spill- in the event of spill hazardous materials evacuate immediately, warn
other personnel, wear appropriate PPE, cleanup spill using appropriate spill kit if trained
 to do so contains fill material in study plastic bag or sealed drum apply a hazardous waste
label.

Conclusion

The experiment resulted with the highest suggested caffeine content of 375 mg/l for
energy drink. It was found out that the energy drink Redbull had the highest concentration of
caffeine. With diet soda and regular soda, as shown in Figure 3 and 4, that they are only slightly
higher as compared to pure caffeine.

As a conclusion, there are varying amount of caffeine in different beverages that we

consume. To our benefits, high performance liquid chromatography (HPLC) is an excellent
method to determine and to have awareness to those amounts of caffeine that we intake. After
all, caffeine is a drug that if not used properly may result adverse effects to our body.

References
https://www.scribd.com/document/388526953/HPLC-Determination-of-Caffeine-in-Soda?
fbclid=IwAR2cJsHtUxsX03zZoR85Ikjd2Mxsjb8A6QBGNyO-Ce9j1n4S3jC3Noxp-hQ

https://studylib.net/doc/7471197/high-performance-liquid-chromatography---hplc?
fbclid=IwAR2ImP56ROxsvnTcT0V6egjJi6uUASJkUJmTVb52iITBhEp1wRmF2mfGbNA

http://classes.kvcc.edu/chm250/HPLC%20Determination%20of%20Caffeine%20in
%20Beverages.pdf

http://bepls.com/?
fbclid=IwAR3aRoTbt0HsC2iNoSuiBYqgBCeMqtcPrVZ13XoG_FagHTr9YbEM9jrTc_E

https://www.hindawi.com/journals/jspec/2020/3716343/?
fbclid=IwAR2iqwLBlHJ7VCuRxJOTu72mtnjPe0t1szU2YfkfDaQ-iUMALG8Jb0Cs3f8

https://www.ajer.org/?fbclid=IwAR1zebgLPCj7gLb-
fe5VaiEm95mejq36APjN1akOCBMDrADbuPIHEjnuDAA

Video Reference
https://www.youtube.com/watch?v=o6Dq9Z59vlg