Fatty flow-rates, direct injection of the sample diluted in

 Fatty acids


acids are important not only as nutritional substances in living organisms, for
example, long-chain polyunsaturated fatty acids (PUFAs), but they are also used
to characterize the quality of oils. SFC offers the possibility of using low
temperatures, high flow-rates, direct injection of the sample diluted in
n-hexane or n-heptane, no derivatization, universal detectors such as flame

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detectors (FID), evaporative light scattering detectors (ELSD) or MSD, and the
simultaneous analysis of fatty acids and other lipid classes (triacylglycerols,
cholesterol, etc.). Hirata and Sogabe pioneered the use of comprehensive
two-dimensional supercritical fluid chromatography (Cao et al2003). They
developed an SFC ×SFC
system with conventional packed columns and FID detector for separating FAMES
from various edible and fish oils. The coupling of SFC and RP-HPLC was studied
by Francois and Sandra (Careri.,2001) They separated the fatty acid (such as
phenacyl esters) of fish oil employing ELSD and UV detectors. Hori et al.
employed SFC coupled with triple quadrupole mass spectrometry to determine 3- monochloropropane-1,2-diol
(3-MCPD) fatty acid esters in edible oils ( Matsubara et al., 2014).


Triacylglycerols (TAGs) are the main
representatives of neutral lipids. The
chromatographic separation of triacylglycerols most typically involves
high-performance liquid chromatography (HPLC) with reversed-phase or silver-ion
columns( Dugo,2004). In addition, gas chromatography (GC) has been used to
separate the methyl esters obtained by trans-esterification of the TAGs (Park etal.,2010).
However, this option has several problems due to the fact that the trans esterification of polyunsaturated
triacylglycerols is not always quantitative(
Milinsk, 2008) and that there is a lack of
information about intact TAG composition. Supercritical fluid chromatography
permits an analysis of TAGs at a high boiling point or when thermally labile,
in short analysis times and without sample derivatization and only by diluting
in an organic solvent. Initially determinationswere carried out using capillary
columns(Lesellier,2000) with non-polar stationary phases (principally
polymethylsiloxane, phenylmethylsiloxane and octyl-methylpolysiloxane) as well as
polar ones (phenyl-cyanopropylpolysiloxane, cyanopropylphenyl-
methylpolysiloxane and polyethyleneglycol). The temperatures employed were 140 ?C or 170 ?C and a
gradient of density. An analysis of triacylglycerols by packed SFC has been performed
by using octadecyl silica (ODS) (Lee,2012)or silver ionexchange (Sandra.,2002)columns


Phospholipids are the most important
class of polar lipids. Different analytical
techniques have traditionally been used for phospholipid analysis, including thin
layer chromatography (TLC), high performance liquid chromatography (HPLC) and
gas chromatography (GC). They have been summarized in several reviews (Restuccia,2012). The
application of SFC to the separation of phospholipids was developed initially
by Lafosse.,et al(1993).They separated phosphatidylcholine, phosphatidic acid,
phosphatidylinositol and phosphatidyl ethanolamine, from soya lecithin, on a
Zorbax silica column (25 cm ×4.6 mm) using isocratic conditions
and evaporative light scattering detection (ELSD). The mobile phase was CO2
modified with 21.6% of a mixture of methanol–water–triethylamine (95/4.95/0.05)
at a flow-rate of 4.3 ml/min, with 27.8 MPa pressure and 45 ?C
temperature. Separation was achieved in 22 min. The research group of T. Bamba
is especially active in the development of lipid analysis methods using SFC-MS
in APCI or ESI modes( Lee et al.,2013). Although most of their work is applied to biological samples,
the separation of complex lipid mixtures including phospholipids, glycolipids,
neutral lipids and sphingolipids is worthy of mention.

Carotenoids and
fat-soluble vitamins

are tetraterpenoidic lipophilic compounds with health beneficial properties (Britton
et al.,2009) widely spread in the plant
kingdom. Carotenoids can be divided in two main classes:

carotenes and xanthophylls. The first class is composed of hydrocarbons
molecules, while the second one contains oxygenated moieties.They
are synthesized by acombination of eight isoprene units and play an important
role in biological processes, not only in plants but also in humans.. In fact, the majority of the published investigations on carotenoids in natural
matrices have been performed after a saponification
step to simplify the analysis, thus losing information on the native
carotenoid profile and probably forming also some artifacts (Rodriguez
et al., 2016). in the carotenoid analysis field there is the need to develop
faster and possibly, also “green” analytical approaches, for both carotenoid
extraction and analysis (Mercadante.,2016) Although recently, SFC coupled
to MS has gained attention as a fast and useful technology applied to the
carotenoids analysis (Giuffrida, et al., 2017) and few recent reports
are available on the SFE of carotenoids (Durante., 2014).More recently
The online coupling of various extraction techniques, including SFE, with LC
and GC has been overviewed  more recently
by Cifuentes and colleagues in 2017 (Sánchez-Camarg et al., 2017)

first study dealing with SFC separation of carotenoids was published by McLaren
et al McLaren, (1968) in 1968. Since then, numerous papers using this technique
have appeared, which indicates that SFC can be a promising alternative to traditional
HPLC methods as it permits separations between the carotenoids that are more
difficult to achieve, such as cis/trans isomers. Lesellier et al (1993)  studied the role of 16 organic modifiers in
the separation of _ and _-cis/trans carotenes using sub-SFC with organic
modifier, UV detection and C18 columns. Matsubara et al (2009) developed an
SFC-MS method to separate a mixture of _-carotene, zeaxanthin, lycopene,
lutein, antheraxanthin, neoxanthin and violaxanthin. Abrahamsson et al (2012)
developed an SFC method for determining carotenoids in SFE extracts of a
microalgae (Scenedesmus sp.)


The dietary supplement has found
wide use in last ten years. Although many of these product claim to be
100% natural, their adulteration has been reported many times. To determine the composition and to guarantee the quality and
safety of dietary supplements, appropriate analytical methods are necessary. Traditionally
high performance liquid chromatography (HPLC) or gas chromatography (GC) has
been the first choice but SFC has emerged as a valuable alternative offering
shorter separation times compared with HPLC. Carotenoids and
isoflavones are found in many dietary supplements. Moreover, carotenoids have
similar structures and exist in a number of structural isomer forms, which are
difficult to separate. UHPSFC with sub-2-?m particle columns was used for the
separation of nine carotenoids (including carotenes and xantophylles) (B et
al., 2015) Polyphenols from SFE grape extracts were determined by
Karnangerpour et al.,(2002)  using
SFC.Some years later Ramirez et al. (2004) analyzed phenolic antioxidants (carnosic acid and carnosol) in
rosemary extracts using a capillary column (25 cm ×500 _m)
packed with silica particles coated with SE-54 (5% phenyl, 95% methyl silicone)
and pure CO2 at 100 ?C and a gradient of pressure (from 15.2 MPa to 37.5 MPa); in this
case analysis time was 30 min. Dugo et al.,(1996) studied the separation of six polymethoxylated flavones
(tangeretin, heptamethoxyflavone, nobiletin, tetra-Omethylscutellarein, hexamethoxyflavone
and sinensetin).



SC-CO2 extraction technology offers solutions for
problems such as product purity, process efficiency, health, and environmental impact
that traditional technologies fail or are unable to solve. Therefore, a
substantial increase in research and developmentactivities has been observed as
summarized above. However, further developments in the areas of fractionation
and particle formation
offer innovative approaches into the future for food and beverage processing,
satisfying the current market demand. With the flexibility offered by
supercritical fluid technology in terms of combining various unit operations of
extraction, fractionation, particle formation, etc., and the know-how developed
over the past three decades, the future offers the development
of novel processes and products based on value-added processing of agricultural
materials for the food and beverage industry

            The extraction of solute will change the phase
equilibrium of the solvent/solute and will, of course, alter the phase diagram
and the critical point of the solvent dramatically. This makes the prediction
and design of extraction conditions very difficult. For the
solubilization of polar solutes, modifiers or co-solvents (also called
entrainers) are often added to CO2 in quantities up to 5(%v/v). The
addition of modifiers or co-solvents, such as methanol, ethers, and
crown-ethers, can introduce many of the disadvantages of liquid solvent
impurities.In addition, the high pressure used in the SCE requires that all
vessels, transfer lines, valves etc. to be designed as unfired pressure vessels
in accordance with the application code.The high cost of compressors, makes the
initial capital outlay for a SCE process very high