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Preservatives are chemical compounds that are used
in a wide range of applications to maintain overall
product quality.
1
Some preservatives act as antimicro-
bial agents, some act as antioxidants, and some can
perform both functions. Of the chemical compounds
commonly used as preservatives, many can be effec-
tively analyzed by high performance liquid chro-
matography (HPLC).
2
Because preservatives include
a number of different compound types, there are a
variety of HPLC stationary phases, mobile phases,
and detectors that can be used.
Chemical preservatives kill or prevent the growth of
microbes either by changing the microbes’ environ-
ment or by reacting directly with them.
3
Antimicrobial
compounds include organic acids, benzoate and
sorbate salts, sulfur dioxide and sulfites, nitrites,
propionates, and parabens. Organic acids, such as
acetic acid and citric acid, can be used to control
the pH of a product. For example, in food products
these acidulants can lower the pH out of the opti-
mum pH range for bacteria, yeast, and/or molds.
Organic acids such as malic acid and citric acid can
be found naturally in fruits, oxalic acid can be found
in spinach and rhubarb, and tartaric acid can be
found in grapes.
Using HPLC, concentrations of these preservatives
can be monitored. However, analyzing polar organic
acids can be difficult on conventional reversed phase
columns, even when using low pH, highly aqueous
mobile phases to suppress ionization of the acid
molecules and maximize retention. The Ultra
Aqueous C18 column provides enhanced retention
and selectivity for challenging applications such as
this. The novel bonding chemistry used for this phase
allows the alkyl groups to remain extended, even in
highly aqueous mobile phase, preventing the chain
folding that occurs with conventional C18 phases.
Therefore, stable and reproducible retention is pos-
sible even with 100% aqueous mobile phases. Notice
the excellent retention for a series of organic acids
using the Ultra Aqueous C18 column and UV detec-
tion (Figure 1).
Products containing fats and oils are prone to lipid
oxidation, which can limit shelf life by promoting
off-flavors, off-odors, and color changes. To inhibit
lipid oxidation, antioxidants can be added to the
product. Phenolic antioxidants include butylated
hydroxyanisole (BHA), butylated hydroxytoluene
(BHT), propyl gallate (PG), and
tert
-butyl hydro-
quinone (TBHQ). These four, plus the tocopherols,
are the primary antioxidants found in foods and bev-
erages produced in the U.S. Phenolic antioxidants,
such as BHT, are regulated by the US Food and Drug
Administration (FDA), and can be added to many
products at levels up to 200ppm, based on the fat
content.
Phenolic antioxidants can be analyzed by reversed
phase HPLC using a Pinnacle II
™
C18 column and
an acidified mobile phase. As with the analysis of
organic acids, an acidic mobile phase is used to
suppress ionization of the analytes. The HPLC sepa-
ration of BHA, BHT, PG, and TBHQ using UV detec-
tion at 280nm shows how effectively these com-
pounds can be separated using the Pinnacle II
™
C18
column (Figure 2).
“Natural” antioxidants, such as tocopherols and
tocotrienols, are used to inhibit lipid oxidation and
to promote general health in the consumer. These
compounds are found naturally in products such as
fats and oils. When used as additives, however, they
are regulated. Antioxidants such as tocopherols can
be challenging to analyze, because they readily oxi-
dize when exposed to light or oxygen. The analysis
of four tocopherols by normal phase HPLC, using a
Pinnacle II
™
Silica column, shows how effectively
these positional isomers can be separated (Figure
3). These compounds can be quantified using either
fluorescence or UV detection.
HPLC is a powerful tool for analyzing preservatives
in a wide range of consumer products. One of its
advantages is that many times only minimal sample
preparation is required. Chromatographic tech-
niques allow analysts to separate preservatives from
other compounds in the sample matrix, improving
the overall quality of the results. For analyzing
organic acids, the Ultra Aqueous C18 column is the
perfect choice, offering superior retention and
reproducibility for polar compounds, even when
using highly aqueous mobile phases. Pinnacle II
™
C18 and silica HPLC columns are excellent choices
for analyses of preservative compounds such as
parabens, benzoate and sorbate salts, phenolic
antioxidants, and tocopherols. Pinnacle II
™
columns
also are available with C8, phenyl, and amino sta-
tionary phases.
1. Fennema, Owen R.
Food Chemistry
(1996), Marcel Dekker,
New York.
2. Nollet (ed.),
Food Analysis by HPLC
(2000), 2nd edition,
Marcel Dekker, New York.
3. Foulke, Judith E. "A Fresh Look at Food Preservatives" in
FDA
Consumer
(October 1993), US. Food & Drug Administration.
by Rebecca Wittrig, Ph.D., Food, Flavors, and Fragrances Innovations Chemist
HPLC Analysis of Preservatives
Using Ultra Aqueous and Pinnacle
II
™
Columns
✔
Minimal sample preparation saves time.
✔
Ultra Aqueous C18 column provides superior retention and reproducibility for polar
compounds.
✔
Pinnacle
II
™
Silica column resolves tocopherol isomers.
Figure 1
An Ultra Aqueous C18 column shows excellent retention of organic acids typically found
in foods, beverages, personal care products, etc.
LC_0140
1
2
3
4
5
6
0
1
2
3
4
5 min.
Peak List:
Conc. (µg/mL):
1. malonic acid
500
2. lactic acid
500
3. acetic acid
1000
4. citric acid
1000
5. succinic acid
2000
6. fumaric acid
10
Sample:
Solvent:
HPLC-grade water
Inj.:
10µL
Column:
Ultra Aqueous C18
Catalog #:
9178565
Dimensions:
150 x 4.6mm
Particle size:
5µm
Pore size:
100Å
Conditions:
Mobile phase:
50mM potassium phosphate, pH 2.5:
acetonitrile (99:1)
Flow:
1.5mL/min.
Temp.:
25°C
Det.:
UV @ 210nm
Questions?
Contact the industry’s best Technical Service
Team at 800-356-1688 or 814-353-1300, ext.
4, or contact your local Restek representative.