A GUIDE TO CAROTENOID ANALYSIS IN FOODS

18 A Guide to Carotenoid Analysis in Foods
Absorbance
sorption peak, designated III, and that of the middle
absorption peak, designated II, taking the minimum
between the two peaks as baseline, multiplied by 100
(Britton 1995). In a few cases, such as ζ-carotene,
III is greater than II, thus the %III/II value is slightly
higher than 100 (Table 7). For conjugated
ketocarotenoids, such as canthaxanthin and echinenone,
the spectrum consists of a broad single maximum,
having no defined fine structure, thus %III/II
is 0.
Wavelength
Figure 11. Calculation of %III/II as indication of spectral fine
structure (%III/II = III/II × 100).
The absorption coefficient A1% of a carotenoid
1cm
(absorbance at a given wavelength of a 1% solution
in spectrophotometer cuvette with a 1-cm light path)
used in the calculation of the concentration also varies
pronouncedly in different solvents (Table 8).
Carotenoids in solution obey the Beer-Lambert
law—their absorbance is directly proportional to the
concentration. Thus, carotenoids are quantified spectrophotometrically.
This quantification, however, depends
on the availability of accurate absorption coefficients,
which are difficult to obtain. The procedure
normally involves weighing a small amount of the
carotenoid, typically 1 to 2 mg, with an accuracy of
±0.001 mg (Britton 1995). An accurate and sensitive
balance is required and the carotenoid should be absolutely
free from all contaminants, including residual
solvent. Moreover, complete dissolution of the carotenoids
in the desired solvent can be difficult. Thus,
some published values may have some significant
level of error or uncertainty (Britton 1995), and some
discrepancies can be noted in the values presented in
Table 8. Because authors choose different coefficients
for some carotenoids (in the same solvents),
this alone can account for a good part of the variations
in analytic results. For the accuracy of both opencolumn
chromatography and HPLC methods , reassessment
of the absorption coefficients is warranted.
Adsorption and Partition Properties
The chromatographic behavior of carotenoids bears
a definite relationship with their structures. However,
chromatographic data cannot be used as sole criteria
for carotenoid’s identity. Nevertheless, these data
serve as useful complementary information. In normalphase
open-column chromatography, the adsorption
affinity depends on the number of conjugated double
bonds, cyclization, and the presence of oxygen
substituents.
The influence of the double bonds is best illustrated
by the adsorption affinities of the acyclic carotenoids,
which elute in the sequence phytoene, phytofluene,
ζ-carotene, neurosporene, and lycopene. Comparing
monocyclic and bicyclic carotenes, δ-carotene elutes
before γ-carotene, and α-carotene elutes before βcarotene.
Cyclization decreases the adsorption affinity.
Thus, β-carotene is much more weakly adsorbed than
γ-carotene, which in turn elutes before lycopene.
The presence of oxygen substituents increases
adsorption, the extent of such increase depending on
the type, number, and location of the functions. This
is demonstrated in a silica thin layer developed with
3% methanol in benzene or 5% methanol in toluene,
in which all carotenes elute with the solvent front and
the xanthophylls distributed in the plate according to
the number and type of substituents present (Figure
12).
The hydroxyl group exerts a great influence on
adsorption; methylation, acetylation, and silylation
markedly reduce this effect. The adsorption affinity
of a carbonyl group is less than that of a free hydroxyl
substituent. The contribution of the functional groups
on adsorption affinity increases in the sequence
(Davies 1976)
-OR < -C=O < 2 [-C=O] < -OH < -COOH
The 5,8-epoxide is more strongly adsorbed than
is the corresponding 5,6-epoxide. Thus, the adsorption
affinity of β-carotene and its epoxides on an alumina
thin layer increases in the following order (El-Tinay
and Chichester 1970):
β-carotene < 5,6-epoxide < 5,6,5´,6´-diepoxide < 5,8epoxide
< 5,6,5´,8´-diepoxide < 5,8,5´,8´-diepoxide
The effects of more than one oxygen substituent
are not always additive; a second substituent in the
same end group tends to have less influence than the
first.
The order of elution of carotenoids in a given
adsorbent-solvent system does not vary but the order
may differ in different adsorbents. For example, β-