Focusing Review

Chromatography, Vol.29 No.1 (2008)

Focusing Review

Development of Environmental Analysis Methods Using Supercritical
Fluid Extraction and Supercritical Fluid Chromatography
Takashi Yarita
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba Central 3, Umezono, Tsukuba 305-8563, Japan
Received for review January 11, 2008. Accepted February 4, 2008

Abstract
Environmental analysis methods have been developed by applying supercritical fluid extraction (SFE) and supercritical fluid chromatography(SFC). The highly sensitive and selective detection of organochlorine pollutants by SFC was demonstrated using an electron-capture detector.
For accurate quantification by SFE, the extraction behavior was investigated in detail using herbicides in soil and polychlorinated biphenyls(PCBs) in sediment as target analytes. For rapid quantification, on-line coupling of SFE and SFC was demonstrated, in which thiolcarbamate
herbicides in soil samples could be quantified within 1 h. As an application of the developed analytical method, the production of sedimentcertified reference materials NMIJ CRM 7304-a and CRM 7305-a, with the use of SFE as one of the extraction techniques for the determination of certified values of PCBs, is also described.
Keywords: Supercritical fluid extraction, Supercritical fluid chromatography, Pesticide, Herbicide, Polychlorinated biphenyls, Certified reference material

electron-capture detection (ECD) in SFC for the highly sensitive

1. Introduction
Supercritical fluid chromatography (SFC) was first attemptedand selective detection of polychlorinated biphenyls (PCBs) [5]in 1962 [1]. Although several papers on SFC were published dur-and organochlorine pesticides [6]. For accurate quantification bying the subsequent two decades [2], their content did not encourage

SFE, we investigated the effects of operation parameters and themany researchers to investigate this technique. After the introduc-

sample matrix on extraction efficiency using organochlorine pesti-tion of fused-silica capillary SFC in the early 1980s [3], many pa-cides in carrots [6], herbicides in soil [7, 8] and PCBs in sedimentpers describing fundamental studies as well as application studies[9] as target analytes. In addition, we applied an optimized SFEappeared. Today, SFC is recognized as being a separation tech-method for PCBs to the production of sediment certified referencenique that acts as a bridge between GC and HPLC.

materials (CRMs) for which accurate quantification methods wereAnalytical supercritical fluid extraction (SFE) was demon-

needed to determine certified values [9, 10]. We also demonstrated

strated in the mid-1980s [4]. Since then, the number of papers de-rapid quantification of thiolcarbamate herbicides in soil using an on

scribing the use of this technique has steadily increased. Because of-line SFE/SFC system [8].

the preferable properties of supercritical fluids as an extraction me-This paper briefly overviews fundamental and application

dium, SFE is a useful alternative to conventional solvent extraction

studies by the author’s group on the development of environmental

as well as an “environmentally friendly” technique.

analysis methods using SFE and SFC.

The author and coworkers have made efforts to develop SFEand SFC. In the field of environmental analysis, we developed
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E-mail:t-yarita@aist.go.jp―１９―Chromatography, Vol.29 No.1 (2008)

Figure 1. Supercritical fluid chromatograms of a carrot sample.
Peak identification: (1), 1α, 2α, 3β, 4α, 5α, 6βhexachlorocyclohexane (γ-HCH); (2), 1,1-dichloro-2,2bis-(4-chlorophenyl)ethane (4,4’-DDD). Detection: (A),electron-capture detection; (B), UV detection at 190
nm. Other chromatographic conditions: mobile phase,carbon dioxide; column, L-column ODS (size, 250 mm×4.6 mm I.D.; particle size, 5 µm; pore diameter, 12nm); column temperature, 55°
C; back pressure, 15 MPa.
Reproduced by permission of Elsevier [6].

Figure 2. Response surface for SFE of 2,4,4’-trichlorobiphenyl(CB 28) in a sediment sample, showing the effect of theextraction temperature and pressure on SFE efficiency.
Other extraction conditions: medium, carbon dioxide;
mode and time, static mode for 15 min then dynamic
mode for 30 min. Reproduced by permission of theJapan Society for Analytical Chemistry [9].

mental analysis, the effect of these parameters on the SFE yield of
the target analyte is not predictable, since the sample matrix has a
profound effect on the extraction efficiency. As one example, we

2. Electron-capture detection in SFC
Although many detectors used in GC and HPLC are consid-

have reported the effect of moisture content in soil samples on SFE

ered to be applicable to SFC, only flame ionization detection (FID)

recovery yields using triazine and thiolcarbamate herbicides as tar-and UV-VIS detection were widely employed. On the other hand,

get analytes [7, 8]. The presence of small amounts of water in thehighly sensitive and selective detection is essential for developing

matrix was considered to increase the recovery yields of analytes

environmental analysis methods. Our first attempt to use SFC forby acting as an internal modifier. In addition, adsorption sites onenvironmental analysis involved developing ECD [5, 6].

the matrix might be removed by the presence of moisture. On theWe connected a commercially available ECD detector, which

other hand, the recovery yields of target analytes from soil contain-

had been designed for packed-column GC, to a packed-column

ing 30% water were poorer than those from soil containing 10-20%

SFC system. The mobile phase of SFC was split (split ratio ap-water. We have also found that the concentration level of the tri-

proximately 2%) and introduced into the detector via a capillaryazine herbicides in the sample also influenced the recovery yield

tube with a small orifice at one end. Having optimized the position[7]. These results indicate that extraction of real samples should beof the capillary tube as well as the detection temperature, we suc-carried out experimentally to permit successful optimization of ex-

ceeded in detecting small amounts of PCBs and organochlorine

traction conditions.

pesticides. Figure 1 shows a comparison of ECD and UV detection

The extraction temperature and pressure are the key parame-

in SFC of organochlorine pesticides in a carrot sample, in which 1

ters that control the physical properties of the carbon dioxide ex-

α, 2 α, 3 β, 4 α, 5 α, 6 β-hexachlorocyclohexane (γ-HCH) could betraction medium. Because the effect of these parameters on extrac-

detected only in ECD [6]. The proposed system is anticipated to betion efficiency is complex, their optimization sometimes requires asuitable for the microchemical determination of nonvolatile halo-process of trial and error. We have evaluated these parameters bygenated compounds.

two-way analysis of variance (ANOVA) using experimental SFE
results for PCBs in a sediment sample [9]. Each corresponding 13C12-labeled PCB was used as an internal standard. The results indi-3. Investigation of SFE behavior
In general, the properties of the extraction medium of SFE,

cated that not only extraction temperature and pressure but also

such as density, viscosity and diffusion coefficient, are controlled

their interaction were statistically significant in most target PCBs.

by various operation parameters. When applying SFE to environ-

We also observed that equilibrium between native PCBs and the―２０―

Chromatography, Vol.29 No.1 (2008)

Takashi Yarita

corresponding 13C12-labeled PCBs was not realized. We regressed
the experimental data using a second-order polynomial that wasused in a fractional factorial design approach [11] and predicted theSFE efficiency, as shown in Fig. 2.
4. On-line coupling of SFE and SFC
Development of high-throughput analytical methods is an important task in environmental analysis. SFE itself is a far more
rapid extraction technique than classical solvent extraction, and theobtained extracts can in most cases be subjected to chroma-Figure 3. Schematic diagram of the SFE-SFC system. (1) and (17),carbon dioxide cylinders; (2) and (15), pumps; (3) and(14), ovens; (4), (6) and (10), switching valves; (5), extraction chamber; (7), pump controller; (8) and (13), restrictors; (9), trap column; (11), injector; (12), SFC column; (16), UV detector. Reproduced by permission ofElsevier [8].

tographic systems. In addition, SFE has the potential to be connected to chromatographic systems, since carbon dioxide can beevacuated by changing the phase from supercritical fluid to gas under normal conditions such as room temperature and atmosphericpressure. We demonstrated on-line coupling of SFE and SFC for
the rapid determination of thiolcarbamate herbicides in soil [8].
The development of an “organic-solvent free” system is anothertask in this study.
A schematic diagram of the developed system is shown in Fig.
3. The extract obtained by SFE was initially trapped in a trap column (9), then introduced into an SFC column (12) together withthe mobile phase by changing the position of switching valves (6and 10). Here, ODS columns prepared for HPLC were used as thetrap and SFC columns. Because the degree of inertness of the column packings considerably influenced the retention of polar solutes when carbon dioxide was used as the mobile phase without amodifier, the inertness of the column packings was an essential
contributor to realizing an organic solvent-free system [12, 13]. We
therefore examined various ODS columns, ultimately concludingthat a column packed with polymer-coated silica-gel packings
showed the best performance. Figure 4 shows a typical chromatogram obtained using the proposed system. We successfully deter-Figure 4. Chromatogram of thiolcarbamate herbicides (each 1 mg/kg spiked) in a soil sample obtained by the SFE-SFCsystem. Peak identification: (1), molinate; (2), thiobencarb. Pressure programming of carbon dioxide mobilephase: 10 MPa for 1 min, then raised at 1 MPa/min, andkept at 15 MPa for 25 min. Other SFC conditions: column, Capcell Pak C18 UG (size, 250 mm×4.6 mm I.D.;
particle size, 5 µm; pore diameter, 12 nm); column temperature, 55°
C; UV detection, 220 nm. SFE conditions:medium, carbon dioxide; pressure, 20 MPa; temperature,60°
C; mode and time, dynamic for 20 min. Reproducedby permission of Elsevier [8].

mined two target herbicides in soil samples within 1 h (20 min forextraction and 30 min for chromatographic analysis) without theuse of any organic solvents.
5. Application of SFE to the production of certified referencematerials
CRMs are playing an increasingly important role in ensuring
the reliability of environmental analysis. However, the variety ofmatrix-type CRMs that are commercially available is limited, andso the development of these CRMs is of great concern. The National Metrology Institute of Japan (NMIJ) has been working on
the development of matrix-type CRMs. To establish highly reliable

analytical results are obtained from true values if equilibration is(possibly traceable to the SI units) certified values, we principally

not achieved, so a highly efficient extraction technique is required

apply isotope-dilution mass spectrometry (IDMS), which has thefor accurate determination of certified values. To avoid any possi-potential to be operated as a primary method of measurement [14],ble procedural bias, we apply two or more independent extraction

for the determination of certified values. However, incorrectly lowtechniques [15]. In the case of the determination of PCB certified―２１―

Chromatography, Vol.29 No.1 (2008)

Figure 5. Analytical scheme for the determination of PCB certified values of NMIJ CRM
7304-a. Extraction solvent: Hex, hexane; Ace, acetone; DCM, dichloromethane;
EtOH, ethanol.

values in sediment reference materials (NMIJ CRM 7304-a andTable 1. Certified valuesa (mass fraction, µg/kg dry mass) of PCB

CRM 7305-a), five independent extraction techniques were appliedcongenersb in NMIJ CRM 7304-a and CRM 7305-aafter the conditions of each had been fully validated or optimized[9, 16-19]. SFE was one of the extraction techniques that operated

NMIJ CRM 7304-a

NMIJ CRM 7305-a

0.311±0.085
2.26±0.24
34.9±2.3
27.1±1.860.7±3.831.9±2.6
18.4±2.013.9±1.1
15.9±1.0
3.62±0.22
9.10±0.69
1.89±0.11
0.476±0.050
1.28±0.20

0.15±0.07
0.31±0.05
2.9±0.2
2.26±0.18
4.0±0.3
2.6±0.3
1.27±0.14
1.92±0.15
3.2±0.3
0.92±0.16
2.4±0.5
0.62±0.13
0.15±0.03
0.16±0.03CB 3
CB 15
CB 28
CB 31
CB 70
CB 101
CB 105
CB 138
CB 153
CB 170CB 180CB 194
CB 206
CB 209under optimized conditions, as briefly described in Section 3.
Figure 5 shows an analytical scheme for determining PCB
certified values in CRM 7304-a [10]. Here, SFE has a characteristicvery different from those of other extraction techniques, so it wasregarded as being suitable as one of the extraction techniques in thedetermination of certified values. That is, supercritical carbon dioxide was used in SFE as an extraction medium without any organicmodifier, whereas organic solvents were required in all the otherextraction techniques. In addition, SFE did not require a cleanup
process of the obtained extracts. Therefore, use of the method
based on SFE ensured that no analytical bias was found during thecleanup process of the other analytical methods.
In most cases, no significant differences were found among
the analytical results of target PCBs obtained by each extractionaResults are expressed as the certified value ± expanded uncertainty (coverage factor k=2)bThe numbering presented is identical to the IUPAC numbertechnique. In addition, the uncertainties associated with each analytical method were comparable to each other. Using all the resultsobtained, we provided the certified values and their uncertainties,
as listed in Table 1. These CRMs are the first to be obtained by
IDMS techniques together with well-validated or optimized extrac-variety of detection techniques as well as the rapidity of analysis bytion processes, and the obtained certified values are traceable to theon-line coupling with SFE. Meanwhile, the methods based on SFE

SI units.

provided accurate determination that was applicable to the production of CRMs. The author expects that SFE and SFC have potential
for routine application to environmental analyses as a complement

6. Conclusions

to classical analytical techniques.

The author and coworkers have carried out fundamental andapplication studies for the development of environmental analysis
methods using SFE and SFC. The methods developed by applying

Acknowledgements
The author acknowledges the fruitful advice of Dr. Akira

SFC demonstrated its advantages over conventional methods in the―２２―

Chromatography, Vol.29 No.1 (2008)Takashi Yarita[8]Nomura. The author also expresses his gratitude to all collaborators

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and coworkers involved with these studies, especially Dr. Shigeo[9]Gonda, Prof. Masami Shibukawa, Dr. Yoshiyuki Horimoto, Dr.

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Akiko Takatsu, Dr. Masahiko Numata, Dr. Kazumi Inagaki and Ms
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Chromatographic Sciences for selecting him as a recipient of theM.; Takatsu, A.; Ishikawa, K.; Chiba, K.; Okamoto, K.
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