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Carotenoids extraction and quantification: A review

Article  in  Analytical Methods · June 2013

DOI: 10.1039/c3ay26295b

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Analytical
Methods
CRITICAL REVIEW

Carotenoids extraction and quantification: a review†

Cite this: Anal. Methods, 2013, 5, 2916
Héctor Arvayo-Enrı́quez, Iram Mondaca-Fernández, Pablo Gortárez-Moroyoqui,
Jaime López-Cervantes and Roberto Rodrı́guez-Ramı́rez*

In recent years carotenoids have represented a good alternative for the pharmaceutical and food industries
and especially for the human health, they prevent different diseases, such cancer, macular degradation and
cataracts. The use of post-harvest residues represents a good alternative for obtaining analytes of interest.
The application of enzymatic pretreatment over plant matrices has resulted in an increase in the level of
Received 30th October 2012
Accepted 28th March 2013
extraction and lowered operating costs. Today, the use of organic solvents such as hexane in this
process is frequent, however, new investigations with novel environmentally friendly solvents (non-toxic)
DOI: 10.1039/c3ay26295b
promise new cleaner technologies. The aim of this review is to present some methodologies and
www.rsc.org/methods technologies used today to obtain carotenoids from plant residues, industrial and post-harvest materials.

1 Introduction 700 known carotenoids, which are divided into carotenes (e.g.
a-carotene, b-carotene, lycopene) and xanthophylls, which
Carotenoids are natural pigments found in all plants and are represent the oxygenated carotenoids fraction (lutein, zeax-
among the most important nutrients in food; they are derived anthin, and b-cryptoxanthin) (Fig. 1). The a-carotene, b-caro-
from acyclic C40 isoprenoid lycopene, which can be classied as tene and b-cryptoxanthin are also the carotenoids that are
a tetraterpene.1 Carotenoids are fat-soluble micro constituents promoters of vitamin A.4 In plants, carotenoids play a crucial
which have benecial effects to human health, including role in protecting chlorophyll with antioxidant activity and
protection against cancer, cardiovascular diseases and macular these properties are one the many reasons that carotenoids are
degeneration.2 They represent one of the most important of interest to humans. All carotenoids exist in plants in the trans
classes of pigments in plants. Today there are approximately geometric form; however, thermal processing can induce trans
to cis carotenoid isomerization.5
Departamento de Biotecnologı́a y Ciencias Alimentarias, 5 de Febrero 818 Sur, Cd.
The content of carotenoids in plants is inuenced by several
Obregón, Mexico. E-mail: [email protected]; Fax: +524100910; Tel: factors, these may be genetic, environmental, or strategies used
+524100900 ext. 2134 to manage the crop during its growth. The latter can result in
† Electronic supplementary information (ESI) available. See DOI: an increase in the concentration of carotenoids. However,
10.1039/c3ay26295b

Hector Arvayo Enriquez Iram Mondaca Fernandez

obtained his bachelor degree in obtained his PhD in Agriculture
Biotechnology Engineering from and Biological Engineering from
ITSON (Technological Institute the University of Arizona, USA.
of Sonora) in Cd. Obregon, He is a researcher-professor
Sonora Mexico. He is presently since 1994, at the Technological
working towards his PhD at Institute of Sonora, Mexico. His
ITSON developing a method- research work is around the use
ology for the analysis of carot- of biotechnological methods to
enoids. His research interests obtain value added products,
are centered in the development such as colorants and biofuels,
of analytical methodology in from agriculture and livestock
utilization of agro-industrial waste. He has participated in
waste from the food industry. projects related to the use of FTIR infrared methods to the detec-
tion of changes in microbial viability, production of biofuels and
the design of microbial biosensors for the detection of soluble
organic matter.

2916 | Anal. Methods, 2013, 5, 2916–2924 This journal is ª The Royal Society of Chemistry 2013
Critical Review Analytical Methods

post-harvest processes can alter the chemistry of carotene,

hence, their bioavailability.6
In the market, b-carotene sales have been estimated to
surpass USD 280 million in 2015. Most of the b-carotene
commercialized in the world is obtained by chemical synthesis
from b-ionone. There are biotechnological alternatives using
lamentous fungi, bacteria, microalgae, and yeast as producers,
or even by extraction from vegetable sources.7

2 Carotenes extraction procedure

The obtained carotenoid-rich extract is usually used in health
foods, food additives, medicines and cosmetics. One of the
problems is the elimination of the residual solvents to obtain a
safe extract.8 It is well established that t low efficiency in the
Fig. 1 Chemical structure of some nonpolar (carotenes) and polar (xanthophylls) extraction process of carotenoids may be due to the difficulty of
carotenoids. Redrawn with permission from ref. 3. Copyright 1994 American the solvent molecules to penetrate the plant tissue and thus
Chemical Society.
solubilising carotene, which is located in the structure of the
chromoplast.9
Several extraction processes involve the use of commercial
Pablo Gortáres-Moroyoqui is enzyme preparations and they are complemented by extraction
professor, since 1987, at the with organic solvents such as hexane and ethyl acetate and
Technological Institute of mixtures, which are approved for use in foods in most coun-
Sonora, Mexico. He obtained his tries.9 The same processes are being applied for the utilization
PhD in soil, water, and environ- of industrial waste, as is the case of the ethanol industry where
mental science from the Univer- waste is rich in corn zein and xanthophylls. These carotenoids
sity of Arizona, USA. His research could be extracted with 70% ethanol (v/v).10 The extraction of
work is around environmental colorants is being applied to the treatment of another kind of
problems such as wastewater waste, such the residues of sheries and aquaculture processes,
quality and treatment, waste- for the extraction of carotenoids. The best results for the
water recycling, air pollution, extraction from shrimp shell waste using various vegetable oils
pollutants transport and fate, were obtained at a temperature of 70
C for 150 minutes using
and environmental impact sunower oil; similar results were obtained with soybean oil.11
assessment. He has participated in several projects related to One of the crucial points in the process of extraction is the
wastewater quality, treatment and reuse, pollutants transport on solvent used; it has the ability to separate analytes of interest
the environment. He has been leading several projects related to from a mixture of compounds due to affinity with the solvent. In
environmental microbiology and biotechnology. He is currently case of b-carotene, hexane has been one of the most widely used
studying micro pollutants and the environment. solvents in the industry, due to its high affinity for carotenoids.

Jaime López-Cervantes is Roberto Rodriguez Ramirez has

Research Professor at the Tech- a bachelor degree in Food
nological Institute of Sonora. He Chemistry from Universidad of
received a PhD in Food Biotech- Sonora, in Hermosillo, Sonora,
nology at the University of San- Mexico and a Masters and PhD
tiago Compostela in Spain. in Food Science from CIAD, A.C.
Professor Lopez-Cervantes has (Food Research and Develop-
developed and optimized a clean ment Center) in Hermosillo,
technology for shrimp biowaste Sonora, Mexico. He has been
processing for recovery and working as a scientist and
separation of chitin, protein and professor at ITSON (Technolog-
astaxanthin. His research ical Institute of Sonora) in Cd.
includes native plants and active Obregon, Sonora Mexico. His
packaging technologies. He has published more than 48 interna- research interests are centered in the development of analytical
tional papers and four international patents. He participated as methodology for solving food authenticity, traceability and
director of thesis guiding for MSc and PhD students. biotechnology issues, mainly based on DNA technologies.

This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 2916–2924 | 2917
Analytical Methods Critical Review

This solvent has been a reported treatment in the extraction of processes of hot air drying, vacuum drying and pressure cook-
xanthophylls from Calendula owers.12,13 ing. It was found that in all the treatments conversion of all-
The extraction of carotenoids should be carried out under trans-b-carotene to 13-cis-b-carotene, with regard to the last two
special conditions, and be performed in the shortest possible treatments had the lowest rates of degradation.24 It is worth
time in order to avoid exposure to light,14 oxygen, high mentioning that during the drying process, carotenoids are
temperatures, so minimizing auto-oxidation and isomer- highly susceptible to oxygen, heat, and light, so easily promote
isation.15 The addition of antioxidants such a-tocopherol (TOC) oxidative degradation. The degradation will depend on the
in a dosage of 50 mg/10 mL of solvent,16 butylated hydrox- protective activity of antioxidant present in the extracellular
ytoluene (BTH) at nal concentrations ranging from 0.1%,17 matrix.25 Other researchers recommend using drying tempera-
0.05%18 and 0.01%19 inhibited this process. It is recommended tures below 70
C in which the b-carotene and lycopene have a
the addition of calcium or magnesium carbonate to the mixture high retention rate regardless of the drying process. The
of extraction solvent with the sample to neutralize organic acids replacement of air by nitrogen during the drying process does
usually found in plant material.20 Investigators suggest the not have advantages in the stability of b-carotene above 70
C.26
application of pre-treatment (enzymatic, thermal, chemical) to
the sample has been shown to increase the extraction of caro-
tenes.13 Some examples can be seen in Table 1.
2.2 Extraction with biological methods
In recent years, research into the application of enzymatic
2.1 Dry pretreatment to analysis and conservation treatments in vegetable matrices to obtain the release of ana-
In many cases, to quantify the carotenoid content of the vege- lytes of interest has been increased. It has been shown that the
table matrix it is necessary to eliminate all the water content application of enzymatic treatments in carrot and dried
because most of the solvents used for extraction have non-polar pumpkin improves the attractiveness of dehydrated products.
properties. It is known that the drying process is used to Usually, the disintegration of the cell wall structure accelerates
preserve foods, including vegetables. There is little information metabolic transformations, allowing undesirable colours and
available about the effect of drying vegetables such carrots on avour changes, but this has not been observed in enzymati-
the process of cis–trans isomerisation of b-carotene, however, cally treated vegetable matrices, the reason being that the
some investigators have conducted research to determine the carotenoids released from cellular structures by pectinase and
kinetics of isomerisation of b-carotene in carrot under cellulase are still bound to proteins, so they keep their state,

Table 1 Chemical and biological treatments applied to diverse matrices

Type
extraction Sample Carotene Conditions Advantages Disadvantages Author Reference

Chemical Tomato, Lycopene, b- Extraction with - Non-toxic solvent.- Expensive Ishida and 16
carrots and carotene and ethyl lactate Environmentally Champman,
white corn lutein friendly - 2009
Chemical Corn Lutein Using ethanol 70% - High yield extraction — Kale et al., 10
and concentration 2007
upper 90%.- Low cost
solvent
Chemical Shrimp Astaxanthin Vegetal oil, ratio - The carotene is - Has been applied Sachindra and 11
waste 2 : 1 oil/waste. conservated in natural only in sea waste Mahendrakar,
Temperature 70
C matrix.- High yield residues 2005
for 2 half hour extraction
Chemical– Tomato Lycopene and 300 bar at 80
C, - Non-toxic solvent.- - High cost of Sabio et al., 21
Physical processing b-carotene using 130 g of CO2 Environmentally extraction.- 1998
waste per gram of sample friendly Infrastructure
Chemical– Carrots b-carotene 120 to 327 bar at - Non-toxic solvent.- - High cost of Salda~
na et al., 2006 22
Physical 40–50
C. Environmentally extraction.-
friendly Infrastructure
Biological Marigold Lutein Commercial enzymes - Recovery yield of - Using hexane as Barzana et al., 2002 23
owers (pectinase, cellulase 97%.- Reutilization of solvent to extraction
(T. erecta) and hemicellulase) the extraction solvent
Biological Marigold Lutein Commercial and non- - Enzymatic treatment - Using hexane as Navarrete et al., 13
owers commercial enzymes increase xanthophylls solvent to extraction 2005
(T. erecta) (produced from yield extraction- The
Flavobacterium IIb, highest content of
Acinetobacter xanthophylls were
anitratus, and obtained form non-
Rhizopus nigricans) commercial enzymes

2918 | Anal. Methods, 2013, 5, 2916–2924 This journal is ª The Royal Society of Chemistry 2013
Critical Review Analytical Methods

This process can improve its efficiency if it uses native or

microbial ora present in the plant material. During the
extraction process losses of carotene of around 50% can occur,
depending on the conditions of silage, drying and solvent
extraction. An alternative in the extraction process is the
application of enzymatic treatments prior to the use of
solvents.23 Applying appropriate pre-treatments to the plant
matrix is possible to increase cell wall permeability, facilitating
the mechanisms of diffusion and mass exchange between the
immiscible phases during the leaching process. There is a
correlation between the application of enzymatic pre-treatment
and the extraction yield of carotenoids.13 Several experiments
have shown that complex substrates have been hydrolysed
under a combination of different enzymes.29 The use of raw
enzymes has advantages over the use of commercial enzymes
because of their cost-effectiveness rate and a substantial
reduction in processing time can be achieved while attaining
high carotene content.13
In industrial processes, some microorganisms are used for
the production of cellulolytic enzymes, like A. terreus with an
enzyme activity of 0.688 U mg1 of protein.30 Aspergillus niger is
considered as one of the most complete multienzyme producers
to produce cellulases, hemicellulases, glucoamylases, pecti-
nases, showing enzymatic activities of 0.99, 15.86, 13.37, and
7.62 U mg1 of protein respectively.29
Water is vital to carry out enzymatic treatment, however,
although it allows the enzymatic hydrolysis to take place, an
excess of this solvent can slow the extraction process due to the
formation of an aqueous phase which prevents the solvent
becoming in contact with the vesicles of intracellular lipid
leading to carotenoids.23 Agitation of the system during enzy-
matic treatment seems to facilitate the diffusion of the enzyme
Fig. 2 Schematic representation of the hydrolysis of amorphous and micro-
from the supernatant (liquid phase) into with the vegetable
crystalline cellulose by noncomplex cellulose system. Redrawn from ref. 33 with
permission from American Society for Microbiology. matrix (solid phase). The rapid adsorption of the enzyme
accelerates the lysis of the cell wall, leading to an increase in the
extraction yield. This is why agitation plays an important role in
which provides stability of the highly unsaturated pigment
enzymatic treatment.28
structure and the colour remains more stable during storage.27
2.2.1 Experimental analysis with enzymes. Some of the
The use of enzyme mixtures generated from cultures of
factors that are involved in enzymatic treatments are the ratio
microorganisms can reduce the processing time by 95%
enzyme/substrate, the temperature and pH conditions. The
compared to treatments where commercial enzymes are used.28

Table 2 Quantification of carotenoids using HPLC in different matricesa

Analysis
Sample Carotene detector Author Reference

Petals (Calendula officianalis L.) Lycopene, g-carotene and rubixanthin DAD-MS Kisimoto et al., 2005 46
Fruits and vegetables (Spain Consumption) b-carotene, lutein and lycopene DAD Go~ni et al., 2006 57
Carrot powder (wastes) Lutein, a-carotene and b-carotene DAD Chen and Tang, 1998 58
isomers
Processed foods and food supplements Retinol, lutein, a- and b-carotene DAD Akhtar and Bryan, 2008 18
Pure standards b-Carotene isomers DAD-UV-VIS Rajendran and Chen, 59
2007
Eggs Lutein, zeaxanthin and b-carotene UV-VIS Hargitai et al., 2006 49
Tomato, carrots, chili bell (green and red), Lycopene and b-carotene UV-VIS Olives et al., 2006 12
watermelon
a
DAD ¼ Diode Array Detector, MS ¼ Mass Spectrometry UV-Vis ¼ Ultraviolet-Visible.

This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 2916–2924 | 2919
Analytical Methods Critical Review

concentrations of enzymes used for pre-treatment of plants industry.10 Some investigations compare the use of hexane,
range from 0.01 to 0.1% (w/w).31 ethyl acetate and the mixture hexano/acetona/ethanol
2.2.2 Use of cellulases. As it is well known, cellulase acts on 50 : 25 : 25 (v/v) to extract lycopene from dry tomato peel.9 For
cellulose, which is present in the main wall below the rst half the extraction of astaxanthin from Hematococcus pluvialis a
layer of the cell wall of plants. The primary wall consists of a mixture of ethyl acetate and ethanol (1 : 1, v/v) was found to be
rigid skeleton of cellulose embedded in a gelatinous matrix the best extraction solvent tested due to its high efficiency and
containing pectic compounds, hemicellulose, and glycopro- low toxicity compared with other organic solvents.43 An alter-
teins.32 Fig. 2 presents a diagram of cellulose hydrolysis by a native to traditional extraction methods is pressurized liquid
noncomplex cellulase system. extraction (PLE) that is currently used to extract biologically
One of the standard procedures for measuring enzymatic active constituents. This system uses conventional solvents at
activity is by using carboxymethylcellulose (CMC) and a colori- elevated temperatures and pressures, and it requires less
metric method (DNS) to estimate the amount of reducing sugars solvent and shorter extraction times, resulting in an efficient
released.34 and environmentally friendly extraction technique. The use of
Cellulases production can be made from agro-industrial and ethanol in PLE may be a useful method for extracting zeax-
domestic waste, inoculated with cellulolytic microorganisms, anthin from Chlorella ellipsoidea, a green microalga which has
which can produce large amounts of cellulases. Some of the more than nine times the total zeaxanthin level than that of red
microorganisms currently used are Aspergillus niger, Saccharo- pepper.44 The same process has been used to extract carotenoids
myces cerevisiae and Trichoderma longibrachiatum.35 from carrot by-products using pressurized hot ethanol.45
2.2.3 Use of pectinase. Pectin is a major constituent of
plant cell walls; pectin structure engineering can be used as a
2.4 Carotene quantication and analysis
tool to modify the structural quality of plant based food prod-
ucts.36 During cell development, pectin is modied by the There are several methods used for quantication of caroten-
enzyme pectin methylesterase that confers different properties oids, and these are described in the AOAC and have been
to the cell wall.37 Pectinases have the ability to break down proposed through the years by various researchers. The analysis
pectin and pectic compounds, the latter polymer of 100–200
galacturonic acids, is in the primary and also in the middle
lamella.32 Pectin is a heteropolysaccharide composed of chains
of galacturonic acid with 1.4-a links. Pectins have a high
percentage of methylated esterication. Pectin degradation
requires a combined action of several enzymes that can be
classied into two main groups: methylesterases, which remove
methoxyl groups pectin, and depolymerases (hydrolases and
lyases), which cleave the bonds between galacturonate units.38
In pectinase production diverse experiments demonstrated that
the addition of glucose increased pectinesterase and poly-
galacturonase production in solid state systems, but in
submerged fermentations the production was markedly
inhibited. However, pectin acts as an inducer for the production
of pectinolytic enzymes by microbial systems.39,40
Concerning the balance between structural properties and
the nutritional value of vegetables, there exists an inverse rela-
tion between the structural quality, represented by the texture
and the carotene in vitro bio-accessibility, indicating that vege-
tables with a rm texture have low amounts of bio-accessible
carotene and vice versa.36

2.3 Extraction with chemical methods

The use of hexane and ethanol in several vegetable matrices
have been evaluated for quantication of carotenoids such as
lycopene, a and b-carotene.41 However, many of these solvents
do not comply with regulations for human consumption. One
alternative is the use of environmentally friendly solvents. Ethyl
lactate is an excellent solvent for extraction of trans–cis-lyco-
Fig. 3 The use of (A) monomeric and (B) polymeric C18 columns, and (C) C30
pene, b-carotene16 and astaxanthin.42 The use of ethanol as “carotenoid column” for the separation of b-carotene in preparations containing
solvent has given good results when used for the extraction of a high percentage of cis-isomers. Redrawn with permission from ref. 3. Copyright
carotenoids, besides xantophylls waste from the ethanol 1994 American Chemical Society.

2920 | Anal. Methods, 2013, 5, 2916–2924 This journal is ª The Royal Society of Chemistry 2013
Critical Review Analytical Methods

has been applied to several parts of the plants. Various inves- identication of carotenoids, however, this methods fails to
tigations have been conducted on the analysis of petals46 differentiate between other carotenoids or the same carotene
leaves,15 fruits, vegetables, roots,41 grains,47 pulp and various isomers (cis and trans forms), so that the use of chromato-
tissues.48 On the other hand, the content of carotenoids in foods graphic techniques in the separation of these compounds
of animal origin such as eggs49 or clinical analysis with refer- represents a good alternative for the quantication of some
ence to human health have been conducted. particular carotene molecules.53 For the analysis of astaxanthin,
2.4.1 Sample handling. Sample handling is one of the most derivative spectrophotometer techniques offer a powerful tool
important factors in the analysis of carotenoids in plants, since for the quantitative analysis of multicomponent mixtures. The
a more accurate quantication of the compounds present in the statistical analysis between rst-order derivative spectropho-
sample can be observed. The use of modied atmospheres of tometry and high performance liquid chromatography (HPLC)
nitrogen and various antioxidants during sample handling by T-testing revealed no signicant differences between these
decreases the rate of isomerisation of carotenoids.50 Some of two methods, which is rapid, convenient, and suitable for
these antioxidants are a-tocopherol (TOC) at concentrations of routine analysis.43 In food industry it is frequent used in the
50 mg per 10 mL of solvent providing optimum extraction yields analysis of total carotenoids content in foods. Some techniques
aer 1 hour, on carrot powder.16 The use of powdered vegetable have been used for this purpose and are classied in two
matrices is recommended, because under these conditions the groups, the rst is a destructive technique including spectro-
extraction time is considerably reduced. photometry and HPLC and the second is a non-destructive,
2.4.2 Saponication process. It is necessary to have enough direct (i.e., no sample preparation required) method including
information about the content of carotenoids in foods to carry resonance Raman spectroscopy, photoacoustic spectroscopy
out trials on the benets of carotenoids in humans for various and colorimetry. The results obtained by the direct methods
industrial applications. The saponication process is correlate well with the total carotenoid content assessed by
commonly used for the analysis of carotenoids in fruits and spectrophotometry and HPLC.54
vegetables for the removal of esteried xanthophylls and One of the conventional techniques for the qualitative and
substances which may interfere in the analysis. However, the quantitative quantication of carotenoids in various tissues of
disadvantage of this process is the extra steps performed during the plant is HPLC. However, the use of this technique is not
sample handling, such as drying (solvent evaporation), the always justied by its cost and handling. In some cases, when it
solubilisation of the sample prior to injection equipment such is necessary to determine the composition of the carotenoids, a
as HPLC, quantication, etc., thus increasing the time and cost simple and inexpensive quantication by spectrophotometric
of analysis.48 methods is a good option as it can be used without making prior
The saponication process is oen used with green vegeta- chromatographic separation. These days the use of HPLC and
bles like broccoli, beans, bell chillies, lettuce, spinach, etc. Some spectrophotometric techniques together are the technologies
researchers describe a method of saponication for quantitative used mostly in the eld of analysis of carotenoids. Some
determination of carotenoids in green vegetables.51 The method detectors used for quantication and HPLC determination of
involves acetone extraction procedures and the selective carotenoids are UV-Vis,53 mass spectrometry and photodiode
removal of chlorophylls and esteried fatty acids from the array detection, the latter represents one of the best alternatives
organic phase using a strong basic resin (Ambersep 900 OH). because it allows the generation of three-dimensional spectra
One advantage of this method is the low level of b-carotene for carotenoids. HPLC coupled with visible spectrophotometric
isomerisation during saponication. In one traditional process and mass spectrometric detection creates a powerful tool for the
of saponication, carotene has a long contact time with examination of composition of natural products.46,55,56 The
hydroxyl ions thus resulting in oxidation and isomerisation due multidimensional nature of scanning diode detectors may be of
to the multiple extractions and evaporation. It exists as a quick great importance for the study of complex natural products for
methodology, which signicantly reduces the saponication the quantication of carotenoids. Some examples of different
time, obtaining a high recovery of carotene and low solvent detectors utilized in quantication of carotenes are indicated in
consumption, with an 80–90% reduction in the cost and time of Table 2. Reliability of data obtained by HPLC depends on the
analysis.4 In the case of samples rich in b-carotene, such carrots, accuracy of the calibration. It exists as a strategy for isolating
the saponication process is unnecessary.12 However, plants or standards by open column chromatography and quantication
fruits that contain signicant proportions of esteried xantho- by HPLC, using leafy vegetables as examples.60
phylls must be saponied for accurate quantication of carot- Some of the solvents used for HPLC quantication of
enoids (provitamin A).48 carotenoids are water, methanol, and methyl tertiary butyl
Working with samples rich in lycopene content, the extrac- ether.55 Methanol is relatively inexpensive and has low residual
tion procedure without saponication can only be employed for toxicity but may cause viscosity in the presence of water. Using
samples containing low amounts of lipids (<1%). However, a methanol and isopropanol it is possible to separate some of the
signicant difference in the amount of lycopene analyzed by most important carotenoids except lycopene which cannot be
either using the extraction method with or without saponica- eluted under these conditions.18
tion is not detected.52 In the selection of chromatographic column is important to
2.4.3 Analysis methods and measurement conditions. consider factors such particle size, shape, size and degree of
Spectrophotometry is a technique commonly used in the pore.56 In all investigated systems a C30 column presents the

This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 2916–2924 | 2921
Analytical Methods Critical Review

best separation and resolution compared to a C18 column.18 reduced by up to 5–10 fold, compared to conventional HPLC
Investigators comparing C18 and C30 columns under various methods. UHPLC methods can therefore be applicable for a
solvent systems for separation of b-carotene and its cis isomers, wide variety of foods.61 Recently capillary electrophoresis (CE)
found that b-carotene isomers were efficiently separated using a has been found to be a powerful separation technique for the
C30 column under gradient elution of methanol and methylene separation of chlorophylls and carotenoids, with advantages
chloride.59 A comparison of the resolution of the chromato- such as rapid analysis time, low volume of sample, great sepa-
grams by using a chromatographic column C18 and C30 are ration efficiency, and higher selectivity.62 Recent advances in CE
shown in Fig. 3 and 4. The use of ultra high pressure liquid have been presented.63
chromatography (UHPLC) method is known to be economical Other available technologies include the use of supercritical
and environmentally friendly due to its extremely rapid analysis uid extraction (SFE) processes. This uses solvents such as CO2
times. The consumption of solvent for the mobile phase can be which offers an organic-chemical-free process that yields high

Fig. 4 The use of (A) monomeric and (B) polymeric C18 columns, as well as engineered C30 “carotenoid column” in separations of carotenoid standards. Redrawn with
permission from ref. 3. Copyright 1994 American Chemical Society.

2922 | Anal. Methods, 2013, 5, 2916–2924 This journal is ª The Royal Society of Chemistry 2013
Critical Review Analytical Methods

quality in food products, compared to traditional extraction 9 R. Lavecchia and A. Zuorro, Eur. Food Res. Technol., 2008,
methods with organic solvents. An important step in SFE 288, 153–158.
processes for plant materials is the measurement and predic- 10 A. Kale, F. Zhu and M. Cheryan, Ind. Crops Prod., 2007, 26,
tion of the solubility of the compound in supercritical uid at 44–53.
various temperatures and pressures in order to optimize the 11 N. M. Sachindra and N. S. Mahendrakar, Bioresour. Technol.,
extraction process. The solubility of carotene is related to 2005, 96, 1195–1200.
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