Detection and Measurement of Carotenoids UNIT F2.

2
by UV/VIS Spectrophotometry
The majority of carotenoids exhibit absorption in the visible region of the spectrum,
between 400 and 500 nm. Because they obey the Beer-Lambert law (i.e., absorbance is
linearly proportional to the concentration), absorbance measurements can be used to
quantify the concentration of a pure (standard) carotenoid (see Basic Protocol 1) or to
estimate the total carotenoid concentration in a mixture or extract of carotenoids in a
sample (see Basic Protocol 2). Considerations for the preparation of carotenoid-contain-
ing samples are presented in Critical Parameters (see Sampling and Sample Preparation).
NOTE: Carotenoids are easily degraded. See Critical Parameters for a discussion of
suitable precautions.
NOTE: All extinction coefficients in this unit assume a 1-cm pathlength.

PREPARATION AND CALIBRATION OF INDIVIDUAL CAROTENOID BASIC

STANDARDS PROTOCOL 1
In this protocol, commercially purchased carotenoid standards are dissolved in a suitable
solvent and the absorbance measured at its maximum wavelength (λmax). Using published
extinction coefficients and taking into consideration the dilution factor, the concentration
of the standard carotenoid is calculated. The spectrum is also scanned in order to evaluate
the fine structure (see Spectral Fine Structure in Background Information). The carotenoid
solution should ideally be assayed by HPLC as described in UNIT F2.3 to establish
chromatographic purity and thus correct the calculated concentration.
Materials
∼1 to 5 mg standard carotenoids (Table F2.2.1 and Table F2.2.2)
High-grade organic solvent (Table F2.2.2)
10- to 50-ml volumetric flask
Additional reagents and equipment for HPLC analysis (UNIT F2.3)
1. Dissolve carotenoid (e.g., ∼1 to 5 mg) in a suitable solvent (see Table F2.2.2). Make
to an accurate volume (e.g., 10 to 50 ml) in a volumetric flask.
A larger volume may be used if desired.

Table F2.2.1 Commercial Sources of Carotenoidsa

Carl
Sigma Extra- Atomergic Indifine Fisher Fluka
Roth
Aldrich synthese Chemical Chemical Scientific Chemical
GmgH
β-carotene X X X X X X X
α-caroteneb
Lycopene X X X X X
β-crytoxanthin X X X X
Zeaxanthin X X X X
Lutein X X X X X
aThe companies may have offices or distributors in other countries.
bAt the time of writing there were no commercial suppliers of α-carotene.
Carotenoids
Contributed by K. John Scott F2.2.1
Current Protocols in Food Analytical Chemistry (2001) F2.2.1-F2.2.10
Copyright © 2001 by John Wiley & Sons, Inc.
 It is essential that the carotenoid be completely dissolved. With crystalline samples,
dissolution can be aided by initial addition of a small amount of a more effective solvent
(e.g., dichloromethane) prior to making to volume for spectrophotometric measurement
(for most commonly assayed carotenoids, no more than 10% of the total volume should be
required; however, lycopene may require up to 100%). Where a carotenoid is supplied in
a sealed ampule, dissolution can be conveniently achieved by adding successive small
aliquots of the more effective solvent to the ampule, and transferring to a volumetric flask.
It is not easy to assess complete dissolution visually; therefore, it is advisable to filter the
solution through a suitable solvent-compatible 0.45-µm filter.
2. Dilute the solution (e.g., 1:50) in desired solvent if necessary to give ∼0.3 to 0.7 AU
as measured on a spectrophotometer at λmax.
It is recommended that at least two independent dilutions be made to ensure confidence in
the measurement.
If a larger final volume is used (see step 1) reduce the dilution accordingly.
3. Warm up the spectrophotometer per manufacturer’s instructions.
4. Zero the spectrophotometer with solvent in a cuvette.
NOTE: Cuvettes must be kept scrupulously clean; avoid handling the surfaces of the cell
(see Critical Parameters).
5. Place a cuvette containing the carotenoid solution into the sample cell holder of the
spectrophotometer.
6. Measure the absorbance at λmax. Take reading immediately.
See Critical Parameters concerning degradation.
7. Scan to allow measurement of the fine structure (see Table F2.2.3; also see Spectral
Fine Structure in Background Information).
The same principles can be applied to measurement of chromatographic fractions.
8. Calculate the concentration of carotenoid as shown in the example below for all
trans-β-carotene.

A × V1 0.5 AU × 50
× C 1% = × 10 mg/ml = 96.5 µg/ml
A1% 2592 AU

Table F2.2.2 Data on λmax and Extinction Coefficients of a Selection of Carotenoids

MW A1% ε1 mM λ(nm) Solvent %III/II

α-carotene 537 2710 145 445 Hexane 55
β-carotene 537 2592 139 450 Hexane 25
β-cryptoxanthin 553 2460 136 450 Hexane 25
Lutein 569 2550 145 445 Ethanol 60
Lycopene 537 3450 185 470 Hexane 65
Zeaxanthin 569 2480 141 450 Hexane 25
Natural carotenoids as food colors
Bixin (Bixa orellana) 395 4200 166 456 Petroleum ether
Capsanthin (paprika) 585 2072 121 483 Benzene
Detection and Capsorubin (paprika) 601 2200 132 489 Benzene
Measurement of Synthetic food colors
Carotenoids by
UV/VIS β-apo-8′-carotenal 417 2640 110 457 Petroleum ether
Spectrophotometry Canthaxanthin 564 2200 124 466 Petroleum ether 0

F2.2.2
Current Protocols in Food Analytical Chemistry
 Where A is the absorbance reading of the diluted sample (0.5 AU), V1 is the dilution
factor (50×), A1% is the absorbance of a 1% solution (i.e., the extinction coefficient;
2592 AU), and C1% is the concentration of a 1% solution (10 mg/ml). Using this
formula, the concentration of the original solution in this example is = 96.5 µg/ml.
9. Subsequent to measurement of the carotenoid concentration, the solution should be
assayed by HPLC to establish the chromatographic purity (see UNIT F2.3).
For example, assuming the same values as Equation F2.2.1, if the total chromatographic
area is 10000, and the area of all-trans β-carotene peak is 9500, then the chromatographic
purity is 9500/10000 × 100 or 95%, and the actual concentration of β-carotene is 95.5
µg/ml × 95/100 or 91.7 µg/ml.

MEASUREMENT OF TOTAL CAROTENOID CONCENTRATION IN FOOD BASIC

COLORANTS, PHARMACEUTICALS, AND NATURAL EXTRACTS PROTOCOL 2
The same principle as described above can be used for the “estimation” of the carotenoid
content of extracts of food colorants, pharmaceuticals, foods, biological samples, or
chromatographic fractions. This procedure employs calculations used for individual
carotenoids of high purity and thus will estimate the “total carotenoids” present in a food
or biological extract, where a mixture of carotenoids would be expected. Greater accuracy
can be obtained as extracts are purified to contain single components (see Commentary).
A spectrum scan is not employed in this procedure as the fine structure of a mix of
carotenoids can only be identified after HPLC separation (see Commentary).
Materials
Sample
Appropriate solvent (Table F2.2.2)
Additional equipment and reagents for sample extraction (UNIT F2.1).
1. Prepare the sample as detailed in UNIT F2.1, taking into consideration the guidelines
detailed in Critical Parameters (see Sampling and Sample Preparation). Dilute the
sample appropriately using a suitable solvent.
Samples containing esterified carotenoids, chlorophyll, or high levels of fat may require
saponification (UNIT F2.1).
2. Warm up spectrophotometer per manufacturer’s instructions.
3. Zero the spectrophotometer with solvent in a cuvette.

Table F2.2.3 Additional Spectral Characteristics of Carotenoids

Common name Chemical name Solvent Absorption peaks

β-carotene (β,β-carotene) Hexane 425 450 478

α-carotene (β,ε-carotene) Hexane 422 445 473
Lycopene (ϕ,ϕ-carotene) Hexane 444 470 502
β-cryptoxanthin (3-hydroxy-β-carotene) Hexane 428 450 478
Zeaxanthin (β,β-carotene-3,3′-diol) Hexane 425 450 478
Lutein (β,ε-carotene-3,3′-diol) Ethanol 421 445 474
Capsanthin Petroleum ether 450 475 505
Capsorubin Petroleum ether 445 479 510
Bixin Petroleum ether 432 456 490
Canthaxanthin Petroleum ether 466
β-apo-8′-carotenal Ethanol 405 430 460
Carotenoids

F2.2.3
Current Protocols in Food Analytical Chemistry
 4. Place a cuvette containing a suitably diluted (i.e., ∼0.3 to 0.7 AU) extract of
pharmaceutical, food, or biological material into the sample cell holder of the
spectrophotometer.
5. Measure absorbance at selected λmax (Table F2.2.2 and Table F2.2.3). Read immediately.
See Critical Parameters concerning degradation.
6. Estimate the total carotenoid concentration of the sample (see Basic Protocol 1, step
9 and Equation F2.2.1).
With the exception of individual food colorants and single carotenoid pharmaceutical
products, it is unlikely that the extract will be composed of only one predominant carote-
noid; therefore, a specific λmax or extinction coefficient (Table F2.2.2) cannot be used. In
this case it is convenient to use a λmax of 450 nm and a typical A1% value of 2500. Alternative
values and other considerations are discussed elsewhere (see Commentary).

COMMENTARY
Background Information
The majority of carotenoids exhibit absorp-
tion in the visible region of the spectrum,
mainly between 400 and 500 nm (see Table
F2.2.2 for examples). A few carotenoids (e.g.,
phytoene) exhibit maximum absorbance in the
UV region. This absorption is due to the long
conjugated double bond system of carotenoids.
The conjugated unsaturated part of the carote-
noid molecule containing delocalized π-elec-
trons is called the “chromophore” and is re-

Abs (AU)

260 300 380 460 540

nm
Detection and Wavelength (nm)
Measurement of
Carotenoids by
UV/VIS
Spectrophotometry Figure F2.2.1 The spectral characteristics of all-trans β-carotene (solid line), 9-cis β-carotene
(dashed and dotted line) and 15-cis β-carotene (dashed line).
F2.2.4
Current Protocols in Food Analytical Chemistry
sponsible for the absorption of light in the
visible region.
The differences in the spectral charac-
teristics of individual carotenoids are often
small, but are of great importance in their iden-
tification; however, carotenoids having the
same chromophore, such as β-carotene and its
hydroxy-derivative zeaxanthin, have identical
spectra. The spectra of cis- or Z-isomers of
carotenoids, while being similar to the all-trans
or all-E form, are different in as far as they
exhibit a change in the λmax to a shorter wave-
length, a decrease in the magnitude of the ab-
sorbance, a reduction in the fine structure, and
additional absorption bands in the UV region
around 340 nm and 280 nm (Fig. F2.2.1). It is
not the intention of the author to go into any
great detail of the molecular characteristics of
carotenoids in this unit, as details can be found
in a excellent chapter by Britton (1995).
II
440 460
Wavelength (nm)
Figure F2.2.2 The calculation of %III/II from the spectral fine structure.
Current Protocols in Food Analytical Chemistry
Extinction coefficients
Carotenoids in solution obey the Beer-
Lambert law, where absorbance (A) equals con-
centration multiplied by extinction coefficient
(A1%), where the extinction coefficient (A1%) is
defined as the absorbance of a 1% (10 g/liter)
solution of carotenoid, in a defined solvent, in
a 1-cm path-length cuvette, at a specific wave-
length (λ). This information can be used to
quantify the concentration of a pure (standard)
carotenoid (see Basic Protocol 1), or to “esti-
mate” the total carotenoid concentration in a
mixture or extract of carotenoids in a sample
(see Basic Protocol 2). The extinction coeffi-
cient may also be expressed in terms of molar-
ity.
As seen in Table F2.2.2, a 1% solution (10
g/liter) of β-carotene has an absorbance of 2592
AU and a molecular weight of 537 g/mol (i.e.,
1 mM = 0.537 g/liter); therefore, the absor-
III
480 500 520
Carotenoids
F2.2.5
 bance expected for a 1 mM solution is: (0.537
g/liter)/(10 g/liter) × (2592 AU) = 139 AU/1
mM β-carotene. Using this ratio, if a β-carotene
solution has an absorbance of 5, then the con-
centration is given as: (5 AU)/(139 AU/mM) =
0.036 mM. It is important to note that due to
the inherent difficulties in procedures for accu-
rate determination of extinction coefficients,
there may be a significant level of uncertainty
in some published values. Small variations
(e.g., 2 to 3 nm) may also occur in published
data of absorption maxima. Whenever possible,
the spectrum of a compound under investiga-
tion should be compared directly with an
authentic pure standard. The spectra of the
unknown and the standard should be identical
for both the λmax and the fine structure (see
below).
Spectral fine structure
In addition to the absorption maxima of the
carotenoids, the “shape” of the spectra provides
important information for identification of pu-
rified carotenoid extracts or pure standard
(while the identity of the standard is generally
not in question, it is a good idea to check the
purity by fine structure analysis). Fine structure
Abs (AU)
250 350
Detection and Wavelength (nm)
Measurement of
Carotenoids by
UV/VIS
Spectrophotometry Figure F2.2.3 The spectral characteristics of β-carotene (solid line) and canthaxanthin (dashed
line). Redrawn from original by Jaffé and Orchin (1962).
F2.2.6
is demonstrated in Figure 2.2.2, and is meas-
ured as a ratio of the absorbance maxima to one
of the shoulders (i.e., %III/II). The longest
wavelength band is called III and the middle
absorption band II. The baseline is taken as the
minimum between the two peaks, and the
height of each peak is measured. Carotenoids
having the same chromophore, such as β-caro-
tene and its hydroxy-derivative zeaxanthin,
have identical fine structure, while conjugated
ketocarotenoids, for example canthaxanthin,
have only a rounded spectrum with no fine
structure (see Fig. F2.2.3), thus the %III/II is 0.
Spectral monitoring during HPLC
The spectral characteristics of a standard can
be monitored during HPLC using a diode-array
detector (UNIT F2.3). A directory of standard spec-
tra can be stored, enabling additional identifica-
tion of sample peaks. The actual absorption
maxima and fine structure will be dependent on
the composition of the mobile phase (see Fig.
F2.2.4). Peak I may only occur as a “shoulder”
with cis-carotenoids, while an additional peak
is observed at around 340 nm (see Fig. 2.2.1).
450 550
Current Protocols in Food Analytical Chemistry
Critical Parameters and
Troubleshooting
Good laboratory practice for UV/VIS
spectrophotometry
While the procedures outlined above are
fairly straight forward, in order to obtain maxi-
mum accuracy, “good laboratory practice”
should be applied at all times.
1. Whenever possible the preparation and han-
dling of carotenoid solutions should be carried
out under yellow/gold fluorescent lighting to
avoid light induced degradation.
2. All glassware should be scrupulously clean
and reagents (e.g., solvents) should be of the
highest quality.
3. The spectrophotometer should be located in
a clean environment away from direct sunlight
and drafts, at a reasonably even temperature,
and free from electrical interference.
4. Any spillage should be cleaned up immedi-
ately.
450
456
475
400 450
Wavelength (nm)
Figure F2.2.4 The spectral characteristics of β-carotene (solid line), lutein (long-dashed line), and
lycopene (short-dashed line) in an acetonitrile-based HPLC solvent (75:25:5 v/v/v acetoni-
trile/methanol/dichloromethane).
Current Protocols in Food Analytical Chemistry
5. When working with organic solvents it is
advisable to use stoppered cells to prevent dam-
age to cell holders and other sensitive parts of
the instrument, and to avoid evaporation of the
sample.
6. It is advisable to have the instrument regu-
larly serviced (i.e., annually) by the manufac-
turer’s engineer.
7. Once they have become familiar with the
equipment, day to day maintenance can be
carried out by the user; however, if in doubt,
consult the manufacturer.
8. Modern equipment displays a range of error
messages, often in the form of a letter and
number, if the instrument fails or is not working
to specification (e.g., failure of light source).
Refer to the user’s manual for details.
9. Cuvettes should be kept clean and the faces
to be placed in the light beam should not be
handled.
10. For normal measurement of carotenoids,
glass cells can be used.
500 550
Carotenoids
F2.2.7
 11. It is not necessary to completely fill the
cells, ∼2⁄3 full is normally sufficient. This helps
to avoid accidental spillage.
12. Before placing the cells in the cell holder,
the optical faces can, if necessary, be polished
carefully (avoiding any spillage) with a fine
tissue.
13. For accurate results, it is advisable to use
optically matched pairs or sets of cuvettes (i.e.,
cuvettes manufactured to a high specification
to ensure they have the same optical parame-
ters).
14. As pointed out above (see Basic Protocol
1), when preparing carotenoid standard solu-
tions, complete dissolution of the solid material
is essential; however, it is always advisable to
filter the solution through a solvent compatible
filter.
15. Stock solutions that have been stored (e.g.,
in a refrigerator), should be allowed to warm
up to room temperature, refiltered, and a “new”
concentration calculated prior to preparing a
new working solution, which should be
checked for chromatographic purity.
16. Spectrophotometric readings should be
carried out immediately after the solution has
been placed in the cuvette to avoid evaporation
of the solvent or degradation.
17. Accurate measurement of standard solu-
tions is a critical factor in the overall analysis
of carotenoids. Inaccuracy of measurement can
be a major cause of intra- and interlaboratory
variation.
18. Experience will allow the analyst to recog-
nize possible problems and anomalies, but a
useful maxim is “if in doubt repeat it.”
Sampling and sample preparation
Sample selection, number of samples, sam-
ple handling, and sample preparation prior to
extraction are important factors effecting data
quality. The type of sample (e.g., vitamin prepa-
rations, fruit drinks, supplemented foods, fruits
and vegetables, biological materials), will de-
termine to a large extent the sampling protocol
and how the samples are handled. In this re-
spect, the main considerations are the degree of
homogeneity of the material and the possible
variation in the vitamin content, not only be-
tween different materials, but also between dif-
ferent samples of the same material. Powdered,
freeze-dried, and liquid materials for example,
are likely to be more homogeneous with respect
Detection and to vitamin distribution than fruits and vegeta-
Measurement of
Carotenoids by bles. With fruits and vegetables, a good rule of
UV/VIS thumb is that levels of vitamin in the outer part
Spectrophotometry (e.g., outer leaves, skin, peel) are generally
F2.2.8
higher, considerably so in some cases, than the
inner parts.
Equally with fruits and vegetables, the vari-
ety, origin, season of year, growing conditions,
etc., will affect the vitamin content. In addition,
consideration must be given to typical ways of
preparation and cooking of the foods in the
home. It is essential that the sampling protocol
and the number of samples collected reflect the
purpose of the exercise, be it to determine
between batch variation of vitamin supplement
preparations, the variation within vegetables of
the same type, or to obtain a typical overall
value for that vegetable. Ideally the time be-
tween sample collection and analysis should be
as short as possible. The protocol should mini-
mize any effects that may cause undesirable
losses prior to analysis.
Frozen materials should be stored at −20°C,
fruits and salad vegetables at around 4°C, and
canned foods at room temperature. Powdered
and freeze-dried materials should be stored in
the dark in their original containers. Storage of
fresh materials should preferably not exceed 3
days. After the initial preparation (see below),
fresh or cooked materials can be conveniently
stored at −20°C for a short time prior to extrac-
tion.
As indicated above, the preparation of the
sample depends on the type of material and the
homogeneity of that material. With dry pow-
dered or liquid materials, the whole or parts of
the samples collected can simply be thoroughly
mixed prior to analysis.
Vegetables and fruits are prepared as appro-
priate for typical “in home” preparation meth-
ods (e.g., by removal of outside leaves, peeling,
coring). Larger items such as cabbages, may be
quartered, and then one quarter from each of
the individual samples cut and mixed. Smaller
items are cut and mixed. Further subsamples of
the individual samples (e.g., 100 g depending
on the number of individual samples collected
initially) of the cut mixed materials are taken
and these subsamples bulked and thoroughly
mixed.
In order to obtain a thoroughly repre-
sentative sample for extraction and subsequent
analysis the following procedure has been used
in the author’s laboratory. Immediately after the
preparation of the composite sample (raw and
or after cooking as appropriate) the sample was
frozen in liquid nitrogen and ground under
liquid nitrogen in a Waring-type blender.
Ground sample was then stored in an air-tight
bottle under nitrogen at −20°C for up to 3 days
Current Protocols in Food Analytical Chemistry
prior to analysis. All manipulations were car-
ried out under yellow/gold fluorescent lighting.
Extraction
Methods of extraction are dealt with in detail
in UNIT F2.1. All manipulations should be carried
out under gold/yellow light, avoiding exposure
to daylight or artificial “white” fluorescent
light. All solvents must be of a high degree of
purity. Cooking procedures such as boiling may
cause disruption of the cellular matrix of vege-
table material making the carotenoids more
readily extractable. Samples containing high
levels of fat, esterified carotenoids, or chloro-
phylls require saponification (see UNIT F2.1);
however, in many instances chlorophylls may
be separated from the carotenoids of interest
during column chromatography avoiding the
need for saponification. Certain saponification
conditions may cause degradation of carote-
noids, particularly the xanthophylls, so the con-
centration of KOH, time, and temperature must
be carefully assessed for the particular type of
material being analyzed.
Solvents
Different solvents and the composition of the
mobile phase used in HPLC may effect lmax.
Generally speaking, the lmax in hexane, etha-
nol, and petroleum ether will show little if any
change, but chloroform, for example, will show
a shift to a longer wavelength. Other factors
which may effect the spectral characteristics are
water in water-miscible solvents, protein in
carotenoproteins, and low temperatures.
Degradation of carotenoids
It must be remembered that carotenoids are
sensitive to light, heat, air, and active surfaces;
therefore, precautions must be taken during
preparation and any subsequent storage to
avoid any detrimental effects such as degrada-
tion, formation of stereoisomers, structural re-
arrangement, and other physicochemical reac-
tions. The conditions of handling and any
preparation prior to storage or extraction are
critical to ensuring that there is no degradation
of the analytes prior to analysis. Where possi-
ble, all manipulations during the preparation of
standard solutions and extracts should be car-
ried out under yellow/gold fluorescent lighting.
In certain instances, degradation can be
rapid, thus it is essential that the chances of it
occurring be lessened or avoided completely.
This is particularly important in the case of
standard carotenoid solutions. It has been re-
ported that lycopene in particular can be rapidly
Current Protocols in Food Analytical Chemistry
degraded in chloroform from certain sources
(Scott, 1992). With all standard solutions it is
important not to assume that a standard has
remained stable during storage.
Carotenoids in chlorophyll containing sam-
ples may be subject to chlorophyll sensitized
photoisomerization, resulting in the production
of significant amounts of cis (Z) isomers even
during a brief exposure of an extract to light.
As an example of a related problem, in acetone,
cis-isomers can be produced as a result of the
production of triplet state carotenes. The pres-
ence of O2 in stored samples, peroxides in
solvents, or oxidizing agents can rapidly lead
to bleaching and carotenoid epoxides and apo-
carotenoids. Unsaturated lipids and metal ions
can also enhance oxidative breakdown. Impu-
rities such as plasticizers, especially phalates,
can produce severe problems in spectro-
photometric analysis, so all contact of samples,
solvents and other reagents with plastic mate-
rials should be avoided wherever possible.
Carotenoids can be converted into mixtures
of geometrical isomers under appropriate con-
ditions, the most common being iodine cata-
lyzed photoisomerization. This produces an
equilibrium mixture of isomers, in general the
all-trans isomers predominates. These isomers
in an isomeric mixture cannot be measured
separately by simple spectrophotometric deter-
mination. The usual method of subsequent
measurement would be chromatographic sepa-
ration, diode-array detection, and spectral
analysis. In the absence of any definitive data
on extinction coefficients for cis-isomers, they
are quantified against the all-trans isomer.
Modern procedures involve the direct synthesis
of cis-carotenoids.
Extinction coefficients for carotenoid
extracts
There are fewer problems if the extract con-
tains essentially only one carotenoid, or if a
single carotenoid has been collected from a
chromatographic separation; however, most
food extracts will contain at least two predomi-
nant carotenoids, and green vegetables in par-
ticular will also contain chlorophylls, which
have absorption bands in the same region as the
carotenoids. Plasma samples will contain a
whole array of different carotenoids, although
only five or six may predominate, but even
these may, depending on the diet, be present at
very different levels in different individuals,
and some may even be absent; however, for
plasmas, it may be a useful tool for screening
individuals with “high” and “low” carotenoid Carotenoids
F2.2.9
 levels. Here again it does not give any informa-
tion about individual carotenoids and samples
indicating a “high” level may be biased in favor
of one or two carotenoids.
The problem relating to chlorophylls can be
overcome to some extent by saponification of
the sample, which will remove the chloro-
phylls; however, care must be taken in the
choice of conditions, as some carotenoids, par-
ticularly the xanthophylls, may be degraded
(see UNIT F2.1). On the other hand, it is possible
to use an alternate wavelength for the carote-
noids. For example most of the major carote-
noids of interest in foods have an absorption
peak around 480 nm, where any absorption of
chlorophylls causes less interference; however,
it is then necessary to use alternate extinction
coefficients (e.g., 2180 for β-carotene).
If the extract or fraction is composed of one
predominant carotenoid, it can be monitored at
the appropriate wavelength and the A1% for that
carotenoid used. If the extract is composed of
more than one carotenoid, the absorbance at
450 nm can be measured and the A1% for
β-carotene used. Results can be expressed as
total β-carotene equivalents. Alternatively a
“typical” A1% value of 2500 can be used for
comparing relative quantities between various
sample extracts. An A1% value of 2500 is ap-
propriate since the predominate carotenoids,
with the exception of lycopene (A1% = 3450),
have λ between 2460 and 2710. Of course if
samples of tomatoes are to be analyzed, which
contain lycopene predominantly, then an A1%
value of 3450 would be more appropriate.
It must be stressed that the value obtained in
this way for mixtures of carotenoids is only an
approximation. For more definitive analysis
column chromatography (UNIT F2.3) should be
used.
Anticipated Results
Using the extinction coefficients given
above, the example for β-carotene, a 1% solu-
tion (1 g/100 ml or 10 mg/ml) would give a
theoretical absorbance reading of 2592 AU. A
2 µg/ml solution should therefore give an ab-
sorbance reading of 0.518 AU; however, as
indicated earlier, the standard solution must be
Detection and
Measurement of
Carotenoids by
UV/VIS
Spectrophotometry
F2.2.10
analyzed by HPLC to determine the chroma-
tographic purity (see Basic Protocol 1).
Time Considerations
The time taken to carry out the procedures
outlined above will depend on the knowledge
and experience of the analyst and their famili-
arity with the equipment; however, once famil-
iar with the procedure, the preparation and cali-
bration of a standard solution of a carotenoid
(see Basic Protocol 1) should not take >1 hr.
The time required for Basic Protocol 2 will
depend on the extraction procedure.
Literature Cited
Britton, G. 1995. UV/visible spectroscopy. In Ca-
rotenoids, Volume 1B (G. Britton, S. Liaanen-
Jensen, and H. Pfander, eds.) pp. 13-62. Birk-
häuser, Basel, Switzerland.
Jaffé, H.H. and Orchin, M. 1962. Theory and Appli-
cations of Ultraviolet Spectroscopy. John Wiley
and Sons, New York.
Scott, K.J. 1992. Observation on some of the prob-
lems associated with the analysis of carotenoids
in foods by HPLC. Food Chemistry 45:357-364.
Key References
Bauerfiend, J.C. (ed.) 1981. Carotenoids as Color-
ants and Vitamin A Precursors: Technological
and Nutritional Applications. Academic Press,
New York.
A comprehensive treatise on carotenoid color tech-
nology.
Britton, G., Liaanen-Jensen, S., and Pfander, H.
(eds.) 1995. Carotenoids. Volumes 1A and 1B,
Spectroscopy. Birkhäuser, Basel, Switzerland.
Workbooks as well as a reference book with practi-
cal guidance and examples.
Goodwin, T.W. (ed.) 1988. Plant Pigments. Aca-
demic Press, London.
Chlorophylls and carotenoids (distribution, func-
tion and analysis).
Pfander, H. (ed.) 1987. Key to Carotenoids, 2nd ed.
Birkhäuser, Basel, Switzerland.
Structural formula, common and chemical designa-
tions, and references.
Contributed by K. John Scott
Institute of Food Research
Colney, Norwich, United Kingdom
Current Protocols in Food Analytical Chemistry