Rodney C.

Salazar October 27, 2018

CHM622M October 27, 2018

High Performance Liquid Chromatography of Caffeine and Benzoic Acid in Soda

Experiment No. 7
Group 3

Abstract

HPLC or High Performance Liquid Chromatography is a fast and efficient way for separation and
quantification of components of foods. Caffeine and Benzoic acid present in our beverages as food
preservatives can be analyzed by HPLC technique. Using Agilent 1200 HPLC system caffeine and benzoic
acid was determined on a soda sample. Proportionality constant obtained from system suitability test was
used to compute for concentration of both analytes. Amount of caffeine for every serving (330 mL) was
determined to be 1.89  0.022 mg. Benzoic acid was not detected on the sample.

I. INTRODUCTION

HPLC is a chromatographic technique wherein mobile phase is a liquid, and either solid or
another liquid stationary phase. It was an improved liquid chromatography, utilizing high
pressure instead of gravity in forcing eluent through column thereby has an advantage of faster
analysis time. HPLC includes solvent reservoir, pump, injector, column and detector (see Figure
1). Column which is usually made of stainless steel, filled with stationary phase with 3-10 µm
particles size, the heart of HPLC analysis where separation of analytes occurs. Molecules of
analytes are being absorbed and desorbed at stationary phase, since different molecules have
different affinities to the stationary phase, separation occurs as molecules of analytes are swept
by mobile phase.

Figure 1. Schematic Diagram of Basic

HPLC Configuration.
HPLC is becoming a method of choice for detecting or analyzing food analytes. Methods that
employs stepwise approach that removes sample from matrix and then separates analytes of
interest and individually resolves them on chromatographic column. Thus, HPLC has a major
role in ensuring food safety. Caffeine and Benzoic acid both food additives can be found on
soda and can be analyzed by HPLC. Caffeine (C8H10N4O2) as the most consumed legal drug
and has pharmacological effects, such as stimulating the central nervous system, eliminating
drowsiness, and increasing concentration is commonly added to soft drinks, typically, from 30 to
40 mg of caffeine per 12 oz (330mL) serving. Meanwhile, benzoic acid (C6H5COOH) prevents
chemical decomposition and prevents growth of harmful bacteria in foods.

II. METHODOLOGY

A. MATERIALS

 Sample
Soda (Pepsi carbonated drink in a can).

Figure 2. Soda sample used in the experiment. At the left

showing the brand and at the right showing the ingredients
of the product.

 Mobile phase
0.5% Phosphoric acid in 40% aqueous methanol.

 Caffeine Standard
HPLC grade, anhydrous, ≥99.0%

 Benzoic acid Standard

HPLC grade, anhydrous, ≥99.0%

B. INSTRUMENTATIONS

Instrument used was Agilent 1200 HPLC System equipped with auto-sampler,
vacuum degasser, quaternary pump and variable wavelength detector. The
column was Agilent ZORBAX Eclipse XDB columns – C18 (reversed phase
column) with length of 4.6 x 150 mm and particle size of 5 µm.

C. PROCEDURE

 Preparation of Sample
Soda sample was transferred to beaker and heated to expel CO2. The solution
was filtered and cooled to room temperature. An aliquot of 10.00 mL was
pipetted out and transferred to 100 mL volumetric flask.

 Preparation of Standard Solution

12.5 mg of Caffeine was dissolved to 100 mL resulting to 124 mg/L caffeine

concentration wherein impurities was already accounted. For Benzoic acid
standard, 5.7 mg was dissolved to 50 mL resulting 56.4 mg/L solution of benzoic
acid. A 5.00 mL and 1.00 mL of caffeine and benzoic acid standards respectively
was mixed and diluted to 100 mL. The mixed solution has final 6.2 ppm caffeine
and 0.564 ppm benzoic acid concentration, it was sonicated to remove dissolved
gas. The solution was used for both system suitability test and as a standard.

 Determination of Caffeine and Benzoic Acid

The mixed solution was filtered and transferred to vial. HPLC system was set to
the following parameters:

Injection volume: 20 µL
Flow rate: 1mL/min
Wavelength: 254 nm
Elution condition: Isocratic, 0.5% Phosphoric acid in 40% aqueous methanol

Retention time and peak area of the standards were determined. Sample solution
was filtered and transferred to vial to which a triplicate measurement was
performed. Chromatogram of samples was compared with standards’
chromatogram to determine concentration of each analytes on the sample
 III. RESULTS

Figure 3. Trials of System Suitability Test.

Mixture of caffeine and benzoic acid was
used to test system suitability. Two peaks
were observed 2.5 min and 4.9 min for each
trial (top and bottom).

Figure 4. Chromatogram of Sample Trial 1. Figure 5. Chromatogram of Sample Trial 2.

Figure 6. Chromatogram of Sample Trial 3.

 Samples were prepared at triplicate (see Figure 4, 5 and 6). Peaks for caffeine were
observed ( 2.5 min) on all trials but no peaks were observed for benzoic acid.
Amount of caffeine per serving (330 mL as per label) was determined to be 1.89 
0.022 mg (see Figure 7).

Trial Volume Mother Peak Area Proportional Caffeine Amount of Average SD, %
of liquor ity constant, Concentra caffeine amount of mg RSD
Sample, volume, area/ppm tion, ppm per caffeine
mL mL Caffeine serving, per
mg serving,
mg
1 10.0 100.0 250.560 5.773272 1.90518
2 10.0 100.0 249.556 434 5.750138 1.897546 1.89 0.022 0.012
3 10.0 100.0 245.036 5.645991 1.863177
Figure 7. Sample information and result on determination of caffeine in soda sample. Serving volume was based from
label on can (330 mL).

IV. DISCUSSION AND CONCLUSIONS

Individual retention times for both caffeine and benzoic were determined to be at 2.5 min and
4.9 min respectively. System suitability test (SST) was performed (see Figure 1), no peak was
detected on benzoic acid, however peak areas of caffeine obtained 2691.67  4.70 which
passed the repeatability limit of  1.0 % set by FDA Center for Drug Evaluation and Research
(CDER). A peak area of 2691 (tr=2.5 min) was set to contain 6.2 ppm caffeine Since no peaks
were observed on SST for benzoic acid the prepared solution was probably at detection limit of
the instrument.

Determination of Caffeine and Benzoic acid on sample was performed in triplicate. Other
component was present at the solution causing unresolved peak of caffeine. Proportionality
constant obtained from SST was used to determine caffeine concentration on sample (k=434
area/ppm Caffeine) assuming that the other variables e.g. flow rate were constant throughout
the experiment.

The results of the experiment coincide with the label of the product in which there are presence
of caffeine and absence of benzoic acid (see Figure 2).
 V. REFERENCES

The Application of HPLC in Food Analysis. (n.d.). Retrieved from

https://www.foodsafetymagazine.com/magazine-archive1/aprilmay-2002/the-application-of-hplc-
in-food-analysis/
Fajara, B. E., & Susanti, H. (2017). HPLC determination of caffeine in coffee beverage. IOP
Conference Series: Materials Science and Engineering,259, 012011. doi:10.1088/1757-
899x/259/1/012011
Franco, F., Jr. (2016-2017). CHM622M/P Advanced Analytical Chemistry Laboratory Manual
(Instrumental Analysis).

(n.d.). Retrieved from https://pubs.acs.org/subscribe/archive/tcaw/10/i09/html/09dong.html