Simultaneous Determination of Chlorophylls,

Pheophytins, ␤-Carotene, Tocopherols, and

Tocotrienols in Olive and Soybean Oils by High-
performance Liquid Chromatography
C.M. SEPPANEN, M. RAHMANI, AND A. SAARI CSALLANY
Food Chemistry and Toxicology

ABSTRACT: A high-performance liquid chromatographic method is described for the simultaneous measure-
ment of chlorophylls, pheophytins, ␤ -carotene, tocopherols, and tocotrienols in vegetable oils using 2 spectro-
photometers in series and 1 normal-phase silica column. Oil samples were diluted in the mobile phase, isopro-
panol-hexane (1.5:98.5 vol/vol), and injected directly onto the column. A programmable UV/Vis spectropho-
tometer was used to measure chlorophylls a and b, pheophytins a and b, and ␤ -carotene at their appropriate
absorption maxima (430, 452, 409, 433, and 452 nm, respectively). ␣ -, ␥ -, and ␦ -tocopherols and ␣ -, ␥ -, and ␦ -
tocotrienols were measured by fluorescence at 295-nm excitation and 330-nm emission. Samples of virgin olive
oil, soybean oil, and palm oil were analyzed.
Keywords: ␤ -carotene, chlorophylls, HPLC, tocopherols, vegetable oil

Introduction
HLOROPHYLLS , PHEOPHYTINS, ␤-CAROTENE AND OTHER CARO-
C tenoids, tocopherols, and tocotrienols are naturally occurring
constituents found in varying amounts in vegetable oils. Chloro-
phylls a and b and their primary degradation products, pheophyt-
ins a and b, are green pigments that are desirable in some oils such
as olive oil, but are typically removed, along with ␤-carotene, from
other vegetable oils during processing ( White 2000). The toco-
pherol homologues (␣-, ␤-, ␥-, and ␦-tocopherol) and the tocot-
rienol homologues (␣-, ␤-, ␥-, and ␦-tocotrienol) are natural antiox-
idants that contribute to the oxidative stability of oils. Exposure of
vegetable oils to oxidative conditions, such as light or heat during
processing, storage, or use, will result in increased oxidation and
changes in the concentrations of the above-mentioned constitu-
ents. The presence of these compounds is important in relation to
oil stability and nutritional labeling and possible health effects
related to the consumption of oils.
High-performance liquid chromatography (HPLC) is widely used
for determining chlorophyll pigments, carotenoids, tocopherols,
and tocotrienols in vegetable oils (Fraser and Frankl 1985; Rahmani
and Csallany 1985, 1991; Mehlenbacher and others 1990; Endo and
others 1992; Minguez-Mosquera and others 1992; Carvalho and
others 1995; Psomiadou and Tsimidou 1998; Ye and others 1998).
Most published procedures use one type of detector and rely on
multiple injections to measure several different components be-
cause of differences in absorption and fluorescent properties be-
tween chlorophylls, carotenes, tocopherols, and tocotrienols. Toco-
pherols and tocotrienols are only weakly absorbed in the ultraviolet
(UV ) region (292 to 298 nm) (Kasparek 1980) but exhibit strong
native fluorescence due to the chroman ring structure (Duggan and
others 1957). Fluorescence detection of tocopherols and tocot-
rienols provides greater sensitivity and specificity than UV detec-
tion. The fact that ␤-carotene and chlorophyll pigments exhibit
strong absorption in the visible region make them suitable for anal-
ysis in the visible region. The general use of fluorescent and ultra-
violet/visible (UV/Vis) spectrophotometers in series has been dem-
onstrated previously (Ye and others 2001). Our objective was to
develop a simple, rapid HPLC method for simultaneous detection
and measurement of chlorophyll and pheophytin homologues, ␤-
carotene, and tocopherol and tocotrienol homologues in vegetable
oils with minimal sample preparation by diluting the oil sample in
the HPLC mobile phase and directly injecting it onto the HPLC
column.
Materials and Methods
Preparation of standards and oil samples
Reagent-grade chlorophylls a (about 98%) and b (about 95%)
and lutein (> 93%) were purchased from Sigma Chemical Co. (St.
Louis, Mo., U.S.A.). Synthetic ␤-carotene (96.8%) was obtained from
Kalsec, Inc. (Kalamazoo, Mich., U.S.A.). Analytical-grade RRR-␣-
tocopherol (= 99.0%) and RRR-␥-tocopherol (= 97.0%) were pur-
chased from Fluka Chemical (Milwaukee, Wis., U.S.A.). RRR-␦-toco-
pherol (about 90%) was purchased from Sigma Chemical and
DL-␣-tocotrienol (about 90%) was purchased from Hoffman-
LaRoche (Basel, Switzerland). Commercial sources of analytical-
grade pheophytins a and b could not be obtained. These pigments
were prepared from chlorophylls a and b by a previously described
method (Moberg and others 2000). Chlorophyll a or b solution (0.04
mg/mL in acetone, 10 mL) was combined with 10 drops of 1 M HCl
and then mixed with 10 mL diethyl ether. The mixture was washed
with water (5 × 5 mL) in a separatory funnel. The ether layers were
combined, transferred to a conical test tube, and evaporated to
dryness under N2 gas. The test tube was rinsed with acetone into a
10-mL volumetric flask and brought to volume with acetone. The
prepared pheophytins were shown to be chromatographically
pure, and the purity was further checked by comparing their visi-
ble spectra and molar extinction coefficients to those of pure pheo-
Chlorophylls and tocopherols in oil . . .
phytins reported in the literature (Fraser and Frankl 1985). Dilu-
tions of the standards, prepared with isopropanol-hexane (1.5:98.5
vol/vol), were stored under refrigeration and were protected from
light during preparation and handling.
Samples (0.5 g) of olive oil and soybean oil were diluted with iso-
propanol-hexane (1.5:98.5 vol/vol) in a 10-mL volumetric flask. The
palm oil sample was prepared by dissolving the contents of a palm
oil dietary supplement capsule ( J.R. Carlson Laboratories, Inc.,
Arlington Heights, Ill., U.S.A.) in 10 mL isopropanol-hexane
(1.5:98.5 vol/vol). This palm oil capsule was labeled to contain ␣-
tocopherol, and ␣-, ␥-, and ␦-tocotrienols. The identification of ␥-
tocotrienol and ␦-tocotrienol in the palm oil was made by compar-
ison of elution profiles to that given in American Oil Chemists’
Society Official Method Ce 8-89 (Mehlenbacher and others 1990).
The samples were stored under refrigeration for no more than 1 wk
and were protected against light during preparation and handling.
HPLC conditions
The HPLC conditions described here were developed from the
work of Rahmani and Csallany (1985) and Carpenter (1979). Nor-
mal-phase chromatographic separation was performed on a 4.6 mm
× 25 cm Lichrobsorb Si-60 5␮ column (Alltech, Deerfield, Ill., U.S.A.)
with a mobile phase of isopropanol-hexane (1.5:98.5 vol/vol) at a
flow rate of 1.0 mL/min. HPLC-grade reagents were purchased
from Fisher Scientific Co. (Fair Lawn, N.J., U.S.A.). Standards and oil
samples were injected into a 20-␮L sample loop. Two detectors were
connected in series for detection of all components of interest: a
variable-wavelength UV/Vis spectrophotometer with wavelength
programming capability for detection of the chlorophylls, pheo-
phytins, and ␤-carotene and a fluorescent spectrophotometer for
detection of tocopherols and tocotrienols. Each detector was indi-
vidually connected to a computing integrator. UV absorption was
monitored at 430 nm for detection of chlorophyll a, 452 nm for chlo-
rophyll b, 409 nm for pheophytin a, 433 nm for pheophytin b, and
452 nm for ␤-carotene by programming the detector to change the
wavelength during elution of the appropriate compounds. Fluores-
cence was continuously monitored at 295-nm excitation and 330-
nm emission for detection of ␣-, ␥-, and ␦-tocopherols and ␣-, ␥-,
and ␦-tocotrienols.
Quantification of standards
Absorption maxima were determined for each pure standard in
the mobile phase. Calibration curves were prepared for each stan-
dard at the appropriate absorption maximum from peak area re-
sponse plotted against concentration, at various concentrations.
These concentrations were: chlorophyll a, 0.22 to 3.4 ␮g/mL
(r2 = 0.999); chlorophyll b, 0.24 to 2.4 ␮g/mL (r2 = 0.998); pheophy-
tin a, 0.81 to 20.2 ␮g/mL (r2 = 0.999); pheophytin b, 0.80 to 20.0 ␮g/
mL (r2 = 0.999); ␤-carotene, 6.5 to 32.0 ␮g/mL (r2 = 0.934); ␣-toco-
pherol, 0.03 to 12.5 ␮g/mL (r2 = 0.997); ␥-tocopherol, 0.02 to 10.0
␮g/mL (r2 = 0.995); ␦-tocopherol, 0.9 to 9.0 ␮g/mL (r2 = 0.981); and
␣-tocotrienol, 0.04 to 2.0 ␮g/mL (r2 = 0.991). Quantification of lutein
was not accomplished because the pure standard did not elute in
less than 1 h.
Percent recoveries for chlorophyll pigments,
tocopherols, ␣ -tocotrienol, and ␤ -carotene from oil
samples
To determine whether the oil matrix affected the quantification
of the compounds of interest, virgin olive oil was dissolved in iso-
propanol-hexane (1.5:98.5 vol/vol) and was spiked with known
amounts of chlorophylls a and b and pheophytins a and b. Similarly,
a solution of a bleached soybean oil was diluted in isopropanol-
hexane (1.5:98.5 vol/vol) and spiked with known amounts of ␣-, ␥-
, and ␦-tocopherols, ␣-tocotrienol, and ␤-carotene. The concentra-
tions of individual compounds were determined identically in the
spiked and unspiked samples. The percent recovery for each com-
pound was calculated as % Recovery = [C/(A + B)] × 100, where
A = ␮g compound found in original (unspiked) sample; B = ␮g com-
pound added to sample; C = ␮g compound found in spiked sam-
ple.
Results and Discussion
T HE HPLC SEPARATION OF A MIXTURE OF 〉-CAROTENE, CHLOROPHYLL
a, chlorophyll b, pheophytin a, and pheophytin b pure stan-
dards is illustrated in Figure 1. This chromatogram shows that each
Food Chemistry and Toxicology
compound is well separated on the silica column and that with the
use of a programmable detector, simultaneous measurements were
made at the appropriate wavelength for each compound. The min-
imum detection was determined to be 0.01 ␮g for ␤-carotene, 0.01
␮g for chlorophyll a or b, and 0.02 ␮g for pheophytin a or b per injec-
tion.
The HPLC separation of a mixture of ␣-, ␥-, and ␦-tocopherols
and ␣-tocotrienol pure standards is shown in Figure 2. Because
commercial sources of pure ␥- and ␦-tocotrienols could not be locat-
ed, the contents of commercially available palm oil capsules, sold
as dietary supplements, were analyzed. The palm oil capsule con-
tained, according to its label, ␣-tocopherol and ␣-, ␥-, and ␦-tocot-
rienols. The HPLC separation of the tocopherols and tocotrienols in
the palm oil is shown in Figure 3. In addition, we also detected ␥-
and ␦-tocopherols in the palm oil capsules. The minimum detection
was found to be 0.01 ␮g per injection for ␣-tocopherol.
Lutein is a more polar compound than ␤-carotene, chlorophylls,
pheophytins, tocopherols, and tocotrienols. It had an elution time
of more than 1 h in this solvent system on a normal-phase HPLC
column. Therefore, lutein was excluded from this rapid determina-
tion of these other oil constituents.
The percent recoveries were determined for each standard
Figure 1—High-performance liquid chromatographic sepa-
ration of chlorophylls a and b, pheophytins a and b, and
␤-carotene pure standards detected by a ultraviolet/vis-
ible spectrophotometer at wavelengths 430, 452, 409,
430, and 452 nm, respectively. A = ␤-carotene;
B = pheophytin a; C = chlorophyll a; D = pheophytin b;
E = chlorophyll b. Separation conditions: Lichrobsorb Si-
60 5␮ column (4.6 mm × 25 cm); isocratic elution with iso-
propanol-hexane (1.5:98.5 vol/vol); flow rate, 1.0 mL/min;
injected volume, 20 ␮L.
 Chlorophylls and tocopherols in oil . . .

mixed with oil to see if the oil matrix influenced the isolation of the Table 1—Percent recoveries of chlorophylls a and b and
previously mentioned compounds. The results of these measure- pheophytins a and b standards from olive oil
ments are listed in Table 1 and 2. These percent recoveries show Sample Chl a Chl b Phe a Phe b
that the oil matrix did not affect the detection of the compounds % Recovery 99.1% 97.9% 99.4% 98.3%
and that the repeatability of this method is very good. A: Conc. of standard 0.218 0.484 0.808 1.0
Samples of soybean oil and virgin olive oil also were investigated. solution used for
Three samples of commercial soybean oil (refined and deodorized) spiking oil (␮g/mL)a
B: Conc. in virgin olive 0.0 0.0 0.080 0.0
and 4 samples of virgin olive oil were examined for the presence of oil (␮g/mL)a,b
the analytes of interest in each oil. Chlorophyll and pheophytin C: Conc. in spiked 0.216 0.474 0.883 0.983
pigments were measured in the olive oil but not in soybean oil be- oil samples (avg. of 3 ± 0.009 ± 0.006 ± 0.02 ± 0.004
cause these compounds are removed during the refining process. replicates ± SD) (␮g/mL)a
Quantification of the various compounds in the oil samples was a Injected volume = 100 ␮L
b Oil concentration = 53.7 mg oil/mL mobile phase
accomplished by comparison of the integrated peak areas to the
Food Chemistry and Toxicology

appropriate calibration curves (n = 4 to 6).

Most samples of virgin olive oil contained no detectable
amounts of chlorophyll a, chlorophyll b, or pheophytin b, and con-
tained only small amounts of pheophytin a, ranging from 1.5 to 8.5 Table 2—Percent recoveries of ␣-, ␥-, and ␦-tocopherols,
ppm for the different oils. A sample of virgin olive oil, which was ␣-tocotrienol, and ␤-carotene standards from bleached
pressed in the laboratory from green olives, contained 1.90 ppm soybean oil
chlorophyll a, no chlorophyll b, 35.73 ppm pheophytin a, and 1.05 Sample ␣ -T ␥ -T ␦ -T ␣ -T3 ␤ -C
ppm pheophytin b. It was expected that small amounts of the chlo- % Recovery 96.5% 97.4% 95.2% 97.3% 99.2%
rophylls and larger quantities of their degradation products, the A: Conc. of standard 2.87 1.54 1.06 3.33 3.93
pheophytins, would be found in the freshly prepared olive oil. The solution used for
presence of the degradation products indicated that the oil in the spiking oil (␮g/mL)a
olive fruit or the pressed olive oil had been exposed to oxidative B: Conc. in bleached 0.0 0.0 0.0 0.0 0.0
soybean oil (␮g/mL)a,b
conditions. The ␤-carotene concentration of the olive oils ranged C: Conc. in spiked 2.76 1.57 1.00 3.24 3.91
from 0.54 to 1.0 ppm. oil samples (avg. of 3 ± 0.15 ± 0.07 ± 0.04 ± 0.12 ± 0.05
Three different commercial soybean oil samples contained 58.3 to replicates ± SD) (␮g/mL)a
174.8 ppm ␣-tocopherol, 300.0 to 763.6 ppm ␥-tocopherol, and 186.6 a Injected volume = 20 ␮L
b Oil concentration = 50.0 mg oil/mL mobile phase
to 399.6 ppm ␦-tocopherol. No tocotrienols or ␤-carotene were de-

Figure 3—High-performance liquid chromatographic sepa-

Figure 2—High-performance liquid chromatographic sepa-
ration of tocopherols and ␣-tocotrienol pure standards
detected by a fluorescent spectrophotometer at 295-nm
excitation and 330-nm emission. A = ␣-tocopherol; B = ␣-
tocotrienol; C = ␥-tocopherol; D = ␦-tocopherol. Separation
conditions: Lichrobsorb Si-60 5␮ column (4.6 mm × 25 cm);
isocratic elution with isopropanol-hexane (1.5:98.5 vol/vol);
flow rate, 1.0 mL/min; injected volume, 20 ␮L.
ration of tocopherols and tocotrienols in palm oil detected
by a fluorescent spectrophotometer at 295-nm excitation
and 330-nm emission. A = ␣-tocopherol; B = ␣-tocotrienol;
C = ␥-tocopherol; D = ␥-tocotrienol; E = ␦-tocopherol; F = ␦-
tocotrienol. Separation conditions: Lichrobsorb Si-60 5␮
column (4.6 mm × 25 cm); isocratic elution with isopro-
panol-hexane (1.5:98.5 vol/vol); flow rate, 1.0 mL/min;
injected volume, 20 ␮L.
Chlorophylls and tocopherols in oil . . .
tected in the soybean oil samples. The values determined for each oil
are in good agreement with published data (Bauernfeind 1980).
Conclusions
T HE METHOD DESCRIBED PRESENTS A SIMPLE HPLC PROCEDURE FOR
simultaneously separating and detecting chlorophylls, pheo-
phytins, ␤-carotene, and tocopherols in olive and soybean oils dis-
solved in 1.5% isopropanol in hexane. The determination of up to
11 analytes (chlorophylls a and b; pheophytins a and b; ␤-carotene;
␣-, ␥-, and ␦-tocopherols; and ␣-, ␥-, and ␦-tocotrienols) allows for
monitoring changes in antioxidant status of the oil due to exposure
to oxidative stresses.
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MS 20030006 Submitted 1/3/03, Revised 2/20/03, Accepted 4/25/03, Received
4/25/03
This work was supported in part by the Minnesota Agricultural Experiment Station and by
the Agency for Intl. Development, Contract nr 608-0160 IAV Morocco Project. The authors
express thanks to Ms. K.L. Fritz for assistance with the tocopherol analysis.
Authors Seppanen and Csallany are with the Dept. of Food Science and
Nutrition, Univ. of Minnesota, 1334 Eckles Ave., St. Paul, MN 55108. Author
Rahmani is with the Inst. Agronomiqe et Veterinaire Hassan II, Section de
Technologie Alimentaire, Rabat – Instituts, Morocco. Direct inquiries to au-
thor Csallany (E-mail: ascsalla@umn.edu).