CHEM 311L, Spring 2009
Lab #11 - HPLC: The Determination of Sweeteners and Additives in Diet Soft Drinks
Introduction and Background
High performance liquid chromatography (HPLC) is one of the most popular and widely used analytical 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). In this experiment, reversed-phase HPLC will be
used to identify and determine the concentrations of sweeteners and additives in a set of diet soft drinks.
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 called “reversed phase,” which is a type of partition chromatography. In
this method, stationary phase particles (usually SiO2) are coated with a covalently-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 bonded layer, they will take longer to pass through the column and elute later.
Relatively polar compounds will not interact as strongly with the stationary phase and will therefore elute earlier.
This mode works well for the separation of water-soluble 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 ionizable organic compounds (such as weak acids), the ionization of 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 some or all of the compounds to be separated are weak acids, 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 (used to make “cola” beverages), coffee beans, cacao beans (used to make chocolate), 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 colas). 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 three of these sweeteners, aspartame, saccharine, and acesulfame K. The table below
gives information on all five of these compounds.
One important aspect of chromatography (both GC and HPLC) is the detection method. A detector must be used to
visualize and quantify the compounds as the exit the column. In HPLC, a number of detection strategies are
commonly used, including molecular spectroscopy, electrochemistry, refractive index changes, and mass
spectrometry. 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 the five
compounds in Table 1 possible.
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