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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 (CO2). Triglycerides, cho-
lesterol, waxes, and free fatty acids are quite soluble in supercritical CO2. The sol-
ubility of polar lipids, such as phospholipids, can be improved by augmenting the
supercritical CO2 with a small addition of ethanol or other polar modifier solvent.
Even though CO2 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
still contribute positively to Green Chemistry. CO2 has the additional advantage
of being nonflammable and less toxic than most organic solvents. For example,
petroleum ether, which is commonly used in fat extractions, can be easily detonat-
ed by static electricity, and diethyl ether can form explosive peroxides. On the
other hand, some fire extinguishers use CO2, which is also commonly found in
foods and drinks such as bread and carbonated drinks. Finally, several common
chlorinated solvents are banned by law, and supercritical CO2 can be an alternative
to these solvents. All of these factors make SFE attractive.
Temperature (C)
Fig. 5.1. Phase diagram of carbon dioxide.
For the process of solvation to proceed spontaneously, the value of the Gibbs free
energy of mixing Gmix must be negative. Because the solvation (dissolution)
process increases the disorder of the analyte-solvent system, the entropy of mixing
Smix is expected to have a large positive value. The spontaneity of the solvation
process ultimately depends on the heat of mixing Hmix. A smaller value predicts
greater solubility (10).
Using the earlier work of van Laar and Lorenz on the vapor pressure of binary
liquid mixtures according to the Van der Waal equation, Hildebrand and Scott
showed that the heat of mixing Hmix can be expressed as follows:
where sn is the partial volume factor, Vs and Vn are the molar volumes for the
solvent and analyte, and as and an are the Van der Waal intermolecular attraction
parameters for the solvent and analyte. They further showed that
a1/2/V = (Ev/V)1/2
where Ev is the energy of vaporization of either the liquid solvent or liquid analyte.
The heat of mixing Hmix 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 , also known as the Hildebrand solubility parameter:
where s and n are the Hildebrand solubility parameters for solvent and analyte
(11). To keep the heat of mixing as small as possible, the solubility parameter of
the solvent should be similar in value to the solubility parameter of the analytes. A
smaller difference gives higher solubility.
The solubility parameter for a supercritical fluid cannot be determined from
the energy of vaporization as with liquids because the liquid and vapor phases cannot
be distinguished (12). Giddings and colleagues assumed that a supercritical fluid sol-
vent can be described qualitatively by the Van der Waal state equation. The intermole-
cular attraction parameter can be related to the critical values of the solvent as
a = 3PcVc2
where Pc and Vc are the critical pressure and critical molar volume, respectively
(13). Upon substitution, they obtained
s = (3Pc)1/2 Vc/V
Vc/V = /c = r
giving
s = (3Pc)1/2 r
s = 0.47 Pc1/2 r
= n /s
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 analytes 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 25C; higher temperatures tend to reduce the ana-
lytes solubility parameter. According to King, a temperature increase of 60C can
reduce an analytes solubility parameter by 1.01.5 cal1/2/cm3/2 (18). As a final point,
solubility is less of a concern for trace-level analytes, such as pesticides in foods. The
above discussion applies mainly to analytes present at high levels in the sample where
solubility saturation could be an issue. Much lower pressures may actually be suffi-
cient for analytes on the ppb level.
Diolein: CH2COO(CH2)7HC=CH(CH2)7CH3
|
CHCOO(CH2)7HC=CH(CH2)7 CH3
|
CH2OH
i ni Ui (cal/mol) ni Ui Vi (cm3/mol) ni Vi
CH3 2 1125 2250 33.5 67
CH2 30 1180 35400 16.1 483
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
ni Ui = 58310 ni Vi = 649
= ( ni Ui / ni Vi )1/2
9.48 (cal/cm3)1/2
According to the Law of Corresponding States, two gases with the same
reduced temperature and reduced pressure are in corresponding states. Both gases
should have the same reduced density. A reduced state parameter is the state para-
meter divided by its corresponding critical value. For example, the reduced tem-
perature is the temperature divided by the critical temperature of the gas. Even
though quantitatively inaccurate, the Van der Waal equation predicts the Law of
Corresponding States (19).
For dense gases, the deviations from the ideal gas law can be treated by the
compressibility factor Z which is defined as
Z = MwP/(RT)
where, Mw, P, T and are the molecular weight, pressure, temperature, and weight
density of the gas, and R is the ideal gas constant. For ideal gases, Z = 1. The com-
pressibility factor can be expressed as a function of reduced temperature and
reduced pressure: Z(Tr,Pr). According to the Law of Corresponding States, gases in
corresponding states should have the same compressibility factor Z value. In the
engineering literature, there are isotherm plots of Z vs. reduced pressure at various
reduced temperatures, which are universal for all gases (20). Using these graphs,
along with knowledge of the critical point for a gas, the weight density of any
dense gas can be determined as follows:
Even though the Law of Corresponding States is not exact, it is sufficiently accu-
rate for practical engineering calculations.
where Z0 and Z1 are obtained from their published tables through linear interpola-
tion. Using this improved compressibility factor Z, a more accurate value for the
density of a gas can be calculated using the formula in the previous paragraph.
Figure 5.3 is a contour plot of the density of CO2 for various temperatures and
pressures based on Pitzers work.
As noted earlier, supercritical CO2 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:
Temperature (C)
Fig. 5.3. Contour plot of CO2 density.
b = xss + xmm00
Mwb = xsMws + xmMwm
where s and m are the acentric factors for the solvent and modifier, and Mws
and Mwm are the molecular weights of the solvent and modifier. The critical pres-
sure Pcb of the binary mixture is first calculated by determining the value of the
compressibility factor Zcb at the critical point using Pitzers tables:
The critical pressure is then determined using the definition of the compressibility
factor Z:
Pcb = ZcbRTcb/Vb
Using these critical values for the binary mixture, the compressibility factor Z can
be calculated from Pitzers tables for a given temperature and pressure (23). The den-
sity and Hildebrand solubility parameter can then be calculated for the new mixture.
Table 5.3 lists the critical values for a few binary mixtures. Isco Incorporated offers a
Microsoft Windowsbased program that calculates the state parameters and solubility
parameters for CO2 and a few binary mixtures. As a final note, this theory does not
adequately treat polarity and hydrogen bonding. This treatment is only an approxima-
tion but has been successfully applied to a wide range of solvent systems.
TABLE 5.3
Critical Parameters for Pure Fluids and Mixtures
V Tc Pc
Fluid (cc/mol) (C) (atm) MW
CO2 93.8 31.1 72.8 0.239 44.01
Ethanol 175.2 243.1 62.2 0.644 46.07
5 mol% ethanol in CO2 97.5 42.9 71.9 0.259 44.11
15 mol% ethanol in CO2 104.9 66.1 70.8 0.299 44.32
Acetone 208 235.1 47.6 0.304 58.08
5 mol% acetone in CO2 98.8 43 71.3 0.242 44.71
15 mol% acetone in CO2 109 66.2 69.2 0.249 46.12
Fluid Source
In SFE, an organic extraction solvent is replaced with CO2. Just as with organic
solvents, the CO2 source must be clean enough so as not to contaminate the sample
being analyzed. As with most solvents, high-pressure CO2 is commercially avail-
able in different grades of purity. For trace-level analyses, such as extracting pesti-
cides at ppb levels, the CO2 must be at its highest grade of purity. Most gas ven-
dors can supply an SFE-grade that is intended for such applications, generally in
Pumps
The vapor pressure of CO2 at room temperature is below its critical pressure. Even
with cylinders charged with helium, the pressure is still too low for most applica-
tions, and a pump is required to increase and control the pressure of the CO2. As
shown earlier, higher pressure produces higher density fluid, which offers greater
solvent strength. The solvent strength of a supercritical fluid can be tuned by its
pressure (see Table 5.1).
Both syringe pumps and reciprocating piston pumps have been used in SFE
applications. Syringe pumps offer smoother pressure control, i.e., no pulsation,
higher flow rates, and more precise volumetric delivery. Syringe pumps, on the
other hand, may have to interrupt an extraction to be refilled if the extraction time
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 CO2. 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 CO2 and modifier solvent must be accu-
rately known to maintain the correct percentage. Modified CO2 can be delivered by
two syringe pumps, in which one delivers the high pressure CO2 and the other
delivers the pressurized organic solvent. In this scheme, the two fluids are volu-
metrically metered and mixed at the final pressure. With syringe pumps, the vol-
Extraction Chamber
The extraction chamber serves three functions. First, it controls the temperature of
the extraction by maintaining the temperature of the fluid and sample. Second, it
contains the high pressure of the supercritical fluid. This function places high
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 150C with
an accuracy and precision of 2C.
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 150C). 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
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
or plug easily, which can ruin an extraction and waste the operator's time.
At the restrictor, the CO2 expands rapidly to atmospheric pressure. As in any
adiabatic expansion, a large amount of heat is absorbed to increase entropy. This
expansion produces very low temperatures; without external heating, extracted
water will freeze and plug a capillary. Water is frequently present in food samples.
In addition, the supercritical fluid rapidly loses its solvent strength during expan-
sion, allowing analytes to precipitate out of solution, which may plug a simple cap-
illary. Heating can help to prevent such problems.
Isco offers a patent restrictor, which is based on a stainless steel capillary. The
stainless steel is less brittle than fused silica and is not easily attacked by modifiers
such as methanol or water. The restrictor is heated from end to end. A controlled
electric current is applied along the entire length of the capillary, so that the capil-
lary serves as an electrical heating element. Because the electrical resistance of the
capillary is dependent on temperature, its temperature can be determined from
measuring its ohmic resistance. This measurement can be used as feedback to pro-
portionally control the temperature of the restrictor. The stainless steel capillary
serves to control the flow of the fluid, to heat the fluid, and to measure the temper-
ature of the restrictor (26). Most SFE vendors offer some form of heated fixed flow
restrictor.
Isco and most other SFE vendors also offer heated variable flow restrictors (27).
Most variable restrictors are based on a needle valve design. To increase the flow, the
needle is withdrawn from the seat, thus opening the orifice. The valve body can be
heated to reduce the cooling effects from the adiabatic expansion. By incorporating a
motor drive to the valve needle, the flow rate of the fluid can be automatically adjust-
ed during an extraction. Unfortunately, the most sophisticated restrictor can still occa-
sionally plug, especially during method development on new samples.
Online Techniques
SFE is quite amenable to direct coupling with other analytical instruments, such as
GC or FTIR. These online techniques offer higher sensitivity and eliminate the
Conclusions
In this review, we examined the fundamental theory behind SFE and the current tech-
nology for instrumentation. The end user can optimize SFE procedures using knowl-
edge of the density of CO2, the Hildebrand solubility parameter, and Fedors method
during research involving SFE. For routine extraction procedures requiring direct
gravimetric determination of fat or oil from food matrices such as oilseeds, meats, or
infant formula, approved methods utilizing SFE were verified and validated.
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