Chapter 5
Analytical Supercritical Fluid Extraction
for Food Applications
Tracy Doane-Weideman and Phillip B. Liescheski
Isco Incorporated, Lincoln, NE 68504
Abstract
In this review, we explore the fundamental concepts of supercritical fluids and
supercritical fluid extractions. Carbon dioxide and other solvents are discussed; the
solubility theory is introduced together with the calculation of the density of carbon
dioxide. The state-of-the-art instrumentation is presented in terms of fundamental
components. The most widely used application of analytical SFE is in the food
industry and this review includes fats, oils, vitamins, and pesticides in research and
routine applications.
Introduction
Supercritical fluid extraction (SFE) is becoming an important sample preparation
method in the chemical analysis of food products, especially for fats and fatty oils.
SFE has been used successfully for over a decade in analyses of food samples
(1,2). The most popular SFE solvent is carbon dioxide (CO
2
). Triglycerides, cho-
lesterol, waxes, and free fatty acids are quite soluble in supercritical CO
2
. The sol-
ubility of polar lipids, such as phospholipids, can be improved by augmenting the
supercritical CO
2
with a small addition of ethanol or other polar modifier solvent.
Even though CO
2
is considered a “green-house” gas, it is ubiquitous in nature and
can be retrieved from the environment and returned clean (3). As a result, SFE can

or sublimation temperatures at given pressures. These curves characterize the tem-
peratures and pressures at which two phases coexist in equilibrium. For example,
the liquid-gas equilibrium curve divides the liquid and gaseous phase regions. On
this curve, the substance coexists as both a liquid and gas (vapor) in equilibrium.
When the temperature and pressure change so that the substance leaves the liquid
phase region and crosses the equilibrium curve into the gas phase region, the sub-
stance boils. As its state crosses this curve, there is an entropy change. In the case
of boiling, its entropy increases and absorbs heat, known as heat (enthalpy) of
vaporization. In the case of condensation, its entropy decreases and liberates heat,
known as heat of condensation. An obvious physical change is seen in the sub-
stance as its state crosses one of these curves.
Fig. 5.1. Phase diagram of carbon dioxide.
Temperature (°C)
Pressure (atm)
Copyright © 2004 AOCS Press
The three equilibrium curves (solid-gas, solid-liquid, liquid-gas) intersect at a
common point, called the triple point. At this point, the substance coexists in equi-
librium with all three phases. Each substance has only one triple point. The solid-
liquid equilibrium curve radiates from the triple point to infinity. The solid-gas
equilibrium curve radiates from the triple point and eventually terminates at
absolute zero and vacuum. The liquid-gas equilibrium curve does not radiate indef-
initely from the triple point but terminates at another important point, called the
critical point. This point is the critical temperature and critical pressure of the sub-
stance. Beyond the critical point, there is no longer an equilibrium curve to divide
the liquid and gaseous regions; thus, the liquid and gas phases are no longer distin-
guishable. There are no physical changes observed as the substance's state crosses
over this region. This region of the phase diagram is sometimes called the super-
critical fluid region.
The critical temperature is the temperature above which the substance can no
longer be condensed into a liquid. Increasing the pressure will not induce conden-

Copyright © 2004 AOCS Press
molecules close together to maintain the molecular attraction necessary for solva-
tion. The pressure must be at least above the critical pressure. Higher pressure
yields higher density at a constant temperature. In turn, higher density yields
greater solvating power.
Advantages and Disadvantages
The most popular SFE solvent is carbon dioxide. There are several reasons for its
popularity. First, CO
2
is inexpensive and commercially available even at high puri-
ty. Second, it is nonflammable, unlike many organic solvents, and is used in some
fire extinguishers. It also does not support combustion, except in the extraordinary
case of burning magnesium. Third, CO
2
is relatively nontoxic, especially in com-
parison to many organic solvents; it is actually present in air, foods, and drinks.
Some caution must be followed with the use of CO
2
. Because CO
2
does not sup-
port combustion or human respiration, it can be an asphyxiant at high concentra-
tions. Fourth, its critical temperature is low, allowing it to be used to extract ther-
mally liable analytes. Its critical temperature and pressure are easily attainable. As
a comparison, the critical temperature of water, 374.0°C, is a challenge for many
materials. Finally, CO
2
is environmentally compatible. Even though it is consid-
ered a “green-house” gas, it is ubiquitous in nature.
Other solvents have been used in SFE, but they have serious drawbacks.

2
when added
in small amounts. Second, the high pressure necessary for SFE is a concern
because some people are still uncomfortable with such pressures. Current technol-
ogy makes such pressure safe to use in the laboratory; however, it renders the
equipment expensive. To reduce the cost of the equipment, sample size is restricted
because smaller high-pressure vessels are safer and less expensive. Smaller sam-
pling size can be a disadvantage though for nonhomogeneous sample matrices.
Smaller sampling sizes can also reduce the detection sensitivity of the analytical
method and increase measurement error. These disadvantages must be addressed to
develop a successful SFE application method.
Giddings-Hildebrand Solubility Theory
The solubility of analytes in a supercritical fluid can be treated by thermodynamics.
The Gibbs free energy of the mixing process can be described by the Gibbs-
Helmholtz equation:
∆G
mix
= ∆H
mix
– T∆S
mix
For the process of solvation to proceed spontaneously, the value of the Gibbs free
energy of mixing ∆G
mix
must be negative. Because the solvation (dissolution)
process increases the disorder of the analyte-solvent system, the entropy of mixing
∆S
mix
is expected to have a large positive value. The spontaneity of the solvation
process ultimately depends on the heat of mixing ∆H

is the partial volume factor, V
s
and V
n
are the molar volumes for the
solvent and analyte, and a
s
and a
n
are the Van der Waal intermolecular attraction
parameters for the solvent and analyte. They further showed that
a
1/2
/V = (∆E
v
/V)
1/2
where ∆E
v
is the energy of vaporization of either the liquid solvent or liquid analyte.
The heat of mixing ∆H
mix
is produced from the breaking and reformation of attractive
forces between solvent-solvent, analyte-analyte, and solvent-analyte molecules.
Intuitively, it should be related to their energy of vaporization. Seeing the physical sig-
nificance of this formula in reference to solvation, they defined it as the solubility
parameter δ, also known as the Hildebrand solubility parameter:
Copyright © 2004 AOCS Press
δ = a
1/2

a = 3P
c
V
c
2
where P
c
and V
c
are the critical pressure and critical molar volume, respectively
(13). Upon substitution, they obtained
δ
s
= (3P
c
)
1/2
V
c
/V
The volumetric ratio can be written in terms of the reduced density as
V
c
/V = ρ/ρ
c
= ρ
r
giving
δ
s

using solubility parameters for analytes estimated by Fedors’ method (15). One
advantage of the reduced solubility parameter is that it is unitless.
Figure 5.2 relates the density of supercritical CO
2
with its corresponding
Hildebrand solubility parameter based on Giddings’ formula. The solubility para-
Copyright © 2004 AOCS Press
meters for a few common solvents are also included for comparison. Table 5.1
contains the Hildebrand solubility and reduced solubility parameters for a few
common lipid analytes as reported in the literature and estimated by Fedors’
method (16). Fedors’ group contribution method estimates an analyte’s solubility
parameter solely from information about its molecular structure (17). Table 5.2
contains an example of a calculation for estimating the Hildebrand solubility para-
meter by Fedors’ method. It can be incorporated into a spreadsheet for calculating
other fatty acids and their corresponding glycerides.
Even though higher temperatures at a given pressure would lower the density of
the supercritical fluid, the overall extraction performance should be enhanced. First,
supercritical fluids can solvate liquids better than solids. Performing an extraction at a
temperature above the melting point of the analyte should improve the recovery.
Second, temperature affects the solubility parameter of the analytes. The values pre-
sented in Tables 5.1 and 5.2 are for 25°C; higher temperatures tend to reduce the ana-
lyte’s solubility parameter. According to King, a temperature increase of 60°C can
reduce an analyte’s solubility parameter by 1.0–1.5 cal
1/2
/cm
3/2
(18). As a final point,
CO
2
Density

2
at 80°C and
Analyte MPa
1/2
MPa
1/2
(cal/cm
3
)
1/2
200 atm 400 atm 600 atm
Pentane 14.5 14.5 7.1 0.71 0.99 1.11
Hexane 14.9 14.9 7.3 0.69 0.96 1.08
Heptane 15.3 15.2 7.4 0.68 0.95 1.07
1-Butanol 23.1 23.2 11.3 0.45 0.62 0.70
1-Octanol 20.9 21 10.3 0.49 0.68 0.77
Stearyl alcohol 19.3 9.4 0.54 0.75 0.84
Hexyl acetate 17.3 17.7 8.7 0.58 0.81 0.91
Methyl oleate 17.7 8.6 0.59 0.82 0.92
Stearyl stearate 17.6 8.6 0.59 0.82 0.92
Tripalmitin 18.6 18.3 9.0 0.56 0.78 0.88
Triolein 1 8.5 18.3 8.9 0.57 0.79 0.89
Tristearin 17.9 18.2 8.9 0.57 0.79 0.89
Distearin 19.3 9.5 0.53 0.74 0.83
Monostearin 22 10.8 0.47 0.65 0.73
Glycerol 36.1 40.9 20 0.25 0.35 0.39
Acetic acid 21.4 22.8 11.2 0.45 0.63 0.70
Butyric acid 18.8 21.2 10.3 0.49 0.68 0.77
Palmitic acid 18.8 9.2 0.55 0.77 0.86
Oleic acid 18.7 9.1 0.55 0.77 0.87

along with knowledge of the critical point for a gas, the weight density of any
dense gas can be determined as follows:
ρ = M
w
P/[RT Z(T/T
c
, P/P
c
)]
Even though the Law of Corresponding States is not exact, it is sufficiently accu-
rate for practical engineering calculations.
TABLE 5.2
Example for Estimating the Hildebrand Solubility Parameter by Fedors’ Method
Diolein: CH
2
–COO–(CH
2
)
7
–HC=CH–(CH
2
)
7
–CH
3
CH–COO–(CH
2
)
7
–HC=CH–(CH

CH 1 820 820 –1 –1
HC= 4 1030 4120 13.5 54
OH 1 7120 7120 10 10
COO 2 4300 8600 18 36
COOH 0 6600 0 28.5 0
Σ n
i
⋅U
i
= 58310 Σ n
i
⋅V
i
= 649
δ = (Σ n
i
⋅U
i
/Σ n
i
⋅V
i
)
1/2
9.48 (cal/cm
3
)
1/2
|
|

Figure 5.3 is a contour plot of the density of CO
2
for various temperatures and
pressures based on Pitzer’s work.
As noted earlier, supercritical CO
2
can be augmented with another modifying
solvent to enhance its solubility for challenging analytes. These binary solvent
mixtures have different critical values and solubility parameters. Their new values
can be calculated using the modified Handinson-Brobst-Thomson equations:
V
b
=
1/4
[(x
s
V
s
+ x
m
V
m
) + 3(x
s
V
s
2/3
+ x
m
V

T
cs
T
cm
)
1/2
+ x
m
2
V
m
T
cm
]/V
b
Fig. 5.3. Contour plot of CO
2
density.
Temperature (°C)
Pressure (atm)
Copyright © 2004 AOCS Press
where V
b
, V
s
, and V
m
are the characteristic molar volumes for the binary mixture,
principal solvent, and modifying solvent, respectively; x
s

s
M
ws
+ x
m
M
wm
where ω
s
and ω
m
are the acentric factors for the solvent and modifier, and M
ws
and M
wm
are the molecular weights of the solvent and modifier. The critical pres-
sure P
cb
of the binary mixture is first calculated by determining the value of the
compressibility factor Z
cb
at the critical point using Pitzer’s tables:
Z
cb
= 0.291 – 0.08ω
b
The critical pressure is then determined using the definition of the compressibility
factor Z:
P
cb

15 mol% ethanol in CO
2
104.9 66.1 70.8 0.299 44.32
Acetone 208 235.1 47.6 0.304 58.08
5 mol% acetone in CO
2
98.8 43 71.3 0.242 44.71
15 mol% acetone in CO
2
109 66.2 69.2 0.249 46.12
Copyright © 2004 AOCS Press
Instrumentation
The instrumentation required to perform a successful SFE is commercially avail-
able. The process begins with a clean source of fluid, which in most cases is a
high-pressure cylinder of CO
2
. A pump is used to increase the pressure of the fluid
above its critical pressure. The working extraction pressure is determined by the
density required to dissolve the target analytes from the sample. The sample is con-
tained in the extraction chamber, which is heated to the desired extraction tempera-
ture above the critical point. The pressurized fluid is brought to temperature by the
chamber and allowed to flow through the sample matrix to extract the analytes.
After the sample, the analyte-laden fluid flows to a restrictor, which controls the
flow rate of the fluid. The restrictor maintains the high pressure of the fluid in the
chamber. At the restrictor, the supercritical fluid loses its solvating strength as its
pressure drops to atmosphere. After the restrictor, the analytes can be collected for
analysis. Figure 5.4 shows a block diagram of a complete SFE system.
Fluid Source
In SFE, an organic extraction solvent is replaced with CO
2

2
. At room temperature, the head (vapor) pres-
sure is ~900 psi or 60 atm. For the pumps to operate efficiently, liquid CO
2
must
be supplied. Cylinders must be equipped with an eductor or dip tube to deliver the
liquid fraction at the bottom instead of the gas head at the top. SFE-grade cylinders
should automatically be supplied with dip tubes. CO
2
cylinders with helium-head
pressure are also available to improve the performance of HPLC-style reciprocat-
ing piston pumps. The helium-head pressure is usually charged between 130 and
200 atm. However, there are concerns about the helium diluting the solvent
strength of the CO
2
because a significant amount of helium dissolves in liquid
CO
2
. It has been reported that helium-tainted CO
2
can reduce extraction perfor-
mance, but the extraction had to be performed well below optimal conditions to
detect a difference (24). Helium-charged cylinders are not necessary for syringe
pumps or head-cooled reciprocating pumps especially designed for SFE. Most SFE
vendors supply pumps that do not require helium-charged cylinders.
The solvating power of CO
2
can be enhanced by the addition of a small
amount of modifying solvent. The purity of the modifier solvent must be ensured.
For trace analyses, GC-grade solvent is satisfactory, whereas HPLC-grade should

is lengthy. They generally are refilled before the start of an extraction to reduce
such interruptions. Reciprocating pumps are smaller and less expensive than
syringe pumps but can be limited in maximum flow rate. The pump head must be
cooled to prevent vapor lock at higher flow rates. Reciprocating pumps are also
less precise in volumetric delivery due to variable fill efficiency. The controlling
software for a reciprocating pump must be sophisticated enough to determine and
compensate for refill efficiency for each stroke of the piston. Delivered volume
becomes an important issue in modifier solvent addition.
Most SFE vendors offer pumps that can deliver up to 680 atm. In most appli-
cations, 500 atm pressure is sufficient. The pumps must also maintain these pres-
sures with both accuracy and precision at maximum flow rate. Most extractions
can be performed in a reasonable time with pressurized fluid flows of >4 mL/min.
If several extractions are performed in parallel, then this number must be consid-
ered in determining the maximum flow rate required. Performing four extractions
in parallel may demand up to 16 mL/min of 680 atm CO
2
. At this flow rate, the
pump must maintain the programmed extraction pressure in all chambers contain-
ing samples.
Most SFE vendors also offer separate pumps to meter and mix modifier sol-
vents volumetrically. Volumetric control is essential in this application to ensure a
consistent mix. Both the volumes of the CO
2
and modifier solvent must be accu-
rately known to maintain the correct percentage. Modified CO
2
can be delivered by
two syringe pumps, in which one delivers the high pressure CO
2
and the other

demands on the material and design of the chamber. Third, it contains the sample
to be analyzed and allows the supercritical fluid to flow through the matrix without
allowing the sample to extrude.
Because the critical temperature of most fluids is above room temperature, the
extraction chamber for SFE must be heated. This can be accomplished with electri-
cal heating elements embedded in the heating block surrounding the extraction
chamber. These heating elements are powered and controlled by a proportional
temperature controller. The chamber temperature is usually measured with a ther-
mocouple, which serves as feedback for the proportional controller. The heating
block typically includes heat-exchanging coils, which serve to equilibrate the tem-
perature of the supercritical fluid before it comes in contact with the sample. The
typical temperature range for an SFE system should be between 40 and 150°C with
an accuracy and precision of ± 2°C.
The chamber should also be constructed to withstand the maximum allowable
extraction pressure with a factor of four safety margin. In other words, if the maxi-
mum operating pressure is 680 atm, the chamber should be designed to withstand
2720 atm at the maximum operating temperature (typically 150°C). This margin
ensures that SFE instruments are extremely safe. The chamber should also be
equipped with safety devices such as a rupture disk or a pressure relief valve that
will gracefully relieve the pressure if it should rise above 150% of the maximum
operating pressure. Although obvious, it should be noted that a supercritical fluid
under high pressure is more dangerous than a liquid at the same pressure. The high
Copyright © 2004 AOCS Press
compressibility of gases and supercritical fluids allows for the storage of a danger-
ous amount of mechanical energy at high pressures. On the other hand, the volume
for most liquids does not change much with high pressure, thus little mechanical
energy is stored. Vessels originally designed for HPLC applications may not be
rated safe for SFE applications.
Stainless steels, such as 304 and 316, offer both the strength and chemical
resistance required for most SFE applications. Alloys, such as Nitronics 50, can be

inlet and outlet sides. An inlet frit would prevent the sample from flowing back
into the pump in the event of pump failure. Check valves can also prevent back
flow.
In the early days of analytical SFE, empty HPLC column vessels were used as
extraction vessels. Even though readily available in a typical laboratory, they may
not be the safest approach because they are designed for pressurized liquids instead
of gases as noted above. They are also not convenient because tools are required to
open and close these vessels during sample introduction.
Thar, Inc. is a vendor of high-pressure vessels for SFE applications. Their ves-
sels are designed for pressurized gases, making them safer to use for SFE. They
have also been designed to be opened and closed without tools, making them more
convenient to use. Isco, Inc. has a patented design that allows the sample cartridges
Copyright © 2004 AOCS Press
to be made from light-weight polymeric material or aluminum instead of stainless
steel. The light weight aids in sample weighing, and these cartridges can be easily
opened and closed by hand. Their polymer cartridges are durable enough to be
reused and are chemically clean and resistant. The polymer was specially selected
not to absorb CO
2
; therefore, contamination and the “bends” are less likely. The
Isco design allows for light-weight cartridges because the pressure of the supercrit-
ical fluid is applied to both the inside and outside of the cartridge during extrac-
tion. The sample cartridge is inserted into a stainless steel chamber, which is also
easy to open and close at hand (25).
Restrictor
The restrictor controls the flow of the supercritical fluid after passing through the
sample. The restriction to the flow maintains the high pressure in the extraction
chamber. It is the most technologically demanding component of an SFE system.
Even though it can be as simple as a length of fused silica capillary, this simplicity
can be a source of frustration. Silica capillaries are inexpensive, but they also break

The final step in SFE is to transfer the target analyte from the CO
2
to another
medium for further analysis. There are several options available depending on the
analysis. The simplest collection scheme involves trapping the analyte on glass
wool. This is particularly useful for gravimetric fat analysis of food products. In a
gravimetric analysis, the collection vial is weighed before and after the extraction
to determine the weight of the extracted analyte. Fats are generally viscous and
nonvolatile; thus they are easily trapped by the wool in the collection vial. Any
extracted water or modifier solvent (e.g., ethanol), that could interfere with the
final weighing, is poorly trapped by the glass wool. After the extraction, the collec-
tion vial can be dried in a microwave or vacuum oven to remove any residual
water or modifier solvent. This simple scheme has the added elegance of being
completely solvent free, assuming that no modifier was used during the extraction.
For analysis methods based on GC or HPLC, it may be convenient to trap the
analytes in a liquid solvent. Because SFE is selective and offers cleaner extracts,
the collected solution can generally be injected directly into a GC or HPLC after
including an appropriate internal standard. The collected solution can be diluted to
volume or evaporated to dryness under a nitrogen stream in a heated water bath to
isolate the neat analytes. The collected solution may have to be derivatized by
saponification or transesterification as in a FAME analysis before proceeding to
the GC or HPLC. To enhance trapping performance, the collection solvent can be
cooled or maintained under a mild back pressure. A back pressure of 30 psig
reduces the size of the expanding CO
2
bubbles, which enhances analyte transport
from the CO
2
to the liquid solvent. It also reduces the evaporation of the solvent
during the extraction or solvent aerosol formation (28). Certain solvents, such as

at the front of the separation column. During the extraction, the column tempera-
ture is kept low so that the analytes focus on the front of the column. After the
extraction, the normal column temperature program is executed to elute and sepa-
rate the analytes on the column (29).
In online SFE-FTIR, the target analytes, while still in solution in the supercritical
fluid, are analyzed directly by a FTIR spectrometer (30). After the extraction chamber,
the analyte-laden fluid is transferred immediately to a heated, high-pressure IR flow
cell through a heated transfer line. The temperatures of the extraction chamber, transfer
line, flow cell, and restrictor should be the same. Some analytes, such as waxes, can
precipitate out of solution at lower temperatures. In this application, zinc sulfide is an
appropriate IR window material. It is transparent to infrared light and strong enough to
contain the high pressure. The high pressure flow cell is mounted in the optical bench
of the FTIR spectrometer so that the spectra of the target analytes can be monitored
during the extraction. After the flow cell, the supercritical fluid is allowed to flow to a
restrictor where the target analytes can be collected for additional analysis (Fig. 5.5).
FTIR is a nondestructive analysis method. CO
2
is an excellent IR solvent
because it does not contain hydrogen atoms. It is transparent in the C–H stretch
band (3000 cm
–1
); thus, it can substitute for CS
2
, freon, and perchlorinated sol-
vents in many IR applications. Finally, the spectrum is collected real time during
the extraction, making this technique useful in extraction kinetic studies (31,32).
Applications of Supercritical Fluid Extraction
This review is intended to be a limited summary of applications and research
involving methods development, verification, and validation using the technology
of SFE in the agricultural and food industries. Early leaders in research had the

at
40–80°C and 245–392 bar to extract valuable oils that are high in unsaturated fatty
acids and phospholipids. The oil extracts, using traditional extraction techniques,
required the removal of extraction solvents (33).
Supercritical CO
2
extraction followed by characterization of cocoa extracts for
the purpose of determining triglycerides, fatty acid composition, unsaponifiable mat-
ter, and aromatics is another broad application. Investigation of SF extracts from
cocoa liquor, nibs, and shells under a variety of SFE conditions (50–80°C and
300–400 bar) was investigated for the purpose of determining industrial applicability
of SFE in cocoa processing. Total fat is determined gravimetrically, whereas the com-
position is determined using fatty acid methyl esterification (FAME) followed by GC,
with the aromatic fraction analyzed by HPLC for pyrazines. This application showed
that the extract obtained from cocoa liquor met the definition of cocoa butter; howev-
er, the portion of triglycerides extracted from the shell contained unsaponifiables that
exceeded the limit allowed. Additionally, it was determined that SFE maintained an
even distribution of aromatics between the cocoa butter and the cocoa powder, thus
maintaining the aromatic properties of the products (34).
The most popular SFE solvent is CO
2
, and triglycerides, cholesterol, waxes,
and free fatty acids are quite soluble in supercritical CO
2
. The solubility of polar
lipids, such as phospholipids, can be improved by augmenting the supercritical
CO
2
by the addition of a small amount of ethanol or other polar modifier solvents.
Phospholipid (PL) content is useful for the investigation of storage conditions of

2
. Studies have shown that the SFE may cause
chemical alterations when sufficient quantities of oil are removed. This alteration
allows subsequent extractions to remove additional oil (39). During sample prepa-
ration for oilseeds, such as grinding, the surface-to-volume ratio is increased, thus
exposing more oil to the extraction solvent. During the process, seeds are uninten-
tionally milled to different particle sizes and the amount of oil extracted from each
fraction (based on size) produces different results (40,41). These studies all indi-
cate that proper sample preparation before SFE has an effect on the quantity of
analyte recovered.
In 1996, the method using SFE was approved by the AOCS. Method Am3-96,
SFE Determination of Oil in Oilseeds, was subsequently adopted by AOAC in
2000 as method number 999.02. The method is based on gravimetric analysis from
a set of oilseeds determined to be representative of the oilseed industry and encom-
passes soybeans, canola, sunflower, safflower, and sunflower. This method refer-
ences AOCS sample preparation methods as determined for each type of oilseed
and allows the user to choose between two variations of SFE (CO
2
alone or CO
2
with a 15% EtOH modifier). The SFE parameters are 100°C, 7500 psi, with or
without a 15% modifier at 2 mL/min with a total extraction time of 30 min (CO
2
only) or 45 min (CO
2
+ 15% EtOH) (42).
The application of SFE for the determination of total fat in meats was investi-
gated by numerous researchers in response to the Nutrition and Labeling Education
Act (NLEA) of 1990, which defined fat as the sum of all fatty acids obtained from
a total lipid extract expressed as triglycerides (43). Snyder et al. (44) investigated

metric results (P = 0.246) using a paired t-test. Additionally it was shown that fatty
acid composition can be determined by GC analysis (after hydrolysis and derivati-
zation) by using an aliquot of the same extract (49).
The application of gravimetric SFE as a direct method for the determination of
crude fat (defined as the components of meat that are extractable with petroleum
ether, without digestion of the sample) from meat resulted in an AOAC peer-verified
method (PVM) 3:2000. The study used nine meat matrices including raw ground beef,
raw pork sausage, raw veal sausage, smoked ham, kielbasa, braunschweiger, sweet
Italian sausage, smoked sausage, and bologna with fat ranging from 6–28% and
involved two peer laboratories. The results were compared with AOAC Method
960.39. Samples for SFE were prepared by mixing 1.0–1.5 g of sample with 2.2 g of
diatomaceous earth. The mixture was transferred to the extraction vessel and extracted
at 100°C, 9000 psi for 45 min. To remove any subsequent co-extracted moisture, the
collection vessels were dried after extraction and before reweighing. This method has
a mean accuracy of +0.22 to –1.41 and mean repeatability and reproducibility of <3.0.
The data suggest that the method should perform well for all meats with fat content in
the range of 6–28% (50).
Infant formula is intended to act as a substitute for human milk. Therefore,
due to health concerns and NLEA labeling requirements, the composition and
quantity of fat in infant formula must be disclosed. Traditional methods for the
analysis of milk-type products include solvent extraction techniques that require
sample pretreatment such as acid or base hydrolysis, e.g., AOAC Method 905.02
Copyright © 2004 AOCS Press
(Roese-Gotttlieb). The application of SFE for milk products that employ gravimetric
analysis compared with accepted gravimetric methods were reported (51–53).
Further investigation and direct correlation between traditional hydrolysis/solvent
extraction techniques and SFE was performed using gravimetric and GC/MS
analysis. Method development utilized a powdered infant formula standard refer-
ence material NIST SRM 1846 (the National Institute of Science and Technology,
Gaithersburg, MD) and nine commercially available infant formulas. Sample

assessing the suitability of the analytical method for any given purpose. They fur-
ther suggest that this technique could be applied to other food matrices (55).
The application of SFE for the determination of fat and oil in food and agricul-
ture products is by no means limited to the matrices discussed in this review.
However, there is a growing body of validated and/or approved methods utilizing
SFE as the method of choice for fat and oil determination.
Copyright © 2004 AOCS Press
Fat-Soluble Vitamins and Other Nutritional Components in Foods
Vitamins are essential to the health and nutrition of all members of the animal king-
dom. Animals cannot synthesize vitamins and instead require dietary intake to meet
the nutritional demands for development, growth, and maintenance during their life
cycle. Vitamins are classified on the basis of their solubility in nonpolar organic sol-
vents (fat-soluble vitamins) and polar solvents (water-soluble vitamins) (56). Lipid-
soluble vitamins are the topic of this section and include A, D, E, and K.
Vitamin A (retinol) in its various forms functions as a hormone and is of
importance in protein metabolism of cells that develop from the ectoderm. Retinol
in the form of 11-cis-retinal (II) is the chromophore component of the visual cycle
chromoproteins in the cone and rod cells of the retina. Deficiency of this compo-
nent produces a variety of conditions such as dryness and thickening of the skin,
retarded development and growth, and night blindness.
Vitamin D (calciferol) is comprised of two compounds, Vitamin D
2
and D
3
.
Although both are absorbed from the diet, D
3
(cholecalciferol) is also synthesized
biologically from cholesterol in the skin by exposure to UV radiation. Similarly,
vitamin D

glycerol and soaps of the free fatty acids. Postextraction analysis includes normal
Copyright © 2004 AOCS Press
or reversed-phase liquid chromatography (LC) with UV-vis or fluorescence detec-
tion, GC with flame ionization detection (FID), or MS.
Traditional extraction methods expose vitamins to potentially degradating
environments and require the use of large volumes of organic solvents. Supercritical
fluid extraction is an alternative to the use of organic solvents in that it minimizes
the risk of exposure to oxygen and heat, while having the added benefit of using car-
bon dioxide as a replacement for organic solvents. Although carbon dioxide is the
most commonly used extraction solvent due to its low critical parameters, relative
abundance, and low toxicity, it is relatively nonpolar. However, most commercial
SFE instrumentation offers the ability to add a polar modifier to enhance the
extraction capabilities of SCCO
2
.
Turner et al. (62) provided an excellent review of the literature for the supercriti-
cal extraction of vitamins. They presented the fundamental research for the extraction
of fat-soluble vitamins from matrices that included milk powder, infant formula, dairy
products, liver, processed meats, meat products, cereal products, oilseeds, sweet pota-
toes, carrots, tomato paste, broccoli, collard greens, corn, and zucchini followed by
postextraction analysis using supercritical fluid chromatography (SFC).
Mathiasson et al. (63) went on to complete a collaborative study that demon-
strated the ability of SFE using CO
2
as a replacement technology for liquid extraction
solvent methods for the determination of vitamin A, E, and β-carotene from
processed foods. The validation included 10 laboratories, and the data suggested
that as with any analytical method, additional information was required to deter-
mine control points and that further validation was required. However, these
researchers showed that SFE is a suitable replacement technology for the solvent