PHYTOCHEMICAL ANALYSIS, VOL.

8, 244–246 (1997)

Supercritical Fluid Carbon Dioxide Extraction of

a-and b-Carotene from Carrot (Daucus carota L.)

Amitabh Chandra and Muraleedharan G. Nairw

Bioactive Natural Products Laboratory, Department of Horticulture and Pesticide Research Center, Michigan State University, East
Lansing, Michigan 48824, USA

Carrots (Daucus carota L. var. Caro Pride) were extracted with supercritical fluid carbon dioxide (SFOC2) under
various combinations of pressures and solvent modifiers at 40°C and analysed for their a- and b-carotene content
by high pressure liquid chromatography. The SFCO2 extraction at 40°C and 60.6 MPa with 5% chloroform as
modifier afforded 111.16 and 148.32 mg of a- and b-carotene per gram of dried carrot, respectively. Also, this
method extracted 92.70% of the total carotenoids present in the dried carrots when compared to the solvent
extraction using chloroform (100%). © 1997 by John Wiley & Sons, Ltd.

Phytochem. Anal. 8, 244–246, 1997

No. of Figures: 1. No. of Tables: 1. No. of Refs:. No. of References: 16.
Keywords: Supercritical fluid extraction; high pressure liquid chromatography; carbon dioxide; carotenoids; a-carotene; b-carotene;
carrots; Daucus carota L.

INTRODUCTION (Stahl and Schutz, 1980). SFCO2 extraction has been

Pigments from carrots are provitamin A and are used as
natural food colourants. Carotenes are prone to decomposi-
tion and isomerization upon exposure to light and heat
(Favati et al., 1988). Conventional extraction methods for
plant pigments involve the use of very high temperatures.
Also, autoxidation is known to contribute to the low
recovery of these compounds (Marsili and Callahan, 1993).
Therefore, milder extraction conditions for carotenoids
from natural sources are important to obtain intact pigments
in higher yields. Supercritical fluid extraction with carbon
dioxide (SFCO2) is established as a milder extraction
method since it extracts solutes at comparatively low
temperatures and pressure. It also preserves the integrity
and stability of most of the thermally labile compounds
w
Correspondence to: M. G. Nair, Bioactive Natural Products Laboratory,
Department of Horticulture and Pesticide Research Center, Michigan State
University, East Lansing, Michigan 48824, USA. E-mail: nairm@pi-
lot.msu.edu
Contract grant sponsor: Crop and Food Bioprocessing Center.
Contract grant sponsor:Center for Plant Products
extensivley used for isolation of several natural products of
consumer interest (Spanos et al., 1993). We have reported
SFCO2 extraction methods for the isolation of thermally
labile biologically active natural products and flavour
components from various plant sources (Langezaal et al.,
1990; Yao et al., 1994; Chandra and Nair, 1995, 1996).
Carotenoids are known to play a vital role in the human
body by acting as antioxidants (Bryant et al., 1992). Among
the carotenoids, b-carotene exhibited the highest provitamin
A activity. Therefore, its bioavailability in food products is
of great consumer interst (Sweeney and Marsh, 1973). Main
sources of carotenoids in nature are paprika and carrots.
However, a large portion of commercially available b-
carotene is synthetically produced (Favati et al, 1988).
Carotenoids are present in the plant cells as chlorophyll- and
caroteno-protein conjugates (Bryant et al., 1992). Forma-
tion of such complexes are considered to enhance the
stability of the carotenoids, especially under high tem-
perature processing.
Recently, carotenoid pigments, especially b-carotene,
have received much attention owing to their biological
activity (Mangels et al., 1993). Evidence of antitumour
activity of carotenoids in humans has revived the interest in
these compounds with respect to their use in food, nutrition
and medicine (Poppel, 1993). Currently, high consumer
demand for these compounds is met by b-carotene produced
by synthetic methods (Favati et al., 1988). Contamination of
the carotenoids with low levels of chemicals used in their
production and the isomeric impurities are of great concern
(Miyahara et al., 1995). Therefore, naturally occurring
carotenoids extracted with SFCO2 should be in great
demand for their use in food and medicine.
This paper describes the SFCO2 extraction of a- and b-
carotenes from lyophilized carrots and their quantification
by high pressure liquid chromatography (HPLC). The
carrots were subjected to chloroform extraction to extract
completely carotenoids, in order to compare the efficiencies
of SFCO2 extraction conditions employed for a- and b-
carotenes.

CCC 0958–0344/97/050244–03 $17.50 Received 23 February 1996

© 1997 by John Wiley & Sons, Ltd. Revised 15 July 1996
Accepted 1 September 1996
 SFE OF CAROTENE FROM CARROT 245

Table 1. Yields of a- and b-carotene extracted from carrots using extraction methods
A–Fa
Carrot Extract
Extraction
methoda mass yield a-Carotene b-Carotene
(g) (mg) (mg) (mg)
A 2.08( ± 0.02) 19.27( ± 2.48) 171.09( ± 17.67) 220.45( ± 24.45)
B 2.10( ± 0.02) 24.60( ± 3.50) 227.54( ± 33.61) 288.52( ± 26.53)
C 2.07( ± 0.01) 22.16( ± 2.59) 174.36( ± 18.40) 228.10( ± 26.74)
D 2.07( ± 0.01) 25.27( ± 2.59) 191.95( ± 20.42) 255.43( ± 26.74)
E 2.04( ± 0.03) 24.40( ± 2.03) 226.76( ± 18.96) 302.58( ± 25.71)
F 2.07( ± 0.02) 46.80( ± 1.00) 235.21( ± 2.89) 344.16( ± 4.34)
a
For extraction protocols see Experimental section.

EXPERIMENTAL processes A to E and solvent extract F were dissolved

General Procedures. SFCO2 extractions were performed on a
Dionex (Salt Lake City, UT, USA) model 703 supercritical
fluid extractor in 8 3 32 mL stainless steel extraction cells
equipped with a Dionex 703M solvent modifier unit. The
HPLC analysis was performed on a capcell-pak C-18 HPLC
column (250 3 4.6 mm i.d.; 5 mm, Dychrom, Sunnyvale,
CA, USA) using a Waters (Milford, MA, USA) photo diode
array detector at 445 nm, equipped with Millennium 2010
software (Waters). The samples were filtered through
0.22 mm filter prior to analysis. Methanol:chloroform
(90:10 v/v) was used as mobile phase under isocratic
conditions (flow-rate (1.0 mL/min) ). The experiments were
conducted in triplicate. The weights of the extracts and a-
and b-carotenes are expressed as their mean values ( ± SD).
separately (2.0 mg/mL) in chloroform:methanol (65:35 v/v)
and passed through a 0.22 mm filter prior to analysis. The
HPLC analyses were carried out at ambient temperature.
RESULTS AND DISCUSSION
Previously, carrots have been extracted using SFCO2 at
34.2 MPa and 40°C. A combination of static mode (20 min)
followed by dynamic mode (10 min) extraction with ethanol
as modifier was necessary to achieve 90–100% recovery of
a- and b-carotenes by this procedure (Marsili and Callahan,
1993). The carrot samples were homogenized with ethanol
prior to the SFCO2 extraction. Also, homogenization of

Plant Material. Daucus carota L. var ‘Caro Pride’, a

commercial variety, was grown in the Horticulture Experi-
ment Station at Michigan State University. The carrots
(1404.95 g) were lyophilized (5°C; 48 h), milled and the
resulting orange powder (168.0 g) was used for the SFCO2
and chloroform extraction studies.

SFCO2 and Solvent Extraction. Liquid carbon dioxide

(99.996%) charged with a helium head space at 13.69 mPa
(AGA Gas Inc., Cleveland, OH, USA) was used as the
extraction fluid; the restrictor was kept at 175°C. All SFCO2
extractions were conducted separately at 40°C (1 h) at
various combinations of pressures and solvent modifiers,
namely A, 40.4 MPa; no modifier; 500–600 mL/min; B,
50.5 MPa; no modifier; 600–750 mL/min; C, 60.6 MPa; no
modifier, 600–900 mL/min; D, 60.6 MPa; 5% hexane;
800–850 mL/min; E, 60.6 MPa; 5% chloroform;
700–800 mL/min. Chloroform extraction (F) was carried
out by soaking the milled plant material in chloroform
(2 3 50 mL) in the dark at ambient temperature for 24 h. All
extractions and analyses were carried out in triplicate under
identical conditions. The amounts of plant material and the
extracts obtained by processes A to F are shown in Table
1.

HPLC Analyses. Standard solutions of pure a- and b-

carotene (500 mg/mL each; Sigma Chemical Co., St Louis,
MO, USA) in chloroform:methanol (65:35 v/v) were used
to generate the calibration curves. The solutions were
prepared by serial dilutions of the respective stock solutions
to afford concentrations of 250, 125, 62.5, 31.25, 15.63,
7.81, 3.91, 1.95 and 0.98 mg/mL, respectively. The calibra-
tion curve was generated by Millennium 2010
chromatography manager software. The extracts from
Figure 1. HPLC chromatogram of the SFCO2 extract of carrots
obtained using process B described in Experimental section.
Key to peak identity: 1—a-carotene; 2—b-carotene. HPLC condi-
tions: column — Capcell-pak C-18 (Dychrom) 250 3 4.6 mm i.d.;
mobile phase—methanol:chloroform (90:10, v/v) isocratic; flow-
rate—1.0 mL/min.

Phytochem. Anal. VOL. 8, 244–246 (1997) © 1997 by John Wiley & Sons, Ltd.
246 A. CHANDRA AND M. G. NAIR
plant materials with the modifier solvent is recommended
prior to SFE in order to extract quantitatively the carote-
noids unless a continuous source of solvent is supplied
through a solvent modifier tank of carbon dioxide or a
modifier pump (Marsili and Callahan, 1993). In our
experiments we have used hexane and chloroform as
solvent modifiers through an online modifier pump, thus
eliminating the need for homogenization of the carrots prior
to the extraction.
The extracts from carrots were dissolved in chloro-
form:methanol (65:35 v/v) and analysed by HPLC without
any further clean up. Gradient systems involving methanol
and chloroform, and isocratic systems with methanol,
acetonitrile, dichloromethane and hexane have been used as
the mobile phases for the HPLC analysis of carotenes
(Marsili and Callahan, 1993; Spanos et al., 1993). We have
carried out the HPLC separation and quantification of a-
and b-carotene using methanol:chloroform (90:10 v/v) as
the mobile phase under isocratic conditions. The HPLC
retention times (Rt) for standard a- and b-carotenes at 26.83
and 28.63 min, respectively, were identical to those given
by the a- and b-carotenes present in the carrot extracts (Fig.
1).
Extraction processes A–C yielded 63.75, 82.63 and
66.27% of b-carotene, respectively. SFCO2 using 5%
hexane and chloroform solvent modification increased the
yield of b-carotene considerably. The combined amounts of
a- and trans-b-carotene extracted under SFCO2 processes
A–E were 67.25, 87.79, 69.46, 77.21 and 92.70%, respec-
tively, when compared to the amounts extracted by
chloroform alone (F). The recovery of these compuonds
from carrots under SFCO2 methods A–C indicated that
process B (at 40°C and 50.5 MPa pressure) was the best
extraction condition in the absence of a solvent modifier.
The density of carbon dioxide at 40°C increases in the order
of 0.97, 1.04 and 1.34 at 40.4, 50.5 and 60.6 MPa,
respectively. An increase in the density of SFCO2 should
increase the polarity of SFCO2 and influence its extraction
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abilities (Myer et al., 1991). However, method C gave a
lower yield of carotenoids (by 18.33%) when compared to
process B. An inversion in extraction capacity of SFCO2
was observed by increasing the pressure by 10.1 MPa at a
constant temperature of 40°C.
Addition of solvent modifiers to SFCO2 increases its
solvating power. Thus appropriate combinations of pres-
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efficiently (Hawthorne, 1990). Therefore, method C was
modified by adding 5% each of hexane (D) or chloroform
(E) into the SFCO2. The yield of b-carotene obtained from
processes D and E was increased by 7.95 and 23%
compared with B, respectively. Also, the total pigment
content was higher in these extracts when compared to the
SFE extracts from A–C. Therefore, SFE for 1 h at 40°C ,
60.6 MPa pressure and 5% chloroform as modifier was
found to extract most of the carotenoids when compared to
the extraction with chloroform alone for 24 h. This method
(E) afforded 111.6 and 148.32 mg of a- and b-carotenes per
gram of dried carrots compared to 113.63 and 166.26 mg/g,
obtained by solvent extraction (F). In other words, the
SFCO2 extraction process E extracted 98.21 and 89.20% of
the total a- and b-carotenes present in the carrots.
These SFCO2 extraction methods can be further modified
or applied as such for the isolation of b-carotene from other
sources rich in carotenoids such as Capsicum spp (Levy et
al., 1995) and other varieties of carrots which possess
higher b-carotene content (Mangels et al., 1993) providing
natural b-carotene for use in food and human medicine.
Acknowledgements
This is a contribution from Michigan State University Agriculture
Experiment Station and was partially funded by grants from the Crop and
Food Bioprocessing Center and the Center for Plant Products at Michigan
State University.
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Phytochem. Anal. VOL. 8, 244–246 (1997)