A. Prin Princi ci ple
4.5.05 AOAC Of ficial ficial Method 2003.05 Crude Fat in Feeds, Cereal Ce real Grains, and For ages ages Randall/Soxtec/Diethylether Extrac Extraction-Sub tion-Submer mer sion sion Method First Action Action 2003 Final Action 2006
[Ap pli plica ca ble to the analy anal ysis of for ages, ages, cereal cereal grains, and ani animal feeds other than baked or ex panded prod products, ucts, dried milk or milk products, prod ucts, fishmeal, fishmeal, or oilseeds oilseeds at concen concentra trations tions from 0.5 to 100% fat. It is ap pli plica ca ble to the same matrixes matrixes as 920.39 ( see see 4.5.01) and 930.09 ( see 3.5.07).]
Caution Cau tion:: Store so solvents in metal contain containers ers in solvent solvent cabinet or solvent sol vent room that con forms to ap pli plica cable safety legis leg isla lation. tion. Ethers are extremely ex tremely flamma flamma ble. Have no open flames in the lab ora ratory tory where the analy analysis is being be ing per formed. formed. Avoid inhal inhaling ing va pors. Use solvents solvents in a properly properly oper oper at ating hood equipped with ex plo plosion-proof sion-proof lighting, lighting, wir ing, ing, and fan. Diethylether Diethylether has the potential to form sho ck-sen ck-sensi sitive, tive, ex plo plosive sive per oxides oxides with age. Check each n ew container of ether for peroxides when it is opened. Also check partial containers of ether that have not been used for several months before using them again. Do again. Do not use not use ether that contains peroxides. Dispose as hazardous material. Stabilized ether may be used. Ground electri electrical cal equipment equip ment and maintain maintain in proper work ing ing or der. der. Follow Fol low manu manufac facturer turer recom rec ommen menda dations tions for instal installa lation, tion, oper op er ation, and safety of all extrac extraction tion equipment. Make sure all solvent is evaporated from cups before be fore placing placing them in the oven to avoid a fire or ex plosion.
See Ta ble 2003.05 for results results of the interlaboratory study sup port porting ing accep acceptance tance of the method.
Table 2003.05. Table method
The Randall modi modifi fica cation tion of the standard standard Soxhlet Soxhlet extrac extraction tion submerges sub merges the test portion in boiling solvent, re reduc ducing ing the time needed for extrac extraction. The sol solvent vent dissolves dissolves fats, oils, pigments, pigments, and other solu solu ble substances, substances, collec collectively tively termed “crude fat.” A dried, ground test por tion tion is ex tracted by a 2-step pro cess: cess: In the first step, the thim ble con contain taining ing the test por tion tion is immersed immersed into the boiling boil ing solvent. solvent. The inter inter mixing mixing of matrix matrix with hot solvent solvent ensures ensures rapid solubilization of extractables. The thim ble is then raised above the solvent solvent and the test por tion is fur ther ther ex tracted tracted by a con tinu tinuous flow of condensed con densed solvent. solvent. The solvent solvent is evaporated and recov re covered ered by con conden densa sation. tion. The result resulting ing crude fat resi residue is deter deter mined mined gravimetrically af ter ter drying. drying. The solu solu bil bility ity char acter acter istics istics of dif fer fer ent ent solvents solvents may result result in slight dif fer fer ences ences in crude fat results. For this reason, rea son, the report should reflect reflect the solvent solvent used. Exam ple: % Crude Fat, Ether Extrac Ex traction. tion. B. Ap pa para ratus tus
(a) Sol vent vent extrac extraction tion system. system. —Multi —Multi ple ple posi position tion extrac extraction tion unit con ducting conduct ing 2-stage Randall extrac ex traction pro cess with sol vent ™ recov re covery ery cycle, cycle, with Viton or Tef lon lon seals com pat patii ble ble with ether. (b) Thim Thimbles bles and stand. —Cel —Cellu lulose lose thim bles and stand to hold thim bles. ( c ) Ex trac traction tion cups. —A —A lu mi num or glass. (Extrac (Ex traction tion tem per ature settings settings may dif fer; fer; consult consult manu manufac facturer’s turer’s oper oper ating ating instruc in structions.) tions.) Items (a)–(c) are available available as Soxtec systems systems from Foss or other Randall-type extrac extraction tion systems. systems. C. Reagents Reagents
(a) An Anhy hydrous drous diethylether diethylether .—Pu .—Puri rified fied for fat extrac extraction, tion, Fisher E492 la beled “For Fat Extrac Ex traction” tion” is also sta bi bilized, lized, or E134-4, or equivaalent. To pre equiv p revent vent ether from absorb ab sorbing ing water, water, pur chase it in small contain containers ers and keep contain containers ers tightly closed. Pe troleum ether cannot can not be substi substituted tuted for diethylether because be cause it does not dissolve dissolve all of the plant lipid mate material. rial.
Interlaboratory re results sults for crude fat in ani an imal feed, cereal cereal grain, and for age, age, diethylether diethylether extrac extraction tion (submer (submer sion) sion)
Mate Ma terrial
Mean
No. of labs
Dehy De hydrated drated alf alfalfa
4.44
10
Corn sil silage
2.09
Mixed bird seed
7.19
Texturized feed
3.20
Fat supple supplem ment
96.38
Mediicated goat feed Med
a
sr
RSDr , %
sR
0.21
4.62
0.23
5.1
0.02
1.02
0.06
3.06
0.19
2.64
0.23
3.17
0.1
3.22
0.22
6.77
10
4.82
5.00
4.82
5.00
1.89
10
0.04
2.09
1.08
4.5
Feedlot Feed lot concen concentrate trate pell pellets
1.53
10
0.14
9.26
0.32
21.0
Cellu Cel lullose (blank)
0.17
10
0.05
0.08
48.3
Calf starter medi medicated
2.96
8(2)
0.05
1.58
0.06
1.9
Calf feed medi medicated
3.42
9(1)
0.05
1.44
0.12
3.50
Meat meal/hulls mix
4.99
10
0.22
4.33
10.55
11.08
Swine feed
2.77
10
0.07
2.61
0.15
5.5
Broiler starter
7.05
10
0.1
1.35
0.16
2.33
High oil corn
7.69
10
0.1
1.31
0.22
2.93
a
9(1) 10 9(1)
26.1
RSDR, %
Number Num ber of labo labora rato tories ries retained retained af ter ter elimi eliminat nating ing the number number of labo labora rato tories ries in paren parenthe theses. ses.
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2006 AOAC INTER IN TERNA NATIONAL TIONAL
( b ) Co t to n.—Defatted. Soak medical grade cotton in diethylether or hexanes for 24 h, agitating several times dur ing this period. Re move and air dry. (c ) Sand . —Ashed (for igni tion boats). EM SX0075-3, or equivalent (CAS 14808-60-7). (d) Celite 545.
Place defatted (with same solvent to be used for extrac tion) cotton plug on top of test portion to keep material immersed dur ing the boiling step and prevent any loss of test por tion from top of thim ble. Pre pare cotton plug large enough to hold materials in place, yet as small as possi ble to minimize absorption of solvent. Adding the cotton plug before the 102° ± 2°C, 2 h drying step is acceptable.
D. Preparation of Analyt i cal Sam ple
Place three or four 5 mm glass boiling beads into each cup, and dry cups for at least 30 min at 102° ± 2°C. Transfer to desiccator and cool to room tem per ature. Weigh extraction cups and re cord weight to near est 0.1 mg (T).
Reduce particle size of samples to fineness of 0.75–1 mm. E. Deter mi nation
Weigh 1–5 g test por tions con taining ca 100–200 mg fat directly into tared cellulose thim bles, according to following scheme: Crude fat, %
Test portion weight, g
<2
5
5
2–4
10
1–2
>20
1
Record weight to near est 0.1 mg (S) and thim ble num ber. Note: If test sam ple contains large amounts of urea salts (>5%) or sol u ble car bohydrates (>15%), glyc erol, lactic acid, amino acid salts (>10%) or other water-solu ble com ponents, remove by water extraction. Weigh test por tion onto fil ter pa per, extract with five 20 mL por tions of water, allowing each por tion to drain. Place filter pa per containing washed test por tion into thim ble and dry at 102° ± 2°C for 2 h. To facilitate filtration, add 1–2 g ashed, acid-washed sand, or Celite to bot tom of filter or mix in with test por tion before water extraction. Dry thim bles containing test portions at 102° ± 2°C for 2 h. If dried test por tions will not be extracted immediately, store in desiccator. Both solvent and test materials must be free of moisture to avoid extraction of water-solu bl e co m ponents such as car bohydrates, urea, lactic acid, and glycerol, which will result in false high values. An absor bent, such as diatomaceous earth (Celite or Super-Cel), can be added to the test por tion when high fat materials, which melt through the thim ble dur ing the predry step, are present. Alter natively, defatted cotton can be added before the predry step to absorb the melted fat. If the material melts at 102°C, place a pretared extraction cup under the thim ble during the drying step to catch any melted fat that was unabsorbed and escaped the thim ble.
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2006 AOAC INTERNATIONAL
Extract, following manufacturer’s instructions for oper ation of extractor. Preheat extractor and turn on condenser cooling water. Attach thim bles containing dried test por t ions to extraction columns. Put suf ficient amount of solvent into each extraction cup to cover test p ortion when thimbles are in boiling position. Place cups under extraction columns and secure in place. Make sure that cups are matched to their cor responding thim ble. Lower thim bles into solvent and b oil for 20 min. Verify proper re flux rate which is critical to the com plete extraction of fat. This rate de pends upon the equipment and should be sup plied by the manufacturer. A reflux rate of ca 3–5 drops/s applies to many extraction sy stems. Raise thim bles out of solvent and extract in this position for 40 min. Then evap orate as much sol vent as possi ble from cups to reclaim solvent and attain ap par ent dryness. Remove extraction cups from extractor and place in oper ating fume hood to finish evaporating solvent at low tem per ature. ( Note: Take care not to pick up any de bris on bottom of extraction cup while in hood. Let cups remain in hood until all traces of solvent are gone.) Dry extraction cups in a 102° ± 2°C oven for 30 min to remove moisture. Excessive drying may oxidize fat and give high results. Cool in desiccator to room tem per ature and weigh to near est 0.1 mg (F). F. Cal cu lations
% Crude fat, diethylether extract =
F
-
S
T
´
100
where F = weight of cup + fat residue, g; T = weight of empty cup, g; S = test por tion weight, g. Ref er ences: J. AOAC Int. 86, 888(2003); 899(2003).
