K. Jinno (Ed.), Hy henated Techniques in Supercritical FluifChrornatography and Extraction Journal of Chromatography Library Series, Vol. 53 0 1992 Elsevier Science Publishers B.V. All rights reserved.

225

Chapter 12

ADVANCES IN ANALYTICAL SUPERCRITICAL FLUID EXTRACTION (SFE)

Steven B. Hawthorne, David J. Miller, and John J. Langenfeld

Energy and Environmental Research Center University of North Dakota

Grand Forks, North Dakota, USA 58202

INTRODUCTION

The ability of supercritical fluids to dissolve low vapor pressure solids was first reported by Hannay and Hogarth in 1879 (l), and numerous process-scale applications of SFE have been developed since the mid 1900s ( 2 ) . In contrast, the use of SFE for the extraction of trace and minor components on the analytical scale has only received attention during the last few years. For example, at the first International Workshop on Supercritical Fluid Chromatography (Park City, Utah, USA) held in January, 1988, only one paper focused on SFE. Since then, the number of investigators developing analytical-scale SFE methods has increased rapidly, as evidenced by the fact that 25 SFE papers were presented at the next meeting held in June, 1989.

Compared to the incredible development of chromatographic techniques and detectors which has occurred since Tswett's first report of chromatography in 1906, relatively few advances in sample extraction/preparation methods have been developed. Indeed, one of the most commonly-used extraction methods, liquid solvent extraction with a Soxhlet apparatus, is still performed in essentially the same manner as it was before Tswett's 1906 report. Since the extraction of organic compounds from sample matrices is often the most error-prone and slowest of an entire analytical scheme, the replacement of liquid solvent extractions with SFE has several potential advantages. These advantages can be summarized as:

Speed: Mass transfer is faster in a supercritical fluid than in liquid solvents because supercritical fluids have lower viscosities (10" vs. N-sec/m2) and higher solute diffusivities (10" vs. cm2/sec). Since mass transfer limitations often control extraction rates, quantitative SFE can usually be completed in 10 to 60 minutes, compared to several hours for liquid solvent extractions.

Variable solvent strength: Since the solvent strength of a supercritical fluid depends primarily on its density (2,3,4), the solvent strength can easily be manipulated by changing the pressure and temperature of the extraction. This allows SFE parameters to be optimized for a target analyte, and provides a method to achieve class-selective extractions (e .g . alkanes versus PAHs) from a single sample by simply extracting the sample at two different pressures with the same supercritical fluid.

Reduction of liquid solvent usage: The large volumes of liquid solvents used for conventional extractions have caused recent concern because of their potentially toxic nature and rapidly-increasing disposal costs. Since most commonly-used supercritical fluids are gases at ambient conditions and SFE effluents are typically collected in only a few mL of liquid solvent (or no liquid solvent for on-line SFE methods), the need for liquid solvents is dramatically reduced. In a similar manner, the need to concentrate extracts prior to analysis of trace analytes is also greatly reduced.

Simplified on-line coupling with chromatographic techniques: The gaseous nature (at ambient conditions) of most supercritical fluids also facilitates the direct coupling of SFE with GC and SFC (as discussed in Chapter 9) - It must be emphasized that SFE is a sample extraction/

preparation technique (analogous to liquid solvent extraction), and has no inherent need to be coupled with a chromatographic technique. While on-line techniques such as coupled SFE-GC and SFE-SFC (discussed in Chapter 9) are viable approaches to many analytical problems, the majority of SFE studies have been performed using off-line collection of the extracted analytes followed by analysis using a variety of measurement techniques including (but not limited to) chromatographic, spectroscopic, gravimetric, and radiochemical. Factors that contribute to the choice of off- line or on-line SFE are outlined in Table I. The major advantage of on-line approaches is the potential to transfer every extracted analyte molecule to the chromatographic system. Thus, on-line techniques are preferred when maximum sensitivity is needed from small samples. However, on-line SFE techniques generally require that the chromatographic system be used for sample collection during the SFE step, thus reducing the number of chromatographic runs that can be performed during a set period of time. Off-line SFE is inherently simpler to perform, since only the extraction parameters need to be understood, and several analyses can be performed on a single extract. The list in Table I should only be viewed as general guideline, since the continued development and refinement of on-line and off-line techniques

227

will likely change the factors that control the choice between these two approaches.

Table I

Comparison of Off-Line and On-Line SFE

Off-Line On-Line

GC or SFC needed for extraction? no Yes 100% transfer of analytes? no Yes multiple injections per extract? Yes no polarity modifiers useful? Yes ?? sample handling between SFE and analysis? yes no maximum convenient sample size? 10-15g 1-3g

Because the development of analytical SFE has occurred almost exclusively since 1986, the related literature has tended to be applications oriented, and the development of analytical SFE methods has been largely empirical. Since pure supercritical fluids that are convenient to use (e.g., CO,) are relatively non-polar, the majority of SFE studies reported to date have focused on the extraction of relatively non-polar and low molecular weight analytes (e.g., those amenable to separation using GC) although there are several notable reports of the extraction of more polar analytes. A survey of the literature also makes it apparent that the processes that control the extraction rates and ultimate recoveries achieved using SFE are poorly understood. Numerous qualitative and quantitative applications of analytical SFE have been reported during the last three years for a wide variety of sample matrices including (but not limited to) environmental solids (5-27), sorbent resins (9,18,28-33), food products and biological tissues (18,22,23,34-55), polymers (30,50,56-59), and petroleum-related samples (17,25,60-64). A review of analytical SFE applications is beyond the scope of this chapter, and the interested reader is referred to the above references and recent review articles (3,65). Instead, this chapter includes a brief description of off-line SFE techniques and instrumentation, and focuses more on the mechanical and chemical principles that need to be considered to successfully develop an analytical SFE method for both non- polar and polar analytes from a variety of matrices. Approaches to extending SFE to more polar and higher molecular weight species including the use of polarity modifiers and in- situ chemical derivatization, and the recent extension of SFE to water samples will also be discussed. Examples will be given of recent results from the literature and our laboratory.

GENERALIZED METHODS FOR SFE

A brief description of SFE techniques and instrumentation is given below. However, since instrumentation and methods are rapidly evolving, the reader should consult individual publications and instrument suppliers for details. A recent review ( 3 ) and the references therein describe various approaches to SFE in detail.

In contrast to a popular misconception, analytical-scale SFE is inherently simple to perform, and need not be unreasonably expensive. (For purposes of this chapter, analytical SFE will be restricted to sample sizes <ca. 10 grams for reasons discussed later in the text). A highly- serviceable SFE instrument can be constructed in the lab in a few hours for less than $10000 US ( 3 ) . Perhaps the most common mistake made by new investigators is to unnecessarily complicate the SFE system by the addition of multiple switching valves, transfer lines, and associated mechanisms. In our experience, simple systems have been much more successful in avoiding leaks and contamination. However, the development of commercial SFE units with multiple sample capabilities should increase the reliability of SFE components so that our present view that Ilsimpler is better" may no longer be valid.

The essential equipment needed to perform analytical SFE is shown in Figure 1. A high-pressure pump is used to provide the pressurized fluid (at a constant pressure) to the sample which is contained in the sample cell. The sample cell is placed in a heater (e.g., a pipe wrapped with heat tape or a GC-type oven) to maintain the temperature above the critical temperature of the extraction fluid. The extraction fluid is pumped through the sample cell, the analytes are partitioned into the supercritical fluid, and the analytes are collected after depressurization of the supercritical fluid. The depressurization step and the flow control is achieved either with a variable restrictor orifice (available in some commercial units) or, more commonly, by a length of fused silica tubing (typically 20 to 40 pm i.d.). Extracted analytes are most often collected in a few mL of liquid solvent SFE) , or the analytes are collected by directly transferring them to a chromatographic system ("on- line" SFE, see Chapter 9) ., Alternate methods such as cryogenic trapping or collection onto a sorbent cartridge have also been used, particularly for instruments utilizing variable orifices for the outlet restrictor. However, these last two approaches are inherently more complex than off-line collection in a small volume of liquid solvent, and have more potential to yield poor analyte recoveries from the collection step. (Off-line SFE collection parameters are discussed later in the text.)

229

Figure 1: Components of a simple SFE instrument. The fluid reservoir (A) is connected using 1/16 inch stainless steel tubing to a shut-off valve (B) mounted on a pressure- controlled syringe pump (C). During extraction, valve (B) is closed, and shut-off valve (D) is opened to supply the pressurized extraction fluid to the extraction cell (E) which is placed in a tube heater (F) to maintain the extraction temperature. The extracted analytes then flow out of the restrictor (G) into the collection vessel (H). For static extractions, an additional shut-off valve (I) can be placed between the extraction cell and the restrictor as indicated on the figure.

Two common modes are used for SFE; dynamic and static. For dynamic SFE the sample is constantly swept with fresh supercritical fluid at a flow rate determined by the extraction pressure and the dimensions of the outlet restrictor. For static SFE the sample cell is pressurized with the fluid and the sample is extracted with no outflow of the supercritical fluid. After the extraction is thought to be completed, a valve is opened at the outlet of the cell (Figure 1) to allow the analytes to be swept from the cell into the collection device. Typically, a static extraction is followed by several minutes of dynamic extraction to recover the analytes. Dynamic SFE continually provides new fluid to the sample, and is more effective when the supercritical fluid is likely to be saturated with the target analytes. Static extraction has the advantages that less fluid is used and that liquid polarity modifiers can be used by simply adding them to the cell prior to pressurization. Absolute criteria for selecting dynamic or static modes are not yet clear, and both have been widely used to achieve quantitative extractions. Additional considerations will be discussed later in the text.

While it is not the purpose of this work to rank SFE instrumentation (a process that would not be useful given the rapid changes in commercial instruments), some general guidelines to consider before selecting SFE pumps and extraction cells are listed in Table 11. In general,

230

characteristics in the llminimalll column will allow essentially the same SFE experiments to be performed as those in the lldesirablell column (with the exception of pressure limits) ; however, the number of extractions per day and the overall convenience will be lower. We have found the m o s t important characteristics of the SFE pump to be fast recovery after pressurizing the cell, fast refill and pressurization to working pressure, and a continuous display of the fluid flow rate. For extractions requiring polarity modifiers (discussed below), dual pumps that can provide known fluid mixtures are convenient, however, a single pump can also be used by mixing the modified supercritical fluid in the pump. Untilrecently, many SFE cells (including commercial cells and those made in the lab) were subject to leaks and had very limited lifetimes. Fortunately, commercial SFE cells are now available at a cost of ca. $100 to $1000 (US) that are extremely reliable, and some cells in our lab have been used for more than 1000 extractions without leaking.

Table I1

Characteristics of SFE Pumps and Cells

minimal desirable

P U P maximum flow rate (at full P) 1 mL/min >50 mL/min recovery time (after 5 min < l o sec

fill rate 10 mL/min 100 mL/min total fill cycle timeb 30 min <5 min upper pressure limit 300 atm 600 atm flow readout (real time) should be Yes modifier mixing (variable and no Yes

pressurizing cell) '

controlled by pumps)

extraction cells pressure rating 300 atm >600 atm no wrenches needed to assemble' no Yes finger tight connections to pumpC no Yes replaceable frits and seals' no Yes

lifetime (number of extractions) 10 > l o o 0 dead volume <I mL <0.1 mL

'Time required for the pump to regain the working pressure after pressurizing the sample cell.

bTotal time required for an empty pump to be filled and returned to the working pressure.

'These characteristics add convenience, but are not necessary.

23 1

SELECTING SFE FLUIDS AND CONDITIONS

Criteria for selecting supercritical fluids and SFE conditions have been previously discussed (3), and will only be briefly addressed here. Table I11 shows a list of several fluids that have been used for SFE, along with their critical temperatures and pressures, Hildebrand solubility parameters, and dipole moments. (Note that the Hildebrand parameters given in Table I11 are maximum values that are approached at very high pressures and are based on a correlation suggested by Giddings, ref. 4. Because of pressure limitations, typical extractions are performed at conditions which yield lower solvent strengths.)

Carbon dioxide has been the most widely used fluid for SFE, primarily because it is relatively non-reactive, non- toxic, inexpensive, and has a relatively low critical temperature and pressure. Unfortunately, CO, is not sufficiently polar to extract many analytes of interest. As a very general rule, organic compounds that can be separated using conventional GC techniques can be extracted at high pressure (e.g. , 400 atm) with CO,, providing that there are no strong interactions between the analytes and the sample matrix (discussed below) . Since the solvent strength of a supercritical fluid increases with its density, and since CO, is rarely too polar, SFE with CO, is generally performed at the highest pressure compatible with the extraction system.

Table I11

Characteristics of Representative Supercritical Fluids Used for SFE

co2 72.8 N2O 71.5

F6 37.1 CHF, 48.0 CHClF, 49.1 ethane 48.2 n-butane 37.5 xenon 57.6

NH3 112.0

31.0 36.4

132.2 45.6 26.1 96.1 32.2

152.0 16.5

10.7 10.6 13.2 7.6 8.7 8.8 8.7 7.7 9.5

0.0 0.2 1.5 0.0 1.6 1.4 0.0 0.0 0.0

'Hildebrand solubility parameter in (cal/cm3)

'Dipole moment.

232

The lack of polar supercritical fluids that also have attractive practical characteristics has been a major frustration for developing SFE methods for polar and higher molecular weight analytes. For example, ammonia would be an excellent SFE fluid from a polarity standpoint, except that it is highly reactive and toxic. Nitrous oxide has some advantages over CO, for some applications (discussed below), and recent investigations have shown some polar freons such as chlorodifluoromethane (freon-22) to have potential for yielding more efficient extractions (discussed below). However, successful applications of SFE to polar analytes have so far relied on the addition of organic solvents to increase the polarity of the supercritical fluid. While methanol has been the most widely used, a variety of organic solvents have been used including (but not limited to) lower alcohols, organic acids, propylene carbonate, acetone, 2-methoxyethanol, and methylene chloride. An example of the use of organic modifiers in CO, for the extraction of the ionic surfactant, linear alkylbenzenesulfonate ( U S ) from municipal wastewater treatment digester sludge is shown in Figure 2 . Note that neither pure CO, nor N,O gave any detectable recovery of the LAS, which is not surprising since LAS is an ionic compound with an average molecular weight of ca. 340 amu. However, when modifiers were added to the CO,, a 15-minute extraction at 380 atm (125 "C) yielded much higher recoveries, and SFE with methanol-modified CO, yielded quantitative recovery of the LAS.

100

so

5' 60

8 5 u

2 40

20

0

a, m C 0

n u a, C a x a

- f

- 2 a

Fisure 2: Recoveries (

- P m

a, x x 0

s

f

.A 2

- 0 s J? Y

I

3

2f LAS from municiDal wastewater treatment sludge using a 15-minute SFE extraction at 380 atm with pure CO, and N,O, and different polarity modifiers in CO,.

233

As shown in Figure 2, the use of different modifiers can yield quite different recoveries, and no clear criteria exist for the selection of the best modifier for a particular extraction. A modifier may act by increasing the solubility of the analyte in the extraction fluid, or by competing with the target analytes for the active sites on the sample matrix. Until a better understanding of the action of modifiers and the factors that control SFE efficiencies is gained, selection of modifiers for a particular extraction will largely be empirical, although a reasonable starting point would be to select a modifier that is a good solvent for the target analyte in its liquid state.

FACTORS THAT CONTROL SFE RATES AND RECOVERIES

While SFE is experimentally simple to perform, the development of quantitative extraction conditions has been severely hampered by the lack of overall understanding of the factors that control SFE extraction rates and ultimate extraction efficiencies. Several of the parameters (grouped loosely into IIExperimental parameters" and I'Sample/analyte- related parameters") that can control SFE recoveries are listed in Table IV.

Table IV

Parameters That Can Control SFE Rates and Recoveries

Experimental parameters: 1 Choice of supercritical fluid and modifier (if used). 2 Extraction T and P (which control fluid density). 3 The flow rate and total volume of the supercritical

4 Extraction time. 5 Volume and dimensions of the extraction cell. 6 Dead volume of the extraction cell. 7 Sample size (related to cell size). 8 Efficiency of the post-extraction analyte collection

fluid used.

method.

Sample/analyte-related parameters: 1 Analyte solubility in the supercritical fluid. 2 Sorption strength of analytes onto matrix active

3 Ability of the supercritical fluid to compete with

4 Physical location of the analyte in or on the sample

sites.

analytes for active sites on the matrix.

matrix.

234

Initial SFE conditions have most often been based on the solubility of the analytes in the supercritical fluid, but it has become increasingly clear that the interactions among the matrix, analytes, and the supercritical fluid frequently control the extraction rates to a much larger extent than can be explained by solubility considerations. Additionally, sample size, extraction flow rate and time, and the dimensions of the extraction cell all contribute to the final recoveries achieved.

When developing an SFE method it is useful to divide the SFE process into steps that are summarized by the following three questions:

Are the analytes partitioned from the sample matrix into the supercritical fluid ?

1.

2. Once in the supercritical fluid, are the analytes swept efficiently out of the cell ?

3 . Are the analytes collected efficiently after

These three factors interrelate to ultimately determine the optimal way to conduct an SFE sample preparation, and to some extent they can be evaluated independently. The last two questions are simplest to address and can provide a good starting point for developing an SFE method for specific target analytes. Once analytes are partitioned into the supercritical fluid, they are swept from the cell at a rate related to the sample size (and total void volume of the cell) and the flow rate. Thus, if the analytes are rapidly partitioned into the supercritical fluid (and saturation is not approached), the extraction rate is limited primarily by the need to sweep the void volume of the sample cell. For such extractions, extracting the sample with a few cell volumes is often sufficient to achieve quantitative recovery. (However, for most samples the partitioning of the analytes from the matrix into the supercritical fluid is not instantaneous, as discussed below.) Based on these considerations, relatively fast flow rates (e.g., >2 mL/min of the supercritical fluid) could be used to extract relatively large samples (e.g., >20 gram) in a reasonable time (e.g., < 3 0 minutes). Unfortunately, the useful flow rate is limited by the need to quantitatively trap the extracted analytes upon depressurization (question 3 ) .

depressurization ?

As previously mentioned, SFE extracts are most commonly collected off-line into a few mL of liquid solvent. This approach is useful for supercritical fluid flows of up to ca. 1-2 mL/min which corresponds to gas flows of ca. 500 to 1000 mL/min. Thus, assuming a 3 0 % void volume for a packed solid sample and a supercritical fluid flow of ca. 1 mL/min, sweeping 10 void volumes through a cell that has been packed

235

full with 10 grams of sample would require ca. 30 minutes. Such flow considerations combined with the difficulty in making large extraction cells indicate that sample sizes are best limited to ca. 10 grams for analytical SFE, unless larger samples are needed to insure homogeneity.

The efficiency of the trapping step (question 3 ) is important to distinguish from the efficiency of the extraction step (questions 1 and 2). Poor SFE recoveries have often mistakenly been blamed on the extraction step, when the actual reason for poor recoveries was inefficient trapping of the extracted analytes. A s shown in Figure 3 by the SFE extraction of n-alkanes from Tenax with collection in 2 mL methylene chloride, the more volatile alkanes were not quantitatively trapped in the methylene chloride during a 30- minute extraction with a flow rate of ca. 1 mL/min supercritical CO, (500 mL/min of gas) at 400 atm. However, switching the solvent to hexane or increasing the volume of methylene chloride allowed the n-alkanes to be efficiently trapped.

lo(1

so e ? a 2 ,

2 40

20

0

Figure 3 . methylene

j 1

Q

m

U

c

B d

4

Collection efficiency of n-alkanes in 2 mL chloride during a 3 0-minute SFE extraction.

Extraction conditions are given in the text.

Whether the extracted analytes are collected off-line into a liquid solvent or sorbent trap, or on-line into a chromatographic system, testing the efficiency of the trapping system for each analyte of interest is imperative. Regardless of the trapping system, quantitative recovery of spikes placed into empty cells (or cells packed with a non-absorbing clean matrix) indicates that the trapping system is efficient, and such spike recovery studies are a necessary preliminary to performing the extraction of real samples (note however, as discussed below, that good spike recoveries do not necessarily mean that the same analytes are quantitatively extracted from

real-world matrices). When liquid solvents are used for analyte collection, an additional simple test of the trapping system is to prepare a standard solution of the target analytes in the proposed trapping solvent, then use a few mL of the standard solution as the trapping solvent for a blank (empty cell) SFE experiment. No significant loss of the standard compounds from the standard solution used as a collection solvent indicates that purging of the target analytes will not occur during the extraction of real-world samples. If quantitative collection can not be achieved with a particular solvent based on this purging test, the collection efficiencies can generally be increased by one of the following methods: 1. Select a liquid with better solvating properties for the target analytes 2. Increase the collection solvent volume and/or use a taller capped collection vial (making sure that a vent hole is provided for venting the depressurized fluid) . 3 . Reduce the SFE flow rate and/or extraction time (if possible).

While the flow and analyte trapping considerations discussed above are relatively straight-forward to understand, the factors that control partitioning of the analytes from the sample matrix into the bulk supercritical fluid (question 3 , above) are far more complex and less understood. At least three general factors can be identified, any or all of which can control the rate of SFE recoveries from different samples. These factors, analyte solubility, analyteimatrix interactions (including the ability of the supercritical fluid to displace the analytes from active sites on the matrix), and diffusion of the analyte in the matrix particles (not in the supercritical fluid) are each discussed below along with examples of real-world extraction data. It must be emphasized that all three of these factors likely contribute to some extent to controlling SFE rates and efficiencies for every sample. The following discussions are presented only as examples where it appears that one of the factors appears to predominately control the extraction, and interpretations of the experimental results may change as the SFE mechanisms are better understood.

Analyte solubility:

Early SFE studies often determined the extraction conditions (pressure and temperature) based on maximizing the solubility of the target analytes. (A recent review contains extensive lists of the solubilities of organic compounds in CO,, ref. 66). While this approach is quite useful when the target analytes represent a large percentage of the bulk matrix (e.g, the extraction of fats from meat products), maximum solubility considerations are less useful when the target analytes are present in minor and trace amounts. In these cases, the analyte need only be soluble enough to be

237

transported out of the extraction vessel, and the concept of threshold solubility (the pressure where an analyte becomes significantly soluble) suggested by King (34) becomes a useful guide.

SFE rates appear to be controlled by solubility limitations for two general cases. First, when the analytes are present in very high concentrations (e.g., fats from meats), saturation of the supercritical fluid can occur. In this case, optimizing the SFE conditions for maximum solubility and increasing the amount of supercritical fluid used will yield higher extraction efficiencies.

The second general case where analyte solubility controls the extraction rate occurs when the analytes do not have sufficient solubility to be transported from the extraction vessel (i.e., the threshold pressure has not been reached). This is frequently the case when the target analytes are polar and/or have high molecular weights. As previously discussed, pure fluids such as CO, and N,O do not have sufficient polarity to dissolve such analytes, and the addition of polarity modifiers is needed. The extraction of the ionic surfactant, linear alkylbenzenesulfonate (US), from municipal wastewater treatment sludge and the extraction of pure abietic acid provide good examples (67). As previously shown in Figure 2, LAS could not be extracted with pure CO, or N,O. However, when methanol modifier was added to CO,, quantitative recoveries were achieved. The extraction of a 200-mg sample of abietic acid (Figure 4 ) was also very slow with pure CO,, and only 4 % of the material was extracted after 30 minutes at 400 atm (50 "C) . However, when 1 mL of methanol was added to the cell prior to extraction with CO,, 100% of the abietic acid was recovered in 20 minutes.

Figure 4. Structure of abietic acid.

AnalyteIMatrix Interactions:

A s mentioned above, solubility considerations frequently do not predict the results of SFE extractions, primarily because they fail to consider interactions between the analytes and active sites on (or in) the sample matrix. For example, both CO, and N,O have similar solubility parameters (Table 111), which indicate that their SFE extraction efficiencies should be similar. However, several comparisons of SFE recoveries have been reported (7,16,27) Which show that, at least for some matrices, N,O yields considerably better extraction efficiencies than CO, when the same sample is extracted under identical conditions. A dramatic example has been reported by Alexandrou (7) for the extraction of chlorinated dibenzo-p-dioxins from incinerator fly ash. While N,O yielded essentially quantitative recoveries of the dioxins, extraction with CO, yielded virtually zero recoveries. Interestingly, if the fly ash was first treated with acid, CO, then yielded good recoveries, demonstrating that analytelmatrix interactions were more important than solubility considerations for controlling the extraction efficiencies.

For many samples, the superiority of N,O over CO, is not so dramatic, but is still significant. For example, the extraction of polycyclic aromatic hydrocarbons (PAHs) from marine sediment (16) shows similar recoveries of the lower molecular weight PAHs from a 15-minute extraction using N,O and CO,, but the N,O yields much higher efficiencies for the higher molecular weight PAHs (Figure 5). The reason for the higher recoveries with N,O is unclear, but is likely related to the fact that N,O has a permanent dipole while CO, does not (Table 111), and therefore N,O can better compete with the active sites on the fly ash and marine sediment matrices.

This explanation is further supported by comparing the extraction rates achieved using CO, (no dipole moment) , N,O (small dipole moment), and CHClF, (relatively strong dipole moment, Table 111) for the extraction of polychlorinated biphenyls (PCBs) from a river sediment and PAHs from a petroleum waste sludge. Each extraction was performed at 400 atm and a few degrees above the critical temperatures of the individual fluids (extraction temperatures of 4 5 'C for co, and N,O, and 100 "C for CHClF,). Supercritical fluid flow rates were maintained at ca. 1 mL/minute. N,O yielded somewhat faster recoveries of the PCBs from the sediment and PAHs from the waste sludge than CO,, but CHClF, yielded much faster extraction rates of all of the PAHs and PCBs than N,O. This is demonstrated in Figure 6 by the extraction kinetic curves for the PAH chrysene and in Figure 7 for the trichlorobiphenyl PCB isomers. Since all of the PCBS and PAHS studied should have more than sufficient solubility in all three fluids, these results indicate that the factor controlling the

239

1.6

1.4

1.2

1.0 ,i g 0.8

2 0.6

0.4

0.2

0.0

Figure 5. SFE recoveries of PAHs from marine sediment with CO, and N,O at 400 atm (45 "C) with a 15-minute extraction. Results are adapted from reference 16.

- - 0 10 20 $0 40 SO 60 70 SO 90

Extraction Time (min)

,CO2 +NZO ,Freon-ZZ

Figure 6. SFE extraction kinetics of chrysene from a petroleum waste sludge using CO,, N,O, and CHClF,. SFE conditions are given in the text.

10 20 30 40 Extraction Time (min)

Figure 7. SFE extraction kinetics of trichlorobiphenyl PCBs from river sediment using CO,, N,O, and CHClF,. SFE conditions are given in the text. Values are normalized to CO,.

extraction rates was more related to the ability of the fluid to displace the analytes from the sorptive sites on the sample matrices.

Similar increases in extraction efficiencies of steroids using CHClF, were recently reported by Li et al. (52). For example, recoveries of estrone spikes were quantitative in 15 minutes using CHClF,, but were only 16% using CO, for 30 minutes (both extractions at 18 MPa).

Diffusion Limitations:

As discussed above, mass transfer in supercritical fluids is much better than in liquids, which is the major reason why quantitative SFE can often be completed in much less time than liquid solvent extractions. However, a recent report has shown that SFE extraction rates can be explained by a model which describes diffusion of the analytes in the sample matrix itself (as opposed to the diffusion of the analytes in the supercritical fluid, ref. 58). The model assumes that the analytes are evenly distributed throughout spherical sample particles, and that the concentration of the analytes in the supercritical fluid is always low. While these assumptions

24 1

are clearly not true for most real-world samples, all of the samples tested (including the extraction of PAHs from contam- inated soil, flavor and fragrance compounds from rosemary spice, the ionic surfactant LAS from wastewater treatment sludge, and Tinuvin-326 from polypropylene beads) conformed to the diffusion model after an initial extraction time.

To test the diffusion model, fractions were collected during SFE of each sample and analyzed. The results were then plotted as the In (m/m,) versus extraction time, where m, is the total mass of extractable analyte and m is the mass of the analyte remaining in the sample after extraction time t. While the model assumes an even distribution of the analytes throughout the matrix particle, distribution of analytes in the sample matrix is generally not known for real-world samples. However, an example that almost ideally fits this theoretical model is the extraction of the polymer additive Tinuvin-326 [2-(3-tertiarybutyl-2-hydroxy-5-methylphenyl)-2-H- 5-chlorobenzotriazole] from polypropylene, since it can be assumed that the additive is evenly distributed in the polymer particles.

Figure 8 shows the extraction efficiency curves that resulted from the CO, extraction of two particle sizes of the ground polypropylene beads. The top of the figure shows the cumulative % recovery of the Tinuvin-326, while the bottom part of the figure shows the diffusion model plots. The extractions show nearly ideal conformity with the diffusion model, and clearly demonstrate that recovery is much faster from the smaller particles (95% recovery at ca. 45 minutes for the c0.6 mm particles compared to ca. 250 minutes for the 0.6 mm to 1.2 mm particles). The original sample was supplied as ca. 5 mm beads, which required more than 80 hours to achieve near quantitative extraction ( 5 8 ) . These results clearly demonstrate the great advantage in extraction rates achieved by grinding samples when analytes are distributed throughout the matrix.

The majority of samples tested, however, showed a faster initial extraction (corresponding to a steeper initial drop in the diffusion model curve) than predicted by the model, indicating that analytes have higher concentrations at or near the surface of the sample particles. For example, after an initial rapid extraction period which removed ca. 90% of the LAS, the extraction rate then conformed to the diffusion model. This is demonstrated in Figure 9 for the extraction of LAS from municipal wastewater treatment sludge. The initial sharp drop corresponds to faster extraction of the LAS during the first 15 minutes than the model predicts, indicating that the LAS was present at higher concentrations at the surface of the sludge particles. However, the plot becomes linear after ca. 15 minutes, and the remaining extraction conforms to the diffusion model.

242

-3

rn particles < 0.6 mm

- \ \ -

I 1 1 I I I

4 0.6 mm < particles < 1.2 mm

I

0 50 100 150 200 250

t (llli 11s)

0

-1

a E E - -2 \

c -

Figure 8. SFE extraction kinetic curves (top) and diffusion model plots (bottom) for the extraction of Tinuvin-326 from two particle sizes of polypropylene. Extractions were performed with CO, at 400 atm (45 "C) at a flow rate of ca. 0.5 mL/min of supercritical fluid. Bottom figure was adapted with permission from reference 58.

243

0 10 20 30 40 50 60

Time, minutes

Figure 9. from municipal wastewater treatment sludge.

Diffusion model SFE plots for the extraction of LAS

Even though the multitude of parameters that can control SFE rates are not well understood, a consideration of the factors just discussed, analyte solubility, matrix/analyte interactions, and diffusion limitations in the sample matrix can help to design quantitative SFE conditions. For example, if an extraction is primarily limited by diffusion in the matrix (e.g., the extraction of Tinuvin-326 from polypropylene discussed above), the extraction rate will be greatly increased by grinding the sample, but will be little affected by changing the extraction flow rate. However, if the extraction is limited by solubility, increasing the flow rate may yield faster extractions, or the analyst should change extraction conditions to increase the fluid's solvent strength ( e . g . , by using higher pressure or adding a modifier) as discussed above for the LAS and abietic acid extractions. Increasing the solvent strength should also increase extraction rates that are limited by analytelmatrix interactions, and the use of fluids with high dipole moments such as CHClF, also appears to be effective.

244

CLASS-SELECTIVE SFE

As discussed above, one of the potential advantages of SFE over liquid solvent extraction is that the solvent strength of a supercritical fluid can be changed by simply changing its density. Thus, the potential to achieve class- selective extractions exists by simply extracting the same sample at two different pressures with the same fluid. For example, diesel exhaust particulates contain pgfg levels of PAHs with mutagenic activity, and much higher concentrations of branched and normal alkanes which can interfere with the determination of the PAHs when conventional methylene chloride extraction is used to recover the PAHs. However, sequential extractions at a relatively low pressure (to extract the non- polar alkanes) followed by a high pressure extraction (to extract the PAHs) can be used to yield a cleaner PAH fraction. Figure 10 shows the relative concentrations of representative PAHs and alkanes in extracts using a 5-minute SFE at 75 atrn with CO, (fraction 1) followed by a 15-minute extraction of the same sample at 300 atm (9). Note that ca. 85% of the alkanes were removed in the first extract, while (with the exception of phenanthrene) >90% of the PAHs were found in the 300 atm extract. Unfortunately, similar approaches to the selective extraction of the same alkanes and PAHs from petroleum industry waste sludges were not so successful, and selectivities of only ca. 60% were achieved compared to the 85-90% selectivities achieved for the diesel exhaust particulate sample.

I00

5 75

t

- u

5 50 Y

25

0

Alkanes PAHs 19 22 26 178 202 228 228 276

1 75 atm CO,, 0-5 rnin

300 atm CO,, 5-90 min

Figure 10. Class-selective SFE of alkanes and PAHs from diesel exhaust particulate matter using sequential CO, extractions at 75 atm (fraction 1) followed by 300 atm (fraction 2). Adapted from reference 9.

245

Class-selective SFE has also been applied to the extraction of target analytes from a bulk matrix (e.g., fat) which is itself highly soluble in supercritical CO, under most conditions. Even though fat components are highly soluble above pressures of ca. 120 atm, selective extracts of non- polar analytes have been achieved by extracting the samples at lower pressures. With this approach, King has achieved quantitative recovery of pesticides from fat samples, yet the extracts were sufficiently fat-free to allow direct GC analysis (34). Selective extraction of lactones from milk fat triglycerides has also been recently reported (55). A single extraction concentrated the lactones by 2 0 to 50 times, while a two-step extraction yielded a concentration factor of ca. 500 times.

When sufficient selectivity can not be achieved by simply extracting the sample under different SFE conditions, class- selective extractions have also been achieved by depositing the analytes onto a sorbent column, then eluting them with the supercritical fluid in an SFC mode. Such approaches have been used to achieve rapid fractionation of alkanes, alkenes, and aromatics from gasoline (17 , 61) , and fractionation of saturates, aromatics, and asphaltenes from crude oil ( 6 4 ) . France et al. have recently described an elegant technique using silica columns to selectively recover pesticides from fat samples (68). For example, the recovery of several pesticides from lard at concentrations of ca. 0.5 to 2 ppm were excellent (Table V) using CO, with 2% (v/v) methanol modifier, yet the extracts were sufficiently fat-free to be analyzed directly by capillary GC.

Table V

Class-selective Recoveries of Pesticides from Lard"

pesticide % recoverv

lindane heptachlor heptachlor epoxide dieldrin endrin 2 , 4 -DDT

103 107 102

95 103

98

'Adapted from reference 68.

Despite the attractive potential of class-selective SFE, relatively few quantitative applications have been described. Experience in our lab has shown that one sample may yield encouraging results, while the next sample will not, even though the same analytes are being studied (as described above for the extraction of alkanes and PAHs). In addition to

246

solubility considerations, the nature of analyte/matrix interactions clearly has an influence on the success of class- selective extractions, and an increased understanding of these interactions would greatly facilitate the development of class-selective SFE.

SFE OF WATER SAMPLES

Nearly all of the SFE investigations to date have focused on the recovery of organic analytes from a solid matrix. However a few recent studies have demonstrated the potential to use SFE to recover analytes from water and water-based fluids (44,45,69-71). Special extraction cells have been developed to ensure that the supercritical fluid (usually CO,) percolates through the water sample as shown by the cell reported by Hedrick et al. in Figure 11. The supercritical CO, enters the extraction cell through the top tube and exits into the bottom of the water sample. Since the supercritical CO, is less dense than water, the CO, percolates through the water sample to the top of the cell, where it exits for analyte collection. The most common application has been the extraction of phenols from water, and quite acceptable recoveries of 80 to 85% with RSDs <lo% have been reported (69- 71.)

CO? in 1

co. out

Figure 11. Extraction cell for performing SFE of water samples. Adapted from reference 69.

247

SFE WITH IN-SITU CHEMICAL DERIVATIZATION

As discussed above, popular supercritical fluids generally do not have sufficient solvent strength to achieve the extraction of polar analytes, and for such extractions the addition of polarity modifiers has been needed to obtain sufficient solubility (as previously described for the extraction of LAS). An alternate approach to extracting highly polar analytes is to reduce their polarity by chemical derivatization, thus making the analytes easier to extract under conventional SFE conditions. This approach would seem to be particularly elegant, since many polar species need to be derivatized prior to performing chromatographic analysis of the extracts. For example, conventional methods for the analysis of acid herbicides (e.g., 2,4-dichlorophenoxyacetic acid) require liquid solvent extraction followed by diazomethane derivatization of the herbicides to their methyl derivatives prior to capillary GC analysis. With in-situ derivatization during the SFE step, the herbicides can be converted to their methyl derivatives (which also makes them easier to extract) and extracted in a single step.

Two examples of SFE derivatizationjextraction are shown in Figures 12 and 13 for bacterial phospholipids from a 10-mg sample of Bacillus subtilis and several polar pesticides from a 2-gram sample of river sediment. To perform the SFE derivatizationjextraction, 0.5 to 1 mL of methanol which contained 0.1 M of the transesterification reagent, TMPA (trimethylphenylammoniumhydroxide), was added to the sample in the extraction cell, and the cell was pressurized to 400 atm with CO,. The derivatization step was carried out for 10 minutes at 80"C in the static mode, then the SFE was continued in the dynamic mode for 15 minutes with pure CO, to recover the derivatized analytes in a few mL of methanol. The extracts were then analyzed without any additional preparation using capillary GC.

The extraction of the underivatized bacterial phospholipids was difficult even when methanol was added as a modifier. However, with the derivatization under SFE conditions, the fatty acid methyl esters from the derivatized phospholipids were very easy to extract, even with pure CO,. Figure 12 shows the chromatogram of the fatty acid methyl esters resulting from the 25-minute SFE derivatization- extraction procedure. Performing the procedure a second time on the same sample showed no additional peaks, indicating that the first extraction/derivatization was quantitative. Additionally, comparisons of the chromatograms of the extracts from the SFE procedure with the conventional procedure which requires liquid solvent extraction followed by a separate derivatization step (a process requiring several hours), show good agreement.

248

i 15:C

\

st SFE

111 2nd SFE

16:o 17:O

1 a17:O

i

L L

:0

L

I

19:o

I Figure 12. GC/FID analysis of the phospholipid-derived fatty acid methyl esters from a 20-mg sample of Bacillus subtilis. SFE derivatizationjextraction procedures are given in the text.

Figure 13 shows the capillary GC analysis using an electron capture detector (ECD) of the extract from the SFE derivatizationjextraction of polar pesticides from the river sediment. (Names shown on the figure are the parent compounds prior to derivatization. Identities of the methyl derivatives were confirmed by GC/MS analysis). Each of the spiked pesticides showed the appropriate methyl derivatives in the SFE extract. Although quantitative work has only been done with the acid form of 2,4-dichlorophenoxyacetic acid (2,4-D), these results are encouraging, since recoveries of the 2,4-D acid as the methyl ester are >90$ with an SFE extraction1 derivatization procedure which requires only 30 minutes.

These initial studies demonstrate that performing derivatizations under SFE conditions can achieve two goals at once: making the target analytes easier to extract, and making the analytes easier to measure. Although SFE derivatization- extraction studies are very limited, a wealth of chemical derivatization techniques are available for liquid phase reactions which should be applicable to SFE conditions. In addition to decreasing the polarity of target analytes, SFE derivatizations could be used to enhance their detection, e.g., dansylation for fluorescence detection, or trifluoro- acetylation to enhance ECD sensitivities. The potential for

249

performing class-selective derivatizations could also enhance the ability to achieve class-selective extractions under SFE conditions.

Dalapon 1 /

/ Dicamba

I

0 c Q) c a 0 & Y 5

\

Pentachlorophenol

:Ioram

\ Dinoseb

ib 2'

Retention Time (min) Figure 13. GC/ECD analysis of polar pesticides from river sediment that were derivatized and extracted under SFE conditions as described in the text.

CONCLUSIONS

The rapid increase in publications describing analytical- scale SFE over the last four years attests to both the interest in, and the need for new sample extraction methods. A wide range of quantitative SFE applications has been reported, and several investigators have demonstrated the ability of SFE to drastically reduce both the time required, and the quantities of waste solvents produced for sample extractions. Further developments in the use of modifiers, the extraction of water samples, class-selective extractions, and chemical derivatizations under SFE conditions will doubtless increase the range of quantitative SFE applications in the near future. However, analytical SFE is still a very new field, and a much deeper understanding of the physical and chemical factors that control extraction rates and efficiencies is needed to support the development of routine and widely-applicable SFE methods, particularly for polar and high molecular weight analytes.

250

ACKNOWLEDGEMENTS

The authors would like to thank the U.S. Environmental Protection Agency (Cincinnati) and British Petroleum (USA) for financial support. Instrument support from ISCO (Lincoln, NB, USA) and Suprex (Pittsburgh, PA, USA) is also acknowledged.

REFERENCES

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

J.B. Hannay and J. Hogarth, Proc. R. SOC. London, 29 (1879) 324.

M. McHugh and V. Krukonis, Supercritical Fluid Extraction; Principles and Practice, Butterworths, Boston, 1986.

S.B. Hawthorne, Anal.Chem., 62 (1990) 633A.

J.C. Giddings, M.N. Myers, L. McLaren and R.A. Keller, Science, 162 (1968) 67.

J.R. Wheeler and M.E. McNally, J. Chromatogr. sci., 27 (1989) 534.

F. I. Onuska and K. A. Terry , J. High Resolut . Chromatogr . , 12 (1989) 357.

N. Alexandrou and J. Pawliszyn, Anal. Chem., 61 (1989) 2770.

B.W. Wright, C.W. Wright and J . S . Fruchter, Energy and Fuels, 3 (1989) 474.

S.B. Hawthorne and D . J . Miller, J. Chromatogr. Sci., 24 (1986) 258.

S.B. Hawthorne and D.J. Miller, Anal. Chem., 59 (1987) 1705.

P. Capriel, A. Haisch ana S . U . Khan, J. Agric. Food Chem., 34 (1986) 70.

V. Janda, G. Steenbeke and P. Sandra, J. Chromatogr. , 479 (1989) 200.

M.M. Schantz and S.N. Chesler, J. Chromatogr. , 363 (1986) 397.

B.O. Brady, C.P.C. Kao, K.M. Dooley, F.C. Knopf and R . P . Cambrell, Ind. Eng. Chem. Res., 26 (1987) 261.

M.A. Schneiderman, A.K. Sharma and D.C. Eocke, J. Chromatogr., 409 (1987) 343.

25 1

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

S.B. Hawthorne, D.J. Miller and J.J Chromatogr. Sci., 28 (1990) 2.

Langenf eld , J.

J.M. Levy, R.A. Cavalier, T.N. Bosch, A.M. Rynaski and W.E. Huhak, J. Chromatogr. Sci., 27 (1989) 341.

S.B. Hawthorne, D.J. Miller and M.S. Krieger, J. Chromatogr. Sci. , 27 (1989) 347.

B.W. Wright, S.R. Frye, D.G. McMinn and R.D. Smith, Anal. Chem. , 59 (1987) 640.

S.B. Hawthorne and D.J. Miller, J. Chromatogr., 403 (1987) 63.

F. I. Onuska and K. A. Terry , J. High Resolut . Chromatogr . , 12 (1989) 527.

M.R. Andersen, J.T. Swanson, N . L . Porter and B.E. Richter, J. Chromatogr. Sci., 27 (1989) 371.

R. J. Skelton, C.C. Johnson and L.T. Taylor, Chromatographia, 21 (1986) 3.

M.W. Raynor, I . L . Davies, K.D. Bartle, A.A. Clifford, A. Williams, J.M. Chalmers and B.W. Cook, J. High Resolut. Chromatogr. Chromatogr. Commun., 11 (1988) 766.

K. R. Jahn and B. Wenclawiak, Chromatographia, 26 (1988) 345.

M. Ashraf-Khorassani, L.T. Taylor and P. Zimmerman, Anal. Chem. , 62 (1990) 1177.

X. Yu, X. Wang, R. BarYha and J.D. Rosen, Environ. Sci. Technol., 24 (1990) 1732.

S.B . Hawthorne, M.S. Krieger and D.J. Miller, Anal. Chem., 61 (1989) 736.

M.W.F. Nielen, J.T. Sanderson, R.W. Frei and U.A.T. Brinkman, J. Chromatogr., 474 (1989) 388.

S.A. Liebman, E.J. Levy, S . Lurcott, S . O'Neil, J. Guthrie, T. Ryan and S. Yocklovich, J. Chromatogr. Sci., 27 (1989) 118.

B.W. Wright, C.W. Wright, R.W. Gale and R.D. Smith, Anal. Chem. , 59 (1987) 38.

J.H. Raymer and E.D. Pellizzari, Anal. Chem., 59 (1987) 1043.

252

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

J.H. Raymer, E.D. Pellizzari and S.D. Cooper, Anal. Chem., 59 (1987) 2069.

J.W. King, J. Chromatogr. Sci., 27 (1989) 355.

K.S. Nam, S. Kapila, G. Pieczonka, T.E. Clevenger, A.F. Yanders, D . S . Viswanath and B. Mallu, Proceedings of the International Symposium on Supercritical Fluids, (1988) 743.

J.W. King, J.H. Johnson and J.P. Friedrich, J. Agric. Food Chem., 37 (1989) 951.

M.A. Schneiderman, A.K. Sharma, K.R.R. Mahanama and D.C. Locke, J. Assoc. Off. Anal. Chem., 71 (1988) 815.

M.A. Schneiderman, A.K. Sharma and D.C. Locke, J. Chromatogr. Sci., 26 (1988) 458.

K. Sugiyama and M. Saito, J. Chromatogr. , 442 (1988) 121.

E.D. Ramsey, J.R. Perkins, D.E. Games and J.R. Startin, J. Chromatogr., 464 (1989) 353.

Q.L. Xie, K.E. Markides and M.L. Lee, J. Chromatogr. sci., 27 (1989) 365.

M. Saito, Y. Yamauchi, K. Inomata and W. Kottkamp, J. Chromatogr. Sci., 27 (1989) 79.

H. Engelhardt and A. Gross, J. High Resolut. Chromatogr. Chromatogr. Commun. , 11 (1988) 38.

D. Thiebaut, J.P. Chervet, R.W. Vannoort, G.J. DeJong, U.A.T. Brinkman and R.W. Frei, J. Chromatogr. , 477 (1989) 151.

J. Hedrick and L.T. Taylor, Anal. Chem., 61 (1989) 1986.

S . B . Hawthorne, M . S . Krieger and D. J. Miller, Anal. Chem., 60 (1988) 472.

W. Gmuer, J.O. Bosset and E. Plattner, J. Chromatogr., 388 (1987) 335.

K. Anton, R. Menes and H.M. Widmer, Chromatographia, 26 (1988) 221.

K. Sugiyama, M. Saito, T. Hondo and M. Senda, J. Chromatogr., 332 (1985) 107.

R.M. Campbell, D . M . Meunier and H.J. Cortes, J. Microcol. Sep., 1 (1989) 302.

253

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

P. Sandra, F. David and E. Stottmeister, J. High Resolut. Chromatogr. , 13 (1990) 284.

S.F.Y. Li, C.P. Ong, M.L. Lee and H.K. Lee, J. Chromatogr. , 515 (1990) 515.

C.P. Ong, H.K. Lee and S.F.Y. Lit J. Chromatogr., 515 (1990) 509.

K. Sugiyama, T. Shiokawa and T. Moriya, J. Chromatogr., 515 (1990) 555.

A.B. de Haan, J. de Graauw, J.E. Schaap and H.T. Badings, J. Supercritical Fluids, 3 (1990) 15.

J. Hirata, F. Nakata and M. Horihata, J. High Resolut. Chromatogr. Chromatogr. Commun., 11 (1988) 81.

Y. Hirata and Y. Okamoto, J. Microcol. Sep., 1 (1989) 46.

K.D. Bartle, A . A . Clifford, S.B. Hawthorne, J.J. Langenfeld, D.J. Miller and R. Robinson, J. Supercritical Fluids, 3 (1990) 143.

R.C. Wieboldt, K.D. Kempfert and D.L. Dalrymple, Appl. Spectrosc. , 44 (1990) 1028.

J.C. Monin, D. Barth, M. Perrut, M. Espitalie and B. Durand, Adv. Org. Geochem., 13 (1988) 1079.

J.M. Levy and J.P. Guzowski, Fresenius Z. Anal. Chem., 330 (1989) 207.

J.M. Levy, J.P. Guzowski and W.E. Huhak, J. High Resolut. Chromatogr. Chromatogr. Commun., 10 (1987) 337.

G. Hopfgartner, J.L. Veuthey, F.O. Gulacar and A. Buchs, Org. Geochem., 15 (1990) 397.

H. Skaar, H.R. Norli, E. Lundanes and T. Greibrokk, J. Microcol. Sep., 2 (1990) 222.

R.W. Vanoort, J.-P. Chervet, H. Lingeman, G.J. Dejong and U.A. Th. Brinkman, J. Chromatogr., 505 (1990) 45.

K.D. Bartle, A.A. Clifford, S . A . Jafar and G.F. Shilstone, J. Phys. Chem. Ref. Data (in press).

S.B. Hawthorne, D.J. Miller, D.D. Walker, D.E. Whittington and B.L. Moore, J. Chromatogr. (in press).

J.E. France and J.W. King, J. Ag. Food Chem. , (in review).

254

69 J.L. Hedrick and L.T. Taylor, J. High Resolut. Chromatogr. , 13 (1990) 312.

70 D. Thiebaut, J . P . Chervet, R.W. Vannoort, G.J. Dejong, U.A. Th. Brinkman and R.W. Frei, J. Chromatogr., 477 (1989) 151.

71 R.K. Roop, A. Akgerman, B. J. Dexter and T.R. Irvin, J. Supercritical Fluids, 2 (1989) 51.