In the Laboratory
Determination of Crude Fat in Food Products by Supercritical Fluid Extraction and Gravimetric Analysis Nicholas H. Snow,* Maureen Dunn, and Sohita Patel Department of Chemistry, Seton Hall University, South Orange, NJ 07079
Supercrit . Fluid 73
Pressure (atm)
Supercritical fluid extraction (SFE) is becoming a widely used analytical method for the removal of organic compounds from heterogeneous matrices such as soils, smoke particles, plant matter, food, and other biological matter. Several recent reviews (1–3) highlight developments in theory, applications, and instrumentation. Computer software (4) abstracts over 700 publications on SFE from the past decade. SFE has several advantages over traditional extraction methods that make it suitable for a wide range of applications and show its promise in the undergraduate laboratory: organic solvent consumption is minimal, drastically reducing waste handling costs and worker exposure to toxins; carbon dioxide is relatively environmentally benign, again reducing exposure risks; CO2 is inexpensive (approximately $200 for 50 pounds of SFE grade, $30–70 for lower grades); the extraction systems are simple to operate and maintain; and compared to many other analytical instruments, SFE systems are inexpensive and versatile. Commercial systems are available from several vendors and home-built systems are possible (5, 6). Recently, there has been a thrust in chemical education toward the generation of realistic scientific exercises in the undergraduate curriculum. Although many efforts have focused on the lower-level courses (7, 8), this type of effort is useful in upper-level laboratories as well. One such approach involves the integration of student training in instrumental techniques into an ongoing research effort in inorganic synthesis and characterization (9). A similar approach to the analysis of food is begun here, with this initial experiment leading to many more for students in future years. In the chemical industry, some of the most demanding problems involve isolation of compounds of interest from highly complex matrices. In traditional instrumental analysis curricula (10, 11), sample extraction, now an instrumental method and often the most difficult part of a real-world analysis, is virtually ignored. A brief review of the theory of SFE will show that it may find a valuable place as a sample preparation experiment for undergraduate analytical laboratories, or as a means to study several phenomena discussed in physical chemistry courses. A phase diagram for carbon dioxide is shown in Figure 1 (12). As the pressure and temperature of carbon dioxide are increased beyond the critical point, a fluid in an intermediate state between liquid and gas is obtained. This is widely termed the “supercritical region”, giving rise to the term “supercritical fluid”. The properties of substances commonly used as supercritical fluids are given in Table 1 (13). The main generalization is that supercritical fluids have dissolving properties similar to liquids (enabling them to carry greater amounts of solute than gases), with diffusion properties and zero surface tension characteristic of gases (enabling them to diffuse into the small pores and crevices present in heterogeneous samples such as soils and foods). Analytes are removed by partitioning into the supercritical
67 Liquid Solid
5.1 Gas 1 -78.2
1108
25 31.1
Temperature (°C)
Figure 1. Phase diagram for carbon dioxide.
solvent phase. It is also noted that the substances shown in Table 1 have critical temperatures and pressures that are easily obtained using ordinary chromatographic pumps and ovens. Models for the kinetics of SFE extraction of material from complex heterogeneous samples have recently been presented by Langenfeld et al. (14) and Pawliszyn (15). A thorough description of phase diagrams was presented by Kildahl (16). Carbon dioxide is the most popular supercritical fluid, as its solubility properties may be considered similar to nonpolar organic solvents. Also, many other potential supercritical fluids are corrosive or potentially hazardous. A typical SFE system is shown in Figure 2. The basic system consists of a cylinder of liquid carbon dioxide, a syringe pump that allows the pressure to be adjusted to a known value above the critical pressure, an extraction vessel contained within a temperature controlled zone, an outlet restrictor which is a small-diameter piece of stainless steel or drawn fused silica tubing, and a collection vessel. For this experiment, SFE-grade carbon dioxide was used, although instrument grade, with cooling of the pump head Table 1. Properties of Supercritical Fluids Compound
Boiling Point (°C)
Critical Temp (°C)
Critical Pressure (atm)
Critical Density (g/mL) 0.448
CO2
{78.5
31.3
72.9
N 2O
{89.0
36.5
71.4
0.457
SF6
{63.8
45.6
37.1
0.752
NH3
33.4
132.3
111.3
0.240
100.0
374.4
226.8
0.344
64.7
240.5
78.9
0.272
Water *Corresponding author.
-56.6
Methanol
Journal of Chemical Education • Vol. 74 No. 9 September 1997
In the Laboratory Extraction Vessel
Restrictor Liquid Carbon Dioxide
Heated Zone
Collection Vessel
Mixing Tee
Pump
Figure 2. Schematic of a typical SFE system.
to facilitate pressurization, is adequate. The pump should be capable of delivering pressures of several thousand pounds per square inch to maintain supercritical conditions over a wide temperature range. Between the pump and extraction vessel, a mixing tee is commonly provided to ease the addition of a second pump, which readily allows the use of solvent mixtures when necessary. Extraction vessels typically range in size from 1 to 10 mL and may be connected to the pump using standard HPLC compression fittings with stainless steel tubing. Finally, the outlet restrictor maintains suitable pressure in the extraction vessel while allowing the carbon dioxide to pass through the collection vessel. Restrictors are typically a narrow-bore (ca. 50 mm or less in diameter) length (ca. 20 cm) of fused silica or stainless steel tubing. Collection vessels may contain a suitable organic solvent that dissolves analytes in the effluent as the carbon dioxide escapes, or may involve a solid-phase trap on which the analytes are sorbed. In this work, since the extracts were not analyzed, the restrictor was placed into an empty 2-L round-bottom flask to prevent aerosol spray from the restrictor from spreading the extract throughout the laboratory. Analysis of the fat content of food is a major concern of that industry as food labeling regulations and the resulting analytical requirements become increasingly strict. The percentage of fat by weight is now reported on the label of almost all packaged food products sold in the United States. Typically, fats are removed from the complex food matrix by a combination of acid hydrolysis and solvent extraction (17), a labor- and solvent-intensive process not suitable for the undergraduate lab. Because of their hydrophobic nature, fats are suitable for extraction by nonpolar carbon dioxide. The literature shows many examples of acid hydrolysis and SFE being used for the removal of crude fats from the food matrix, with an especially thorough comparison of two methods shown by Huang, et al. (18) and an SFE study by King et al. (19). Experimental Procedure All supercritical fluid extractions were performed on an SFX-2-10 extractor with syringe pump (Isco, Inc., Lincoln, NE) and a 10 mL/min stainless steel restrictor. Carbon dioxide was SFC/SFE grade with 1500 psi helium headspace and dip tube (MG Industries, Allentown, PA), or refrigeration grade (AGL, Linden, NJ), which is much less expensive. When the refrigeration-grade CO2 was used, the syringe pump was cooled to 10 °C using a cold-water circulator. Dynamic extractions were conducted at 8000 psi and 80 °C. All instrument components are connected using high-
pressure compression fittings of the same type employed in HPLC. Candy bar samples (SnickersTM, M&M Mars, Hackettstown, NJ) were obtained from local vending machines and analyzed as follows. All handling of samples and the extraction vessel was performed wearing gloves or with tongs. Approximately 1 g of candy was accurately weighed (to the nearest milligram) on an analytical balance (Mettler, Toledo, OH) and mixed loosely with a few grams of Celite (Fisher). This mixture was placed into a 10-mL stainless steel extraction vessel (Isco) and the remaining vessel volume was filled with Celite. The assembly was accurately weighed. The vessel was then placed into the extractor and extracted with the full syringe pump volume of supercritical carbon dioxide. The volume (approximately 70 mL) of pressurized carbon dioxide was recorded. After each extraction, the vessel was removed from the extractor, allowed to cool to room temperature, and reweighed. This extraction and weighing procedure was repeated until a nearly constant vessel assembly weight was obtained from the vessel. The percentage of fat was then calculated from the difference in weight of the vessel assembly before and after extraction. Blank samples containing only Celite showed a loss of approximately 3 mg per pump volume of carbon dioxide passed through under the above conditions. Mass calculations were adjusted for this loss. At the conclusion of the experiment, the extraction vessel was cleaned thoroughly with water and allowed to dry. It is noted that the waste from this experiment, a mixture of Celite and candy, is not hazardous. Results and Discussion Results from a typical candy bar analysis are shown in Table 2. It is immediately noted that a complete extraction requires that several full syringe pump volumes of carbon dioxide be used. This reminds the student that kinetic factors may affect extraction recoveries, along with partitioning. It is seen that nearly 100% recovery of the fat is obtained after about 400 mL of supercritical carbon dioxide has passed through the extraction vessel. To ensure complete extraction, several more pump volumes of carbon dioxide were passed through the sample. A finely divided film of fatty material was observed coating the walls of the collection vessel and the entire laboratory took the odor of chocolate and caramel. The results from 11 groups of students are summarized in Table 3. It is seen that the mean percentage of fats found in the sample was 21% with a 95% confidence interval of ± 2%. If the package labeling of 21% is taken as the population mean it is seen that the analytical method is in agreement with the “literature”, as the population mean falls within the confidence interval. It is noted that values ranged between a low of 17% and a high of 26% for individual analyses. This variation may be due to the heterogeneous nature of the samples. For example, some samples contained an entire peanut, while others did not. This provides a realistic illustration of the sampling problems that may befall a real-world analytical method. There is also variation in the final volume of carbon dioxide used in the extractions, but this showed no correlation to the final percentage of fat extracted, probably also due to the heterogeneous nature of samples and of student lab technique. For each extraction, the percentage recovery is calculated as the ratio of the total crude fat removed from the sample to the total amount of fat expected to be present. This is typical of the type of calculation expected from analytical extractions, and similar calculations are shown in many of the references. It is noted, as above, that several of
Vol. 74 No. 9 September 1997 • Journal of Chemical Education
1109
In the Laboratory Table 2. Typical Results of Fat Extraction from Candy Bara Total CO2 Volume (mL)
Trial
Total Mass Extracted (mg)
Percentage of Candy Bar Removed
Fat Recovery (%) 50
1
77
111
11.0
2
153
140
13.8
66
3
225
176
17.4
83
4
295
209
20.7
99
5
365
224
22.2
106
6
436
237
23.4
111 112
7
505
239
23.6
8
575
239
23.6
112
9
649
240
23.7
113
10
723
240
23.7
113
a
Total mass of candy: 1.011 g; mass of candy, vessel, and Celite assembly: 101.380 g; mass of assembly following 700-mL CO 2 extraction: 101.109 g; expected fat percentage: 21%.
Table 3. Compiled Results from Crude Fat Extraction Mass of Fat Removed (mg)
Percentage of Fat Candy Bar Recovery Removed (%)
Group
No. of Extractions
1
9
183
16.5
2
7
182
18.2
87
3
6
279
25.8
123
4
6
234
23.2
110
5
4
228
24.3
115
6
4
244
24.3
115
7
4
204
20.4
97
8
4
189
18.5
88 98
79
9
5
212
20.6
10
6
185
18.5
88
11
10
240
23.7
113
216
21.2
101
31
3.1
14
Average Standard Deviation
120
Percent Fat Recovery
100 80 60 40 20 0 0
200
400
600
800
Carbon Dioxide Volume (mL)
Figure 3. Fat recovery from SnickersTM bar versus carbon dioxide extraction volume.
1110
the recoveries are greater than 100%, again a result of sample inhomogeneity or variable lab techniques. The percentage recovery expressed as a decimal gives a value for the efficiency of the extraction and reminds the student that few extractions, especially those carried out in a single step, achieve 100% recovery or total efficiency. An excellent development of extraction recovery theory as it relates to another modern extraction method, solid phase extraction, is shown in a recent extension of liquid–liquid extraction theory (20). Figure 3 shows a plot of the extraction recovery versus the volume of carbon dioxide passed through the extraction vessel. Data points were obtained by reweighing the extraction vessel assembly after each full pump volume of carbon dioxide has been passed through. Immediately, it is seen that, as with any analytical extraction, extraction time has a profound effect on recovery. Although the graph shows recovery plotted against carbon dioxide volume, which is the quantity easily measured by the student, this is easily converted to extraction time using the known restrictor flow rate. The complete extraction requires 400–700 mL of carbon dioxide, or 40–70 min at 10 mL/min. Qualitative examination of the data indicates that this extraction follows similar kinetics to those observed by Langenfeld et al. (14) and calculated by Pawliszyn (15) for analytes adsorbed onto soils. It is seen, therefore, that the removal of materials of interest from a complex matrix involves various forms of transport, including diffusion through the matrix to its surface, transfer across the boundary between the matrix and the extraction fluid, and diffusion within the extraction fluid. It is also noted that many analytes will partition between the fluid and matrix more than once, indicating that analyte transport in SFE from this complex matrix is a combination of a chromatography-like elution and desorption from the matrix. Further studies of the temperature effects on the recovery and rate of the extraction, to be performed by future classes, will provide more definitive evidence. Operation of SFE instruments is very straightforward. Undergraduates at freshman and junior levels were able to collect the data presented here with a minimum of supervision. Although high pressures are employed, safety is maintained by the use of high-pressure stainless steel compression fittings throughout the instrument. Restrictor venting into a large-volume vessel or through a solvent prevents contact with high-velocity effluent. The cost for a commercial system is similar to that of a well-equipped gas or liquid chromatograph. If an HPLC pump and temperature control, such as an oven, are available, the remaining components (extraction vessel, high-pressure fittings, restrictors) can be purchased for a few hundred dollars. Slack et al. (5, 6) describe configurations for home-built systems. Carbon dioxide is readily available from many distributors. Both expensive SFE grade and inexpensive refrigeration grade CO2 are appropriate for this type of bulk extraction. If nonpressurized cylinders are purchased, compression of liquid CO2 is aided by cooling the pump head to 10 °C. Several extensions of this, along with other SFE experiments, are possible with a basic system. For analytical courses, studies such as the effects of sample homogeneity (perhaps the best method would be to completely homogenize an entire candy bar and then collect a sample of that mixture), mixing agent, sample matrix, and extraction conditions on the recovery and volume of carbon dioxide required would allow the students to perform method development exercises similar to those encountered in industrial laboratories, and to have a “discovery” type of experience. Recently, Moore and Taylor showed a comparison of recoveries from different SFE effluent-trapping methods (21), a type of experiment easily
Journal of Chemical Education • Vol. 74 No. 9 September 1997
In the Laboratory adapted to the undergraduate lab, as many types of glassware or materials can be used to trap materials eluting from the extraction vessel. The gravimetric analysis presented here may also be extended to a complete qualitative analysis of extracted material by examining the extracts by GC or GC/MS. SFE is also applicable to physical chemistry courses. The study of several physical conditions (such as temperature, CO2 density [pressure], and the addition of polar modifiers to the CO2 on the extraction recovery and kinetics) illustrate many of the topics encountered in physical chemistry texts while providing modern instrumentation for the study of these classical problems. King et al. (22) have observed an effect on fat solubility due to the presence of dissolved helium in cylinders pressurized for SFE work with helium headspace. A study of this effect, recently reported in the literature and not fully explained, can be accomplished in an undergraduate laboratory. Finally, this technique can be easily extended to other foods and materials. The procedure given here can be easily employed in the analysis of any fat-containing product. The literature cited herein describes many examples.
1108, especially Leotha Francis, are acknowledged for extracting several candy bars and providing excellent reports of their results. Literature Cited 1. 2. 3. 4. 5. 6. 7.
8.
9. 10.
Conclusions
11.
The use of supercritical fluid extraction, a modern instrumental technique, in the undergraduate laboratory has been shown. Specifically, the crude fat content of a SnickersTM candy bar was determined to be 21 ± 2%, consistent with the package labeling. It was also demonstrated that SFE is a straightforward, versatile, and relatively inexpensive technique for advanced undergraduates. SFE may be easily incorporated into the instrumental analysis curriculum and may serve as the basis for experiments that provide integration between the physical and analytical chemistry laboratories and “discovery”-type research experiences as part of the standard laboratory course training for undergraduates.
12. 13. 14. 15. 16. 17. 18.
19.
Acknowledgments
20.
We gratefully acknowledge Isco, Inc. (Lincoln, NE), especially Dale Messer, for the loan of the SFE equipment and technical support. The students of CHEM 2216 and CHEM
21. 22.
McNally, M. E. Anal. Chem. 1995, 67, 308A. Taylor, L. T. Anal. Chem. 1995, 67, 364A. Hawthorne, S. B. Anal. Chem. 1989, 62, 633A. SF Searcher; ISCO: Lincoln, NE, 1994. Slack, G. C.; McNair, H. M.; Wasserzug, L. J. High Resolution Chromatogr. 1992, 15, 102–104. Slack, G. C.; McNair, H. M.; Hawthorne, S. B.; Miller, D. J. J. High Resolution Chromatogr. 1993, 16, 473–478. “Highlights: Projects Supported by the NSF Division of Undergraduate Education”; Hixson, S. H.; Sears, C. T., Eds.; J. Chem. Educ. 1995, 72, 639–640. “Highlights: Projects Supported by the NSF Division of Undergraduate Education”; Hixson, S. H.; Sears, C. T., Eds.; J. Chem. Educ. 1995, 72, 533–536. Brabson, G. D. Integrated Chemical Experimentation; Kendall-Hunt: Dubuque, IA, 1993. Sawyer, D. T.; Heineman, W. R.; Beebe, J. M. Chemistry Experiments for Instrumental Methods; Wiley: New York, 1984. Skoog, D. A.; Leary, J. J. Principles of Instrumental Analysis, 4th ed.; Saunders: Fort Worth, TX, 1992. Castellan, G. W. Physical Chemistry, 3rd ed.; Addison-Wesley: Reading, MA, 1983, pp 266–268. CRC Handbook of Chemistry and Physics; Weast, R. E., Ed.; CRC: Boca Raton, FL, 1982; pp F76–F77. Langenfeld, J. J.; Hawthorne, S. B.; Miller, D. J.; Pawliszyn, J. Anal. Chem. 1995, 67, 1727–1736. Pawliszyn, J. J. Chromatogr. Sci. 1993, 31, 31–37. Kildahl, N. K. J. Chem. Educ. 1994, 71, 1052–1055. Official Methods of the Association of Analytical Chemists; AOAC: Arlington, VA, 1984; Methods 14.104, 16.136. Huang, A. S.; Robinson, L. R.; Gursky, L. G.; Pidel, G.; Delano, G.; Softly, B. J.; Templemen, G. J.; Finley, J. W.; Leveille, G. A. J. Agric. Food Chem. 1995, 43, 1834–1844. King, J. W.; Hopper, M. L. J. Assoc. Off. Anal. Chem. Int. 1992, 75, 375–378. Hughes, D. E.; Gunton, K. E. Anal. Chem. 1995, 67, 1191– 1196. Moore, W. N.; Taylor, L. T. Anal. Chem. 1995, 67, 2030–2036. King, J. W.; Johnson, J. H.; Eller, F. J. Anal. Chem. 1995, 67, 2288–2291.
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