Chapter 2 Soxtec: Its Principles and Applications Shirley Anderson Foss North America, Eden Prairie, MN 55344 Abstract The classical Soxhlet method provides the fundamental basis for a modern-day sol- vent extraction system, the Soxtec TM . Using the Randall modification, sometimes called the submersion method, the Soxtec provides a faster approach to solvent extraction for the gravimetric quantitation of fat and oil. Typically, the Soxtec meth- ods require only 20–25% of the time required for traditional Soxhlet extraction. Sample preparation, general extraction procedures, method considerations, and opti- mization are addressed. By definition, the procedure to determine “crude fat” is an empirical method in which the result is determined by the conditions of the procedure. Several aspects of the extraction process, such as solvent type, time, and temperature, are explored. Several standardized Soxtec methods are discussed, including the recently approved AOAC method for determining crude fat in feeds, cereal grains, and forages. Many Soxtec applications are routinely used in food, feed, industrial, and environmental laboratories for the measurement of fats, oils, semivolatiles, and other solvent “extractables.” For the determination of crude fat, descriptions are given for various sample pretreatment and extraction procedures. Practical guidelines for han- dling challenging samples as well as general suggestions are presented. History The foundation of today’s automated solvent extraction systems can be traced to 1879 to a German chemist, Franz Von Soxhlet. He devised a liquid/solid extrac- tion apparatus in which a sample is placed in a cellulose thimble and stationed over boiling solvent. Condensed solvent would then drip into the sample, solubiliz- ing extractable material and then siphon back into the boiling solvent, where this cycle would then repeat. After several cycles over many hours, the apparatus is disassembled and the solvent, now containing extract (fat), is evaporated off, leav- ing the residue for further analysis. The Soxhlet procedure remains the most exhaustive extraction technique, and today it is still widely used. Over the years, there had been some improvements to the basic technique but the procedure remained long, tedious, and prone to variability. In the early 1970s, Edward Randall (1) developed an accelerated extraction technique that cut the extraction time to as little as 30 min. In the Randall method, the sample is lowered Copyright © 2004 AOCS Press and totally immersed in the boiling solvent. The simple principle is that the materi- al to be extracted, in this case, fats and waxes, is more soluble in hot solvent than in cold or room temperature solvent. His procedure included this new boiling step followed by a rinsing step to flush residual extract from the sample (Fig. 2.1). Demonstrating excellent agreement with Soxhlet and improved precision, the Randall method has become the basis of many automated extraction systems. In 1975, Tecator AB of Höganäs Sweden acquired the rights to what had become known as the “Randall modification” of the Soxhlet method. This was first commercialized as the RaFaTec and later became the Soxtec systems of today. Figure 2.2 is a photograph of the Soxtec TM Avanti, from Foss-Tecator, an automat- ed Soxhlet extraction system using the Randall submersion technique. Procedural Overview of Soxtec, Automated Soxhlet for Crude Fat Automated extraction methods using the Soxtec have gained widespread accep- tance and have a number of regulatory agency approvals worldwide. The Soxtec method has been used on literally hundreds of different sample types for many extracts. For our purposes, this discussion will be limited mainly to crude fat extraction. Crude fat by solvent extraction is classified as an empirical method (2). This means that the final result can be arrived at only according to the terms or variables of the method. It therefore becomes critical that all aspects of the proce- dure be followed strictly. Sample Preparation. Proper handling of the sample and attention to detail are extremely important parts of the analytical process. An improperly or sloppily pre- FIG. 2.1. Original Soxhlet (left) and Randall Extraction Apparatus. (a) Condenser (b) sample thimble (c) solvent flask (d) siphon tube (e) solvent vapor tube (f) thimble positioning mechanism (g) heater (not shown on the Soxhlet). In the original Randall method, the thim- ble is positioned by use of the slide rod (f). Lowering the thimble (b) into the boiling solvent for the boiling step, then raising it out of the solvent for the rinsing step. In both stages, condensed solvent is flowing continuously through the sample and thimble back into the boiling solvent. Copyright © 2004 AOCS Press pared sample can invalidate even the most carefully performed extraction proce- dure. Depending on the type and nature of the sample, the sample preparation may incorporate different procedures. For grinding and weighing, the sample should be homogenous and finely ground, usually to pass through a 1-mm sieve (~18 mesh). Particular attention should be paid to the type of grinding mill used. The mill or milling process should not contribute to any loss of moisture or fat from the sample. Samples should be weighed using a calibrated 4-place analytical balance and in most cases, can be weighed directly into the cellulose thimble. The weight of the sample is dependent on its approximate fat content. Table 2.1 can be used a guide- line. Because only a few grams of sample are normally used for the analysis, it is critical that this small sample be representative of the larger sample lot. Pretreatment Drying. Most samples should be predried to optimize the fat extraction. Water in the sample can decrease the efficiency of the solvent extraction, resulting in low fat recoveries. Conversely, water-soluble components in the samples such as urea, car- bohydrates, salts, and glycerol can be extracted with fat yielding falsely high recoveries. Samples are weighed into the extraction thimbles and are then typically dried at 102 ± 2°C for 1–2 h (3–5,7). Because the samples are weighed before drying, FIG. 2.2. The Soxtec TM Avanti 2050 automated extraction system. Copyright © 2004 AOCS Press the results are on an as-is basis. Results expressed on a dry matter basis must be calculated from a separate moisture determination. For very moist samples, sand may be mixed with the sample before drying. This prevents the sample from becoming caked during drying and improves the solvent flow for optimal extrac- tion (see below: Crude fat in meat and meat products). Hydrolysis. Samples that have been processed, cooked, or extruded often have fat that is bound to proteins, carbohydrates, and/or minerals, making it unavailable for solubilization. Acid hydrolysis, in which a sample is boiled with hydrochloric acid, breaks these bonds, allowing the fat to be solvent extracted (see below: Total fat). Water Rinse. Samples that contain a large amount of water-soluble components may exhibit poor solvent extraction efficiency. A preextraction with water, fol- lowed by a thorough drying step can be used to obtain an acceptable recovery by removing these water-soluble components. The procedure specifies washing the weighed sample with 5 aliquots of 20 mL of deionized water. The sample is dried and the extraction procedure is carried out as usual. (3,4) Solvent Extraction Once the samples have been properly prepared and pretreated, they can now be placed in the Soxtec for fat extraction. The weights of clean and dry extraction cups must also be obtained for later use in the final calculation. The samples and extraction cups are then positioned in the extractor. The solvent is added through a closed-loop addition process and the extraction begins. The steps of boiling, rins- ing, and evaporation/solvent recovery then proceed in an automated manner. At the end of the cycle, an alarm signals completion. Boiling. In this step, the sample and thimble are lowered and totally immersed in the boiling solvent contained in the extraction cup. The solvent vapor refluxes against a water-jacketed cooling column and the condensed solvent flows back continuously through the sample returning to the boiling solvent (Fig. 2.3). The boiling step is the key to accelerating the extraction process compared with the Soxhlet method. The solvent simply solubilizes the extract faster in hot solvent, thus decreasing the time required for extraction. TABLE 2.1 Expected Fat Content and Sample Weights Fat content (%) Sample weight (g) 0–10 2–3 10–25 1–2 >25 0.5–1 Copyright © 2004 AOCS Press To ensure optimal extraction, the level of boiling solvent must be higher than the sample in the thimble. A plug of defatted cotton is frequently placed on top of the sample to keep it in the thimble during extraction. With the Soxtec Avanti sys- tem, 70–90 mL of solvent is used. Typical extraction times range from 20 to 40 min depending on the solvent and sample characteristics. Immediately after the boiling step, the rinsing step begins. The sample is raised and suspended over the boiling solvent. During rinsing, residual traces of the extractable material are flushed out of the sample and are retained in the extraction cup. This step is usually 10–20 min longer than the boiling step to ensure complete extraction. The last step in the crude fat extraction process is evaporation/solvent recovery. The condensed solvent continues to boil and evaporate and, using an internal valve, the condensate is redirected out of the condenser. The evaporation step is complete when all solvent is driven from the cup, concentrating the extract. This usually requires 7–10 min depending on the solvent. Excessive drying may oxidize the extract, causing weight changes and erroneous readings. The Soxtec Avanti stores the evaporated solvent in a common collection tank for reuse. The Soxtec Avanti offers an optional fourth, cup predrying step, i.e., the extraction cups are raised a few millimeters off the heating surface allowing radiant heat to complete the drying cycle. This step is used in applications in which the extract is extremely heat labile. Postextraction. Once the extraction process is completed, the cups are taken off the Soxtec and placed in a drying oven at 103°C for 30 min to drive off any mois- ture or solvent residuals. Extended drying, especially at higher temperatures, should be avoided because it can cause oxidation of the fat extract and falsely high results. Extraction cups are cooled completely to room temperature in a desiccator before final weights are taken. FIG. 2.3. Three-step extraction procedure. The Foss-Tecator Soxtec Avanti automated extraction system is based on the Randall modification of the Soxhlet technique. In the boil- ing and rinsing steps, solvent is refluxed within the condenser. During evaporation, solvent flow is blocked from returning to the extraction cup and flows out tube (a) into a collection tank (not shown). Copyright © 2004 AOCS Press Calculation of Results. Crude fat by solvent extraction is a gravimetric method. The final result is calculated from the original sample weight and the weights of the extraction cup before and after the extraction. % Fat = (W 2 – W 1 )/W 3 × 100 where W 1 = weight of the extraction cup W 2 = weight of the extraction cup + extract W 3 = weight of the sample All weights should be recorded to 0.1 mg (0.0001g). Optimizing the Extraction Process The Soxtec/Soxhlet extraction method for crude fat relies on separating sample components on the basis of physical and chemical (solubility) properties. There are several factors that influence the extraction; the most significant of these is the spe- cific solvent that is being used. Nevertheless, the influence of sample preparation, extraction timing, and general Soxtec operating conditions is important. Keeping in mind the empirical nature of the analysis, consistency with all aspects of the proce- dure is strongly recommended. An often overlooked aspect of a fat extraction method is the predrying of the sample. As mentioned earlier, water in the sample can contribute to error in two ways, i.e., it can act as a physical barrier preventing dissolution of the fat into the solvent, thus generating low fat recoveries; it can also contribute to falsely high apparent fat recoveries by allowing water-soluble compo- nents such as urea or carbohydrates to be co-extracted with the fat. Unfortunately, in the interest of time and productivity, many laboratories do not predry samples. In these instances, the error potential for each type of sample should be investigat- ed fully by carrying out extractions both with and without predrying. Figure 2.4 illustrates apparent fat recovery on dried vs. undried samples. Moisture in the samples ranges from 5 to 25%. Some samples show a “water effect” more than others. Samples such as the texturized feeds, which contain molasses, and the feedlot concentrate, which contains urea, are examples in which failing to predry the sample can have a marked effect on recovery. Note that this is also dependent on the solvent used. [%Recovery is defined as (%crude fat from the undried sample/% crude fat from the dried sample) × 100]. Solvent Choices. The versatility of the Soxhlet/Soxtec extraction method allows for the use of various classes of organic solvents. These include ethers, aliphatic, aromatic, and chlorinated hydrocarbons, as well as alcohols. Due to the different solubility characteristics of various solvents, a sample extraction will have some- what different fat yields depending on the solvent. For crude fat extractions, diethyl ether and petroleum ether are most commonly used. The peroxide-forming Copyright © 2004 AOCS Press nature of diethyl ether causes it to be a less than desirable choice for routine use in the laboratory. This has caused many laboratories to look for an alternative solvent. Commonly, petroleum ether is directly substituted. However, petroleum ethers or ligroin, are not true ethers but mixtures of aliphatic hydrocarbons and can be pur- chased in various formulations and boiling point ranges. Further complicating the “pet ether” issue is that solvents are often recycled and reused in the Soxtec. This can cause a change in the properties of petroleum ether because its more volatile components may be driven off. This can cause a change or drift in the fat results. Considering the innate variability of petroleum ether and the relative lower recovery of plant-based lipids, it is not a suitable substitution for diethyl ether. An experiment was undertaken (3,4) to compare the recovery of three common sol- vents, petroleum ether, hexanes, and pentane, to that of diethyl ether in terms of crude fat recovery. The objective was to find a solvent that is safer and has recov- ery statistically equivalent to diethyl ether. The results are shown in Table 2.2. From these results, it can be seen that hexanes yield a recovery equivalent to that of diethyl ether with an R 2 of 0.9925. It should also be noted that for meat and bone meal, petroleum ether also yields a good recovery. This is consistent with the use of petroleum ether in the AOAC method 991.36 (7) for crude fat in meat and meat products. Extraction Times. In Soxtec extraction procedures, the timing for the boiling and the rinsing steps is important. If the boiling or rinsing step is too short, the extrac- tion will likely not adequately recover the fat in the sample. Most method develop- ment protocols will define the extraction times at which the results closely match FIG. 2.4. Apparent fat recovery from dried vs. undried samples with moisture content ranging from 5 to 25%. Dehydrated Alfalfa Corn Silage Mixed Bird Seed Texturized Feed (Molasses) Fat Supplement Medicated Goat Feed Feedlot Conc. (Urea) Calf Starter, Medicated Calf Feed, Medicated Meat Meal/Hulls Mixture Swine Feed Broiler Starter High Oil Corn Diethyl ether Hexanes % Recovery Undried vs. Dried Copyright © 2004 AOCS Press those obtained by classical Soxhlet methods using suitable reference materials. The automation of the Soxtec offers consistent extraction timing for each batch of sam- ples. Extraction Temperature. The temperature of the extraction system should be set to the recommendations provided by the manufacturer. This helps ensure optimal condensation or reflex rates. Ideally, this is typically 3–5 drops/s coming off the condenser. Condenser Temperature. The temperature of the condenser cooling water plays an important role in establishing the condensation or reflux rate of the solvent. Cold tap water, <20°C at 2 L/min, should be used so that the condensers feel cool to the touch. If the water temperature is too warm, it will usually cause slow reflux rates and can result in low fat recovery. In some cases, warm condensers can cause the loss of solvent during the boiling and rinsing stages. In areas in which cold tap water is seasonal or not obtainable or in which water conservation is an issue, refrigerated circulating water baths are a convenient way to regulate the tempera- ture and flow. Robustness of a Method. Using the statistical model described by Youden (8) the “ruggedness” of the extraction method was evaluated. The purpose of this exercise was to determine whether the method can tolerate minor variations in the proce- dure that might be encountered in the laboratory. In this model, several variables or factors are identified and are selectively used in a defined protocol or matrix. This is described in Table 2.3. For the extrac- tion of crude fat, sample handling, weight, extraction parameters, and solvent types were defined as variables. For example, sample weight of 1 g is described as vari- able “E” and 3 g as “e.” Using the schedule in Table 2.3, and implementing the variables in this manner allows the evaluation of the ruggedness of the method while minimizing the number of determinations. TABLE 2.2 Crude Fat Recovery of Four Common Solvents Diethyl ether Petroleum ether Hexanes Pentane Sample (% Crude fat) Alfalfa hay 1.29 1.00 1.36 0.97 Beet pulp 0.30 0.24 0.25 0.19 Meat/bone meal 10.52 10.40 10.69 10.54 Cattle protein supplement 3.10 2.65 2.88 2.54 Corn 3.59 3.06 3.16 3.00 Average 3.76 3.47 3.67 3.45 R 2 — 0.9878 0.9925 0.9880 Copyright © 2004 AOCS Press The results from three different samples, cattle, swine, and mixed feeds, from three different laboratories are seen in Table 2.4. Values for the individual factors are averages from all results in which this variable was used. Considering the aver- ages of the differences, the most significant variable is the choice of solvent. Other variables in the method do not contribute to a significant variation, thus making the method robust. Common Applications Crude Fat in Meat and Meat Products: AOAC Method 991.36. A 2-g sample of homogenized meat is mixed with acid-washed sand and dried at 125°C for 1 h. The sand is added directly to the thimble at approximately double the sample weight. A glass stir rod is used to thoroughly mix the sample and sand together. The glass rod is left in the sample during drying and subsequently used to break up any sample/sand clumps before extraction. The sand is used to maintain porosity of the sample after drying for optimal solvent penetration. A petroleum ether Soxtec extraction is then performed with 25-min boil and 35-min rinse periods. Table 2.5 compares recovery and repeatability of classical Soxhlet to Soxtec (6) showing that the Soxtec offers bet- ter precision with the same results as the classical Soxhlet method. Crude Fat in Feed, Cereal Grains, and Forages: AOAC Methods 2003.05 and 2003.06. A 1- to 5-g ground sample is weighed into an extraction thimble. If the sample contains quantities of water-soluble components such as >5% carbohy- drates, >15% glycerol, lactic acid, or amino salts, or >10% of other water-soluble TABLE 2.3 Variables Used to Determine Method Robustness Combination or determination number Factor value 1 2 3 4 5 6 7 8 A or a A A A A a a a a B or b B B b b B B b b C or c C c C c C c C c D or d D D d d d d D D E or e E e E e e E e E F or f F f f F F f f F G or g G g g G g G G g Observed result S T U V W X Y Z The chosen variables (factors): A Sample predry 103°C, 2 h a Sample predry 103°C, 4 h B Boil time, 20 min b Boil time, 40 min C Diethyl ether c Petroleum ether D Rinse time, 30 min d Rinse time, 60 min E Sample weight, 1 g e Sample weight, 3 g F Cup dry, 103°C, 2 h f Cup dry, 103°C, 4 h G Solvent drip rate, 2/s g Solvent drip rate, 6/s Copyright © 2004 AOCS Press components, the sample is washed with 5 aliquots of 20 mL deionized water. The sample is dried at 102°C for 2 h. Extraction is performed with either diethyl ether or hexanes using a 20-min boil and 40-min rinse cycle. The Soxtec method was compared with the AOAC Soxhlet method on data from 90 AAFCO check sam- ples. Regression analysis generated an R 2 correlation coefficient of 0.9946, slope TABLE 2.4 Evaluating the Robustness of the Crude Fat Method Using Cattle, Swine, and Mixed Feeds from Three Different Laboratories Laboratory 1 Laboratory 2 Laboratory 3 (% crude fat) Feed type Cattle Swine Mixed Cattle Swine Mixed Cattle Swine Mixed Average S 11.61 2.45 10.50 11.82 2.41 11.16 11.99 2.99 10.97 T 11.30 2.10 10.24 11.63 2.35 10.25 11.55 2.49 10.64 U 11.79 2.46 10.60 11.98 2.81 10.84 11.84 2.93 11.05 V 11.02 2.17 9.88 11.76 2.70 10.78 11.57 2.46 10.50 W 10.87 2.60 10.67 11.61 2.52 9.86 11.87 4.13 10.94 X 11.11 2.00 10.00 11.28 2.10 10.28 11.24 2.38 10.43 Y 11.62 2.55 10.60 11.49 2.64 10.53 11.94 2.82 10.91 Z 10.73 1.91 9.82 11.19 2.10 10.01 11.74 2.51 10.73 Predry 2 h 11.43 2.29 10.30 11.80 2.57 10.76 11.74 2.72 10.79 4 h 11.08 2.26 10.28 11.39 2.34 10.17 11.70 2.96 10.75 Difference 0.34 0.03 0.03 0.41 0.23 0.59 0.04 –0.24 0.04 0.16 Boil time 20 min 11.22 2.29 10.35 11.59 2.35 10.39 11.66 3.00 10.75 40 min 11.29 2.27 10.23 11.61 2.56 10.54 11.77 2.68 10.80 Difference –0.06 0.02 0.13 –0.02 –0.22 –0.15 –0.11 0.32 –0.05 –0.02 Ether Diethyl 11.47 2.52 10.59 11.73 2.60 10.60 11.91 3.22 10.97 Petroleum 11.04 2.04 9.99 11.47 2.31 10.33 11.53 2.46 10.58 Difference 0.43 0.47 0.61 0.26 0.28 0.27 0.38 0.76 0.39 0.43 Rinse time 30 min 11.32 2.25 10.29 11.53 2.38 10.49 11.81 2.70 10.81 60 min 11.20 2.31 10.29 11.66 2.53 10.44 11.63 2.98 10.73 Difference 0.12 –0.06 0.00 –0.13 –0.16 0.05 0.17 –0.27 0.08 –0.02 Sample wt 1 g 11.31 2.20 10.23 11.57 2.36 10.57 11.70 2.70 10.80 3 g 11.20 2.35 10.35 11.62 2.55 10.36 11.73 2.98 10.75 Difference 0.11 –0.15 –0.12 –0.06 –0.20 0.22 –0.03 –0.27 0.05 –0.05 Cup dry 30 m 11.06 2.28 10.22 11.60 2.43 10.45 11.79 3.02 10.79 2 hr 11.46 2.28 10.36 11.60 2.48 10.48 11.64 2.66 10.76 Difference –0.40 0.01 –0.14 0.00 –0.04 –0.02 0.15 0.37 0.03 0.00 Drop rate 2/s 11.34 2.29 10.24 11.59 2.46 10.69 11.69 2.66 10.70 6/s 11.17 2.27 10.33 11.60 2.45 10.24 11.75 3.02 10.84 Difference 0.17 0.02 –0.09 –0.01 0.02 0.45 –0.06 –0.35 –0.14 0.00 Copyright © 2004 AOCS Press [...]...TABLE 2. 5 Average Recoveries and Relative Standard Deviations (RSD) for Meat Samples with Petroleum Ether Extraction Average recovery RSDr Average recovery Soxhleta Meat sample 1 2 3 4 5 6 Average RSDr Soxtecb (%) 4.6 28 .35 28 .21 34.98 34.10 26 .81 26 .18 2. 63 8.75 5. 52 2.09 2. 25 1.74 3.83 4.34 27 .29 27 .95 34.51 33.57 26 .20 25 .64 2. 44 1.95 2. 32 2 .21 1.01 1.55 1.91 aSoxhlet: bSoxtec: 4-h extraction, 2- h... in soil Extraction of resins from paper pulp Extraction of finish from textiles Extraction of starch containing finish from textiles Extraction of surfactant from detergents Extraction of paraffin from detergent Extractable matter in leather Extraction of petroleum source rock Extraction of explosives and propellants Extraction of plastics and polymers Extraction of rubber and rubber compounds Extraction. .. extraction References 1 Randall, E.L (1974) Improved Method for Fat and Oil Analysis by a New Process of Extraction, JAOAC 57: 1165–1168 2 Codex Alimentarius Commission (1986) Procedural Manual, 6th edn., p 139, Food and Agricultural Organization, Rome, Italy 3 Thiex, N., Anderson, S., and Gildemeister, B (20 03) Crude Fat, Hexanes Extraction, in Feed, Cereal Grain, & Forage (Randall/Soxtec/Submersion... Extraction of finish oils from textiles and synthetic fibers Extraction of migration components in plastic packaging Extraction of organic dyestuffs Extraction of leather Extraction of core material in petroleum exploration Extraction of tobacco Extraction of solubles in paper pulp Extraction of fecal fat sub notes (ASN) are available from Foss Tecator Copyright © 20 04 AOCS Press TABLE 2. 7 Common Approaches... 2. 6 Application Sub Notes (ASN) are available from Foss Tecator Copyright © 20 04 AOCS Press FIG 2. 5 The 20 47 SoxCapTM hydrolysis system TABLE 2. 6 Summary of Soxtec Extraction Methods Used in Industrial Applicationsa ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN aApplication 3516 36 02 3603 3604 3605 3606 3607 3608 3611 36 12 3613 3614 3615 3616 3617 3618 3619 3 622 3700 Extraction. .. added together Sample becomes impacted during extraction • Mix equal volumes of acid-washed sand or Celite and sample in the extraction thimble This allows for better solvent flow through the sample Solvent overboiling • Use 3–5 boiling beads in the extraction cups • Decrease the temperature setting on the extractor Moist samples • Mix sample with sand and predry • Mix sample with equal weight of sodium... same extraction procedure and sample protocol Conclusion The long history of solvent extraction has led to automated Soxhlet extraction systems, such as the SoxtecTM Avanti They offer convenient and useful tools with which to improve productivity in the laboratory Modern instrumentation provides application flexibility and improved economy as well as enhancement of the precision and recovery of the extraction. .. containing the sample into a preweighed extraction cup Any sample that melts out of the thimble will be retained in the cup Incomplete fat recovery, high-fat seeds • A two-phase extraction protocol is used: After the first extraction boil and rinse, the samples are removed from the thimbles, ground with a mortar and pestle and returned to the thimble for a secondary extraction The results are added together... Collaborative Study, JAOAC Int 86: 888–898 4 Thiex, N., Anderson, S., and Gildemeister, B (20 03) Crude Fat, Diethyl Ether Extraction, in Feed, Cereal Grain, & Forage (Randall/ Soxtec/Submersion Method): A Collaborative Study, JAOAC Int 86: 899–908 5 Official Methods of Analysis of AOAC International, 16th ed., Chapter 4, p 25 , section 4.5.01 AOAC Official Method 920 .39, Fat (Crude) or Ether Extract in Animal... M.L., and Gonzales, S.E (19 92) Soxtec Fat Analyzer for Determination of Total Fat in Meat: Collaborative Study, Kansas State Board of Agriculture, JAOAC Int 75: 28 8 29 2 7 Official Methods of Analysis of AOAC International, 16th ed., Chapter 39, p 3, section 39.1.08 AOAC Official Method 991.36, Fat (Crude) in Meats and Meat Products, AOAC International, Gaithersburg, MD, 1997 8 Youden, W.J., and Steiner, . (%) 1 4.6 2. 63 4.34 2. 44 2 28.35 8.75 27 .29 1.95 3 28 .21 5. 52 27.95 2. 32 4 34.98 2. 09 34.51 2. 21 5 34.10 2. 25 33.57 1.01 6 26 .81 1.74 26 .20 1.55 Average 26 .18 3.83 25 .64 1.91 a Soxhlet: 4-h extraction, . 11 .28 2. 10 10 .28 11 .24 2. 38 10.43 Y 11. 62 2.55 10.60 11.49 2. 64 10.53 11.94 2. 82 10.91 Z 10.73 1.91 9. 82 11.19 2. 10 10.01 11.74 2. 51 10.73 Predry 2 h 11.43 2. 29 10.30 11.80 2. 57 10.76 11.74 2. 72. 2. 26 10 .28 11.39 2. 34 10.17 11.70 2. 96 10.75 Difference 0.34 0.03 0.03 0.41 0 .23 0.59 0.04 –0 .24 0.04 0.16 Boil time 20 min 11 .22 2. 29 10.35 11.59 2. 35 10.39 11.66 3.00 10.75 40 min 11 .29 2. 27