CHEM 311L

Quantitative Analysis Laboratory

Version 1.0

An HPLC Analysis of Sweeteners in Beverages

In this laboratory exercise we will perform a separation of the components of diet soft drinks
using reversed-phase High Performance Liquid Chromatography (HPLC). This will allow us to
confirm the presence of Caffeine, Benzoic Acid and the sweeteners Aspartame, Saccharine, and
Acesulfame K in the soft drink. Once separated and identified, the amount of each sweetener
present in the soft drink will be determined.

HPLC is one of the most popular and widely used chromatographic techniques today. This
method uses a solid or “bonded” stationary phase and a liquid mobile phase to separate mixture
components in time. The method yields information about the identity of mixture components
(qualitative information) and their concentrations (quantitative information).

”HPLC uses high pressure to force solvent through closed columns containing very fine particles
that give high-resolution separations. The HPLC system ... consists of a solvent delivery system,
a sample injection valve, a high-pressure column, a detector, and a computer to control the
system and display results.” (Quantitative Chemical Analysis, 7th Ed. by Daniel C. Harris;
Freeman) A column packed with very small particles increases chromatographic efficiency
dramatically. This is because diffusion in liquids is very slow, so small particles provide a large
surface area for equilibration of the analyte between the mobile and stationary phases of the
chromatographic system. However, high pressures are then needed to provide a reasonable flow
rate through the column.
A large number of possible stationary phase/mobile phase combinations exist for HPLC, each
defining a specific mode of HPLC. For example, adsorption mode HPLC uses a polar, solid
stationary phase such as SiO2 or Al2O3 and a nonpolar mobile phase, such as hexane or
chloroform to separate compounds by their polarity. Other modes, such as ion-exchange
chromatography and size exclusion chromatography separate compounds by other mechanisms.

One of the most popular modes of HPLC is reversed phase, which is a type of partition
chromatography. In this method, stationary phase particles (usually SiO2) are coated with a
chemically-bonded layer of some type of non-polar molecule. Commonly used bonded layers
include 18 and 8 carbon-long straight chain alkanes (C18 and C8) and phenyl groups. The mobile
phase is typically water mixed with some fraction of miscible, polar organic solvent (usually
Methanol, Acetonitrile, or THF). During the separation, analyte molecules partition between the
mobile phase and the bonded layer of stationary phase. Since relatively non-polar molecules will
dissolve more easily into the stationary phase, they will elute last. Relatively polar compounds
will not interact as strongly with the stationary phase and will therefore elute first. This mode
works well for the separation of Water-soluble, non-volatile organic compounds. Since these
compounds are not usually amenable to analysis by gas chromatography (GC), reversed phase
HPLC is an excellent compliment to that method.

When reversed-phase HPLC is used with ionized organic compounds, the ionization of the these
analytes must be limited or minimized. Otherwise, these compounds are simply too polar to be
retained by the non-polar stationary phase. In the case that the compound is a weak acid, the pH
of the mobile phase is controlled with a buffer. Ion-pairing reagents can also be used to partially
neutralize the charge of the analytes and allow them to be retained. In this experiment, the pH of
the mobile phase will be buffered at pH = 4.2 using Acetic Acid to allow two of the compounds
that are weak acids, Benzoic Acid and Aspartame, to be adequately retained and separated.

Soft drinks frequently contain a number of additives that affect the beverage’s taste and
characteristics. Caffeine, one popular ingredient, is a natural xanthine alkaloid stimulant that
exists in many plants as a natural insecticide, including kola nuts, coffee beans, cacao beans, and
tea leaves. In some cases, it’s extracted from the natural ingredients as part of the beverage-
making process (e.g. in coffee and tea and in cola beverages). In other cases, it’s added by the
manufacturer (e.g. in some citrus-flavored sodas). Often, even if Caffeine is naturally present, it
is elevated or controlled at a consistent level by the manufacturer (e.g. in many colas). Benzoic
Acid is also added to many soft drinks and other foods as a preservative against microbial
growth. Diet soft drinks often contain low calorie artificial (man-made) sweeteners in place of
sugar or corn syrup. We will analyze for Ceffiene, Benzoic Acid and three of these sweeteners,
Aspartame, Saccharine, and Acesulfame K in a soft drifnk. The table below gives information
on all five of these compounds.
Common names IUPAC name M.W. Structure
caffeine 1,3,7-trimethyl-1H-purine- 194.19
2,6(3H,7H)-dione

benzoic acid benzoic acid 122.12

aspartame N-(L-α-Aspartyl)-L- 294.30

(NutraSweet®) phenylalanine methyl ester
(Equal®)

saccharine 1,1-Dioxo-1,2-benzothiazol-3-one 183.18

(Sweet'N Low®)

acesulfame potassium 6-methyl-2,2-dioxo- 201.24

potassium oxathiazin-4-olate
(Ace K)

Finally, as the analytes exit the column, they must be detected by an appropriate detection
system. In this experiment, a single channel UV-Visible absorbance detector will be used. The
detector will be set to 254 nm, a wavelength where aromatic rings absorb light strongly, making
the detection of our five compounds possible.

Thus, we will separate each of our five analytes in a soft drink using a reversed-phase HPLC
methodology. Each component will be detected using a UV-VIS absobance measurement. The
signal response of the UV-VIS detector will allow us to determine the amount of each analyte
present, when calibrated against appropriate standards.
Pre-Lab Calculation
Suppose your analysis for the Saccharine standards yields the following data:

conc (M) peak area

1.00E-03 1.41E+06
5.00E-04 6.61E+05
2.00E-04 2.72E+05
1.00E-04 1.34E+05

What is the concentration of the Saccharine in your soft drink if your analysis gives a
chromatographic peak area of 3.66 x 105?
Procedure

1. Due to the limited time available for this lab, you’ll be provided with a solution containing
a mixture of all five compounds at known concentrations. Additionally, you’ll be provided
with the retention times of the pure compounds and a set of calibration data (peak area vs.
concentration for each compound), both determined earlier by the TA.

2. The TA and/or instructor will set-up the HPLC and start the mobile phase flow before
class. Approximately 30 minutes of run time is needed to establish a stable system
baseline, prior to any injections.

3. Obtain 4-6 diet beverages. Note the presence and absence of any of the compounds in
Table 1 in the list of ingredients on the beverage container.

4. Bring the beverages to room temperature and (if carbonated) degas them by letting them
stand for a few hours or by sonicating them. Filter ~10 ml of each using a syringe filter
and syringe into a clean, labeled vial.

5. Inject each of the filtered and degassed diet drink samples and the five standard mixture
into the HPLC one at a time.

6. Using the HPLC software, determine the retention times of each compound. Also,
integrate each chromatogram to determine the peak areas of each compound in each
standard or sample.

7. For each analyte, conduct a linear least squares analysis on the concentration and peak area
data for the standard mixtures. Give the slope, y-intercept, and associated standard
deviations for each analyte. (Attach a copy of a graph of the data to your assay sheet.)

8. Using these results, determine and report the concentrations of each of the five compounds
in the beverage you analyzed. An error estimate for each concentration should also be
provided.