A GUIDE TO CAROTENOID ANALYSIS IN FOODS

28 A Guide to Carotenoid Analysis in Foods
The possibility of degradation varies with different
stationary phases (adsorbents) and increases as the
chromatographic process is prolonged. Rechromatography
of an impure fraction may sometimes be necessary,
extending the analysis time and increasing the
danger of carotenoid decomposition.
Common adsorbents are magnesium oxide–
Hyflosupercel, activated or not, and in different proportions
(e.g., 1:1 or 1:2), and deactivated, neutral
alumina. Magnesium oxide was found to be least likely
to cause carotenoid alteration (Tanaka et al. 1981,
Rodriguez-Amaya et al. 1976b), although the contrary
was observed with magnesium oxide activated
according to the Association of Official Analytical
Chemists (Rouchaud et al. 1984). Isomerization, degradation,
or both are more likely to happen in an alumina
column, so magnesium oxide–Hyflosupercel
should be the first choice. Magnesium oxide is usually
diluted with celite or Hyflosupercel to lower adsorption
affinity and thus prevent irreversible adsorption
of polar carotenoids. Also, when used alone,
magnesium oxide is sufficiently basic to catalyze aldol
condensation and cause polymerization of acetone.
Silica gel is not a popular adsorbent because its inherent
acidity may cause carotenoid isomerization and
degradation (Taylor 1983, Tanaka et al. 1981,
Rodriguez-Amaya et al. 1976b). Many solvent combinations
have been tried, but the most common is
petroleum ether or hexane containing increasing
amount of diethyl ether and acetone.
Commercially available adsorbents are known to
vary in their adsorptive properties, and even minute
amounts of impurities, especially polar substances,
alter the solvent’s eluting strength. Although variations
are greater between brands, lot-to-lot differences
also exist, and these variations tend to be
greater in developing countries where quality control
of laboratory materials may not be as rigorous. Therefore,
a laboratory’s first attempt may not duplicate
reported separation, and adjustment of the chromatographic
conditions may be necessary.
The adsorption capacity can be increased by
activating the adsorbent for 4 hours at 110 o C or decreased
by increasing the proportion of Hyflosupercel
(e.g., magnesium oxide–Hyflosupercel, 1:2). The
composition and volumes of the eluting solvents should
also be optimized. For example, to increase the separation
of α- and β-carotene, activated magnesium
oxide–Hyflosupercel (1:1) can be used, the volumes
of the initial solvents (i.e., petroleum ether and 1%
ether in petroleum ether) can be increased, or both
can be done. Because carotenoids are colored, alterations
in the eluting solvents can be made without
resorting to the collection and scanning of numerous
fractions, which would be necessary for colorless
compounds.
To separate cis and trans isomers by OCC, especially
of the provitamin A carotenoids, each fraction
separated by the magnesium oxide–
Hyflosupercel column is rechromatographed on a
smaller (dimensions depend on the amount of carotenoid)
calcium hydroxide column, using 0%, 2%, and
4% ethyl ether in petroleum ether to elute the isomers
of β-carotene and 10% and 20% acetone in
petroleum ether to elute the isomers of β-cryptoxanthin
(Godoy and Rodriguez-Amaya 1998, 1994,
Rodriguez-Amaya and Tavares 1992, Bickoff et al.
1949). Although quite laborious, this traditional method
is still considered the most effective and practical
way of separating isomeric mixtures of carotenoids
in quantity (Tsukida 1992).
In OCC, a column has to be packed for each
analysis. A definite improvement in HPLC is the possibility
of reproducible separations with a reusable
column, under controlled conditions, without undue
exposure to air or light. Reversed-phase HPLC on
C 18 columns is clearly the preferred mode. Reasons
for the popularity of the C 18 column are its weak
hydrophobic interactions with the analytes (thus it is
expected to be less destructive than the polar forces
in normal-phase OCC), compatibility with most carotenoid
solvents and the polarity range of carotenoids,
and wide commercial availability.
Many different C 18 reversed-phase materials are
available from different manufacturers and vary in
the degree of carbon loading, end capping, and the
nature of the bonded phase (i.e., monomeric or polymeric).
Lack of reproducibility is a persisting problem.
The properties and quality of the same kind of
column differ considerably between brands, between
batches of the same brand, and even within the same
batch (Pfander and Riesen 1995). Thus, some adjustments
are often needed when published methods
are adapted.
Most carotenoid separations have been carried
out with 5-µm C 18 spherical particles packed in a 250
× 4.6 mm column. Some laboratories are already using
shorter and narrower (narrow bore) columns,
smaller particles, and a C 30 stationary phase. Most
commercial reversed-phase columns are now end
capped to minimize polar interaction of the silanol
residues with the analytes and thus diminish tailing
and improve column reproducibility.
Monomeric phases are simpler to use and more
reproducible. Polymeric C 18 phases, on the other hand,
have been found to have excellent selectivity for structurally
similar carotenoids, as in the difficult separation
of geometric isomers of β-carotene (Craft et al.