Journal of Chromatographic Science, Vol.

31, October 1993

Comparison of a Liquid Solvent Extraction Technique and

Supercritical Fluid Extraction for the Determination of
a- and β-Carotene in Vegetables

Ray Marsili a n d Daniel Callahan*

D e a n F o o d s C o m p a n y , R e s e a r c h D e p a r t m e n t , 1 1 2 6 K i l b u r n A v e n u e , R o c k f o r d , Illinois 6 1 1 0 1

with 200 m L ethyl ether, followed by a second extraction with an

Abstract
additional 200 m L ethyl ether; washing the combined ether ex­
tracts with water; evaporating to near dryness on a steam bath;
A n ethanol-pentane solvent extraction procedure and a
quantitatively transferring the extract concentrate to a volumetric
supercritical C O extraction procedure are compared for the
2

high-performance liquid chromatographic determination of α-

and β-carotene in vegetables. The vegetables tested included
carrots, collard greens, turnips, turnip greens, kale, mustard
greens, broccoli florets, zucchini, and squash.
Homogenization of the sample prior to liquid or supercritical
fluid extraction significantly improved recovery of the
carotenoids. A combination of static and dynamic modes of
extraction with ethanol modifier at 338 atm and 40°C was
necessary in order to achieve optimum recovery with the
supercritical fluid procedure. β-Carotene results with the
supercritical C O procedure averaged 2 3 % higher than results
2
flask with hexane; and finally analyzing for carotene by non­
aqueous reversed-phase high-performance liquid chromatog­
raphy (HPLC).
Numerous researchers have reported other solvent extraction
procedures for β-carotene and provitamin A carotenoids before
H P L C analysis. Zakaria and Simpson (2) homogenized a mixture
of tomatoes and acetone in a blender; the acetone extract was
then extracted with petroleum ether. Gatautis and Pearson (3) ex­
tracted carotenoids from plasma with ethanol and hexane. Sadler
and co-workers (4) extracted lycopene and β-carotene from

for the liquid extraction procedure. Only corn and carrots tomato paste and pink grapefruit using a mixture of h e x a n e -
contained detectable levels of α-carotene, and, in both cases, acetone-ethanol. Rouseff and co-workers (5) determined the
liquid extraction yielded slightly higher results. Liquid concentrations of β-carotene and other hydrocarbon carotenoids
extractions were performed in approximately 90 min, and in red grapefruit cultivars using a slightly modified version of
supercritical fluid extractions were performed in 30 min; Sadler's extraction method. Khachik and co-workers (6) ana­
however, the supercritical fluid extractions procedure required lyzed the major carotenoid constituents of broccoli, green beans,
less than 10 min of an analyst's time while the liquid extraction spinach, and tomatoes by chromatographing their tetrahydro-
procedure was labor intensive. furan extracts. Also, in an earlier work, Khachik and co-workers
(7) compared the efficiencies of three solvent systems (tetrahy-
drofuran, petroleum ether-acetone, and diethyl ether-methanol)
for e x t r a c t i n g the m a j o r c a r o t e n o i d s of b r o c c o l i , c a b b a g e ,
spinach, Brussels sprouts, and kale.
Introduction
At least two researchers have investigated the use of solid-
phase extraction as a technique to simplify sample preparation
As a result of the November 8,1990 passage of the Nutritional
procedures for the determination of carotenoids in foods. M o s -
Labeling and Education Act (NLEA) by Congress, the National
quera and co-workers (8) used octadecyl ( C ) disposable ex­ 18
F o o d Processors Association estimates that 158 billion food
traction columns as a clean-up procedure for the analysis of
packages will need to be relabeled. Many, if not all, food prod­
chlorophylls and carotenoids in virgin olive oil by H P L C . A
ucts will require nutrient analysis. Considering the relatively
similar C solid-phase extraction procedure was used by Fisher
1 8
short t i m e a l l o w e d for food c o m p a n i e s to c o m p l y with the
and Rouseff (9) in the analysis of β-cryptoxanthin and α- and β-
N L E A , there is an urgent need for accurate, more rapid testing
carotene in orange juice.
procedures for vitamins and other nutrients in foods, especially
T h e major deficiencies of all these extraction techniques are
for β-carotene, the major contributor to the vitamin A activity of
that they are time consuming and require large amounts of or­
fruits and vegetables.
ganic solvents. Use and disposal of organic solvents are expen­
T h e official Food and Drug Administration procedure for the
sive and potentially dangerous.
determination of β-carotene in foods is cumbersome and time
The present work attempts to minimize the amount of solvents
consuming (1). It involves the following steps: saponifying with
used for the extraction of β-carotene from vegetables. Two ana­
K O H ; adding 50 m L ethanol; extracting in a separatory funnel
lytical approaches are d e v e l o p e d and c o m p a r e d : a s o l v e n t -
*Author to whom correspondence should be addressed.
solvent extraction method that minimizes the use of conven-

422 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
Journal of Chromatographic Science, Vol. 31, October 1993
tional solvents (requiring only 10 m L ethanol and 16 m L pentane
per sample) and a supercritical fluid carbon dioxide extraction
(SFE) procedure. The solvent-solvent extraction procedure pre­
sented in this work is a modification of an extraction procedure
previously reported in the literature (10) for the analysis of ter-
penes and sesqueterpines in orange juice.
S F E is a highly desirable alternative to conventional solvent
extraction for several reasons: speed of extraction, complete­
ness of extraction, elimination of the analyte concentration step,
cost savings, nontoxicity of carbon dioxide, and simplification of
the analytical method. Tonucci and co-workers (11) reported
that the p e r c e n t r e c o v e r y of c a r t e n o i d s from food s a m p l e s
(tomato paste, canned pumpkin, fresh spinach, red p a l m oil,
butter, and Monterey Jack cheese) varied depending on the type
of sample. Generally, the greatest yields were at pressures and
temperatures at or above 4,000 psi and 40°C, respectively. Com­
pared with the dynamic m o d e , the static m o d e yielded larger
amounts of carotenoids. One percent ethanol, methanol, and iso-
propanol modifiers greatly enhanced extractability of carotenoids
from food samples.
We compare a - and β-carotene results for carrots, broccoli,
yellow squash, zucchini, collard greens, kale, mustard greens,
corn, and turnip greens using two new p r e - H P L C extraction
techniques: one method is based on solvent extraction and the
other method is based on SFE.
Experimental
Solvent extraction
Vegetable material to be tested was macerated in a Waring
blender for 3 min at high speed. Approximately 5 g of this mate­
rial and 10 m L ethanol (Formula S D A 3 A) were added to a 50-
m L glass, round-bottom centrifuge tube. The contents of the tube
were then homogenized with a Biohomogenizer (Biospec Prod­
ucts; Bartlesville, OK) for approximately 3 min. For the first ex­
traction, 8 m L pentane was added, and the mixture was reho-
mogenized a second time for an additional 2 min. (As a safety
precaution, the Biohomogenizer was retrofitted with a vent to
permit nitrogen purging of the motor when extracting with highly
volatile organic solvents.) After centrifugation at 7000 × g for 3
min, the upper carotenoid-containing pentane layer was removed
with a Pasteur pipette and added to a second 50-mL centrifuge
tube.
A second extraction was performed on the vegetable material.
Approximately 2 g sodium chloride and 5 m L distilled water were
Table I. SFE Conditions
Chamber temp. 40°C
Pressure 338 atm (.93 g/mL)
Flow rate 1.5 mL/min (liquid)
Static time 20.0 min
Dynamic time 10.0 min
Restrictor temp.
Extraction 45°C
Rinse 40°C
Trap temp.
Extraction 10°C
Rinse 40°C
added to the centrifuge tube that contained the original 5 g of
vegetable sample. The tube was shaken gently. An additional 8 m L
pentane was added. A second extraction was performed by shaking
the tube vigorously for 2 min. (Agitation and homogenization
with the Biohomogenizer was not conducted in this second ex­
traction.) After centrifugation, the pentane layer was removed
with a Pasteur pipette and added to the first pentane extract.
T h e combined extracts were then evaporated (if necessary)
with a stream of nitrogen to permit dilution to an appropriate
v o l u m e in a volumetric flask (e.g., 5.0 m L or 10.0 m L , d e ­
pending on the concentration of β-carotene in the sample). D i ­
lutions with pentane were m a d e so that the final concentration of
β-carotene in the vegetable extracts was near the middle of the
range of concentrations used for the H P L C working standards.
Supercritical fluid extraction
All SFE experiments were performed with a supercritical fluid
extractor (7680A, Hewlett-Packard; Wilmington, D E ) . T h e in­
strument was equipped with a variable restrictor, which allowed
independent control of flow and pressure and reduced plugging
when there was large sample throughput. T h e extracted c o m p o ­
nents were deposited onto a solid-phase trap packed with H y -
persil octadecylsilica (ODS) material placed at the end of the
variable restrictor and rinsed from the trap into a 2-mL vial with
HPLC-grade hexane.
S F E samples were prepared by weighing 1 g of sample and 2
g ethanol into a 3 0 - m L polypropylene beaker. T h e sample was
blended with the Biohomongenizer for approximately 3 min. In
order to absorb the liquid portion of the sample, approximately
2 g of Hydromatrix was added to the beaker and mixed thor­
oughly with the sample. T h e sample and H y d r o m a t r i x w e r e
transferred to a 7-mL extraction thimble and extracted under
the conditions shown in Table I. T h e dead volume of the extrac­
tion thimble containing the sample was measured by water dis­
placement and was found to be approximately 2 m L . T h e ex­
t r a c t i o n p r o c e d u r e c o n s i s t e d of a 2 0 - m i n static e x t r a c t i o n
followed by a 10-min dynamic extraction at 4 0 ° C and 338 atm.
Depressurization of the sample chamber prior to the rinse step re­
moved any residual ethanol from the collection trap. Quantitation
of the β-carotene was performed by H P L C .
HPLC-UV assay
Extracts of the vegetables were injected into a Rainin H P L C
(Woburn, M A ) equipped with a 20-μL loop injector, a Vydac 201
T P 5 4 C reversed-phase column (5-μm particle size, 4.6 m m x
1 8
250 m m ) (Hesperia, C A ) , and a Hewlett-Packard model 1040A
multidiode array detector. T h e detector wavelength was 4 5 0 nm,
Table II. Effect of Various Sample Treatments on the
Recovery of β-Carotene in Broccoli Florets
Relative
Experimental treatment* ppm β-carotene % recovery †
Acetone-pentane 1.16 ±0.03 ‡
100.0
Ethanol-pentane 1.24 ±0.03 107.0
Enzymes; ethanol-pentane 1.09 ±0.04 94.0
Saponification; ethanol-pentane 1.03 ±0.05 88.9
Homogenization; ethanol-pentane 2.39 ± 0.03 206.2
* Broccoli florets macerated in blender for 3 min, and 5 g of macerated material was
subjected to the various experimental treatments.
†
Normalized to value of acetone-pentane, the Association of Official Analytical
Chemists' recommended solvent system.
‡
Uncertainty represents the average deviation of duplicate determinations.
423

and the flow rate of the mobile phase was 1.5 mL/min. Integra­
tion and control of the H P L C were done with Rainin D y n a m a x
M e t h o d M a n a g e r software (version 1.2) and a Macintosh com­
puter. Isocratic analysis was performed using a mobile phase
consisting of 6 5 % methanol, 2 7 % acetonitrile, 4 % methylene
chloride, and 4 % hexane. This mobile phase was a slightly mod­
ified version of the m o b i l e p h a s e used by K h a c h i k and c o ­
workers (6).
Results and Discussion
HPLC studies
In tests with more than 20 commercial columns, Quackenbush
(12) found that a Vydac TP-201 C reversed-phase column was
1 8
the most effective at resolving α-carotene from β-carotene and
cis- from trans-isomers of β-carotene.
In our work, three different C reversed-phase columns were
1 8
evaluated for their ability to separate α-carotene from β-carotene:
a Waters N o v o - P a k column (60Å, 4-μm particle size, 3.9 m m ×
150 m m ) (Milford, M A ) ; a Supelcosil column (5-μm particle
size, 4.6 m m × 250 m m ) ; and a Vydac 201TP column (5-μm par­
ticle size, 4.6 m m x 250 m m ) . T h e Vydac 201TP column pro­
vided the best resolution between α-carotene and β-carotene
and was selected as the column of choice for the analysis of β-
carotene in vegetables.
Initially during the development of a suitable HPLC method for
the extraction of β-carotene, persistent noisy baseline problems oc­
curred because the use of highly volatile solvents in the mobile
phase tended to cause cavitation. T h e cavitation problem was
eliminated by slowing the intake and compression cycles of the
p u m p . The Refill and Compensation settings in the D A program
(of the Rainin H P L C Method Manager software) were adjusted to
5 and 2, respectively. Lowering the Refill setting prevented va­
porization of volatile solvents during the pump intake stroke. The
Compensation setting controlled the duration of the rapid recom­
pression portion of the p u m p cycle; lowering this value also im­
proved baseline noise for the solvent system selected.
Standard calibration has always been a major source of error
in the determination of β-carotene in foods. Inaccurate stan­
dards are often responsible for substantial differences between re­
sults from different laboratories. This problem has been known
to contribute errors of 2 0 % or more in analytical results (13).
Carotene standards are highly susceptible to oxidation and degra­
dation, so commercially available carotene standards are often
less pure than expected. Therefore, stock standard solutions of α-
and β-carotene were routinely checked for purity with a spec-
Table III. Effect of Various Modifiers on Solubility in
Supercritical CO 2
Modifer Area of β-Carotene
None 13,000
Water 13,000
Ethanol (50 μL) 90,000
Ethanol-Water (100 μL each) 164,000
Ethanol (100 μL) 2,800,000
Methylene Chloride (100 μL)*
Hexane (100 μL) †
- varying amounts of methylene chloride modifier: A, no methylene chloride
- added; B, 10 μL added; C, 50 μL added; and D, 100 μL added. The sam­
* Significant decomposition and isomerization.
†
Significant portion of sample lost to waste.
424
Journal of Chromatographic Science, Vol. 31, October 1993
trophotometer using B e e r ' s law and published values for the
1% absorptivity coefficients of a - and β-carotene as discussed by
Rouseff and co-workers (5).
Using an internal standard calibration method proved unnec­
essary. N o improvement in precision was noted when β-apo-8 -
carotenal was used as an internal standard. Furthermore, this
compound, which is frequently used as an internal standard for
carotenoid analysis, tended to coelute with chlorophylls and
xanthophylls in some of the vegetable samples tested. Although
modification of the m o b i l e p h a s e m a y h a v e p r e v e n t e d this
problem, the external standard calibration method was found to
be simpler and more useful.
Five working calibration standards were prepared from the
s t o c k s t a n d a r d s o l u t i o n . T h e c o n c e n t r a t i o n r a n g e of t h e s e
working standards was 0.25 to 10.0 p p m α-carotene and β-
carotene. Linear calibration curves were routinely observed ( R 2
≥0.998). Positive identification of a - and β-carotene peaks was
accomplished by comparison of retention times with authentic α-
and β-carotene standards (Sigma; St. Louis, M O ) and by exam­
ination of photodiode array spectra of chromatographic peaks (5).
Solvent extraction studies
Autooxidation and cis-isomerization are k n o w n to contribute
to significant recovery losses in the analysis of provitamin A
compounds. Minimizing exposure to air and to prooxidant metals
such as iron, copper, and their salts is necessary to control auto­
oxidation. cis-Isomerization of trans-pro vitamins occurs in ex­
tracts or solutions and is accelerated by light, heat, or strong
acids. Therefore, exposure to air, iron and copper, light, heat, and
strong acids was minimized for all samples tested.
T h e extraction efficiency of two different solvent systems
was evaluated for β-carotene recovery using broccoli florets as
a typical vegetable sample. T h e two solvent systems studied
were ethanol-pentane and acetone-pentane. The procedure for
the ethanol-pentane extraction has been described; the procedure
for the acetone-pentane extraction is identical, except acetone is
substituted for ethanol. These two solvent systems were selected
for study because they are recommended in the Association of
Official Analytical Chemists' (A.O.A.C.) procedure for the anal­
ysis of carotenes in fresh plant materials and silages (14).
The ethanol-pentane extraction procedure gave higher recov­
eries than the acetone-pentane method for broccoli analysis.
Figure 1. Extraction of 100 μL of 40 ppm β-carotene in hexane with
ples were spotted on Whatman #40 filter paper and extracted under con­
ditions described in Table I.
Journal of Chromatographic Science, Vol. 31, October 1993
Approximately 7 % more β-carotene was extracted from broccoli
with the alcohol-pentane system than with the acetone-pentane
combination. A study was conducted to determine whether three
extractions per sample rather than two improved recoveries.
Analysis of the broccoli, corn, collard greens, kale, and zuc­
chini revealed that less than 3 % more β-carotene was extracted
from samples in the third extraction.
Initially, tetrahydrofuran and ethyl ether w e r e considered
promising solvents for the extraction of β-carotene from veg­
etables. T h e solubility of β-carotene is approximately 16 times
greater in tetrahydrofuran than in pentane and 2 times greater in
ethyl ether than in pentane; however, tetrahydrofuran and ethyl
ether are known to develop peroxides that can rapidly degrade β-
carotene. Although some researchers have circumvented this
problem with the addition of antioxidants (e.g., butylated hy­
droxy toluene) to the solvent, Quackenbush and Smallidge (15)
have warned that using strong antioxidants can promote au-
tooxidation of β-carotene. Therefore, T H F and ethyl ether were
excluded from this study.
Chloroform and methylene chloride have been used by some
researchers for extracting β-carotene from foods and beverages
(15). Although β-carotene is more soluble in these solvents than
in pentane, using chloroform and methylene chloride solvents is
inadvisable. Discoloration of β-carotene standard solutions pre­
pared in chloroform and especially in methylene chloride was ob­
served. H P L C analysis of these standards revealed significant
cis-isomerization and degradation of the trans-β-carotene. De­
pending on the purity of the solvents, isomerization and degra­
dation occurred over a period of a few minutes to several hours.
Further evidence of the tendency of these solvents to degrade β-
carotene has been recently reported in the literature (16).
In addition to solvent type, three other experimental variables
w e r e investigated to i m p r o v e extraction recoveries: e n z y m e
treatment, saponification, and homogenization before extraction
with organic solvents. Again broccoli was used as a typical veg­
etable. Results from this study appear in Table II and were in­
valuable in developing an effective SFE procedure.
The enzymes studied were Cellulase A C , Hemicellulase Con­
centrate, and Clarase 40,000. All e n z y m e s were obtained in
powder form from Solvay Enzymes Inc. (Elkhart, IN). Enzymes
were dissolved in p H 6.5 buffer to make three separate solutions
containing 5 m g / m L of enzyme. Broccoli florets (5 g macerated
in blender), 2 m L of the cellulase solution, and 2 m L of the
hemicellulase solution were incubated under nitrogen stream in
the dark for 2 h at 50°C. After the 2-h incubation period, 2 m L
of the Clarase 40,000 solution was added, and the incubation was
continued for an additional 2 h. Because no improvement in β-
carotene recoveries was observed, enzyme treatment was con-
Figure 2. Frontal peak broadening and distortion of the β-carotene peak
in standard solutions. A: 5 ppm β-carotene standard solution in hexane.
Β: 5 ppm β-carotene standard solution in methylene chloride.
sidered ineffective and not used in the procedure.
T h e A.O.A.C. method for quantitation of carotenes and xan-
thophylls in dried plant material uses a methanolic K O H hy­
drolysis step. Saponification of plant extracts prior to analysis has
often been used to r e m o v e chlorophylls and lipids that can in­
terfere with chromatographic detection (17).
Saponification of broccoli prior to solvent extraction w a s ac­
complished by adding 2 m L of 10% methanolic K O H solution to
5 g of sample and incubating under a nitrogen stream at r o o m
temperature for 90 min. A s indicated in Table II, significant
alkali-induced losses in β-carotene were observed. Results for β-
carotene were approximately 17% higher in the unsaponified
sample than in the saponified sample. Khachik and co-workers
(7) reported a 6% loss of β-carotene in broccoli attributed to the
saponification step. T h e authors reported an average percent
loss caused by saponification of 5 4 % for nine other carotenoids
monitored in this study. Based on the experimental results re­
ported here and results previously reported in the literature, al­
kali saponification was considered inappropriate for the analysis
of β-carotene in vegetables. Saponification, however, m u s t b e
used to evaluate the presence of carotenal esters if these c o m ­
pounds are of interest.
T h e most effective preextraction sample treatment was h o ­
mogenization of vegetables with the Biohomogenizer. Recovery
of β-carotene from broccoli was 9 9 % higher with h o m o g e n i z e d
samples than with unhomogenized samples.
Supercritical fluid extraction studies
The ideal SFE method would be rapid, quantitative, and require
little or n o additional sample preparation. In an attempt to meet
these criteria, the effects of several variables were evaluated.
Modifiers. The addition of modifiers to C 0 can greatly im­
2
prove extraction efficiency by increasing solubility of the analyte
in C O or by reducing its interaction with the sample matrix or
2
both. In order to determine the effect of different modifiers on the
solubility of β-carotene in C O , approximately 200 m g of crys­
2
talline β-carotene was placed on filter paper and extracted with
and without modifier for a 10-min static extraction followed by
a 10-min dynamic extraction at 40°C and 338 atm. Modifiers that
were evaluated included ethanol, methylene chloride, hexane,
and water. The results are shown in Table III.
Although β-carotene is soluble in h e x a n e , the addition of
hexane modifier did not significantly increase β-carotene solu­
bility in supercritical C O . β-Carotene is highly soluble in methy­
2
lene chloride, and the addition of methylene chloride modifier
significantly increased the solubility of β-carotene in supercrit­
ical C 0 . The addition of methylene chloride modifier, h o w -
2
Figure 3. Degradation and isomerization of β-carotene by impure hexane
rinse solvent. A: Elution of β-carotene from the trap with fresh LC-grade
hexane. Β: Elution of β-carotene from the trap with LC-grade hexane ex­
posed to air and light for 24 h.
425

ever, induced significant degradation of β-carotene as shown in
Figure 1. In addition, a significant portion of the extracted β-
carotene (and its degradation products) passed through the trap
and accumulated in the waste line.
In general, the removal of water from vegetable material fa­
cilitates SFE. Water is usually removed by drying the sample in
an oven maintained at or near 100°C. Removing water in this
manner is not r e c o m m e n d e d for β-carotene analysis because β-
carotene is readily oxidized. Because the vegetable materials
contained 7 5 % to 9 2 % water, the effect of water on the solubility
of β-carotene in supercritical C 0 needed to be determined. It
2 Figure 3. This problem was eliminated by rinsing with LC-grade
was found that w h e n added as the modifier, water neither sig­
nificantly increased nor decreased the solubility of crystalline β-
carotene in supercritical C O . This does not account, however,
2 trap was rinsed with hexane that had been sitting in the rinse
for the matrix effect of water.
Although β-carotene is less soluble in ethanol than in hexane,
the addition of ethanol modifier significantly increased the sol­
ubility of β-carotene in C 0 . T h e addition of 100 μL of ethanol
2
modifier increased the a m o u n t of β-carotene extracted by a
factor of 215. A s a result of these findings, ethanol was used as
the modifier.
β-Carotene collection. The extracted material was deposited
onto a solid-phase trap upon decompression of the supercritical
C O . T h e trap can b e packed with either stainless steel beads or
2 from those that were blended and then homogenized. Because ho­
the O D S material and can be heated or cooled independent of the
restrictor temperature. Although stainless steel beads were ca­
pable of trapping β-carotene when little or no ethanol modifier
was used, a significant portion of β-carotene was found in the
waste line when m o r e than 200 μL ethanol modifier was used.
The O D S material was capable of retaining all β-carotene when
large amounts of ethanol modifier were used, provided the trap
temperature w a s maintained at a temperature no higher than
10°C. T h e ethanol modifier that remained on the trap was re­
moved by depressurizing the extraction thimble before the rinse
step. It was necessary to condition the O D S trap prior to the first
extraction of the day by rinsing with ethanol and hexane.
Rinse solvents. Because the O D S material has a high affinity
for β-carotene, it w a s necessary to rinse the trap with a solvent
that readily dissolves β-carotene. Methylene chloride and chlo­
roform readily eluted β-carotene from the trap, but often in-
Figure 4. Effect of homogenization on the recovery of β-carotene from
sweet peppers. Duplicate analysis for blended and homogenized samples.
426
Journal of Chromatographic Science, Vol. 31, October 1993
duced significant (greater than 50%) decomposition. Further­
more, because β-carotene is m u c h more soluble in methylene
chloride than in the L C mobile phase, quantitation of β-carotene
was difficult because of the broadening of the front edge of the
peak as shown in Figure 2.
Although β-carotene is significantly less soluble in hexane
than in methylene chloride, 1 m L of hexane was sufficient to elute
all β-carotene from the trap; however, some precautions were nec­
essary. Low-purity hexane induced isomerization of β-carotene
and promoted artifact formation, as seen in the chromatogram in
hexane. It is important to maintain the purity of the hexane.
Degradation and isomerization of β-carotene occurred when the
solvent reservoir for more than 24 h. This problem was alleviated
by changing the rinse hexane daily, protecting the hexane from
light with aluminum foil, and purging the hexane with helium
prior to the first extraction of the day. G C - M S analysis of the
hexane before and after prolonged exposure to air and light re­
vealed the formation of low molecular weight oxygenated c o m ­
pounds that may be responsible for the β-carotene degradation.
Particle size. Figure 4 shows the rate and recovery of β-
carotene extraction from sweet peppers that were blended and
mogenizing the sample significantly reduced the size of the par­
ticles, homogenized samples were extracted much more rapidly.
For some samples (i.e., corn, carrots, and sweet peppers), quan­
titative extraction could not be achieved without homogenization.
Static vs. dynamic. A s indicated in Figure 4, it is necessary to
allow time for the ethanol to modify the sample matrix. Figure
5 shows the effect of static time on recovery of a - and β-carotene
from homogenized carrots followed by 10 min of dynamic ex­
traction. Samples that were low in β-carotene or were high in
starch or solids (e.g., corn) required a m i n i m u m of 20 min of
static time as well as thorough homogenization. For most of the
green leafy vegetables (i.e., kale, mustard greens, turnip greens),
m a x i m u m extraction of β-carotene can be achieved with static
times under 10 min. Increasing the time of the dynamic extrac­
tion did not yield results that were statistically different.
Condition-plumbing test. Before the first extraction of the
Figure 5. Effect of static extraction time on the recovery of β-carotene from
homogenized carrots. Static extraction was followed by a 10-min dy­
namic extraction.
Journal of Chromatographic Science, Vol. 31, October 1993
Figure 6 . Results of α-carotene and β-carotene analysis of various veg­
etable samples. All vegetables were commerically prepared. Carrots were
fresh. Collard greens, turnip greens, diced turnips with greens, kale, mus­
tard greens, and diced yellow squash were wet-pack frozen. Broccoli,
corn, and zucchini were individually quick frozen. Results reported as
the average of three extractions.
day, the plumbing of the instrument was tested to ensure that β-
carotene was quantitatively extracted and retained on the trap.
This was accomplished by extracting 100 μL of a saturated so­
lution of β-carotene in ethanol from filter paper. T h e extract
was rinsed from the trap with 1 m L hexane and analyzed by
H P L C . A n additional 100 μL of the β-carotene solution was di­
luted to 1 m L in hexane and analyzed by H P L C . A difference in
the areas of the β-carotene peaks of more than 5 % was usually
indicative of a problem with the SFE. T h e most c o m m o n prob­
lems were insufficient conditioning of the collection trap, im­
purities in the rinse solvent, and small leaks in the instrument.
Comparison of solvent extraction and SFE analytical results
As indicated in Figure 6, results for β-carotene tended to be
higher with SFE than solvent extraction. For the 10 vegetables
studied, SFE extracted an average of 2 3 % more β-carotene than
solvent extraction. T h e largest percent difference between the
t w o m e t h o d s was for zucchini in which the S F E result was
8 1 . 3 % higher. The tendency for SFE to provide higher β-carotene
results may be caused by the superior diffusivity of supercritical
C 0 compared with liquid solvents. Enhanced diffusivity may be
2
responsible for more efficient extraction of β-carotene from the
plant cell chloroplasts.
Of the 10 vegetables studied, only corn had higher β-carotene
results by liquid extraction than by SFE. As mentioned previ­
ously, corn posed a difficult problem for SFE because of its high
solid and starch content. Adding 1 m L ethanol to the extraction
thimble and reextracting the sample was usually sufficient to ex­
tract the remaining β-carotene. Only corn and carrots contained
detectable levels of α-carotene, and in both cases, liquid extrac­
tion yielded higher results. By performing a second extraction as
described above, the remaining α-carotene could be recovered.
One shortcoming of the SFE method is that most of the ethanol
modifier is removed from the extraction cell within the first 2 - 3
min of the dynamic extraction. Using a tank of C 0 modified 2
with ethanol or an external modifier p u m p to constantly deliver
ethanol to the extraction cell should provide more consistent re­
sults and eliminate the need for a second extraction of difficult
samples.
Although precision, as measured by the average deviation of du­
plicate determinations, was excellent for both methods, the preci­
sion for the solvent extraction method was slightly better. This may
be due to the fact that sample weights used for liquid extraction
were 5 to 10 times larger than sample weights used for SFE.
Homogenization of the sample prior to extraction was critical
for both liquid and S F E extractions. F o r s a m p l e s that w e r e
blended but not homogenized, extraction rates were much slower.
For corn, carrots, and some other high starch and high solid sam­
ples, quantitative extraction could not be achieved without h o ­
mogenization. Results for β-carotene extracted from sweet pep­
pers that were not homogenized were 2 5 % lower than the results
for sweet peppers that were homogenized. Tonucci reported 7 8 %
recovery of lycopene from tomato paste (11). Preliminary exper­
iments performed in our laboratory indicate that homogenization
of tomato paste would lead to more quantitative extraction.
A l t h o u g h h o m o g e n i z a t i o n of v e g e t a b l e s p r i o r to S F E is
strongly recommended, it m a y be possible to quantitatively ex­
tract the carotenoids without homogenization if a continuous
source of ethanol is supplied via an ethanol modified tank of C 0 2
or through the use of a modifier p u m p . For liquid extracted sam­
ples, slightly higher recoveries of carotenes could be obtained if
the macerated sample-ethanol mixture was incubated at r o o m
temperature in the dark under a nitrogen atmosphere for 1 h
prior to pentane extraction.
Conclusions
SFE provides recoveries of a - and β-carotene from vegetables
that are equal to or greater than recoveries obtained with tradi­
tional solvent extraction techniques. Furthermore, accurate quan­
titative results can be obtained in less time and with minimal sol­
vent usage. T h e results suggest that S F E may b e an important
analytical tool for the study of other types of food carotenoids
that are currently being studied because of their cancer-preven-
tative properties.
Acknowledgments
T h e authors would like to thank Dennis Gere and R o b Smith
of Hewlett-Packard for their suggestions, ideas, and support
with the SFE.
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Manuscript received December 1 2 , 1 9 9 2 ;