![Moisture Content AOAC 984.20 PDF [PDF]](https://pdfs.asia/img/200x200/moisture-content-aoac-98420-pdf.jpg)
12 0 6 MB
418 0ilS and Fats David Firestone and Mattn Peter Yuraw● z,Cttrer帥 us,F00J and prIJg Ad何碑おfrari19,
俺
41.1.03
41.1.01
AOAC Omcla‖ MoLture 3■
AOAC Omcta,Method 031.11 C)ils and Fats
Method 984.20 (D:33 and Fa皓
Ka"F3scheF MethOd First Actlon 1984 Final Adon 1988
Preparndon tt Test Sampte Procedure F3再 R Adttn 1381
_AcAC mor rsc/rcmc,f― Utt clear sedimntetee liquid― ple diFeCdy after inverting the container several血 .rliquid smple∞ n述 ぉ 貿慮 ment,reLase ドα appliCable tt alkdine or oxidzed dis and fats.) ali sedment fron wal13 0f COntaittr and dstribute unifomy e m a t t e F . 阻 o n o f 航 s 価, a n d v o l 調 t h r o u g h o u t d l e c i l f o r輸 抽蔵 ed by possible F o r d e m 述皿 ぃ h w h i c h F e S u l t t n i g h t にbcet痢 A,APParatws anJ Reagm体 ・ S m p l e a s f ollowtt Addan― P I e S C I I C e o f H 2 0 g●" 1 0 d i n e v t t u e j , 的 h y d r o u s N a 2 S 0 4 i n 甲Ⅳd o n o f l - 2 g p e r 1 0 g s m p l e , a n d h o l d h o拓 arr F泣 れを″r″r4rわ"徳 s抑 冴み_Manual or automated, oven at 50° C,Sdr vlgorously and fllter to obtain clear nlmte,PFe― with stttr.
r 絶 ガ F 持がた, r F a g α W S O l i d ― p l e f o r m t t O n o f m t t s m e a n d v o l a t i l e m a t t e ●) 止―べ 術 l i z e d , w i d l H 2 0 e q u i v a l e n t aS diFeCd in 92t12律β41.1.磁),For oher deteminations,Inelt CaS mg H20/mLreagent.Attilablecomly orprepm asf abovemp.If t e s t s u l e i n d F y i n g O v e n a t a t e m p e r a m a t l e a s t 1C0 ° lowst Dissolve 133 g 12 in 425 ■ lL dry pyridine in dry 血 脚田 部 I d i r d y . r m i d o r c O n t a i n s 申 山 距 L 蝕 t e r t e s t s a n ‐ bottie. Add 425 mL dry ethylene glycol m t i c n s w t t c h t t g h t b e a r e c t e d glass,stopperod by dle P l e i n s d e o v e n ` F o r di ― . g " i t t n e v a l u e j a , t 陀 p o r t i o n o s f t に s m p l e 、 mGl10Elethyle鴎 CooltoK4°Cinicebahandbubblein 102-105g wsence ofmols8● 距 C0 °a b o v e m p , a d d i n g l - 2 g m y d r O uS 0s 2 ' M i X W d l a n d l e t s t a n d 1 2 h . R e a g e n t i s r e a s o n,abbulty s t a b i n t t t i n g o v e nC ,actF5 01°
N%S04 per iO g sanple,Mix test smple thorou抑呼 and fllter h 距i赴け,keep dもandfatsincool place and arying OVen.Tc ttaFd醐 Protect fFOn ttght and血
restandardize for each s9mes of detettnations.Scandardize&遠ly widlsodiulntaHInte・ 2H20・ l mgsodiuntamte・
2H20‐ 0｀ lS66mg
H20・AStematively,standardize widl weighed H20 1■medlyl alcc‐ holasfo1lowtt TransferaccwatelywttaH10unt(50 mg)H20的
Refennce:JAOACa,429(1981).
timtiOn vessel and ttrate to el∝ M皿 週由c end Point・ Calculaに C= ng H20/d reagenほ
41.1.02
0&ガ CⅢ l).
AOAC O御 ctal Method 026.12 Mb3sture and Voindl● Mater in 0383 and Fa低 Vatuum Oven― od F3Rtt Actlon 1926
Fおcttr rcagttr Jil″ を,■_2 Medloxyethancl― pyttdne
"_,Anhydrous CHC13 mettl dcchol(1+1 (d)挽 “ 姥 MJツタ or 2+1). a De―
日nat Atton
rnarOn
Weigh,to nearest O,01g,5-25 g prepared test sample,contain‐ sOm鵬 叫 れ rne_,wgende監 wm sonencun曲 ぬ oFOumy曲 Wagh5士
. 4は 判 巨Cat nOt崎確 lt孟 胡 戴 ve田 諭 航 d述 組 .
住2gpFepndtestsmpleintoAlmoismedshca5cm
diameter and 2 cm tt widl dght― flt dぃ ver COVer.Dry to con, stantwelghtin vacuumovenatunifomtempemme 2い
25°C above
b P O f H 2 0 ■的 由 略 フにs s u r e , w h i c h s h o u l d 1b0c0≦n l m H g (13.3 kPaj.Coolinefflcientdesiccator30min andwetht COnstant weight is amined"“en su韓 韓iVe l h dryl■ 8 pe的赴 軌。wな m‐ t i o n a l l o s s 0o5f%凱, R e p o ■ 坊l o s s h w e i g h t a s 時 m dasn■ d vola‐ dle matter. Refemncest五減 駒 ど.Cttn,1軌
ing玉 100 mg H20,into titration vessel,dissolve in anhydrous CHC13 medlyl alcchol.Titrate with undiluted or diluted(lⅢ l) Karl FischeF reagent to electronetric end Pcint・ CaHy cut blank test using same alnounts ofrengent,thiuent,and solvents.Subtract blank titer.
戦α % = 辮
を「Pyridinebfree Karl FischerFeagents are avttlable frOm iabt 仰θ oratory reagett suppliers.)
1347(1926).
泌9AC 14 247(1931);lS,560K1932).
ReFerence:JAOAC 67,299(1984j.
0 2002 AOAC tNTERNATtONAL
AOAC OFFiCiAL METHODS OF ANttYSS(2000)
01LS AND FAT Chapter 41,p.3
T a b i e 9 8 5 . 1 9 D(egnr3m1Oり 守 f H20 at dmbrent
41.1.07
temperature3(°C)
Tempera‐ ture
AOAC Offtc,a:Method o21.08 index of Refmct:on of Oits and Fats
Tempera‐ 0.99913
16
0。 99897
17
0.99880
18
0。 99862
19
0.99843
20
0.99823
21
0.99802
22
0。 99780
23
0.99757
24
0.99733
25
0.99707
26
0。 99681
27
0,99654
28
0.99626
29
0,99597
30
0.99568
31
0。 99537
２ ︲ ２ ６ ９ ３ 範 勢 賛 ３ 研 範 ３ 範 ４ ４ 範 招 福 範 好 組
15
0,99505
40
0。 98852
0.99473
50
0。 98807
0.99440
51
0.99406
52
0.98715
0。99371
53
0.98669
0,99336
54
0.98621
0,99299
55
0,98573
0。 99262
56
0,98525
Fir3t Actton 1921 Final Action A
0.98762
0.99225
57
0.98475
0.99186
58
0.98425
0,99147
59
0.98375
0.90107
60
0.99066
61
0.9鶴
62
0.98220
0.98982
63
0.98167
0。 98940
64
0.98113
D r r a t r O n s
reading oils at20° or 25° C and fats at40° C .PlaceinsmmentsO that み
総
品 に
セ
ぐ
艦
C)H20 hOugh prisms.Approximate teElperature perature仰 .2° corections ofbuけrOrefractometerreadingsmybemadettf01low_ ing fomula: R=R'+【 24
0。 98272
0.99024
C e w a r
Detemine index ofrefraction e)witt any standard instrulnent,
I f v o l u ro 距 ftt is measured attemperamre r
(r'― め
whereR=reading reducedtO standardtemperature,R'=re記 且 ng Ob_ tainedattemperature■ T=standard temperature,and【 =0.55 for fats and O.58 for olls. R e a d i n g s o f i n s m m e ndtttt ctdtyt cgainv eb■e r e d u c e standard temperature by subsd価 はng factor O.000365 foF O.55 and O.000385 for O.58 in fomula As temperature rises,■ falls。 lnsm‐ m e n t u s e d m y b e s t a n d a r d i z Ce ,d t hw ei ot rl e tH i2 c0 a la ,t O f2 H20 at ttttemperame ttng l.3330.Any corectton found shOuld be made on alirettngs.Index ofrefracton vaFieS With density and c 1 0 sin e salne t o ■ direction. "ytt R3PaCtOmerer
ら Kg/mLj=DT'(g/mLj+0,00068(r―
り
Tochargeinsmment,OpendOubに pFiSmby means of韓礎w head and Place few drops smple on pFiSm Or,if prefered,open"sIIIS where correctoncoettcient O,00M8is aPproximttofaCmalcOef‐ slightly by tuming scttw head and pour few dropsに 立smple intO icient for sample is known,it should be usedj. 血 nel‐ shape aperture between pnsms.C10se pris耐鼠皿 ly by dgiぃ ening screw head.Letinsmment stand few min before reading,sO ( b ) C d C u l a t e s p e c i f l c g r a v i t y ( s p e C i n c g r a v i t y T j o f s a m p l e 筑 m temperamre Oftest sample and ins― ent will be sane.Clean temperamre ras fol10w主 oil o賊 widl soRcloth,then with PriSEISbetween readingsby wiping COtton pad moisに ned wih solvent g"trichloroettlene,toluene, e・ Specttc gravityT=DwaI,Or ortttroleurnetherj,and崎 的 .
MethodofmeasurementisbaseduponObservationOfpositionof b o r d e r l i n e o f t o t。 anl irne nreoecnlは 述t o f a c e s o f n i n t g i a s s p r i 』■0ィ=density of H20 at r(frOm Table 985.19). BFing tMs border line ink)Fleld of vision of telescope by dng rO伍 double prismby means ofalidade h folbwing manner Hold se l f s a m p l e a n d H 2 0 a r e W e i g h e d i n s a m efltty p y andmove c n o m ealidade t e r , abackwardor t s a m e forwarduntil fleldofvision is 砲mperatu峰 (0, divided into light and dark Portion.Line dividing these po `拍田 Ю よI line,"and,as arule,will not be sharp line but 虹 band ofc。 Colors are eliコ ninated by rotating screw head of cOmpensator until spedflc gravityT=7_p1/Hi。 ShW,C010rless line is obdttd.AdiustbOrderline sotttitfalison
where DT=weight per unit volume(g/mL)ofsample at rand
of substance d缶 沌cdy on POint Of intersectton of cross hairs.Read″
絶坪 α拘う″り,一DifFerence between results of minations 2 deに scale of sector,esdmating 4dl decimal place.Take≧ 3 re調ほ ngs,ap‐ done simultaneously or m quick succession by same analyst should rsecdon alternately from one neld 。 t。 ther,and aver― PrOtthng inに nct exceed 2 units of4は decimal place. Referenctt Sran筋だ M2れ て 泳 ル r ttβ Aゅ s着ザ θお ,F何島 切 ″ Der:ッ″ど 、 セs,6th Ed.,1979,Pergamon Press,New York,NY. Taylor,J.K.■ 彪α施 を切 A,aryrたα′晩 醜おょ り,I.M. Kolthoff&P.J.Elving(Eds),1967,hterscた nce Pub‐ lishers,New York,NY,Part I,Sec.D‐ 4,Vol.7, Ch.81,“ Measurement of Densiけ and speciflc Grav‐ ity,"pp 4561-4610.
age.Range of readings should be却 .0002.Check correctness of insmmentasin A,or widl quartz plate that accompames it,using monobromonaphthdene,K420tC=1.6587),and make necessly cor― recton in reading. a gyzerg3日
utyroreractOnerer
Place 2 or 3 droPs flitered tesl smple on surface oflower pns血 Close prisrs and触 的ust mirOr until it gives sharpest reading.If reading isindistnctaFterrunningconstanttemperature H20hOugh insmment fOr someよ me,test saH耳〕 le is unevenly distributed on
◎ 2000 AOAC tNTERNATiONAL
01LS AND FAT Chapter 41,p 2
AOAC OFF,C8AL METHODS OF ANALYSiS(2000)
41.1.04
41.1.06
AOAC Ottcial MethOd 920.242★ specnc cravitt tAppagentl of OitS
AOAC Ofncia‖ Method 98S.19 (Apparentl weight per untt votumo and specttc cravitt of Fatt and o3,3
Pycnometer Method FiFSt Action 1920 Final Ac8o■ Repeated Fi『st Action 198S Surp,us 1934
Pycnomet hthod Ftrst Act,on 1985 Finat Acdon 1994 ′ SO―AO広 C日 ●めoJ
6α″rわ″i sでをAppenttx B,safety notes on chrom拒 駈 c acd.
延id and sul‐
A , Pn 「 c r P r e WeightofgivenvoluHle ofliqddfat atdesired temperatuFe iS de‐ teminedinpycnometeFpreViouslycalibratedatsametettratureゃ
A, StanJaRttz脅 宙o口ofP/cPcmerer Care的 1ly ciean pycnometer(ca 50 mL capacity,臨 mble Class
B , A p p a F B r u S
lnc,,No.15123‐ 50,or equivaent)by ttling with chromic acid
d仰 配 宙 hcapandaleト (a)わ "例 材 ユ コ 働 50nLcttdty,斡 noneter gradmed in ol° c(臨 mble Class hc.No.15123‐ 50,or clean pycnOmterby n■ i ng wihch叩 直c acid i n s e t h o r o u g h i y w i d l L Ol;l虚W i t l r e c e n d y b o i l e d H 2 0 p r e V i o uequlvdenty.caremlly sly d鯛 直ng sddon andletting stand several hon.Enw PycnOWter c o o l e d t o c a C2,0a° n d p l a c e i n c o n s t a n t t e n l p e m m e b a t h Ca.t 2 5 ° and由nse horounly宙 h H20・ ARer 30 min,adiust H20 1eVel to rOper pOlnt on Pycnclneter and cleamng solu歓 〕 n and letting standseveFal h.Ewty pycIIclttterand
●)脇 er ba統.屯 onstanc temperature,held at temperature s. t o b e m a d e く い C ) a t W t t C h d e t e m i n a t i o n iっ
s t o p p e , r e r l o v e f r o m b a t h , wり i pwei凸 th clean cloth oF tOWel,and ° w e i g h . E m p t y p y c n o m e t e r , n n s e s e vコ e区r将w a li直 thalooholandthen
a carrbrB」On crPycnOmetr
ether,letdry completely,relnoveed胸 『 vapor,and weを h.醜 輸mまne F i l l p y c n o m e t edrI,Foe,C覗 ently ttled H20 preVioudy coo weightofcontttnedH20at25° C by subtrndtt weight Pycnometer io ca 5。 C below constanttenperam badl,(b),Carefttly ttsert and fr o m its weight when illed wih H 2 0 ・ sett hemometer avoidlng any ttr bubbles,and Place in collst
temperatuFe bath.After l h,attuSt H20 1eVel to prorr point temperature(りt00.1°C.Renove Fili ciean,dry pycnometer widltest sample rViCuSlypycnOmeter,stopper,read Cooled to PyCnCHleter fronbah wtte dry wm ciean clodl,let cool if neces―
B, Der―
fnatrO,
ca 20° C,Piace in constanttenperame bath 30C,翻 mh じ uSt at 25°sary,and wdgh的 はl ng.Empty pycnometeF,nnSe several unes O n l e v e l t O p r o p e r p o i n t o n P y c n o m e t e rwith , a alcchol n d s tanddleneher,let o p p e F . R e m dry o v ecompletely,remcve frOm ether vapor, bath,wipe dry,and weigh as in A.Subttact wdght empけ pycnometeF frOm its weight when illed with oil md dhttde differ‐ ence by waght H20 at25°C,asdeteminedinA.卸 面 entiS Specinc
FeplaCe themometer and caP,and weigh to O.l mg. Vdime(mLj ofpycnometer a temperature ri yT yT=〔 G7■ ,伊ゃお毛Qが
C(appareno. gravity at 25/25° Ifspedac gravity at 20720 直 isd r判 Prowdas above andasin A but subtract 5° C from each tempeFature specifl乱
where W and W=weight c)ofpycnometerempty and fdled with rr20,reSpectively;れ 0丁 言denSity(grnLj ofH20 attemperature く り,frOmTable 985.1,;and a=mean coefrlcientOfcubic exPansion of pycnomter e=0・ooOo10 foF bOrOSillcaに glass,0.000025 for soda line giass). ' Der―
41.1.05 AOAC OfRcta3 Method 020.213 Spec18c enVtt Of Tmpedure Co― tlon
olL
ftrst Actron 1920 F輸臨:ActSon Repea8ed First ActBon 1985
lⅢ 。(T一Dl 【
ragFOn
メ″ a r 的0 乱姥″ 陥二 Weigh Kto O,l Hlg) ( o O : な a n 2 r a r s“ empty, cllan pycncmeter wi`h cap and thermometer. Fill pycnoneter w血 峰si sample wtth is tt tempeFtture below ttt of
脇r:θ constant temperanR bah and proceed as under Carゎ ″ゲ え ″ θ 控 "を ,メ ∽河″宅rrar″″.―Meit fat attemperattre ca ●)Fars prir at粛 10 above its mel曲 lg"nt miX Well,and let stand at elevated tem, 脱崎 aiF bubbles.Proceed as in(a). lF spccnc gravity of ollis de腕ぱ惑at odler dlan standard tem‐ perame to eli闘 確 5°C mttbecdculated at 2占 perature,approximate specirlcgraviけ as follow賞
C=C'+0,00064(r-25°
C)
whcre C=sPeclflc gravゅ at 25/25° C,α =spcclnc graviけ at r/25° C,T=temperature at which speciflc wvlけWas detettned, and O.00064=nean coFreCdOn for C. l° 0 2000 AOAC tNTERNAT10NAL
E CarcFrwOns
(O CalCulate waghtper unit volume DもOf Sample attempera― m r e r i n g / m L a、s f o l 蕊 外 tg/mL淳 学
A
where W and,7=welght(g)。 f py● nOneteF empty and fliled wih test smples VT=volume ofpycnchetcr(InLh attemperature
0 にS A N D F A T Chapter 41,p.4
Tabie 921.08
AOAC OFFC,AL METHODS OF ANALYSS(2000)
Buwrorerractometer rB8dino3 and tndtces of refractton !ndex of
41.1.08 AOAC Otttc`at Method o20,156 Meiting P。:nt Of Fate and Faty Actds
index of
W31ey Method Ftrst Actlon 1920 Finat Actlon
40.0
1.4524
60.0
1.4659
40.5
1.4527
60.5
1.4662
41.0
1.4531
61.0
1.4665
A 用 田卸 !
41.5
1.4534
61.5
1.4668
42.0
1.4538
62.0
1.4672
A ぢ∽ 力θr _ w " 夕r " 加 “″. ― べp e c i n c g r a v i t y s h O u l d b e s a m e a s u l a t o f t t t o b e e x a m i n e d . P r e P a r e b y S e p m t e b o l l i n g
42.5
1.4541
62.5
1.4675
43.0
1 . 4 6 4 5
63.0
1.4678
43.5
1.4548
63.5
1.4631
44.0
1,4552
64.0
1.4685
44.5
1.4555
64.5
1.4688
45.0
1.4558
65.0
1.4691
45.5
1.4562
05.5
1.4694
46.0
1.4565
86.0
1.4697
46.5
1.4569
66.5
1.4700
47.0
1.4572
67.0
1.4704
47.5
1.4576
67.5
1.4707
48.0
1.4579
68.0
1.4710
48.5
1.45鶴
08.5
1,4713
49.0
1.4586
69.0
194717
49.5
1.4590
69.5
1,4720
50.0
1.4593
70.0
1.4723
50.5
1.4596
70.5
1.4726
51.0
1.4600
71,0
1.4729
51.5
1.4603
71.5
1.4732
52.0
1.4607
72.0
1.4735
52.5
1,4610
72.5
1,4738
53.0
1.4613
73.0
1.4741
53.5
1.4616
73.5
1.4744
54.0
1.4619
74.0
1.4747
54.5
1.4623
74.5
1.4750 1.4753
55.0
1.4626
75,0
55.5
1.4629
75.5
1,4756
56.0
1.4633
76.0
1.4759
56.5
1.4636
76.5
1.4762
57.0
1.4639
″.0
1`4765
57.5
1,4642
77.5
1.4768
58.0
1.4646
78.0
1.4771
53.5
1.4649
78.5
1.474
59.0
1.4652
79.0
1.477
牌艦督 蹴総辞総 紺lil程 附蹴総鉛淵￨ 艦 撤督翻 撒 蹴 1撤鉛離 艦 mre unnt fOr use. ユ Det―
rnarOn
艦 蝸 鮒 甘縦 盤 総 鰐 総 襴 鰍 1-1.5 cmdianeter and weighca200mg.Removediskswhensolli 狙d let Stand 2-3 h to obtain no― lコ対,・
げ 鞘繊隅欄鮒艦 端翻総胡艦 鮒唱 &鞘温盤 鷲 樹 品淵冊盤 淵 謎 艦 鮒艦 路盟 品 艦鮎 Plt and remove disks. Place 30 x3.5-3.8cmに st tube,containing dcchOl― H20 miXture, inta1135x10cmbeakercOn面 胡ngた eandH20,andleaveundmix‐ 艦 紺 盤 艦 鷲品 i 棚 艦 淵 騨 樹 批 itsown.Loweraccurate dlemomece,dlatcanbeゃ adtoO.1°C,into testtube undibulb isjust abovedisk.To secure even temperatw in dl paFtS 9falCOh01■ H20 miXture around disk,shr gently widl dler_ nometer.Slowly heat H20 in beaker,constantly sdrring widl air s的 にam or odler suitable device. When temperature ofalcohol―H20 miXture dsesto ca6°C below mp of的 ,disk ofttbegins to shrivel and gradually rolis up intoト やgularmss.Lower曲 げHlometerundi fat脚配icle is even withcen‐ terofbulb.Rctate themoneterbulbgendy andsO regulateheatthat ca10minisFequiredforlast2° CincFeaSeintempenture.Assoon as 如 massbecottssPheFiCa,配 adttmometer.msis wiに y mp.At his point,temperawe ofbath mustbe=1.5° C above mp ofsample. Conduct 2 additional demin倒 はOns exacdy as above.Second and third results should agree closely. Ifedge ofdisk tOuches side ofmbe,make new dem菰
natiOn.
41.1,09 AOAC OffBcial MethOd 920.157 Meiting Polnt of Fats and Faty Actds prlsm surfaces.As,is greatly affected by temperature,use care to keep ttmperature constant.Caremlly adiuStinsmment,using stan‐ dard fluid supplied with it,convertinsmmentreading tO,fromTa‐ ble 921.08. Referencett Lewkowitsch,勧 卸 比沼r確 統″ ,。 gメa″″A″ rysおげ θ'生Faな 伽 ″W佐確s,6h Ed.,1,312(1921). ユSθa C陸 閉.あa26,512(1907). 泌 OAC 48,128(1965).
0 2000 AOAC,NTERNATiONAL
Caplitary Tube Method First Action 1920 Ftna!Acticn
Draw ca 10 mm melted and nltend fatinto dlin― wall capi■ tty albe, 1_n sealendofmbewitlfatin sMll aaHle.Do notbum tt Ho t l l b e s c o n t a i t t的 n g邦 fc aa t 1 o6 判 めi n r e t i g e d o年r1■0 C°. A ト 触れtllbetotturatetlemometer卸組mtoo.2° C,so ttlowerend is even就h bottom ofHg bulb.Suspendin 600 nLbter&述 ffllled
AOAC OFttCtAL METHODS OF ANALYStS(2000)
OILS AND FAT Chapter 41,p.5
With below
H20SO mp
tlat
t l e m o m e t e r i s i I I I n e r s e d c a 3 0Cm l n , S t a r t i n g
o incFeaSe o f s a . m p l e , a ps pc l ya s h et ■
8-10°
b暉 a t lc a t t m p e 劇
O . 5C° /min,agitating H20 h batl by small sttam ofair or witt slow st的.Take as mp temperam at which substance becoIIles mnspart av‐ e n t . M a g n i t t i n g g l a s s i s u s t t t o d e t e c t c o鴫 m p.l)eRteepmoerlt宜 erage of3 deteminations tshOuld agree C).wihin O.5° References:J吹OAC 69,247(1986). E/SDA β″ぇCれを閉.,ガ と,13(IV),P,448. 2秘た】 打″ 晩 wkowitscL C乃 cttragy ωttA″ 研 体ザ θ協島r物なatt w竹彪s,6th Ed.,1,325(1921).
Wiley,P所 ″″r2S att P″ rar cr確ゲAgた″ ″″ A″rysな ,2nd Ed.,3,309(1906r10.
AOAC Offictat Method 042.18 Ttter Tost for Oil● and Fat8
!的
Omerm
ryP2.―Etched sにmP glass. 駒 滅 ― Mercuy. Rangをα"″s″ た「―お江inus 2 to+68° あ宮httsわ ChO.2° C. -385-390 mm. 駒 勉"鶴 抗 S,c確.―Consmcted of suitable dlemometer ttbng of eidler plain or lens frontけ pet Dねmete,plain tontけpe:6r7mm;diaEle‐ ter,lens frontけ pe:CrosssectionofstemmustbesuchdlatitwHlPass through 8 mm Fing gage but not enter 5 mm slot gage. 助 肱 20C atoms)
rcθ ヌ ″ rれ″ ?sったCれ4ク セrイr,″ .2〕 r]陀
acids are present. (C)PaCttg.― Acid‐washed and silanized diatomaceous earth, with narrow range(25 μ m)grホ n s12e betWeen 125-250 μ m (No.60-120),Average graln size is inversely related to id and di― recdy related to column length.Coat with 5-20%p01yestertype
po‐
◎ 2002 AOAC tNTERNATiONAL
0'LS AND FAT Chapter 41,p.24B
AOAC OFF'C:AL METHODS OFANALYStS(2002)
rc伽続確stt C切 姥rイr,p.2〕 rTc・
⑥ 2002 AOAC iNTERNATiONAL
AOAC OFFCIAL METHODS OF ANALYStS(2000)
Tab!o983.22
C)3LS AND FAT Chapter 41,p.25
Conditlons to● !ute meulyl stearate within ca 15 min at≧ 2000 theoretica,ptates Concentration of
2
15-25
3
20-40
4
40-60
５ ０ ５ ︲ ︲ 郷
1側 '淵 Cdumntt mm 撚私 Car盟 錯材
Column ternperattre, Oc
C18,and C18 medlyl esters appear in order:stearate(18:の ,Oleate (18:1),lin01eate(18:2),and hnolenate(18:3).C20 Saturated ester
b8 e: f3 orに e, sb に ( a r a C M d i C , 2 0a:pop euasrusd 1け ut
m a_y
be砲
versedon some collHnns,orpOsitions may change withcolumn use. 比 carcurarrOns
175 180 185 185
E Perramance sPcrPcarrOn3
Use method Ofnomalization,which assumes ali components of test wiion are represented on chromatogram,sc hatsum Of areas under peaks represents 100%Of consttuents(tOtal elutton)。 Ifinsmment is equipped with integrk)L use igures shOwn.If ■ot,use mangulation:Draw iines for each peak tangenttO sides and i n t e s encgは b a s e l i n e . C a l c u l a t e a r e a o f r e s u l t‐i n g
tri
plying height Kccrrected for a■ y change in attenuation)by%base. Feo r a u t o m a t i c d l y a t t e n u a t e d p e a k , o b t a i n p e a k w i d d l b y d r a w i n g
P e r f o m t t d y sxitsu roen 航o fl mS eに a的 r a t e a n d1 m0 e1 的 eat in caequal proportions ctgⅢ methyl esters froncocoabutteoo Ad‐ tangentstoOutersidesofPeakKdleSemustbefullchartspan,and up just sample size,coluIIln temperature,and carier gas now sc Per%Ofpeakmustbeusedjandintersecttgbaseline.Caculate that area me的 lSにarate peak is recorded ca 15 min after solvent peak,ca% by muttyttg height tcoreCted for atten full scaleo Measure base widtt in mm of methyl stearate(ッ If signiflcant alnounts of cOmpOnents withく 12 C atoms are ab‐ 1)and methyl oleate(w2)betWeen points of intersechon widl baselines eof nt,calculate percent by weight of each component,express t a n g e n t s d r a w n t o i on ni e pc oはi n t s o f c u r v e s . A l s o m e a s u r e r e mt e tn h‐y l e s t e r ,
はo n d i s t a n c e i n I I I m ( D f r O m s t a r t t o p e a k H l a x i m u n f o r m e t h y l
s t e a r a t e a n d d i s t a n c e i n m m b e t w e e n p e a k m a1 x i m u n f o r m e 的 q=q x100s ci stearate and me的 1 01eate,ユC』culate the餌 胡cal plates,″ ●館‐ w h e r e q = a r e a o f p e a k c o m s p o n d i n g t o c oq m= p o n e n t i ciency),and res01ution,R: suHl oF areas under dl peaks. ″=16(S/ッ 1)2 I n ∝的占 n c a s e s , e . g . , i n P e s e n c e o fく c 1o 2m p Co n ae tn ot ms s ,w i 血
i n ,m ao nl de t pc ru el sa er n cw ee t ot fh 低s e c O grouPs,cOrmtion factors must be used tt 慮 cOnvert庫 areas intO w e i g h t p e F e n t . D e t e m i tn ie oc no f―a c t o r s b y a n a l y t t n g k n o w n Select condidons to 6btain,2000 and R≧ 1.25。in addiは on9 erence standards of rttdlyl esters of ccmposidOn linolenicacid(183)mettlestershouldbeseparatedfromarachidic q干ng cOnditionsi For Feference standard, aCid00:OmdgadOlettacid(20:1)eSters.Columnswillshow sample grad‐under identical 脇宜 R=2//Kwiキ
large
W2j
ua1loss in R宙 th use;when value becomes≦
1.25,replace.
dfFerences
Percent by weight componenti=曳 xlo08 31
ぇ D e r e r m r n a r r O n
=weight Of compOnenti in reference standard
where■ gi= With apparams showing stable baseline,lnJect O,1-2Ⅲ 5-10% tod weightOfali componentsinreference standard.Calculate f heptane soluは on of methyl esters,96933A Gcc 41.1,28).If mce ChFOmatOgram: components are desired,smple Hlay be increased≦ 10x Pierce seP tumofinletportandquicklydschargemedlyl esters.Widldrawnee‐ Percent(area/距 Σq つOf COmponenti=q x10け dle and ncte on chart飢 臣出 peak due to airor solven与 mrking start r e f e r e n c e p o i n t , A d i u s t m e t hryalmeosuに n t s o m t t o r p e a k iost■ a ‐ from which calculate co「 factor for each component ection tenuated>8、preferably less.Change setting ofattenuatoras neces‐ sary tokeeppeaksonchartpaper.Markattenuatorsetttlg onc権 氏 =(芯罪 3)X(Σ qゴq) 止. For deteminatiOn of acids《 312,10Wer cdumnに mperature is Deteminecorectionfactorsrelativetopal血 dctttr16=1,SO脱 needed;for>C20,higher.Temperature programttng is useful in such cases,e.g.,with acidsく て, C12,inieCt at 100° C and raise temperat = ―― X・ ture 4-8° 9min to Opdmunは or prograln up to a nxed temperamre 【16 andcontinue atconstanttemperature until aucompOnents areeluted. If apparatus does not use progrmmed heating,operate at 2 rlxed Then to calculate percent of each cOmponent(as methyl esters), にmperamres between 100° and 195°C. multiply its areaby appropriatecorection factor,andsun corected a rgenrrrrcatrOn
areas:
Analyze reFerence standardmixturesundersame operatingoondi‐ Percent by weight componenti=(rix c)x lo幌 (rixq) t i o n s a s f o r 鵡O t ens,tM脚 easure retention dstances(D fOr known In certain cases,e.g.,when all cOmponents are not eluted,use h‐ esters.Plotiog S as function of nuttber of C atoHぃ of acids.Under temal standard,S,such as CぉorCw methyl ester,and detemine its isotherlnal cOnditiOns,graphsOfsmightchainestersOfsanedegree COFreCtion factor.Then, of unstturation should be straight lines,approximate,Parallel. Identify peaks from test portion from these graphs,interpolating if PeFCent by weight oFcomponenti as metlyl ester= necessary. Avoid condidons which penmit“ Inasked peaksr'1.e., (Ws/W)X(IPrで OX(crcs)x loo which are not suFflciently resolved. Esters appearin order ofincreasing number ofC atOHls and ofin‐ w h e r e w s i n g i n t e m a l s t a n=dtaortda la nmdg″ t e s t p o H よ on,and creasing unsamratiOn for sarne number of C atoHlst C16iS ahead of subscnpt S refers to intemal standard compOnent.
0 2000 AOAC iNTERNAT10NAL
01LS AND FAT Chapter 41,p.26
AOAC OFFICI札
Reportresults
to
following
s i g n i n c a n t f l g u r e s , wTiatbhl oo n e9 9 r1 l. 3 g 0u r ie nb te e‐H a b o r a t o r y e t u d y r e 3 u : t e f o r f a t y a c i d 8 i
品 紹 品R払 培 麒Pd 鞘韻 弘:名
yond decilnal pointin all cases:3 for>10%,2 for l-10%,and one for く1%. Faty acid ,P「 ecfs′ 研 (a)R?夕 α勉うJ′ 均ん― TWO Single deteminations performed on same day by salne operatorwith same apparatus on salne sample for Ot differby>3%relative,wih an 珂 OrCOmponents(>5%)should■ absolute value of l%. 一」 TWO Single deteminations perfomed in (b)Rη ″れをめ'Iゎ。 different iaboratories for maJor components should not differ by >10%relative,with an absolute value of
sR
1967),57,336(1970);
62,709(1979). R2ν体を″=March r997
0.58-0.98 0.44-1.91 0.55-2.59 0.05-0.42 0.23tO.68 0.05tO,13 0.04-0.17 0.07-0,24 0.02-0.17 0。15-0.45 0.01-0.08 0.03-0.07
0.04-0.07 0.07-0.19 0.06rO.15 0.43‐2.06 0.02-0.38 0.14-0.80 0,08-0.17 0.04rO.08 0,10-0.32 0.69-1,44
ⅢAdopにd as a Codex ReFerencc Medlod cypeII)fOracid ttdrolytts,bo‐ rOn Hnuttde oflinoleate on the fOm Ofglycerides)in special foods.
41.1.30 AOAC Officta:Method 991.39 FaHv Acids in Encapsutated Fish 01:o
a n d F i s h O i l M e t h y : Ea sn td e rE st “
22:5nt6 22:5n-3 22:6n-3 24:0 24:1 同
0.02-0.14 0.07-0。 73
8.31-13.12 0.49 5,3 5.66-10.02 0.54 2.9 6.80-26。73 0.51 4.3 3.05-14.43 0`06 1.8 1.92-6.71 0。 19 1.6 2.37-10.69 0.01 1.2 3.86-23.14 0.04 5.6 2.43r6.30 0.04 1.5 7.78-34.25 0.01 5.5 4.46-18.13 0。 13 7.3 10.29-27,71 0,01 8.6 16.08-67.73 0.03 17.4
27.47-47,93
0.01
8.86t43.83 0.02 4.91-13.77 0.04 5.48-9.78 0.25 11.77-141.42 0.02 8.88-17.24 38.29‐ 03.55 13.14t50.60 8.84-16.20 7.50-16.09
3,1 2:5 3.5 1.9 8.9
0。 11 7.9 0.06 26.3 0.03 18.6 0.13 6.7 0。 29 3.7
48。38-102.04 0.01 10.9 41.22-78.73 0,03 7.4
s h o l l s , a b s o l u t e w e i g h t ( m g / g t e slto np)o 成 5。 38-19.75 4.24r12.60
7.17 3.68
３ ５
2.98t31.10 2.60-13.27
９
20:5n-3 22:6n-3
Ethyt ester concentrate,area percentage
能 夕Table 99139 for the results of dle intedabomtory study sup‐ 14:0 Porting acceptance ofthe nethod. 16:0 A P r r n c r p r e 16:1 Tese portiOns are weighed into Tenon_lined screwHcap giass 18:0 18:1 tubes that contain appropriate intemal standards.FatけacidS Ofoil s m p l e s a r e d e F i V a t i z e d b m e t t l ;eHsltee的 ぉ1 研 e t t l e s t e r s a I I I ‐18:2n-6 18:電 賄■3 ples require no derivatizationt Prepared mettl esters are mlyzed 18:4n-3 by CC instrument equipped with fused silica colunn coated wi血 20:0
bondedPolyglycolliq岨 dphase,oxygen scrubberin cariergas line, andflalne lonizadon detector,Medlod detemines areapercentages
20:1 20:2n-6 20:3n-6 Cお‐ 5,8,11,14,17-eicosapentaenoic acid or 20,5n-3)and DHA (all― 20:3n-3 x aoei■ c a c i d o r 32 )2 .: 6 n ‐ ( a l l , C f S - 4 , 7 , 1 0 , 1d 3o ,c 1o 6s ,a 1h 9e ‐ 2014n-6
of 24 fatty acids and absolute weights tmgrg sample)Of EPA
204nt3 20:Sn-3 (a)CaS統 ″閉αr9graPれ― with aalne loniztton dete的 ,cap11‐ 22:0 lly coluIIln inieCtiOn system(sPlit mode preferred at sPlit ratio of 22:1 l:50),and Suitable data processor.Ndef ln ishoils analysis,san‐ 22:4n-6
a ApParar崎
ples are usualy sumcienttO pemit operation in sPllt mOde.)Oper― 22:5n-6 ‐ ating conditions: temperatures― lnJection po■250°Ci detector 22:Sn-3 temperature 270°C;oven progra― ed from 170 to 225° C at 22i6n-3 l° C / m i n t n o i n i t i a l o r i n a l h o l o . H e l i u m O r h y d r o2g4e:n0 c a r n e r (99.99%pure,or bette,With Oxygen scrubber h line.
RSDr,%
５
AOCSrAttC m
22:0 22:1 22:4n-6
__
Sr
／︲ ヽ ヽ
Oa3 ChRDmatOgraphic Method First Action 1991 Fina:Actlon 1995
RSDR,%
Ftth dに,area pettentage
14:0 16:0 16:1 18:0 18:1 18:2n-6 18:3nt3 3%. 18:4n-3 ｏ ﹁ お い 巾 巾 伸 市 。 ２ 抑 卸 卸 卸 抑 如 如
References:IUPAC 2.302,7th醜 . 】AttC 46,146(1963);50,21気
METHODS OF ANALYSiS(2000)
24:1
0.06 0.11 10 0。 0.15 0.50 0.06 0,08 0.15 0.03 0。 45 0.10 0。11 0。 07 0.09 0.15 1.54 0,07 0450 0。 41 0.14 0,36 1.40 g a 0.11 s 0.23
17.07 12.24 30.96
7.00 5.72 7.93 12.85 7.62 10.12 3.19 32.42 35,22 29.32 7.37 7.86 5.鶴 73.94 4.68 84.99 43.08 8.52 7.51 158.70 35.16
Ethyt ester concentrate,absolute weightて mq/g test portion) 卯 `―Fused silica9 30 m x O.25 mm(or O.32 mm) (b)CC oθ′ 20:5n・3 20140 9.15 coated witt bonded Polyglycol,based on Carbowax‐ 20M(e・g., 22:6n,3 14.15 8.97 Supeicowax‐10,Supelco,Inc.,Bellefonte, PA 16823,USA,or a SR and RSDRfornSh。 ‖ s are ranges of va:ues obtained in the collaboratMe eqdvalent coluIIln that provides salne eludon pattem as that inus_ study or 4 dlrerent Rsh o13.RSDR Values are etevated for analttes that trated inFigure 99139 andbaselhe separation of21:5n‐ 3,23:0,and rarety exoeed O.1-o.2拷 ot total analytes(20:0,2013n‐ 6,22!0,22:4n‐ 6. 22:召 h‐6). 22:5n‐ 6,24:0,and 2411)
◎ 2000 AOAC iNTERNAT10NAL
AOAC OFttC!札
M目 猟)DS OF ANttYttS(2000)
0,LS AND FAT Chapter 41,p.27
０ ・〓い ”田的
０ Ｃ ０ ・ い ”的田
０ ・エ ロ ””的
罵 守石 “
ｏこ 守 ”０何
〓 ０ ・ “ ぃ０付
罵 字品 “
︱ ” ” 田 付 一 ［ ］ ］
” ・ｔ守 ”０付
Ｏ
・〓 ” 嵐 雨
Ｏ Ｌ〓 ■ ０ ︼
０ ・こ付 “０ 一
い０ ” ︺ ０ “”“
” ・ＣＯ ”的田
？占 ” 罵
” ｏこマ “０ ﹁
卜 ・ｔ ｉ 口 ”
０ ”０ ↓
０ ・Ｃ ””０ ”
Ｃ 卜 ・ ︼”０ ”
０ ぃ０ 一
０ “マ ︼
０●ＥＯ﹂００Ｅ
硫
側
mtlon tt menhattn tt fatyお d methw eSteぉ 。 n印 o対 bL ttsed 罫 協 思 捕試掛1品 樹 培 品 嵩 盟 艦 呂 苫 播 温 景 品 辞 cra触″ ツαセ″腕 抗 _Maintained at l∞° (c)a%Mttr,2切 ″ C. Dry heater bl∝ k may be used.
ume with isOoctane.駒醇 1・ O mL PortiOns into screw‐ cap glass m b e s a n d e v a P o n t e s o l v e n t h Sg te On Fd ee ms bm em S O if n N 能 ezer ifnot to be used imElediately.
c)crass rzba_16x125 mm.With leak‐ dght Teflontlined screw caps. - 2 m L , w i t h s c r e w c a p o r c r i m p c a P K f O r a u t o s t t見w 最阿 l e r柳 jめ角 . q四 膚rOn arlJAnarysfg 。 O yiaな . G 的l g . ( O A ″ け施 a ル α: 御c a ― A c c u r a t e t o 却
rcc (9Dけ れ曲 8鶴 SO″ (h)0ね SdWa″.二Volumetric aasks,25 and 100 mLi volume血 低. Pipets,l and 2 nL;Pasteur 障 p的 aR餌
寧"ra
一 御 虎 r`oュ"apPtts tO all encapsulated materids,in‐ O仇 cluding nonesterifled faty acids,widl excePtton of ) Accuntely weighca 25 alg enl mg)diintO glass ttbe contな ぃ i n g m e t h y l e s t e r I S , E . A d d l .5 5M m Le Ot 。 hanolicNaOH,blanket with nitrogen,caP,mix,and heat 5 min at 10o° c.Co01,add 2 mL BFain methanol,C(a),blanket with N2,Cap tightly,mix,
“句 α″οガル .―BF3,12%in methanol.2nLmberglass (a)β οttθ and heat 30 1nin at 100° C.Cool mixture to 30-40° C,add l mL a n P o u l e s ( S u P e l c O , I n c . , C a t . N o3.032‐ 0,or equivdent reagent, isoocta■e,bla■ket with N2,CaP,and shake vigOrously for 30 s sealed in amber giass mpoules for extended shelf hfe)。(C例 材;例f while still warm. BF3 in medlanol is a corrosive Mgent and mutt be handled with care.思 ddeye andskincontactbyuseofprontiveshieldandrub‐ c 側 禾 モ 辮 靴 脳 ber gloves.Use cnly h Propedy Ottndng fume h00d。 鞘 ) isooctane layer separates from aquecus lower Phase, transfer ●)2Jfθ M力 り′御ゼ β佐メ 容伽 .―Reagents Of99Ⅲ %「 直け as isooctane layer to a cLan glass tube,blanket with N2,and Cap. detemined byTLC and CC analyses m chekPreP,Inc.,Elysian, 鵬 ,Ci No.N‐ 23‐M Enethylester3 andCat.No.N‐ 23‐E Eethyles_ ter3,Or equivalenり,oわ ″f On lMluest,the CharlestOn Laboratory, Soudleast Fish済絶s cen玉 、IWadonal Marine Fisttedes Service,PO Box 12607,Charleston,SC 29412,USA,will Prov間 にcapsules of collaborative sttdy sampに よ[Steam― deodoFiZed mettaden oiJ fOr u s e h o P t i m i t t n g G C e)q u i P m e n t ・ 一 sOdum hydroxide,methanol, (C)絶 宅 c“rg拓 所2c確 碑途 、 isooctane,and sodium chloride.(働 ″r′ ο″r s22物 財 歳 β,Safety notes on scttum hydroxide,methanol,and isooctane.) a PrParaFron o′
sOrurrOns
.-0.5M.Dissolve 2.Og NaOH (a)Arcoれ θrたsθガれ"ゎ だ拘I:あ夕 in methanol and dihte to ltXl mL with methanol. ―一Saturated soludon.Dissolve 36 g NaClin (b)Sο tt cれ 勉
欧m c t a q u e O u s l o w t t P h a s e a S e c o n d l mL isooctane.Combine isooctane exmcts and concenmte tO ca lmL hshamばdry N2・
d E l e
InieCt l-2 1tL into CC system. (b)財 閉り′θre的 I夕sr2rs._Accurately weigh≦ 15 mg仰 .l mgj methyl or ethyl esterinto glass ttbe contthing aPProPnate ester IS, E.Add l mLisooctane,blanketwithN2,Cap,andmよ
thorougtty.
InieCt l-2に into CC System.Ifpeak height of ls is幻 .5 th■of EPA or DHA peaに repeat malysis using 2.O mLIS. a carcwfatrOns G ) A 万 2 α P c にC 純 8 a 一 C a l c u l a t e a r e a p e r c e n t a g e s o f f a t t y a c i d methyl esters or ettl esters as follow試
100 mL H20・ 二 PrepararFan orSranJarJs Accurately wttgh ca25 mg仰 .l mg)of23:O methyl orethyl ester intemal standard(IS)int0 25 mL volumedc nask,and dihte to vOlt
Area percent fatty acidx=tAx/tAT― As)]x100 where Ax=area counts of methyl or ethyl ester X;AT=total area counts for chromatogram;and AIs=area counts of IS.
0 2000 AOAC tNTERNAT10NAL
W i
AOAC OFFICtAL METHODS OF ANALYSiS(2003)
01LS AND FAT Chapter 41,p.28
― ttarθ With name 10nization detector ,一 C)OaS Cれ rθ graPた 41.1.29)or equivalent system].Electrottc integrator [963.22B タ Gタ iS Preferable.
(b)確 をれ′げ EPA a″ ガD「 Aれ θ:rs._5% 10 mL dhyl edler.Stir and mnsfer cOntents to separate columns or should not exceed 2.2g/1oo g sample or 5%of deに funneis,3(o,caCh COntaining ca l g silica gel.Extract mehyl esters mined value,
:言 盟 獄 ま 」 格 韻艦 ‖ 竹 辞 揖 置 村 よ ヽ ま 嵩 嵩 裾 !告 品 長 督 樹 ‖ fず 獄 『 指 古 苫 f監 』 将
us唯
ナ and
3研 four 10mLpoEtionsofewld比
撒
棚
想
oolect血
ェ
5%,wd_ 鮒盤拠rater.ForewicacH く wstt h ar
強 ぃ 1∞ nL
娯艦
evapoFate au sottent with N2 Stream methyl esにお 斑 botmm of tubes.Dissolve methyl esに 、 in ca 20-50 11L hexane.
References:P″″ APPi C確 開.54,2759(1982)iSi 302(184). hood to concentrate ユ As縦 .Pめ こ と A脇 町sぉ18,77(198o.
under
CAS‐112‐ 86‐ 7 cmcic ac10
見 Cas Cttromarurapn/
Sク 9 6 3F.G2`22B4― 1 . 1 . 2 9 ) , e x C e41.1.32 p l i tAOAC t eO付c t,oiallMethod - 2 975.30 1tL hexane Docosenolo Acid in Oits and FatG 盤縄縄紺s鞘瑞陥瀞 艦劇機路‖ 繊￨ Gas Chromatographic Method areas mm chromtOg皿 艦古 漁洗辞 路拙盤 を F3RBt Action 1975 Finat Action 1984
A . P r r n c r p r e
岱 猛蹴器替齢 堵 『盤 鑑機脳 濫柵
1
mcic ackェ,an isomerofdocosenoic acid,is characteristic acidof rapeseed.01l or fat is converted to medlyl esters,which are deter‐ mined by GC,with methyl tetracosanoate as intemal standard.
◎ 2000 AOAC t卜 『ERNATtONAL
AOAC OFFiC:AL METHODS OF ANttYSiS(2000)
C),LS AND FAT Chapter 41.p.30
ユ A P P a r a r w s
41.1.33
統″秘西θ Packard HeWlett‐ OC密 g門肱 二 Varian Model 1740tlα ねndemtorand3生 Q.9mlX 5700的能s,orequlvalent,witlaal.eiOd刻 od stainless steel coluEm COntaining 15%dietlylene l旭ho.2mゅ glycol succinate(DECS)on 30r100 1nesh Chromosorb W,acid washed,Operating condtionsi temperatures― lnJector 250° C,de‐ tector 290° C ,coluHln 190°C;He carier gas flow rate adiuSted to ltime ofca 17 min for mehyltetracosanoate.Measure give retentiα
AOAC O宵 io,at Method 977.17 Poiymers and Oxldation Products of Heated Vegetable 01:s Gas Chromatographi● wethOd fOr Non‐EluHon Materla18 Ftrst Action 1977 Final Action 1984
r
Accurately weigh ca40mg Oiland 10 mg ttheptadecmdn intemal
peak areasby electronicintegrator,diskinttgrator,ordangultton.
Apphed Science Laboratories,Inc.,No.21322 Eno rttcar筋メ西た-125 mL conical iask with stan‐standard(Alltech‐ (b)rraな否r夕 longer avお lable])into 50 mL Erlemeyer.For ease of handling, dard taper 24/40 joint and neck elongated and constdcted to empty entre vial inにmal smdard supplied(100 mg)into tared 8-10 mm id.
100 mL volumedc aask,weigh accmtely,dissolve triglycer hexa■e,anddiluteめVolume.Tc avoid volume changes,immediately mnsfer lo mL aliquots to nine 50 mL Erlemeyers and caremlly (6α″rわ″i S2をAppendix B,safety notes on distillation,sodium metal,hexane,magnesium,and methanol.) e v a p o r a t e s o l v e n t u n d e r N 2 S t r e a m . F l a s k s an町 at O° C if desixtt Weigh test portion dimtly intt nask after (a)HC盟確.一つistil reagent grade hexane and dry‐over stored drous Na2S04 befOre use. tempemure equlllbrium.Add4 nL O。 5M alcoholic NaOH solution and boilhg chip,and reflux 10 min witl H20 COndenser.Add5 mL 統御ο4 α″り″″匹. ―D i s d l w i t h M g t u m i n g s . ( b ) 財夕 BF3‐ metlyl alcohol,Preparedash96933De)G空41.1,28),trough (C)S述 ″閉 加夕1祐え確 sθれ!筋 .―APproximately l%in anhy‐ a Feagents
condenserandboi12価n.Add2-5mLPetroleumedlerttP30-60° C) and boil l min more.Cool mixttlre and mnsfert0 30 mL sepmorP washtt aask several dmes widl total of 10 mL petrolem edler. Shake l航 n to extract memyl esters into petrolem edler.Drain C)MC的 こ`r解 ″勤 勉 ″ J加 筋 .-2 mymL hexane.Dis― s o l v e 1 0 0 m g n e t t l e r u c a t e s i g E l a C h medlanolicKlowerjげ温韓inttsecondseparatoFandeXmctagainwidl emical Co.,99.5%C22::; eher extracts,and wash N o . E 3 5 1 0 ) i n h e x a n e i n 5 0 m L v otlou mVeotlr‐ i10c nLpetroleumether.Combine a a s k a n d d i l upemleun に with5 mLPortiOns H20unは l wattungS are acid‐ ■ee whenに stedHおdl ume with hexane. mehylred.Dry widlttdrOusNa2S04,preferably by pouring s ″.-2 ng/mLhexane. 確cθs物 姥S々 脇滋材 sοれ,jθ (0〃 夕′ り `Cを narow glass colum containing Na2S04;WaSh colum D i s s o l v e 1 0 0 m g廿a Cm Oe Sh ay nl oにa t ce h _o Au Pl pにl i e d S c iPle e n trOugh ce wid15mLttpetroleumeher.EvaporatesolventunderN2HAdlaidof ( Labor菰抗es,Inc.,or Sigma Chettcal Co.,lignOcenc acid mehyl s t e a m b a t l . B e c a u s e o f s m a l i v dおu, tm tn s of fe er s に dry esters b esに対 997o C24;No.L l126 1replaCed by No.6766])in heXane in l nLに st tube‐ shaped volumetric nask or cone‐ shaped vial to fac■ i‐ 50 mL volumettc flask and dilute to volulne witt hexane. tate FeH10Val wih synnge foriniechOn Onto CC column, E DerermrnarrOn Analyze sample wih aame iOnization detector as in 963.22B― C Pipet s2ヵCrOrrar価 (a)降 igル″→ βθ″ 的 [統 OS御餌宅 R22:1・ を41.1.29)。MeaSure peak areas of chromatogram by eidler (s夕 l,2,and3 mLmettylerucate standardsolutionhtoseparate 25 mL planimeter or electronic integrator. drous methyl』 cohol.Clean ca l g Na metalin hexane,的 With nl_ ter paper,and dissolve in 100 mL anttdrous lnethyl acchol h 250 mL conical iask ntted with slllca gel drying mbe.
conical nasktt nttth3 mL Hlettltetracosanoate立 如dardtthtion into each nask and hexane tomake 8.O mL total volume.ItteCt2 μ L ofeach solution into gas chromatograPh.
Nonelution mtedal,%=
R22:1=(馬 私 1′ 晩 0(P24/P221) Where R22,1=Weight response factor for erucate relative tetracosancatei町 221=ng docosenoate(eruCate);町 24=mg t e t r a c o s a n o a t e ; P 2 4 = p e a tk r aa cr Oe sa aにn c a t e , a n d P 2 2 1 = p e a k docose■oate. ― Accurately weigh ca な れ ヵ な α″ど0:な。 (b)D釘 使 ″οた αc′ 50 mg oil into dry transestedflcation nask,add 3 mL methyl
to where PA'=peak areaofintemal standardi W'alld W=weights in‐ s t a n d a r d a減 n do n t9 e sr te 脚 sPechvely:and PA=total area chromatogram。
at re emaa l
Reference: 拡 OAC S8,898(1975).
tetracosanoate standard sohtion and 10 mL NaOCH3 SOlution,and renux l h.cool,add 5 mLhexane,swirl,add7 mLlM HCl,stopper, and
shake
vigorously
l
min.Add
H20
until
41.1.34
hexane
s t t c t t d n e c k a n d t t he ec xt a2 nⅢe l a y e r h t o g a s c h r o m a t o g r a p h .
Docosenoic acids,%=律 2ノP20(比 〃手 略D R221 X 100 where略 “= n g
AOAC Officia:Method 982.27 con― Polar Components tn Frying Fats
reaches
Oil.
ChromatogFaphic Method First Actlon 1982 Final Action 1984 rt/PACrAOAC Mo:わ
oど
References:JムθAC 57,1161(197o:58,488(1975).
A Prrncrpre
CAS-112‐ 85-6(docoSenOic acio CAS-112‐ 86t7(eruciC acio
Metlod assesses dete貞oration of used frying fats,and is applica‐ ble to all fats and oils.Polar components are those conlponents of
◎ 2000 AOAC tNTERNA刊
ONAL
o
AOAC OFFЮ
IAL METHODS OF ANALYSS(2000)
0 1 L S A N D Chapter 41,p.31
・
・●
▲ワ
●●
ａ ｌ ｌ Ａ Ｔ ，
f a t s dmeiに nedby coluEln chromatography underconditions sped‐ fied, and include polar substances such as monoglycerides, diglyceddes,free fatty acids thatoccurin unused fats,as well as po‐ lar transfomatioa products fomed duttng tthg Of foodsmffs and/or dwing heating.Nonpolar components are mosdy unaltered t r i g l y c e r i d e s . F r y i n g f a t s a r e s e p a r a t e d boyg rcaoPlhuym n c h r o m 江 on silica gelinto nOnpolar and Polarcomponents.Polarcomponents are detemned indirectly by subtractng concenmtiOn OfnonPolar
F A T
components.QualiけOf Separation can be checked by thin layer chromatograp町. a Appararws 協 ″. ―G l a s s , 2 . l a ) 6 θ′ and ground‐ glassjoint.
cm
id
x
45
cm,with
Teaon
stopcock
(b)TLCPrares._PrecoatedsllicagelⅢ id10utnuOrescenceindi‐ catoめ,20x20 ctt layer thickness‐ 0.25m.
1
2 1 2 1 FRACTrON
2
FiOure 982.27-Ewa!uation of efnciency of fractionat:on by TLC ceparatlon of po3ar and ROnpOiarfraction:Fracr tio■l contains nonpolar componente,and Fract`on 2 200m (a)紀 S。だ比納は一式斑lLa gel ttL paHiCle size O.063tO。 ontaine p。lar oomponents. 鋼 , M m k N o . 7 7 3 4 , o r e q u l v a l e n t , a d i u s t t O H● σO - 2 3 0 m e s h A め 20 contentof5%容 f o l l o w t t Dlr&yl主g e l コ l h i n p o F C e l a i n d s h i nC 1 6 0 ° oveni cool in desiccator to room tempe― e,Attust H20COntentめ let sample solutton ttdn to level 5 % , e . g " w e i g h 1 5 2 g s d i c a g e l a n db o8t tgo mH 2undercolulm,open 0 h 5 0 0 mstoPcock,and L r o u n d ‐ o f s a n d l a y e r . E nl Ou nにP o l a r c o m p o n e n t s w i t h 1 5 0 m L p e t r o l e u n aask win ground_glass stopper and― iCally shake l h. ehertedler(87Ⅲ 13)contボ 距d i n 2 5 0 m L d r o p p i n g f u n n e l . A d i u s t !燿江れ加″脇 ― Petrolem e臓 ぃ 40-m° ctler (b)酎 叫唯 Sθ aOw rate sc hat150 nL passes dlough cOlum within 60-70 min. K87キ 13). ARer elution,wash any substance adhering tooutlet ofcoluコ a―Analy血劇 reagent grade;pttfled by acid and m into O SCa―Sa″ r o u n db‐ otton nask widl petroleum edlertetler(87+13). calcined. a
F e a g m t e
C)"ray reagttL対 o,bdOphosphOric acid,10%in dcohol, a
P r e P a r a r勃 r a n体ゴ Wamlserll‐liquidandsolidfats toteHlpemture slighdy abovemp and mix dlorougtty;avoid overhettng.Remove visible tEttHdes by flltration;ifH20 iS presen,use hydrophobic fllter. 二 PrePararrOn Or oOrrrmn
h same mlln研 ,elute polar components into second 250 mL round‐botoHl flask with 150 mL ethere Discard silica gel.
Remove solvent from each■ acはOn widl a rotary evaporatorand 1 6 0 C° H 2 0 b a t h o r w i t h N 2 S t r e a n i n 2 5 0 m L a a s k o n Avoidlosses due to foaning.Ifrotary evaPoratoris used,shortly fore end of ev響 げ狐 On,introduce N2 intt System.Co01 residue to mbienttemperature and introduce N2 intO aask.weigh aasks.
nll cOlulnn with ca 30 mL petroleum edlerteher(87+13).PlaCe wadofcotton woolinbotttmofcolumn and realove airby pressing a catwrarrOng with glass rod. In 100 mL gtts beake,Prepare siutt of 25 g silica gel and ca C a l a l a c P o l a r o o w O r m t stPt wa/sV )pWei― h mula 80 mL petroleumether― etler(87+13)and pOur slu『 y into colunn hЮ ugh8 cmglassttmel.Rinsebeaker,funnel,andsides ofcolulnn with same solvent.Open stopccck anddrain solventto 10cmabove 豆l i c a g e l . L e v e l t t l i c a g e l b y t a p p i n g c o l u m n . Add ca 4 g seatsand through fumelinto colunln.Drain solventto where A=g nonPolar fracdon,ど =g smple in 20 nL aliquct sand layer.
=雫 規∞ Polar∞ mpone醇
(ca1 9.RepOrt result to olle decimal place.
比締紹 chromaragraphy 輸iayer Enrrcrenc/ 騨榊
見 C "鋪 「 β3 如
y T o d価 e 俺 n e p o l a r c o m p o n e n t t b y d i f f e t t n c e , o n l y n o n P o l 打 fracton is used.HoweverP if separation is controlled by TLC,botl Dilute polar and nonpolar fraction(lⅢ 9)in CHC13'Apply 2 Ⅲ polar and nonpolar fracdons are requlFed,Separadon may also be withpetroleum c o n t r o l l e d b y c h e c k i n g r e c o v e r y o f s a m p l e , b u t f o rSPOtSusingcapillattdispensingPipet.Developplate smples conttin‐ edlertCH3C00H(70+30+2)in ta■ k lined with ilter paper ing substantial amounts ofpolar material,recovery may be incom― ether― for ca 35 min(ca 17 cmp.Remove plate and let solvenl evaponte. plete because smali amounts of highiy polar material,generally l-2%,are not●1■ ted under condidons specifled. Spray Piate Wit110%molybdophosPhoric acid.Afterevap Accurately weigh 2.5主0.lg(的 0,001g)test portion into 50 mL of alcohol, heat plate in 120-130° C drying oven. Fraction l volumetric aask,and ttssolve in ca 20 mL petroleum ether― cher ( n O n p 0 1 a r j s h o u l d b e f r e e O f p o l a r Fs iu gb us rt ea 算 2n 2c 7e )s . G g を
艦色 督 Ⅲ 轍
酷陥補ξ ::協
鑑
Reference臣 炉移rr2 S修 ″Attrrを 筋 80,106K1978). !ル
20 HL sample aliquot to coluIIIn,witlout disturbing surface. Dry tw。250 HL round‐ bottom flasks in 103± 2° C oven,cool to room temperature,and accurately weigh to O.001g.Place one flask
JAOAC 64,1329(1981)i6S,375(1982). IUPAC 2.507,7th Ed. P″″ APPi Cれ を秘.貿 ,2Z陽(198分 .
◎ 2000 AOAC iNTERNATtONAL
01LS AND FAT Chapter 41,p.32
AOAC OFFICtAL METjtODS OF ANALYSS(2000)
41.1.35 AOAC Official Method 979.19 。す Methy:ene interrupted ら crs「 Po,yunsaturated Faty Acids in tDlis Spectrophotometric Method First Action 1979 Finat Action 1984 A,Peagenお ″“うo「 はこう宅βをみ-1.OM,PH 9,0.Dissolve 61.9g (a)Pο ttSSれ H3B03and 25,O g KOH inca800 mL H20 bysは 対ng and heating. Coolto room temperamre and adiusttC PH 9・O With l.OM HClor l.OM KOH,as required.Dlluteto l L wih H20 and mix. ″ みο″セ う域枠ぇ-0・ 2M,pH 9.o.Dllute (b)D】 ″夕Pοrass,″ 200 mL l.OM buffer,a),to l L with H20 and mよ .
steamofN2・ Pipetl mLalcoholicKOH,c),inmeachnask,aushwitl N2,and Store stoprred in dark 5 h or overIE"ヒ ` Add20 mL l.OM borate buffer,(a),50 mL H20,andl mL O.51M H C i t o e a c h , d i l u t e t o v 0 1 u n e w i t h H 2x0.,Bapnedt血 four 3 mL aliquots Ofeach standard soludon into test tubes,incubate,and pro― ceed asin C,ガ形セ"句筋αガθ″,begianing“ To 2 mbes(blanks)...''. Plot average A at each level aganstg μ thlinolein/mL.
i撤 >協号 枇脇鮒i船鞘総 粗瀧艦 not differ by>2.2%.
References:五AθAcも 0,895(1977);61,1419(1978).
41.1.35A
AOAC Ofmoial Method o94.15 !肋 西.一翌 )並 転 w:″!Jθ な一担Lssolve 10 ng (0と ゃ帆 材徳g sο Totat crs and rrarPetoctadecenoic tsomers lipoxidase,from soybean,50 000 units/mg(ICN Pha― ceudcals, and Generat Faty Acid Composition Inc.,Life Sciences Group,Nuttdonal Blochenicals Divisio■ ,or ln Hydrogenated Vegetabte Oils and Antrnal Fats equivalent)in 10 nLice‐ cold dilute borate bufferD(b).Refrigerated Caplilary cas chttmatographiHn陥 画 solution is stable 30 days.マ rrrわ″._Mix 2,O mL stock )Wθ たれg sθ Spectrophotometric Memod solution with 8.O mL ice‐ cold dilute borate buffer,● ).r large Firet Action 1994 n u m b e r o f a n t t y s e sb eapFeer fⅢ o m e d , d i5l,uOに m L s t o c k s oolnu は Final ActBon 1938 to 25 mL with ice‐ cold diluにborate buffeL(め .(3)あ αctta″″ sθ:″ r:θ ″.ギ ipet4mLworking ena′ me s01u敵)n,(2),to 10mLvolu‐ (Applicおle to脚鵡dly hydrogenated vegetable olls andにrestrid animl tts containing汚 %rran fatty ac赴 .Methodis not appl拒a― mettc flask and hold 5 min in boiling H20・ ble tottdrogenatedmarinecils andpartia■ y hydrogenatedish Oiis, わJた 放 碑 加滅姥 寛 力 FttL-35M.助 ssolve l.40g c)A'あ 抑 り whichcmttn large levelsofcな ‐and rra西‐ isoElerS OfC16,C18,C抑 KOIII h acoholand dlute to 50 mL tt alcohol.臨響 距 純 sh daily. and C22 Cittn lengths.) ユ PreparatrOn Or sampre 挽βTable 994.15 for the results of thedaboratory inに study sup― Accurately weigh ca 100 mg vegetable oil alld ttallsfer wih p o r t i n g a c c e p t a n c e o f t h e m e t h o d . ― ″hexane into 100 mLvolumetric aask,whichhasjustbeeniushed
with N2・ Dilutt to volume widl tthexane or acetone,Pipet i nL di‐ A P r r n c r P r e luted soluton in的100 mL volumetric aask and completely evapo‐ Total rrα“s isOmer content consists of r,α ,s fatty acids rate solvent under N2・ 船 ‐ 。Ctadecencate(18:lr);H10no‐ r西 α西‐ Octadecadienoate(18:2c, t'万 _ O o r r c , d e s c r i b e d a s 1 8 : 2 め 廊 ; ! 瑠 ,rn船 ctadecadienoate(18:2,o;and a DetermrnatrOn mono‐ぃぃ ‐ octadeCatrienoate(18:3 ccr,crc,and rcc,described as Add l mL alcchollc KOH,(d),S01utbn to solvent‐ free residue in l & 3 つ] , w h i C h o C c u r i n h y d r o g e n a t e d v e g e t a b l e o i l s a n d t e H e s d a l volumetric flask.Heat gendy on stealla bath to dissolve.Flush with animal fats. Tctal r,α ″s content is deteimined by infrared N2and h01d stoppered,in dark,mini■ lurn of5 h to oveHllght.After speC廿oPhotomety(IR)using methyl elaidate as extemal standard. saponiflcation is complete,add 20 mL l.OM borate buffer,(a),and VariorSiSOmers of18:2rr,18:2r,and 18:3r are resOlved;their weight 50 mL H20,and mtx.Addl mL O.5M HCl,dilute to volume wih percentagesaredetermittdbygtt chromatography.BasedonIRde‐ H20,and mix, temination,weight percentage of 18:lriscalculated from equ組 on. Pipet 3コ nL saponifled solution into each offour 13 x 100 Elm test Difference betteen total methyl octadecenoate(1811,assum of】1 tubes.To2tdbes(blankS),addO.10mLinactivatedenzymesolution, 18:l peaksin CC)and Calculated 18:1,gives weight percentage of octadecenoate(18:lo. (c)(3),and miX Well.To remaining duplicate tubes,add O.10 mLcな‐ w o r k i n g e n z y m e s o l u n o n , ( c )M (i 2x ) 。 by shaking vigOrously 30 ユ s A P P a r a r t J s i― ediately afteradding enzyme solution,andletali mbes standex― 二With name ionizadon detectoL ″rograPみrcc).‐ cん え,″ posed to air atroom temperature 30 min.Zero spectrophotOmeter (a)CaS at 10o.Operttng con― 2 3 4 n m w i t h b l a n k t u b e s a n d m e a s u r e A o caplllattcolumniniectiOnsystem(splitratio■ freacted oils. dttons:inJection Port 225° Ci dettctor 250° C.TenpeFature pro― For oil,calculate g polyunsaturated fatty acid(PUFA)as C,progralnrate l.0° (yttn,flna1 200° C,「 lnalhold gramiinita1 150° trilinoに in/100 g oil=WxDFxlド 胡流nal ,where witttrilim師 20 min.的 !ef Operator may change opettng conditions to obtain soludon from standard curve,and DF=dilution fltor. optimum settndon ofisomeric fatty acid mettl a PrepararrOn Or sね 何Jara Curye hydrogencamergas(299。 99%puFity)withOXygenscrubberinline. Weigh ltXlmg cれcむ‐ trilinolein,99%suCheCkPrep,POBox295, ●)CCcθ れ脇解.―-100mxO.25 mm fusedsilicacaplll町 coluIIln Elydan,MN 56028-0295,USA,orequivalenty,transfer quantittdvely coated wih SP‐256tl(available from Supelco,Inc.,Bellefonte,PA to 100mL volulnettc nask,anddluteめ VOlulne wi血″‐ 16823,USA)Or other suitable capillary column coated with hexane orace‐ tone.Pipet 10 mL aliquctinめ10D mL volumetric flask and ttlute to cyano―alkylpolysiloxane(e,gⅢSP‐2340,CP SIL‐ 88)that prOvides volume wih salne solvent.Pipet 2,4,こ 8,10,and 12 mL ttqmttinto sane elutton patteコ n as in Figure 994,lS, s e p a r a t e 1 0 0 m L v o l u I I l e t t c a a s k t t a n d e v a p oガ rみ a t.一Maximunvolume e e a c h t t d10 r yμ ttss h L,graduatedtoO.1卜 止. O CCリ g夕 ⑥ 2000 AOAC tNTERNATrONAL
AOAC OFRCttt METHODS OF ANALYSS(2000)
01LS AND FAT Chapter 41,p.33
T a b l o 9 9 4 . 1 5 3 n t e H a b o r a t o r y s t u d y r e s u k s f o r g a s c h r o m a t o g r a p h i o r i n f r a t t d e t e r m i n a t l o n oonf roaft yP aarctitda ,c3oym p o 3 田 hydrogenated veOetabl● o‖3
16:0 A
10.3
0.3
2.4
0.03
0.25
0.08
0,7
B
9.1
■4
2.8
0.13
0.25
0.36
0.7
Cl
9.7
1.1
3.5
0.11
0.34
0.31
0.95
02
9.7
1.2
4.0
0.21
0.39
0。 59
1,09
Cl and C2
9.7
■0
4.0
11 0。
0.39
0.31
0.10 0.95
D E R
10.7 9.7 10.8
■0
3.2
0.10
0。 34
0。 28
3.7
3.7
0.13
0.36
0。 36
1.01
■3
2.6
0.02
0.28
0.06
0.78
18:0
A
3.8
0.8
■9
0.03
0.07
0,08
0.20
B
7.0
1.6
3.3
0 . 1 1
0。 23
0.31
0.64
Cl
6.7
1.2
2.4
0.08
0.16
0.22
0.45
C2
6.7
■2
3.9
0.08
26 0。
0.22
0.73
Ct and C2D
6.6
2.6
4.8
0.17
0.32
0.48
0.38
D
7.3
0.6
■2
0,04
0.08
0.11
0.22
E
6.9
2.9
3.6
0.20
0.25
0.56
0.7
R
5.8
■8
4.4
10 0。
0.25
0.28
0,7
Total satlarated faw acldS A
14.9
0.6
2.5
0.09
0.37
0,25
1.04
B
17.2
0.7
1.6
0 , 1 1
0。 27
0。 31
0。 76
Cl
17.6
2.0
2.9
0。 35
0.51
0.98
1.43
C2
17.3
1.0
4.6
0。 17
0.79
0.48
2.21
Ct and C2め
17.6
1.1
2.3
0.19
0。 40
0.53
1.12
D
19.0
0.8
1.7
0。 15
0.32
0,42
0,90
E
17.5
2.8
3.4
0.50
0.60
1.4
1.68
R
17.4
1.4
3.4
23 0。
0.58
0.64
1 . 6 2
13:l rtsOrners A
4.9
5.2
範.4
0.25
1
0.7
4.96
2.1
9.5
0.32
1.41
0,90
3.95
12.5
0。 91
2.18
2.55
6.10
10.3
0.73
1.81
2,04
5.07
10.9
1 . 2 0
1.90
3.37
5.31
9.6
0。 96
2.55
2.69
7.14
1.9
7.3
0.61
2.53
1.71
7.08
4.3
9.7
0.83
1.87
2.32
5.24
B
14.9
Cl
17.4
5.3
02
17.5
4.2
01 and C2D
17.4
6.9
D
26.6
3.6
E
32.6
R
19.4
.
7
18:l o isorlers A
24.9
1.1
3.8
0,28
0,95
0.73
2.66
B
24.7
2.4
7.1
0.59
1.75
1.65
4.9
Cl
28.1
3.2
6.9
0.88
1.94
2.46
5.43
C2
28。 2
3.3
7.1
0.93
2.01
2.60
5.63
Cl and C2D
28.3
4.2
6.5
1.19
1.84
3.32
5,16
D
34.3
2.6
6.1
1,02
2.11
2.86
5,91
E
34.3
1,9
10.5
0.66
3,61
1.85
10.11
R
32.2
i9
6.5
0.61
2.10
1.71
5.88
53.0
0.2
■0
0,10
0.53
0.28
1448
41.5
1.0
1.4
0,40
0.59
1.12
1
18:2{nい 6) Ａ Ｂ
.
③ 2000 AOAC iNTERNATtONAL
6
5
01LS AND FAT Chapter 41,p.34
Tabl● 004.lS
AOAC OFFCt札
MttODS OF ANALYSS(2000)
(● 。ntrn口。り
Samplea
X,%
RSD"%
Cl
33.6
0.7
1.7
0.22
0.57
0.62
C2
33.5
0.6
1.1
0.19
0.38
0.53
1.06
Cl and C2
33.6
1.8
2.0
0.59
0,66
1.66
1.86
D
13.6
1.2
2.2
0.16
0.30
0。 45
0 . 8 4
E
1 1 , 2
2.0
2,3
0,23
0,26
0.64
0.73
R
26.5
0.8
2.5
0.22
0.66
0.62
1.85
0,25
RSDR,% 1 . 6 0
18:3(何 ‐3)
A
0.9
B
0.8
Cl
0.8
C2
0.8
5,2
Cl and C2
0.8
7.7
D
0,9
2.5
E
0.8
6.8
R
1,0
3.1
7.3 li8 5,6
9.6
0,07
0.09
0。 20
20.2
0,09
0.50
0.25
1.4
8.5
0.05
0.07
0。 14
0.20
8.7
0.04
0.07
0。 11
0.20
0.06
0.16
0.18
0.46
0.02
0.08
0.06
0.22
0,05
0.14
0。 14
0.39
0.03
0.08
0.08
0.22
19.9 8.6 17.5 8.4
18:2r tsomers A
ND°
NAど
NA
NA
NA
NA
NA
B
0.1
40.2
92.1
0103
O,07
O.08
O.20
Ct
0.2
19.8
96.3
0,03
15 0。
0,08
0.42
C2
0.01
25.0
78.9
0.02
0,07
0.06
0。 20
Ci and C2め
0.2
79.3
101.7
12 0。
0.16
0。 34
0.43
D
0。 3
25.8
100.5
0.07
0.26
0.20
0.73
E
0.03
28.5
66.9
0.08
0.20
0。 22
0.56
R
0。 1
80.0
100.7
0.09
0。 12
0.25
0.34
18:2!isorners
A
0。 3
25,8
49.8
0.08
0.16
0。 22
0。 45
B
0.7
3
55,0
0。 24
0.39
0.67
1.09
Cl
1.5
17.2
34,5
0.25
0.51
0,7
1,43
C2
■6
2.1
35.5
0.03
0.56
0.08
1.57
Ct and C2D
1.5
6.0
34.2
0,09
0.51
0.25
1.43
D
3.3
5.1
22.6
0.17
0.75
0.48
2.1
3
.
1
E
3.1
2.6
27.3
0,03
0.84
0.22
2.35
R
2.3
4.3
20.3
0.10
0.48
0.28
1
A
ND°
B
0.03
.
3
4
18:3risomers NA」 152.3
NA
NA
NA
NA
NA
193.7
O.05
O.06
O.14
O.17
Cl
0.1
8
1
136.6
0.04
0.06
0,11
0.17
C2
0.04
75.0
141.6
0.03
0.06
0.08
0.17
Cl and C2
0.04
93.81
133.8
0,04
0.06
0.11
0.16
D
0.2
55.4
76.9
0.12
0.16
0.34
0.福
E
0.2
31.7
79.7
0.06
0.15
0.17
0。 42
R
0.1
0.00
0.08
0.00
0.22
6
.
0.0
150。4
a卜晩喜 Ы endS ous闘 tt ∝ va市 Jttextrattdttm2dttmttattnesoattR瞥 付neS)diluted Httth unhydrogenated corn ;D― o前Eョ blends offat extracted from盟 冊鞘鑑営ヤ 肝‖ 占 ‖ 鞘鞘協雛濫転脚 。 (partia‖ y hydrogenated soybean soiけ
b Blind duplicates. ° NDョ “
notdetded(content ot total faty acids(0.01%).
NA i not apptcab:e
⑥ 2000 AOAC iNTERNATiONAL
線縄眠桶e
AOAC OFF,CIAL MttMODS OF ANALYSiS(2000)
0,LS AND FAT Chapter 41,p.35
30
23
ｒ 湾！ ・ ︵ 翻Ａ
“ 18 3 1 1 7 !
34
3o l 32
0S TIME(MIN)40
30
38 ° 1371♀ 40
45
Figure 994.1-18 region ofthe gaa chromatogram 的 o frt haec偽 artinl:y hydrOgen soybean t d m e t h y l e s toemr 3p竹 o13,using 100 m x O.25 inm fused stiica cap‖:ary●o:umn coated wlth SP‐ 2500.
稲樹脱群「 母 セ 麒 】 響報総ず 岩 と
ω ccaα `初 れ筋 θれ,一Ittect l-2 HL mettles俺 職 (in hexane or heptane soludon)1沌 m test sを m口 le into GC.Compare retention
t i m e s o f t e s t s a t t l e w i t h t h o s e o f C C rnegf‐ erence
n e s s c e l i s O . 1 - 1 . O m m w i t h N a C l o r K B r w i n d o w s . A l l ure i n s ttM,15). trumnts mustbecheckedforwavelength accuracy andphotometricscale ac‐ 見 r F D e rr ― n a t r O n O r T O r a r! curacy according to Mnufacturer's insmcdOns.Chart paper inust be linearin eitler wavelengdl or wave number and calibrated in ei‐ Perfom asin 994.14 Gca 41.1.36A). dler
t r a n s m i os rs i ao bn S, O■r b a n c e , A .
a Feagenrg ‐and rrans‐ CC確 ″″確 Sran協泌 .―Mixttre of cお isomers of known compositlon;available from Nu Chek PrePj lnc.,Elysian, MN5駅 ル8,or Supelco,Inc.,Bellefonte,PA 16823,USA.
t r a n g
C o n 怖
a caturarrOng
Calculate weight percentages of fatty acid medlyl este sumlng unity response factoribr each component: 晩 ,%草 Px/PT x 10tl
,PreParatran Or Mbrnyr Egrers Melt solid fats or free fatty acids at temperature≦ 10°C above melungPOintandmix.Ifcloudy,魚 11erhЮ ughniterpape■ Ifdiluted sample is cloudy due to H20,add smali portion of航 町 drous NttS04tO melted smple,mix,and let settie before taking pOrtion for methyla飲〕 n.Using ca400r500 mg fat,pFepare methyl esters as in 96933(影 を41.1.28).
where Px=GC area counts Ofspecinc methyl●
ster peak;PT=total area countt of ali fatty acid methyl ester peaks in chromatogran. caculate weight percentage of 18:lr isoHlers,Wl&ば 71&1,%=博
、はs―(1.74 x W18あ ) 0・84(M比 82,十 Wi&3か
where ttra■==tOtal rrans cOntent deに mined by IR;W18:2rr=tOtal weight percentage of江 1182″ isomer peaksin CCi 7182=tOtal 一 Iniect l-2ル methyl es‐ (a)CC PCつ rma″確 SP変 "∽ 筋 ″S・ weight percentage of江 1 182risomer peaks in GCi路 歓争 =tOt州 ters from CC reference standards,C,into GC,Select GC conditions weight percentage ofa11 18:3risomer peaksin CC:1.74 and O.84= to obtttn resolutionoflnethyl esters atleast equlvalentto thatinFig_ c o r r e c t i o n f a c t o r s f o r r r a n s , r r a n s rf ra at t ty fa ac ti td ys ure 994.15. acids,respecは vely. 二 CC Anarysrs crFatt/Acfa ConPcsrtrOrT
◎ 2000 AOAC iNTERNAT10NAL
tt
AOAC OFFICiAL MgrHODS OFANALYSS(2000)
(｀ OLS AND FAT Chapter 41,p 38
Calculate weight percentage of 18:lc isomer,Wi8:ici
6α材!わみi Scc Appendx B,safety notes on handling Of special ―carbon disulrlde.最 chendcal hazards‐ %Material Safety Data Sheets,or equivalent,for each lcagent. DispOse of waste solvents according to applicable envi‐ d l e 1 8 1 lr o ni mseonmtea rl rp ue laekss a n d r e g u l a t i o n s .
W i & l c , % = &路1 - W i & 1 ′ w h e r e佑 抑1 = t O t a l in CC.
weight
percentage
of
all
r
S22 Table 96S.34 for he results ofthe interlaboratory study sup―
Reference主 ユAθACあ ,7軌 783(1995). ユ Cれ万0閉燿rag∴sc,28,633(1990). JAOCS67,804(1990)Ⅲ 69,95(1992).
porting acceptance of dle method. A , P r r n c r p r e ln most naturally occurring vegetable Fats and oils,unsalurated constiments cOntain only isolatet ice.,non‐ coniugated,double bonds in cお con「lguEぶon.The cな bonds lnay be isomeFiZed tO rra,s cOnFlguraticn duing extracton and Processing procedures, due to oxidaはon,converslon during headng,and/or partial hy‐ drogenation,A村ml and marine Fats may∞nttnmeasmble anounts ofn― lyoccuringrran isomers.Isolatedr_bondsinlong― chAn fatty 4cids,eslers and triglycerides inay be measured by infrared spectrome的 げ.Absorption band with maximum at ca
R?yな夕か Marchゴ タ99
41.1.36 AOAC Ofrlctat Method 965.34 1301ated rrans:somers in Margarines and Shortenings inttared Spectomerlc Method Firet Actton 1965 Finat Actio■1982 Rev18ed Ftret Action 1997
966 cm 1(10.3 μ m),ariSing from C‐ H defomation about r昭,s double bond,is exhibited in specm of ali compounds containing isolated rra″ s group.This band is nct observed in spectraofcore‐ AOCStAOAC〕 brnc」 sponding cおand saturated compounds,Measuremeitofintensiけ l ttsお sorption band under analyncally Controlled conditions M e t h O d i S a p p l i c a b l働e 耐胡 t o 徒o n o f i s o l a t t d r t t b m d s i n n a d mof is the basis for qumtitative method for deten口 Ination of isolated 研 Focessed longttn wids,esm,andtFiglyuins widlr万 船 levels ! ″酒 C O n t e n t . F o r h i g h a c c u r a c y , c o m m o n i n t e r f e h n g a b s o r p t i o n s t L c aObnlley owriお 挙. 磁. M e t l o d t t n o t atが航 h specinc時 associaにd widl glycerol backbone cf triglycerides and carboxyl
c a u t i ofnastめ s a n d t t s c o n t a i n i n g げc l toen iqい mtidtt tOVer5の groupoffatty acidsmustbeeliminatedbyconversionofthesesaEl‐ .g,■ コじOiめ ;tO materials containing mdiOnal gated unsattmtion● JeSめ their metttl esters prior tO anttysis. ntensity ofC‐ H defomation aboutr″ 附doぃ groups which m田 町戸 丘 APParattr3
blebond[e.g"Castoroil containing五 cinoleicacidoritSgeometrical
isomer,Hcinelaidic acid(12‐ hy仕oxy‐ 9‐ octadecenoic aci● ];to nixed glycerides having long and short chain moieties(e.g,, diacetosteariゅ ;Or in general,to any matenal contalttng consutu_
辮絆讐書 艦鞘輝盟濫艦輪能 齢群総 品 966 cm l band of C‐ H deformation ofisolated rran double bond, For direct analysis of triglyceFideS use procedure desc減 脱 dh
!骨 麟離盤選 総1臨群摘辞 紳 齢 艦 結: : 憾 ぷ8 冊 艦 品 艦 鞘 ミ SPeCtrometers should employ TCS or DTCS detectors orinsure lineariけ .
965.35 ts22 41,1.61].)
Tab!e965。 34 interlaboratory sttdly resu!t3fOr determinatlon of fnn3 380mer3 by infrared 8pctrOmtrlc method・
__ No,oflabs ５
Sample
_Mean,%
６
Metl」
６
Tg‐1・
５
Tg‐2′
Tg‐4g ６
Tg‐5角
T9‐6′
_上
4
宮
_
◎ 2000 AOAC tNTERNATfONAL
RSD..%
RSD。
.%
ヤ
R・
1.30
2.40
6.42
1.37
3.64
6.09
0.22
0.60
3.65
9.82
0.62
1.68
1
0.18
0.44
13.86
0.20
1.19
1.21
7,38
16.02
0。 38
1.07
2.範
6.69
1.09
3.00
30.91
0.46
1
6
1,49
6.03
1.29
5.21
1.22
1.71
2.59
2.27
3_42
.
3
3
.25
0.81
Based on coWaborative study resuRs ofthe rnodi“ed method. D r‐ 2 0 x sr ・ R=2 8x sR・ 」 Me,1‐ Methyl elaidate‐ memyi stearate with 21.lSん rrans. ・ Tg‐1=Trielaidin in zero‐ trans peanut oll with 6.14,6材 ans. ′ Tge2‐ Canola o引 。 TO‐3 and Tg‐4=ShoRening n T9‐ 5ョ Margarine r Tg,6=Par fry o‖
sn
0.49
16.14
６
Tg‐39
Sr 20.23
.
8
32.78
0.50
1.23
0.56
3.33
`
AOAC OFRC'AL METHODS OF ANALYSS(2000)
01LS AND FAT Chapter 41,p.37
E c a r r D r a t r O n (b)rnrra″ ″ ど 竹″泌 M印 !れgα !な.―Equipped with NaCl or KBR windows and flxed Pah lengtt ofl mm.For use in null‐type For each standard soluは on,calculate the exact weight Of medlyl insmments,餌 rs of Cells matched to wittn O.01 absorbance unitselaidate diluted in 10 mL solvent.Analyze each solution and deter‐ are required.In spliい bean type lnsmments widl both cells illed IIllne baseline corected infrared absorbance at 966 cm l as de‐ with solvent,electronic balance of 2 beams to within these limits s c b五e d i n C a n d H . should be attained. (a)乃 r勉 酒 働 確 所 10%),Calculate weight equivalent of methyl elaidate h smple as ●)確 的 !挽 協 確 Sracた ざθJzr助 .-20 mgrmL.Accurate呼 weigh ca 2000 mg nchyl elaidate tpurity刃 follows: 9%)tO the nearest
Methyl elttdate,g= Ac―
intercept
(O MCttr dca郷 成 鋭 施 卜 刻 理 価 β wareas h● )us‐ i n g m e h y l d e a t e t p u r i t y p 9 9 %週 ) h SO に fmehyl daidate. 規 ∞
RepOrt
results
to
the
Reference革 泌 θAC 48,437(1965). AOCS Methods Cd 1461如 R夕νな夕な ル灯 αrch F998
⑥ 2000 AOAC,NTERNAT!ONAL
O。 l mg hto 100 mL volumettc nask,dluteto volume wih CS2 SO‐ lution,and Hux thoroughly.
nearest
O.1%.
d Cd 1495.
う 白 何jθ ″Jθ !″ 』 わ 明 じ.判 .8,1.6,4,8,12,16,and20 mg mdlyl (d)例 ど elaidagmLin 19.2,184,1612,8,4,andOmynLmethyloleatWS2 solution,時 s「湾tvely.Accurately add l,2,5,lα 15,2Q and 25 mL
methyl elaidate stock solution,(め,intO sepane 25 nL vol― tric nasks using plpets.Dilute contents of flasks to volume wm mthyl
oleate stock solution,c).VeriSWdghtperntagesofmethylelaidate andmthy1 61eate by ttPlllatt GC analysis ash卿 41S G2241.1.35A). Iweightpercentages ofmedlyl elaidatedfFerl沌 mexPected valuesby 汚 %KfOrsmplescm成 拭ngpercentmdlylelaidatejor混%tfOrsam‐ 10%mehyl elぶ dateboF>2%(foF― pleS containing ples cOntainingく
AOAC OFFiCiAと
METHODS OF ANALYStS(2000)
0'LS
AND
FAT
Chapter 41,p.39
on ofisotated rrang unsaturated faty ac旧 8 3n parttatiy Table 994.14 8nter,aboratory study resuR3 rOrinfrared determina‖ hydrosenated vesetable oti3 Samolea A
x.%rra,s 6.2
RSD,1%
%
RSDR。
S,
SR
r
R
4.8
34.6
0,3
1.8
0.84
5.04
B
15.5
4.2
11.3
0.7
1.8
1.96
5.04
Ct
18.9
4.6
33,7
0。 9
2.2
2.52
7.06
C2
19.1
3.7
10.3
0,7
2.0
1.96
5.49
Cl and C2b
19.0
5.8
10.5
1.1
2.0
3,07
5.61
D
3 0 , 1
3.0
0,9
2.7
2.52
7.06
E
34,6
1.0
0,3
3.9
0.84
5.04
21.6
4.1
0,9
■9
2.52
R
9,0 11.3 8.8
l 」 穏i嫌 艦側洛 博艦紺艦蹴描隅鮎肥晋 驚認群 堺齢隅 淵鍋品 緒 結 甜 描 掛 erence sample(pa'tatty hydrogenated 30ybean 80iけ
襴
7.06
群挫甫_
1 6ateS B‖ nd du伊
ons.PerFom >107o ndhyl elai的 め,Prepare ttsh calibralion sol岨 ofCS2・ step E l―edately because ofhigh volatiliけ
at 967 cm 1.This can be Obtained by supenmposing 2 spectra to draw basd施 .) Draw anothersmighthnePdLitottnateandpassinghough , P r e p a r a r f O n O r T e s r 軸″ apex ofanalyttal band asin ngure994.14.KLine meets zerolineof M e h s o l i d f a t s o r f r e e f a t t y a c i d s o n s t e a n b a h o F i n Ochat V e n aatt tpoint e m ‐ z apeX at 4 and baseline tangent at X.) For transnlssion spectunl,measuFe dStances XZ and yZ Calcu‐ above meldng ttint.r melted fat is doudy,81ter p e r a t u r e 1C 0 ° 述 山 o u g h n i t e r p a p e r e U s h g c a 4 0 0 - 5 0 0 m g,的 p r e p a r e m e t t l e s t e r s lateおsorption of cd厳 on solution,AF
ル:Accurate results depend on ttmy asin 9ゆ 節 ●解 4 1 . 1 , 2 8 x 距 Ai i log tXZ/海 ら of fatty acd medlyl esters.BefoFe R analysiS,remove excessive uSing levels of impurities[etg.,nonsaponiflable matter,polymers〕 To calculate absoFptiOn of calimtionlutioL s。 At,read Ax at X, suitablecleantuppFOCedurete.g"SaponacatiOnfo1lowedbyextrac‐ andAY at比 layer or column chromato控 don of nonsaPoninabL matter,hill‐ Ai=AY… Ax 1 urately wdghca400-500mgundilutedfatty acidme的 phy].)A∝ l mginto25 nLvolumetFiCaaSk.Dilute esにrSurl)totlenearestO。 ユo t n g n e t t l e l a i d a t e r m L c d i b r a t i o n s o l u t t l t t a b s c tovolumewithCS2S01uticn.PerfomstepEimmediatelyttcauseof sus coHeswnding Ai vttues as ordinate.Draw bett smight line high volatility of CS2・ mugh 7 pOints Plotted.For better accuracy,detemine linear re‐
丘 rfPヵ コ胡 DerermrnarrOn
● ０宮 口０ と０ ●ａ ｔ
辮灘 酬 胡瑞 撒電性 選認酬 群雷
〓●●﹄ ●ュ︶ｏｏＣ８一 一 ” ︵ ■Ｅ”宮８岸
Fill cell with CS2 SOlution,and mtthing cen widltest smple fFOm D or cdibration solution ton C(0.USe hypodemic syinge witlblunted needle,andcen upnght,lnJect tombottomsobubbles beam lR,place cell wih pass up hough ceu.when using double‐ CS2inreference beam.Place cdi wihtett smple oFCaibratiOn so‐ lution in sample beamo Scan spectrum(T or A)fron 1050 to
md store itin memoFy OfinstruHlent datahandling system.Scantest smple or calibration solution in same range as that for reference. R江loobtainedspecmmagttnstbackgFOundSpecmmtoobtainme on soluは cns in oFder Ofincreas― ror A,Measure spectra of calibraは lng concenmiOns. 見 carctrra〔 腕 s
For each specmm,draw basdine tangent to c e n t t o 9 6 7 cFml gl唖 G9 ″ 9 4 . l o o″:oIMtθ i s i m p o r t a n t t o d r a w
peak
ttnina
tta‐
corect baseline because of Hleasurement of basehne corected ab‐ ,0● 0 00● sorption.Absorption minima mightslightly vtt between smples. Frequency,cm‐ ￨ Concentration andamounlofrra,sunsaturationmay ini■ enccPosi‐ tion of absottton ddm Bestresults are obtained when baseline F i g u r e 9 9 4 .J1R4 卜 spectra cf paRialty hydrogenate forsampleisdrawnexacdy asbaselinein specmmofOneofcを 出敬竹 tion in CS2D・ c a n o 3 a o l l m e t h y : e %s ●o t e r:su像 tion standards having approximately same intensity of absorption
0 2000 AOAC tNTERNATiONAL
0にS AND FAT Chapter 41,p.40
AOAC OFFiCIAL METHODS OFANttYSls(2000)
ttf Onceobtained,calibratloncurve ltdatat(Nθ gression equationto「 does not have to be repeated aslong asinsmmentsettings,parts,or cells have not been changed.)
is used,it must have linear resPonse With adequate sensidvity and satisfactory baseline correction.
where〃 1=amount tion,Ing.
a PreParafrOn OrsarTPre
a Feagearg D e t e m i n e a b s o r p t i o n o f, tA e8 s' tu 唱 sS a山 s ma pm に e procedure as as.二He(prefered for betterresolution and coluIIln (a)6α rrJ2,ど forcalibration solutions.Usingcalibrationcurveorlinearregresslon l i f e j o r N 2 d r i e dn ianngdζ 1 0c omngt ぶ Ong. equaton,deterlmne aIIlount M2)。 f mg mehyl elaidate/mL test g いc s , 一 H 2 , 9 99 ・ %free fromorganic impurides.Airor ( b ) O r Lr夕 sample soludon,D,coresPonding toA3・ CalCulate peFCentrratt un‐ 2 ppm hydrocarbons equivalent s a t u r a t e d f a t t y a c i d c o n t e n t a ss t sm me pt lt α l e l a i d a02,free t e i nfrom に organic in,wittes(く tO CH4)・ %,″ 酒 unsaturated fatty acid content(as Elethyl eladate) 鶴 確 s仰 あ ras._Mixttre ofcお andrrarumehylesters oR瞭 =100x財 ノ〃1 of known composition close to that of marga口 nes. of
fatty
acid
methyl
esters
in
″
solu‐
互 OC OPrarrng cOndfJong
Rビッなタ ガf拘脇rch r999
Carrier gas flow depends on carrier gas and packing density. Following aow rates are recommended:He,15-20 mlymini N2' 8-10 mL/min.Flow ofH2tO detectorshouldbe halfthatofcarrier
41.1.37 AOAC Offtcial Method 985.21 Totatrrans Faw Acid isomers in Margarines
gasi now ofo2ShOuldbe ca5-10 dmesthatofH2・ ItteCtOrshould b e 斑2 0 - 5 0 ° C higher ttan colulm temperamre and detector should be at 250-300° C.
Ca8 Chttmatographic Mcthod Ftrat Actlon 1985 Final Action 1992 (Not apphcable to margttnes containing hydrogenated marine oils.) A
sample
lsolate fatby 118●夕 92仇 を33.6,07).Using ca 350 ng fat prepare methyl ester by 96,。 3 3(s2241.1.28).To methyl esters in stoppered flask,add volullle ofheptane to yield l-109ら solu‐ glass‐ はono Swirlto dssolve.
Reference臣 ユ Cれ″初aragぇ 最克 28,633(19901.
JAθ6S 67,804(1990j;69,95(1992). ユA θA C れ , 7 軌 7 8 3 ( 1 9 9 5 ) .
test
P r r r r p r e
Methyl esters of ttty acids from margarlnes are separated and measured by CC to detettnetotal,脇酒 unsamrat10n content(rrans isomers ofunsaturaにd18 C actts).Resultsby this method are com‐ 夕41.1.3o. parable to dlose obtained by IR medlo4 96534●θ
見 6C Ph所 口研旧礎 う口鈍W化回敵閉じ Perfomanalysisofmixtureofmethylelaidateandmethyloに ate, Adiust methyl ester amount,coluHBn temperamFe,and carrier gas nOw so that nettl elaidate peak is recorded ca 30 min aneriniec_ Hon and方 ヲ full SCale.Measure base widths in mm of medlyl elaidate(,1)and Eletlyl oleate(w2)OetWeen Pcints ofinteFSeCは On wldlbaseline oftangents drawn tO innecは 。n pOints ofcurves.Also measuredistanceinttbetweenpettFLaXimumformedlylelお date and metlyl oleate,比 Calculaにresolution,R: R=
ユ A p p a r a r t r s (a)CaS Cれ ″肥 rograPれ.二With flame bnization detector and minimuln dead sPace in inJecはo■system.Maintain column tenper‐ ature within±1°at ca 220° C. _6.lm(20 ftj x 2 Hlm id giass or stainless sに (D ccr″ れた。
Se19ct conditions to obtain R≧0.5. a Det_rnarrOn
el
tpOlyunsaturated components with>3 double bonds my decom― pose in stainless steel columR). 帆 (C)Pa枕 れg.― Acid‐washed md silanized diatomaceous e卸 lα卜120 mesh,coated with 15%OV-275 stationary phase(15% OV‐ 275o■ 100r120 mesh Chromosorb P AW‐ DMCS is available fron Supelco).OV‐ 275 col― s are extremely sensiは veto 02,par‐ dcularly at operating temperature.Carier gas and alllines must be free of02・COnditionnewcolumnsby purgingwidlc狐 】ergas over― nigh at room temperamre and then gradually increasing tempera‐ ture(1-2° 9航 n)tO temperature 10-20° C higher than ope劇 tng temperame but nO hgherthan recomended HElitS.Ifcolumn h岱 been lcmoved fFOninSmmentOF eXpOsed to air,purge coluEm wih c2田ter gas for l-2 h before heating. 一 Maximum volume 10ぃ O rSyringを 。 ,gFaduated的 0.1ル . C)R2Cθ rダタ∴-0-2.5 or5.O mV range,く 1.O s response rate(dme for pen to pass froln O to 90%following momenttty introducよon of 100%signal),25 om mininumpaperwid曲 けand25-100 cm/hpaper sPeed;equipped withattenuatorswitch to change mge.Ifintegrator
◎ 2000 AOAC iNTERNATtONAL
2y 路 ヤ晩
With apparatus showing stable baseline,lnJect O。1-2 μLl-10% hepme s01uttn of nethyl esters.Change setting of attenuator as necessary tt keep peaks On chart paper. Analyzereference standardmixtures undeF Same Condidons as for sample.Measure retentlon distances of known esに rs and identify peaks from sampに by comparison witlretention distances ofpeaks
from standard mixttres.ngure 985.21 」 shows aけ _ ca ChrOmat。 graPhic separation of mehyl esters ofmarganne fatty acids. 胤 carcuratrOn3
Use mehod ofintemal standardization(■ omalization),whch assumes all components of methyl esters are FepreSented on chroHlatogram,so that sum of areas under peaks represents 100% of constituents(tOtal elution).Obtttn total rran cOntent by addi‐ tion of ali rra,s isomers. ,PrarsrOn (う Re陣 腕
】り .―TWO Sirue deteHninations perfomedon sanrle
(
day W Sane Operator wm salne appmms on sane smple foFtOta r 脇な c o n t e n t ( 1 0 - 3 0 7 o ) s h O u l d n o t d f f.e6r pbeyl対 centage umt.
AOAC OFF,C,Aと
0,LS AND FAT Chapter 41,p.41
METHODS OF ANALYS,S(2002)
ration du五 ngextractionandprocessing procedures,as aresuit ofox‐ idation,converslon during heating,and lttdal hydrogenation.Ani― m a l a n d m a r i n e f a t s m na y m ec ao sn ut rぶa b l e a l n o u n t s o f n a t u r a l occumng rran fとtty acid geomenc isomers.Isolated′ ratt double t t t y a c i d s t, y的 a c d m e h y l e s t e r s ( F A M E ) , b o n d s i n lcoMnng ‐ SOaPS,and triacyiglycerols may be measuredby infrare troscoPy.A unique absorPtion band wih amaximumatca966cm 1 S i n g f r o m aH Cd― e f o m a t i o n v i b r a t i o n a b″ oざ ut a r負 ″ ( 1 0 . 3 1 ,t 宜 Iゅ double bond,is exhibited in the spectraofali compounds contぶ mng ″sgroupittsbandisnot6bservedinthespecraofthe an isolated rttα
c o r e s p O n d i n g s a tuunrsaatmerda taendd fcaお tty acids nsity of tlis absorption band detemines total iso― ment of he inに lated仰 筋ど f a t t y a c i disst ■ not necげ e stsoコ c O n v e n f a t a n ds to i l に samples to FAME before analysis, This method is not aPplicable to fats and olls contalning>1%。f COniugated unsaturation(e.g.,mng Oil),tO materials containing funcはonal groups tlat modify dle intensity of dle HC― deformation vibration about tle r,節dOublebond(e.g.,Castoroil whichcontAns hydroxy fatty acids),or,in general,to any matedals contaittng con‐ sdmentsthathavefunctionalgroupsthatttvedSetOspecincabsOrp‐ tion bands at,or sufrlciendy close to interfere widl,dle 966 cm 1 3脚 m)band Ofthe C― H defomation vibration of tle isolated (10。 rra2,dOuble bond. a
A p p a n f u s
(a)Fθ ″rigr F■ 弱 o閉 ボ fatt rrrR,sP2は 的確 統 一Resolぃ insm‐ tion of 4 cm l in dle spectral range of 1050-900 cm he 1.■ ment data handling system should pemit conversion ofdle spectra to absorbance,scale exPansiOn of the x and y axes,readout of Time wavenumbers to the nearest i cm l and absorbance tO dle nearest on of Figure 985.21-Typica:chromatogmphic sepam付 O.0001 AU,and integration ofthe area under the absorption band at m e t h y t e e t e r s伯“ oy 「a c i d s f r o m m a r g a t t n e s . c = c r S , t966 = cm 1.The F「 IR spectroEleter is equipped wldl a deuterated 18:コL18:2ctや t and rrans.Notshown are componen低 n g l y c i n e s u l f a t e ( D T C S ) d e t e c t o r f o r 20:0,which e3ute bemen 18:lc and 18:2cc.
.―TwO Single deteminaは ons perFomed in (b)Rマpr`ガ距:所!:ゥ different laboratories for total rraな content(10-30%)shOuld not differ by>1.5 percentage unil. tt Soc.54,279(1977). . References:ユ Aれ θ】Cたβ
JAttiC 68,弧 1985>69,247(198o.
AOAC Ofnciat Method 2000.10 DeteHnination of Tctaitsolated tans unsaturated
Faty Acids in Fats and Oils ATRfTlR Spectroscopy First Action 2000 (ThiS methOd is applicable to namal or prOcessed ons and fats conSsは ng oflong,chAn fatty acids,esters,and triglyce拭 des wih r″四 levels of≧5.0%.) Sを2 Table 2領対.10forresultsoftheinterlaboratorystudysupport‐ ing the acceptance ofthe method. A. Pガ ,cripre
線 滞 だ脳 謄 温 古鮒 縦 鞘 ぷ 認 o f ≦0 . 0 0 0 5 A U . 陀 コ966cm l bandfora l%trielaidinKTE)standard must yield a signal― to‐ noise ratio(S/N)of>10:1. ど ゎね′Rヂ 2c筋 4 rATR,苺 こ″ど ご夕″.一With a (b)Ar筋 ″arを ZnSe(Orequivalent)cryStal.Thecapacity ofthehorizontal ATRcell isca50 μL,andthe cell mustmintainaconstantに
mperature of65土
2°C.
はngO.3±0,0001g. α配材協,c2.-60gcapacity;wei」 (c)A,αウrた ― ・ For transferFing 50 11L test ωatt p脇豆cP"2お (d)Dゆ
41.1.37A
g
portions to the ATR cell. (e)S=“ "Wα 姥″うα統.一For Hleldng fats. a Reagents TE and宜 olein(TO)Witl p航 ty of>99% Pri艇 ヮ sra取ね必 .一」 (avalぉ le from Nu‐Check Prep,Inc.,PO Box 295,Elysian,MN 56028). ,Prepara″
o何0′rans carrb胸 何。打Sね ,JarJs
Weigh,to he nearest O.0001g,(0.3-メ 10 mL beaker, whereメ
)g To andメ =TE into a equals O.0015, 0.0030, 0.0150, 0,0300,
0.0600,0.0900,0.1200,andO.1500g,to prepare O.5,1,5,10,20,30, 40,and 50%r万 御 s calibration standards,respectively.
i cattb角 ″o何 ln most naturally occumng vegetable fats and olls,unsaturated constituents contain only isolated double bonds in the c,s conttgura‐ For each rttα な standard,calculate the exact%r汚閉じ,exPressed as に the inに‐ tion.Thesectsdoublebondsmaybeisomenzedk)the rhaれ S COnflgu‐ TE,%of total fat.Analyze each standard and detem曲
◎ 2002 AOAC tNTERNATiONAL
01LS AND FAT
AOAC OFFiCIAL MEl情
ODS OFANALYS,S(2002)
Chapter 41,p.42
Table 2000.10. rra,s Lovei3 0ftrietaidin in cotd‐
b M M u にN α
Addet%
1
pressed,bl● ached,degu口
Mea吼
%
0.8
Ret
t
o,8
wned 30ybean oila
RS既
%
RSD島
%
100
75
21
100
105
29
102
2.3
`′
HORRAT 5,0
2
■0
3
5.0
4
10.0
10.3
103
36
51
5
1540
15.6
104
22
3.3
1.2
103
4.5
50
2.0
100
3.5
3.5
1.5
6
20.0
7
___
400
1.0 5,1
20.6 40.1
7.3 3.1
1.0 1.8
PreaS30n 8
0.4
11
68
9
0.1
56
134
10
39.6
11
0,1
12
2.1
4.5
13
14.7
14
1 . 5
15
23.7
16
203
3.4
16 2.7
5,3
0.6
4.0
5,9
12.4
2.8 1
1.5
143
.
1
■7 1.5 3.3
4.2
1.7
4.7
1.8
23.7 The nrst 7 mixtures(Nos l through 7)are‖ Sted in Tabte lぃ ,AOAC加 484,1147(2001)〕 in the same sequence but under dttrent namesithe的‖ owing 1 0 m l x t u r e s ( N o s a t h r O u g h 1 7S )t ae rd ei ‖ n T a b l e 2 A OμA C r n 4 8 4 , 1 1 4 8 ( 2 0 0 1 ) l i n t h e s a m e s e q u e n c e b u t u n d e r d i r e r e n t n a r r l e s A c c u r a c / s t a n a」βs T s e v e n p a t r s o f b t t n d d u p : i cコr a t ea cayαs t a n d a r d s ( M i X t u r e N o s . 1 - 7 ) c o n s i S t e d porfe scsoeldd,‐ bteached,degummed sOybean ol and 二Ten pairs of bttnd dupttde(unknOwn)test Samp3es(MiXbre Nos k n o w n a m o u n t s o r T E T h e s e s t a n d a r d s w e r e p r e p a r me ed t rg kr 海 a!萌l y . T e s r sぃ a品 、
′
8 - 1 7 ) c o n s i S t e d omfm∞ ercial vegetable 「 obnlse。 n d e t T h eu ie 『 nmes were・ r e s p e c t i v,e駅 け 〕 r a p e s e e d io ‖h y d r o g e n a t e d s o y b e a: np a ol ‖ y b e a n oi出 m kernel ol,i a blend of soybean oit,cotonseed oil,and hydrogenated scybean olli a Palrn blend kemel o「 oil and hydrogenated soybean oili 2 blend3 0f SOybean di and h y d r o g e n a t e d sio yab ebalne nod‖ o f r a p e s e e d a n d h y d; ra on gd e ns au tn en d何 Oゃ w se or y。b e a n o ‖
、 、 ′′ ′，︲︲・ヽヽ
grated and H.
area
underthe
absorption
band
a t fledmaterial 9 6 6 c n l forr惚 a s″s‐ dfo■ e sifledに c r i bstsamples,and(3)an ed h G appropri_
ate rra″ J‐ free lnaterial such as the refined,bleached source cil for Using a rlrst_。 rderregression analysis,det銅 ne dle slope andin‐ test samples. tercept Ofthe line that best fits the plot of tle area of dle r比 材ばband Using adisposable Jpet,transfer(WihOut weighng)ca50 μ f o r a l i t hぇ es rs″ L of tandards(y aXiS)aS a funcdon 府 O f(%xr 昭 axiS). the neat(undluted in any solvent)reference background matehal. Once a calibration curve has bcen established,check it pedodcally Place he reFerence background mate五 al on dle horizontal(faCe_up) to ensure hatit has not sMfted, ZnSe sampling surface ofttt ATR cell.The test on pol■ mustcθ加_ 見 Prepa臼すo"of resr sampres llect and save pた セry cOver tle horizOntal sface of dle crystalt C。 Melt sohd fats gently on steani badl andx.Treat Hよ test samples t h e s i nb ge la em ‐s p e c m m t O b e u s e d a s b a c k g r o u n d . C l e a n thatappearcbudy because ofthepresence ofwaterwith ttdrOus tal by wiping Offthe test portiOn with a disposable soft lint,free or Na2S04 untilthey are clear and dlen ilに r. low‐lint tissue paper.In general,to IIllnimize concalnlnation,apPly 6わ
frareJ De絶口研す ,atron
he
St pO■ l on,and tlen wiPe it offthe crystal before re‐ partOfthe nextに a p p l y i n g t h e Lc a t e5 s0 t μ lpoon■ f o r a n d y s i s .
S e t IuRp otpheer aFt「 ing Parameters according to the 鴛 梢譜館:瀞 瑞着挑鮒ぼ器】 盟鷲総 httzontal ZnSe cryst』 播品糧 .It must∽ 姥セリcover the surface ofthe 々β 盤縦i楯 鮮品 総嫌盟錯温樹濫 芦群先路 鰐継告 総批鮒盤出 麟器艦 鞘樹鮎脳ざ t群 緒鷲!i跳 縦塩ゴ 撚 触縦 増嶺督甜総ぶ 路 盟艦 鑑濫撚訳盟 s艦 濫損 艦 fats,maintan2° the ATR響齢 cell at 65± C. Place
c a L5(0w iμ thOut
weighing)ofthe
neattest
crystalo Collect and save the single― bcani spectrum of the test w t i o n . Rl■ o t h es に t s a m p l e sbienagml es‐ pecmm againstthat reference background,and convertto absorbance.Save the abso中
portiOn
ofthe
tion spectrum.Repeat for otherに stsmples. 比 carcuragons
Materials for measuring:he reference background single‐ beam spectrum are(r)To for calibration standards,(2)the unfOrti‐
◎ 2002 AOAC iNTERNAT10NAL
偽 late
the
襴 linear
ふ 萎 麒 鮮 r e g r e s s 1 0 n e q u a d o n亜 f op rl to ht e o af rt eh ae v s % 貯
r″閉 じcalibration standards.
on
t
AOAC OFFICIAL METHODS OFANttYS崎 (2002)
0にS AND FAT Chapter 41,p42A
Using the slope and intercept generated for rratt stttdards,cal‐ umnis heaにd at analyHcaltemperature(ca 130r135° C).AdiustCar_ culate dle%r″ 円s fOr test samples by substimting the value ofthe der giass now sO thatretention time for buけ Hc"id iS Ca 5 min.) Peak taling,even aFter column is conditioned,can somedmes be intcgratcd area of the ,s r招band in the fouowing equationi reduced or eliminated by inieCtiOn 2 μL ttmethylchlorosilane 何 MCS)OntO column` rrans m益 班 ,%=響 a Reagて 加ts Report results to dle nearest O.1%. References:の c朋 拘修伽 αtt R釘Om2滋 Pttcrた容 (1999) 5h Edt,Amedcan Oil C陀 口Ists'Soctety,Cham‐ paign,IL,Cd 14d‐99. 泌 θい 7 7 , 4 5 7 - 4 6 2 ( 2 0 0 0 ) . ユAて弘 Cあ r.MⅢ l144(2001).
41.1.38 AOAC Omclat Method 990.27 ButyrSc Acid in Fatt Containing Buttettt Cas Chromatographic Method F↓ RBt Acuon 199o Fina:Acuon 1993
閉 りかoメ f』 2 sθ r 24だ 例.― CaO.5M in edlanol.Dis‐ (a)ルrtts筋 solve 4.5 g KOH in 100 mL etlanol.
‐ -5托 ぃrnaqueous solution. 脇θ ω ο wたoric ac広
ね材 sθ防 わ″.-0.4 Elg/nL H20・ Dis‐ 律)ル β24yric actt sra取 solve 400 mg 4‐ buty五c acid refeFenCe Standard in 100 mL H20 and dilute 10 mLofttts soluは onto 100 mL wih water.Soluよ onmustbe freshly prepared. ‐ v : 山能 抵」 乱 H 2 0 ・ 0 五 確 m a r s 胸滋 材 w : “, :Lο―対 . 2 5 m g ″ ― Dissolve 250 mg″ valttcacdin 100nLH20anddlute 10mLoftlis so皿 onめ 100 mL witt wtt Solution mustbe mhly prep孤 迅 ,PrepanJ〔
加 orSね ″JaH curve
Use graduated Pipets to transfer to indvidual test tubes O.2,0.5, 1.0,2.0,3.5,and 5.O mL podOns Ofbutyric acid smdard sOludon,
C(C).TC each test tube,add eric acidmal inに pet 2.Obyが mLvお
stttdard solution,C(d),and,Iespectively,4.8,4.5,4.0,3.0,1.5,and O mL H20・StOpper test tubes and gendy mix each solution.Solu‐ dons in test tubes contain,resPectiVely,0.08,0。 2,0.40.8,1.4,and 的 n of butyric acid content of milktt or (Applicable to detemi田 2.O mgbuけ Hc aCid and O.5 mg valenc acid.
rUPACtAOAC ttettoJ
butterfat oF ttXtures of fats contalung milkfat or butterfat.)
Stabilize column ≧ 30 min at analytical temperature (ca 1 3 0 - 1 3 5C° ).USettcrosynngetoiniectCalltLaliquctsofPrepare
Resulいofthe interlaboratory study ngsuppo対 he acceptance ofthe s t a n d a r d s o l u t i o n s i n t u m . M e a s u r e p e a kc ha en id g h t t method: valedc acids tonearestO.5mm.PlotratiosG旭 けricaCid7Valericacio ゴ0 0 % β ″r r g t t r vs corespOmよ ngweightsofbuty=c acid.的 ref Adiustmplinerat_ s,‐0,104i sR=0。242:RS,=3.0%;RSDR=7.0% t e n u a t i o n s o t h a t p e a k h e i g h t f o r b u t y i c a c i d f o rHhCg haecsitdb u け C確伽 朝宅を 確うル】 うLc配効 比 α防 ″,5θ%所 rTar standard is ca 80%fuli scale.) sr=0.043;sR=0・161;RSDr=2.4%:RSDR=9・ 0%
R研″ど確肋ツα加″あ比 5%あ明rr2rar
f
E DeremrnarFon
=0.008t sR=0・ 024;RSDr=4.5%;RSDR=13.1% s『
Accurately weigh ca loo mg test pOrtion into 50 mL beaker. Pipet3 nL ethanolic KOH solution into beaker and add a few glass beads`Coverbeaker with watch glass,PlaCe on bolling water batl, F a t i s s a p o n i n e d w i t h K O H s o l u nt et do n w ia tn ld d l e n i s a c 拙 and heat≧10 1nin or until fat globules are no longer visible on sur‐ H3P04 tO liberate fatty acids,Water‐insoluble and water6soluble Face.Remove watch glass and continue heating until ethanol has f a t t y a c i d s a r e 1s 観 e瓶o p an r. aB tu HeけCd ab cy i品d ri ts t nd ee dに a s complecely evaPorated.Let beaker cool.Pipet 5.O mL H2()intO t h e f r e e a c i d b y g a s c h r o m a mt ao lg r sa tp ah ny d au rs di .n g a n i n に beaker,cover with watch giass,and gendy swirl beaker tO cOm‐ B.Appattttrs pletely dissolve soapt lf necessary,warm mixture gently to cOm‐
A , P r r n c r p r e
_lo mL with grounい (a)確 Sr配 な 。 giass stoppers. (b)P,2rs.traduaに
42-5 mL.
(C)材 軌 り 勅 呼 .-l μ L.
plete diss01ution,Pipet 5.O mL H3P04 S01ution into beaker and gendy swiri to coagulate precipitated fatty acids.Filter solution through small,auted,rapid paper.Pipet 5.0 1nL iltrate into test
tube and add by Jpet 2.O mL valedc acid intemal standard
tion.StOpper testtube and mix contents. (d)CaS Cれ ″"α!θ grapれ.―With nalne lonization detector, 01血 ■analyticaltemperamKca 1304135° o. on‐ coluEm or all glass iniectiOn systett and record駒 e F湖 , ize coluHHl鴻 UsemicrosyringetoinieCtCa l「 止 品nalsoludonontocolumn.Measure -61ass,ca21n x3 mm id,魚 1led with 10-15%sta‐ (e)6θ 肋 ″い tionly phase suitable for free fatty acid analysis on 80-llXl mesh acid,washed slla胡zed suppo■ 。(FFAP or SP‐ 1220 is suitable sta‐ tionary phase;迅 idon ofl%H3P04 Can improve resoluは on ofbぃ :yric acid and solvent Peaks.Chromosorb W is suitable support.) Condition and stabilize coluHln for use at ca 130-135° C.
減c and V2山鹿 樋ids to neanst O.5m. peak heights for buけ Catty out 2 deteminations in rapld succession. 姥r(F)Rinse syinge thoroughly with water between every 脚θ 2 andyses and at completion of analyses widl dhted soap s01ution to minimize coroslon due to H3P04・ (2)After senes of sample in‐
jections,iniect One Or more butyAc acid/valattc acid standard solu―
修f COndition column≧ 48 httca 180° C.Ifon‐ columttecton OV例 tions and check calibration curve vs corespondng peak heig比 is not uset setitteCは On port at≧ 175° C.If butyric and valeric ttd ratios obtお ned from standard sOludons.(3)CaprOiC and caprylic 加of coluIIlns containing ,resolu血 peaks are notsepmted atbaseli確 acids may eluに after valeric acid and cause interfering peaks in sub― a n H 3 P 0 4 S t t O n a r y p h a s e c a n b e i m p r o vL eq du ba yn t‐t e Csequent t n g lanalyses.Be μ sure that dlese acids have elutedttfore another tities of2.5%weight/volume H3P04S01ution into column whle col‐ test solution is ittected.〕 ◎ 2002 AOAC iNTERNAT:ONAL
AOAC OFFiCiAと
0,LS AND FAT
METHODS OF ANALYSiS(2000)
Chapter 41,p.42B
Tabl●990。 27 Repeatabinty tr)and mpttducibili守(R)vatueS 95)for buttHC aCtd deteHninatlon in fats tPヨ 0。
41,1.39 AOAC Omciat Method 933.03
Residue tunsaponlf3ablo) of 01ls and Fa低
Far Stattstica No ofiabs
11
10
Ether Ex“■ct3on Method FiRBt Act3on 1933 Finat Action
11
Mean value,%(w柿)
3.46
1.79
0.18
r vatue(2.3 x sr)
0.29
0.12
0.02
R vatue(2.8x sn) 007 068 0.45 a sr=0104,0043,and 0 008 and sR=° 242,0161,and O.024 brfats l, Vely. 2,and 3,resF8対 ° 1:100%butterrati 2iend b‖o「 cream and vegetable oiに ontaining about
Accurately weigh 2-2.5 g fatinto sapo直 flcadon nask(200 mL
Erlenmeyer with standard taper24/40outerjointis recc― ended).
Add 25 nL alcoholand l.5 mL KOH soludon(3+2).SapOnify by boiling,withoccasional swirling,on steanbah30 min underreaux 50%m‖ k f a t 3 : r e n n e d t a l i o w t lc o n t a i n i n g 5 % ね air condenser.(No loss of alcohol should occur during saponirlcatiOn.)TranSfer alcoholic scap sOludon while still wam to 250 mL separator,ushg total of50 mL 闘nSe H20・ saPonincation ,s ,carcuraめ aask with 50 mLether and add etherto seParatOr.Shake vigorously, and letlayers separate and dttfy.Drain bweFiayer and pour edler Fittpeakheight胡 oOfbuwiC acid/valeric掘 andreadtondi‐ layeF mOugh tOp intO second separator containhg 20 mL H20・ height配 bration cwve weight httC acid equivalentto that"よ 施. Rinse powing edge with鵡 ,adding rinsings tO second sepmtor. C a l c u l a t e b u t y ,i %c w ea ic g遇h t / w e i g h t , i n s 2 m p l e a s f o l l o w 既 欧 tractsoaPsolutlonwtttwo50mLPoltionsetherinsame manner. Make total oF 4 extractions for marine ctis or other oils of ttgh unsaponiflable content.
hい嗣影=甲
Rotate combined edler extracts gently with the 20 mL H20 where Wb=weight,mg,ofbutyric acid read from calib両 lon cutte,
(VigOrous shaking atthis stage my cause troublesome emulsions)。 and Ч Let layers separate and drain aqueous layere Wash wih o江 tヤ H卜 3=Weight,mg,oftest ttdOn. ilityvalueoiSSaは sfactory,analresultis IIleanof2de‐ tiona1 20 nLPortiOns H20,Sttking vigorously.Then washedlerso‐ Ifrepeatお teminations.If Fepeatabdity requirements are nct曲IIleヒ 鯛配 re‐ l u t i c nm e 3s はw i t l a l t e r n a t e 2 0 m5 LM pa Oq ru te io ou ns s K cO aH O 。 sults and cttqy out another 2 deteminationstle o■fat. and H20,Shakhg vigorously eachはIle.If emulsion foms during wasMng,drain as muchaqueous iayeras Possible,leaving emulsion a Repettbi,町 anJReppJwcrbrrrw in separator wih etherlayerj and Proceed widl next washing.Af Rη 夕"力 】り .―When the mean ofduplicate deにminttons lies 岨 rd KOH treament,wash eher solution successively wid1 20 mL between any 2 mean valuesin Tおle 990.27,dle difference between POrtiOns H20 until washings are no longer alkaline to results of 2 decminations caried out in rapid successbn by dle phenolphtlalein. 正test same analyst,using the same apparatus,for analysis ofittnd鹸 Transfereher solu血鴻to 250 mL町 騨丸 ∞お∽l beaker,五 nse sep‐ materia shouldnotbe greaterthanthe rvalue thatcoresPondS ttthe arator and i低 脚 ng edge宙 h eher,and add ttsings to main solu― higher of tle 2 mean values. t i o n酌 、a p O r a t e m c a 5 m L afnedr― q u t t d t t v e l地 y,は 韓v e d ― When he means ofduplicate detettmtons, R?乃 れ c,う 】:り・ s m a l P O H i O n s e t h e r , t o 5 0 n L f a t f l a s k o r E r l e n m eけ yer previoぃ o b t A n e d i n 2 d i f f e r e n bt il ea sb o f嗣o r a n a l y s i s o f i d e n t i c a l t e s t m a ‐ 仕ied and welghed witlsimilar nask as tare.EvapoFate edler.When fference t e r i a l , l i e b e t w e e n a n y 2 n e a n v a l u 附2 e s i7n, tThaeb ldei勇 nearly all cher has been removed,add 2-3 mL acetone,and wttle between the mean resul低 obtainedby those laboratories shoulanot heating on stealnorH20bah,completelyFemOVesolventingendeair be greater than dle R vdue血江 cottspOnds to dle hgher of the current.Dry at 100° C for 30 mh penods to constant welght. 2 mean values. Dissoive contents of flask in 2 mL edler,add 10 mL neutralized ReFerencett P″ ″ 約2β 16ル れ 58,1419(1986). alcoh01,andは m te wit10.lM alcoholic NaOH(or (phenOlphthaleiゅ ンヽ こ 次C72,8∝ 1989)。 KOH)。 (≦ 0.10 mL is usually required.)COrec〔 weight residue for CAS‐ 107-92-6 outyriC acd) f r e e f a t t y a c i d p r e s e n t ( l m L O . laMc iaol.k d i = 0 . 0 2 8 2
r cο ″!れ″を sθ,Cあ4βセ″イメ ,″.43) rT2・
◎ 2000 AOAC tNTERNAT10NAL
OILS AND FAT Chapter 41,p.43
AOAC OFFiCttt METHODS OF ANALYSS(2000)
nducting C O F r e C t W e i g h t r e s i d u e f o r r e a g e n t nbeldabnyk coo脱 determnation siindarly but Orlltting fat.
References:A″ りsr S8,203(1933). JAOAC 28,282(1945);29,248(1946j.
41.1.40 AOAC Officiat Method 943.04 Squalene in O::s and Fats Tltrimetric Method Ftrst Actlon 1943 Final Action A.Peagents θ一 D i s s o l v e 6 0 g ( a ) 働 座 印 ' 確冴 ″θt t s ' 切￨ り 加 冴滋 W ! ″r : 払 KOH in40 nL H20・ r:θ れ.― Dissolve 28 g KOH ●)D:r″ 2 Pθ!ωs:跡たり ″raI:ル Sθ!″ in H20 and dilute的 lL. 63-7げ C),研 eい Vale配・ 2虎 ぇ―ぺ kellysdveBい (C)PCttte跡
H 2 0 b r O u g h t d o w n b y s w i n h g s e p a r a t o r . P o u ru pm e t rh oe に rsolu‐ tionfromtopofseparatorintolipttdconicalbeaker.Rinse separator with petroleum ether and add rinsings to beaker.Add few pieces of brokenporcelainorSiC mdevaporate almostallofsolventon steam bath.Remove lasttraces of solventin curent of C02 0r Otherinert gas while warmingbeaker.To avoidoxidttonofresidue,do notex‐ p o s e t o a l 1r l w wh ai ml .e s は Dissolve unsaponiflable matterin 5 mL petroleum edler and mns‐ fer to adsorption colum.く Eluate,which is caught h 250 mL
glass,stoppered 12naSk,shouldemerge dropwise,cal mWnin butno fastei gende pressure ttng used if necessly.)When sOlution has been nearly drawn into column,add ca 5 nL petroleum etttr‐pre宙 e beakert Cα l dnw adding solvent,prevlously used ousiy used to iぃ to insebeaker,in 5r10 mL poHions,always keeping srace ofcol― uEm COVered witlliquid,until total of 50 mL has eluttd(rCOlum witt Teflon stoPcOCk and top reservolr is used,Promd aS abov trough addidon of 5 mL rinse:dlen dnse beaker witt remaining 40HLpetroleume鹸 、addtoreservdL andletPasstOughcolumn.) Add ttw Jeces OfbrOken porcel血
or SiC and remove most of
.―Adsorption alu‐ 2″ 480-2御 秘asれ (oA筋 ″れ切 θメ滋 aJsο,う solventon steambadl.Finally pass current ofC020r Cherinert gas vttent. 述 n a f o r c h r o m a t o g r a p h i c a n a y s i5s4,0F,iosrh eerq uAi‐ through heated flask undHast traces oF solvent are expelled.Cool Keep in tighay c10sed container,away fron lnoisture. 1l traCes of residue to room temperature under inert attosphere.い (C)わ r:沈,2S″Jra確う″秘:た s】″!われ.-0.lM.Dissolve 8 g Br2 Prepare another solutionby gradually adding, in20 mLCH3C00H・ with cooling,5.45 mL H2S04tO miXture of20 nL CH3COOH and 8.15 mL pyridine.Mix 2 soludons,cool,and diluに t01 L with CH3C00H・
solvent must be removed before detemination is condnued.)
m L C H C L a n d a u n t t ss io dr ub ee d h Ю5 5 0 % e x c e s s ( 1 0 m L i s pyndine sulfatebromide solutton to pFOVide≧ Let mixtutt reHlaln in dark 5 min and dttn add usually adequate)。 roughly, 5 m L 1 0 % K I s o l u t i o n , t o g e t t F W i t h 4 0 MmiLX Hd2l0o・ _o.o5M.Prepare tttly れ 〃 wr2,わ rratts,a滋 0ヽ 鵡 閉"挽 わS″ wash down any free 12 0n StOpper,and ttrate with O.05M Na2S203・ 0.lM solution,942:27A.1.13). Ggβ by diluti増 Toward end of timtion add starch indic斑oL(mix ca l g soluble ユ APPararus starchwithenoughcoldH20tOmakethinPaste,add 100mLboiling ― P r e p ‐f r e s h c o l u H l n f o r e a c h d e t e m i n a tH20 「ing),Shake vigorousiy,and con‐ sは i o n and boll ca l mm while 取 Attο 例 初 ?ガ k de価 na‐ iHlmediately before use.Place small wadofcottn tt constrictedend hnue dtration to disappearance of blue.Conduct bla■ larly and calculate on siH五 tion on pyddne sulfate bromide soluは ofglass mbe,8 mm d and30cmlong.corCOnvenience,column may m L O . 0 5 M N a 2 S 2 0 3 e q u l V d e n t t o a b s o r b e d h a l o g en.Blank detemi‐ 的 nL capacity.) have Teaon stOPcoCk in stem and top reservoir of浮 cally no halogen con‐ nation on all reagents used should show pracは Add alumina adsorbent in ca 10 small portions until col―is ca ・ 71 mg squalene.Report resuits s u m p t l o n . l m L O . 0 5 M N a 2 S 2 0 31‐ 10 cln high.Apply gende s岨 側 and tamp each匹 由 on dumina as lng squalenJ100 g sanPle. lightly wih aattened end ofheavy ttlass rod.Place出wad sE盟 ofcoト ton on top Ofcol― and tt lightly.Wash coluHln witlca 15 mL petroleurn edle,remove sucd価 ,and keep toP of c01uHln covered witl shallow iayer ofpetroleum eher until ready for use. a per_rnarrOn able (Cttrわ″,SタタAppendix B,safety notes on distllation,fla― solvents alld petroleum ether.) Accurately weigh(±20 mg)ca 5 g sample into 125 mL
助s s d v e
References:拡 θAC X,4男 )(1943j;28,282(1945); 29,247(1946):48,127(1965). νな夕体 M α「 転 玉妨 R を CAS‐111-02-4(squalene)
41.1.41
Erlenmeyer wih standard taperjoint,add 3 nL concentrated KOH solution and 20 mL alcchol,and boll under air condenseF 30 inin, shaking occasionally.Cool somewhi and wttle still warm,add 50 nlL petroleunl eher;HIx,and transfer k)separator.Rinse flask with 20 mL alcoholandthen wit140 mL H20,adding nnsings to so― lution in scParatOr.Shake vigorously,let separate completely,and rom toP of on.Pourpetroleun ether extracば slowly dHttn sOap soluは mL H20・Repeat ex‐ separator into another separator ng20 condぶ traction of soap solution wi血50 mL petroleum edler.Rotate com‐ bined exttacts gently with the 20 mL H20 and,after letting iayers separate,discard wash H20・ Repeat washing by shaking vigorously w i h 2 0 n L H 2 0 na n d i sa cg aおr d l o w e r l a y e r Washpetroleumehersolution with 20 mLdihte KOH solution and hen with 20 mL portions H20 undl wash liquid is alkali‐ free,shak‐ ing vigOrously each time.After Flnal washng,drain iast droPs of
AOAC Ottictal Method 986.19 Trigtycerides in Fats and Oiis Cas Chttmatographic Method First Action 1986 Finat Action 1992 rJPA朗
OAC筋 倒腕oJ
Ao Prrncfpre Triglyceride
groups
having
sallle
C
nunber
a fgas t e chromatography r s e p a r a tofi solution o n t of oil or fat,under tempera‐
ar
d c m t t ,t a施n d t t t t t e d b y r e f e r e n c e 雌 ‐ p r O g r ae ― triglyceride solution.Triglycerides having same C nmber are not de― temttned individually.Contentis detemined by peak狙 胡 o.
◎ 2000 AOAC tNTERNAT10NAL
OtLS AND FAT Chapter 41,p.44
AOAC OFttCIAL M日 耐ODS OF ANttYStS(2000)
,APParar“ ●コ打どReagents on‐ iniec‐ (a)効 S勧 加 宅 raPれ二 With facilities for column tion,oven temperature progralming up to 350° C,如 d,preferably, electronic integration.All‐ giass systenl is preferable.
ing same C number expressed as percentage relative to tOtal 面giycendes cOntent by fomula
KATc松,x100
(b)CO'″ 秘″.―Glass,ca O.5rO.6 m long,andみ 4 mmid,船 led w h e r e A _ : = c o r e C にd p e a k a r e a o f t r i g l y c e r i d e s g r o u p : ; A T = t o t a l with 3%(orleSS HlethylPolysi10Xaneonacidbwashedsilattzedsup‐ c o r e c dに p e a k a r e a golfy cte減 ddes groups contained in smple C肛拭ergas aow 50 mmin,He is FeCOm‐ Po止 (OV‐1 lS Suitable)。 “T = Σ A N ) . mended as ctter gas but N2 may be used with some loss in 助 fference between results of 2 deteEttnadons camed out On r e s o ol nu .はC o n d i t i o n c o l u m p nC o rf o3tr6o≧ hu swei talt 3 5 0 ° salne day by sane analystushg salne apparatus forsame test nate― w r a t e o f c a 5血mn口 =Equivalent results may c a n e r g a soユ .降 tを 五狙and foF triglyceddes Present>107o should not exceed l%abso‐ bc Obtained witl use oFshort(≦ 6正めC01ulm.〕 ofく luに.For tFiglyceHdes present at levels 10%,difference should s.―Pudty 99%.Prepare standard sdution in not exceed O.5%absolute.Triglyceddes present atく (c)rrigryCタガガタ 5%are deter‐ CH3Cl(Or diiSOpropyl edler)c側 面 ning ca 10 mgrd each Of Hllned iess accurately. triCapFin,tricaprylin,trilaurin,timyhsun,tripalmitin,andtistearin.
Referencα】 物″ 叩 ,C陸 れ SL 1515(1985).
a mrernfnatron or rrrgrycerfres CarrecrrOn FgctOrg With 2 1tL microsyFinge,iniectCa l l止 triglycerides standard sot 41,1.42 l u t i o n w i t h i n i e c t i O n a n d d e t e c Ct o ar nt de m p e r a t u r e s s e t a t c a 3 7 5 ° AOAC Offtclat Method 955.34 initial oven temperamre ca 220° C.I― ediately commence pro‐
C/mintbut grammng oven ttmperame tOincrease ttrate 5° ofca年 not>5°C)andCOninueanalysisuntiltemperaturereachesca350° C. Manttn this temperature until all ttglycttdes have eluted mm coluHln. AssumethattriladriniscompletelyrecoveredfFOmC01umn and calculate corection factor,五 ,for each remaining ttglyceride from 五=(qノ Oθ Xは」Aか where
AL=peak
triglycendei cL=COnCent述
area
foF
Fats(Vegettb:o in Butterrat
Sterct Acetate Melting Point Method Firet畑 !on 1955 Fina,Actlon fDHSC― AOAC Mernc」
A
A p p a r a t w s
(O SPgCね ′秘た円 炉陸 ぇぺ eC Figure 9SS34A. 二 Heavy R宙 Fe,hammered aat,tO ca (b)P脇 筋 ″秘 SPar2ra。 3 Hlm wide x15 mm long,on one end.Mountin《 hssecting needle trilaurin:An=peak area for standard holder.
側 Kng/mLjoftFilauriniCh=COncen‐
破ldon(mg/mLj oF standard triglyceride f. DeteminertOn】 21可ecは 。 ns Ofstandard soluは cn.Plot graphof average vdues forrfor each triglyceFide agttnst correswndng C nunber.Corec故 鴻 factor>1.l is unsatisfactory.Decrease station‐ ary phase loading orincrease camer gas flow rate to achieve accept‐ able corretton factors. Plot vaues of retention dme for Oach standard dglycende peak agalnst coresponding C nunber.S宙
よght line should be 6btained
from which expected retention dmes for odler dgttcerides can be detenEIned. ,PreparaJt加
ゴ Sampre sorutrOn
Warmに st sample as necessary to comPleに ly liquefy.Homoge― nize liquid test sample by gendy shaking container. Prepare 5 0 m g / m L os no l ou fはt e s t s a m p l e i n C H 3 C i K O r For exmple,transfer ca l.25 g hquid sample to 25 nL graduated
diiSOpropyl
etleo.
flask usingぶ pet and diSsolve smple(while Still止 quid)in 2-3 mL solvent.Dllute widl salne solvent,and mttx.Iftest sample is known to contain signiflcant amounts ofmonot or diglycerides or free fatty acids,remove hese according to 96S.3S(sを241.1.61)befOre pro‐ ceedingwithanalysisasdescibedfordetemnationoftriglycerides co「ection factors. 丘 De拒 宮 研inatron
F`Oure 055,34A口 JGlats『 ntcro■ Rer for steroi acetate
l d e n t i f y e a c hよ辞b y u s i n g i d e n t i n c a t i O n g r a pmhi.nDee に peak l mL口 B: precip的 ぶo3.A:Top poAlon offBRer,capac町 a r e a s o f e a c h g r o u p o f t r i g l y c e r i d e s . C a l c u l a t e c o r e c t e d p e aLower k a r eportio■ as of■ 8ter,Ground suHttces between A and b y u s i n g c o r e c t i o n f a c t o s d e t e m i n e d e i t h e r b y c d c u l a t i o n o r b y iBn ― hold f3iter pad.A and B are heid together by sprlngs, terpoltton from graph of corection factors obtained for standard D.C:Filter fiask.E:Wlre twisted around stopper to hold triglycerides.Dekメ コinequantity ofeachgroupoferiglyceFideS hav‐icwer end of spr3ngs.
0 2000 AOAC iNTERNAT10NAL
01LS AND Chapter 41,p.4S
AOAC OFFIC,AL METHODS OF ANALYSS(2000)
ｕ 除 もも
W 込
グ
/ ミ
色々
デ ｀
Figure 955.34E卜 ● rystalline fom8 0f free sterolo.A:Cholesterol,B:Phytosterol.C:Mlxed cholestero卜
ュ Der― rnatrOn To 15 g flltered fatin 150 mL beaker add 4 g KOH dissolved in 4 mL H20・ Add 20 mL 957o alcchol,cover with watch glass,and ring occasionally. heat O.5 h on stean bath,sは
FAT
●hytoStero:.
Repettrecrystallization and iltratlon third andfoun time;then dry ° precipitate l h筑1∞ C. DeteminempofrecFystanizedmedsterolacetates(temperature at which liquid flrst staHs torun,detemned when heated atrate of
A d d 6 0 n L H 2 0 , m i X , a n d p o u r i n t o 4 0 0 m L b拭enagk e r c o n 面 0ぷ け r mpis辺 。 C higheF han dlat ofpure butter sini‐ a n d a d d 4 0 m L l % d t t i t O n i nO.5-1,0° 1 8 0 n L 9 5 % a l c o h o l . W a m t o c a 4C0 ° larly trett vegetable tt is itticated. Strandlet in alcchol,(Heatmay benecesstt todiSS01vedigitoninぅ stand ovemightin rettgerator. a M r c r O c w t a r 問
Fliter cold ttxture with strong suchon on rapidqualitttive ll cm Disscive sterol acetate remalmng frommpdeteminationin 2 nL paperin Buchner.WhealiquidhasPassedtroughPOur50mLH20 alccholin 20 mL beaker and add 3 drops 40%aqueous KOH.He江 over paper witlout stopping suction.Swin occaslonally.Condnue on steambath 5 min.Add 10 mL H20 and mnsferliquidto 125 mL to apply strong suction(H20 nlters rather slowip until all H20 haS SepaFatOr.Add 25 mL edler and shake.1,t layers separate,then washoutmostofsoaps.Pour50mLalcchol passed throughPaperk〕 over Paper and condnue suction untn a11liquid has passed drough, drain and discard aquecus iayen Wash edler widl dree 5 mL por‐ tiOnS H20 and evaporate edler to dryness in 50 mL beaker. 鵡ons e瞳 ,lethng eachpor‐ Finally wash paper with four 50 mL脚 はon pass tough compleに ly before adding next, 陀 Co Separate preciP的 Dry paper and precipitate 15 min at 100° from paper. Crush or crumble preciPitate, and Piace it in C 18x150mmttsttube.Add2nLaceticanhydFideandheatin 130°
Add 10 mL 70%alcohol to residue and heat to dissolveo Cool, place drop ofclear solution on slide,and examine drop microscopi‐ 200x for typical crystals of phytosterol or cally at 100… rol mixture G″田じ 陀 955.34B). rol,cholesに phyttsに
(PdPitate shoulddissolve hca5 mini donot glycerolbath 15 mdn。 usedirectheat,車ncespatteringmay occurandmatettalmaybelost) ,DrgrrOnrn白 "。yery―ProwJure Coolto ca 70°C. 直 ons and add enough Co品 Ыne iltrates lttn dgitonide precipi田 caremily add4nLalcoholandmix.Filterhotsolutionby gravity combine with all dgitonin pres― cholesterol dissolved in alcohol的 regitype),receiV‐ t h r o u g h p l e d g e t o f c o t t o n i n m dncgrtou bnei tKeP拭 ent.晩 t航 xture stand 3 h or ovemight.Fnter off pttciJtate and ing flltrate h20mLbeaker.Placebeakeronsmallhotplateandcare‐ wash with H20'alCchol,and edle,hen suck dry.Cmsh precipitate fully bFing liquid to gende bDil.Add H20 drOp by drop tthisterol onthinble,Suspend thimble in and tampitiighdy into paperextracは on atbp. acetateisjustabouttoprecipitatebutsturemainsinsoluは standard taper Erlenlneyer closed byflux I● condenser and contttn‐ 晩t cool,stirring occaslonally witl Pt spanla,fOr 15-20 mdn or i n g s m a l l a n o u n t確 .o Hf e黎a t x y l e n e t o b p a n d i e t t h i n longer.niterOn smlldiskofpaperinmicroBtthnerca 15mmdi… contents hang in hot vapors ofboiling xylene 16 h. alneにつ。 Suck dry andseparate precipitate frompaper.Place precipib tate in 5 mL beaker and heat wi血l nL 95%alcohol to dissolve Remove dttmble and dry at 100°C undl xylene has evaporated. completely.Cool beaker by setting in Pett dish ofice‐ H20・When Remove digitonin residue,weigh and transfer tt beaker,Dissolve thoroughly dhilled,mattal usually sets to semisolld crystalline r e s i d u e i n e n o u g h H 2 0 t O m a k e c a 2 % d i g況. i t Ao dn di n c as %o l u 敵 slury. ‐ volulne alcchol and heat on stealn bath.Addl mLれ a賦りl alcohol
Transfer slury to sPecial mttroniter,using Pt spatula,and apply suction.As liquid is drawn ttЮ ugh rliter,cOmpact precipitate by tamping with nat end Of glass rod of suitable size.(PreCipitate can thenbe deanly and completely removed from paperin fomofsmall button or tabld.)Redssolve precipitate in salne 5 mL beaker additttna1 l mL hot alcohol(or O.5 mL ifprecipitate is very sma11), and aner chilling to recrystallize,fliter second hme on lnicrofllter.
Kreagentgrade),C001,and 81ter ordgitonin compoundon Btlchner ofsuitable size.Suck dry andtransferprecipitate to watch glass.Dry at 100° C until ali amyl alcchol is volatilized.DigitOnin may dlen be pulverized and is ready for reuse. wi血
. N財: R e f e F e n C e S !i み 夕性D α: りJ . 9 , 2 6 1 ( 1 9 5 5 ) . JAOAC 38,338(1955);41,268(1958).
0 2000 AOAC iNTERNATtONAL
01LS AND FAT
AOAC OFFiCiAL METHODS OFANttYSIS(2000)
Chapter 41,p.46
are cbtaned On successive itteCは Ons ofidendcal volumes of stan‐ dard mixture.
41.1.43 AOAC Official Method 970.50
Fats
ttegetable)in
Oas Chromatographlc Method Firet Action 1970 Finat Action fDH30-AOAC Mり
B u t t e(め r PrgPara筋″ fat
防ar
A,PreParatrOn Or sanPre Obtain fatfrombutterby,如 .117Gをを33.6■5).Weigh5-10gf11‐ tered fat into 150 mL beaker and proceed as in,5534B G22 41.142),beginning“ ….add4g KOH.… ''.(Sterol acetaに need not be recrystallized udess mp is also desired.) a用 似 gtttrs ″閉″ Pacたれg.―― ″り ″れas夕.―― JXR,or (a)CC Cθ ′ (F)S,αガθ′ OV-1,or OV-101 dimethylpolysiloxane,or OV-17,or OV-22 -100-120 mesh methylphenylPolyslloxane.(2)S″ PPθrr.―
`
一 Pack glass column,1.8m(6 fty x4 ― ・ ザ∽′ m id,wih commercid l-3%stationtt Phase on 100-120 mesh Cas‐ ChromQOrdssolve O.年 1.2 g pOlysi10xane in 200 mL toluene Or CH2C12rt01uene(lⅢ l).Heatto dssolve(polysiloxane dissolves slowly in solventmixttre).(Ca″ だ。″r Slloxanes are toxic,Weardis‐ Posable gloves and use effective fume removal device when han― dling.)Add solution to 40 ChromQandletstand g Gas‐ 10minwi血 occasional gende stirFing.DryinFOtaFyeVaporatorheldh50° Cbath orheaton steambatl with occasional gende sは 胡 ng and renove re― sidual solventin vacuum oven at 50° C. Carefully wash inside of colurm and small amount glass w001 with 5%solution dc出 駅 斌 metり lsilane in toluene,inse widl mehyl alcohol undl insings are neutral to indicator Paper,and ttr dry.Plug coluコ m exit with small plug of silanized glass wool and
t r o u g hh ‐ ole septum,and plug iniectOn side arm wih%hOle
Gas‐Chrom Q.CO―
ercially prePared Packing of l or 3%station‐ tum.Addcoatedpacking materialhoughiniectiOnport,using im‐ 叩 phaSe available from AlletchLAPplied SCience LaboratoFieS, nel ttd Jtttt tubing and tapping colum very gently during Inc.,or Supelco,Inc. 畑 tion.Add%paCking mateFid tt time,remove funnel,and apply
(b)剛 り αご夕惚″ 一 Distilled in glass(BurdiCk and Jackson Labo oratones,Inc.,or equivdenめ.
ca 5-10 psi(34.5-69 kPaj N2 tO itteCtiOn Portwhile tapping gen to settle packing.Pack to 2.5 cmbelow inieCは On side arln and Plug
wih sdanittd glass wool. 地滋丹 ″ W!″われ-0.4ロ ト Wdgh 40.O mg O ChO陀 眼 切伍 ― Heat≧8hat260°C wmcast10psi cholestanestandaFdKAlltechaAppliedSctteLaboratories,Inc.)into 0∽ 越曲 前 昭 ザ 御ど tO VOlullle witl ehyl acemte. (34.5-69 kPaj N2 nOWing hough colum Shut off PFeSSure,dse 100 mL volutttric aask and diluに にmPeratureto290° C,andconはnueheatingttL Reducettmperature 夕 ar s″ 比協rtr sθ !″ゎ″.-0.2 Hg/μ L.Dlluに ざ 1髄 れ々r■ (d)9修 ′ め 260° C,劇 ust N2 t0 5r10ド i,and heat放 阻組ona1 8-12h じ 10.O mL standard solunon,(0,t020,O mL with ethyl acetate.
“―C h r o m a t o g r a p h o ‐ sc ia t O2的Sぃ l俺a c e t a t e o ん r r o ,4初 L.Wdgh ゎざ ″″′acgratt sraFra″ wttj体_2.O Hg/μ (Opざ ど standard solution tt detemine retention tines and FeSOlut 22.2ngp_sitosterolacetatestandard(ICNPhamaceuticas,Inc.,Life unn.Mittmumof1600theottdcalplatesisrequlredforp_sitosterol es GrouP,Ca90%purejinto 10mLvolumetricflaskanddilute Sci9岡 acetate peak. to volume wih ethyl acetate.Comlnedal pbsitosterolacetateismix‐ and 破 o f c m p e s t e D l a c e明tmaitneo rt eCaOrmlpioenre neth)は Theoredca Jates=α ガアx16 ‐ 純concentralionofβ SitOSterol acetatein p_sitosterol acetate,De腕 腕 五 陀 standard tt chromatogゅ h噌 2-3● standard solution,暁 =Hmp_sitosterolacetatepeakfFOmtteCdOnpoint andJ= whereと いand 距a OfCampesterol ace!分 p_sitostrolpeaks acebい by drawingElm triangulatedbase widthofp‐ sitosterol acetate peak. l i n e s t a n g e n t tso osfkpた eak andintersectingbaseline.Detemine ar‐
e a s o f r e s u l t i n g t r i a n g l e s b y m u l t i p l y i nag DetrmrnarrOn hettht by%base. C o n c e n t r a t t t p _ s i t o s t t=d器a c e t 捷
Pipet l.OmLcholestaneintemalstandardsolutioninto3 dramvial contalning sterol acetates,rotate vialto wash down sides,
的dssolve,Iniect2-3ぃ Lsample soluは on and 2-3●standardmix‐ ‐ ture,0,斑 leaSt in duplicate.Identitt SitOSterol β acetate peak in where Q=ng ster01 acetate standard/mL,4=areap_sitosterol smple ac‐ from re俺 耐on dHle in standaFd miXture.rheight ofsample =area campesterol acetate peak. etate peak,4nd兵 peak is>60%fuli scale,add additona1 1.O nL intemalstandard so‐
_β _s統 lution to smple solution,andrechromatograph smple and standard ″ 容ra4夕 。 S確″!α確ねたs勉再協材 ″競′ ″に判 .2躍 (ゆ勧ο Hlixture Measure peak heights of cholestane and c h o l e s t a n e pa,nsdi tlo.s0t距 M i x e q u a l v o l u r n solutions. es tい rol acetatゴ (O and(0. p_sitosterol acetate peaks in mm. a ApParar“ 3 (a)Gtt cれ ,omarog砕 れ_Barber‐ Colman Co.Mode1 5側 , Searle Analytc Series 4740,orequivalent,widlH2flamelo減zation
‐ ng β SitOSterol acetate/100 g smple= 何 /″ x)X(Cノ
Ci)X(SJSi)X(Or2)x10o
p_立 where■and亀=height(m)ChOLstane andtosterol acetate spectivew,h standard mixture;最 and島=heightぃo peaks,降 peaks,respecは smplα h i g h p u r i t y g r5 a dp es ji ,( 21 03 屯 8 - 1 7 2 k P a j t o e l u t e pp_立 _ stoster01 i t o s t e acetate r o l a c tand cholestane vely,in ‐ SitOSterol acetate and L,respecavely, cholestane/μ qand q=μg β datein 16r20 min;H2'Ca 40r45 mW航 対 air,3∞ 屯 40 mWmin, detector and l mV strip chartrecorder.Temperatures(° C〉 c01umn, 220-260;flash heater and detector,240-270,flow ratett N2(ultta
i n s t a n d a r d m i x t u r e ig oc lh =o μ lestane/1tL in sample;and o=mg ‐ Attustelec廿 Ometer sensiHvity so that 2.5円 事β sitOSterol acetate smplerltL. gives ca 50ワうdeflection(ltr9-lo_lo amp Full scale deflecdon w i t h l m V r e c o) rR 住 S3,623(1970,S4,643(1971). e p e a t i l l i e C t i C n s u n t i l c o n s t a n t p e a k hReferences:J■OAC eights
0 2000 AOAC iNTERNAT10NAL
AOAC OFttCiAL METHODS OF ANttYSiS(2000)
01LSAND FAT Chapter 41,p.47
″‐ hexane andlet now mOughpackingundl″
41.1.44 AOAC Offtctat Method 067.18
BetaoSito3terO:in Butter O:1 Cas Chromatographic Method First Action l範7 Fina'Actlon 1980
_hexane reaches tOp Of
Packing material.Use column immediately.Do notlet dry. E PreparatrOn Or resr sanPre
Dissolve 900 mg butter oll in 3 mL″ ‐ hexane.Quantitatively tansfer solution,uslng dispOsablepipet,to digitonin― Celite column andletpassthroughcolumnuntil solutionhasenteredPacttng mate‐ g f r e e p _ s i t o s t e r o 1dal.Wash / 1 0 0 gsmple b u t beakertwice ‐ with 2 mL″ ‐ ( A p d i c a b l e t O s m p ln ei sn cg ot nt tm お hexane and add each ter oil.) wash to column,rinsing sides oftube.Wash c01umn with flve 2 mL hexane.After all hexane has entered cdumn,wash wih portions″‐ A , P r r r T c r p r e flve 2 mL portiOns benzene.When iast portion benzene reach Free 3‐ p_OH ster01s are renoved from butter oil by complexing l cm above top ofPacking田 逮京al,removerubbeFtubing and wash with digitonin,androls sに are then removed froHl digitonideaceliに itterandoutersufaceofcolumn dPthoroughly widlbenzene tore‐ column by eluton with dimethyl sulfoxide● MSO).(6α ″,こ 0■f move traces of fat.(Fお lure to wash colum sides and column dp DMSOcanbeharlnful.Avoid skin contactbywearingheavyrubber wim sOlvent will resuitin Poor chromatograms due to interference gloves.Use effectve fumeFemOVal device.)Butter dl has apparent fromtriglycerides.)Discardhexaneandbenzene.BegineludOn with ― range ofO-l mgβ sitOSteroW100 g andice crealnhas apparentvalue 10 mL DMSO before benzene falls below top ofpacking material. ofca4 mg/100 g Fat from emulsiflers. Collect DMSO eluate in 15 mL screw‐cap cenmfuge tube. Add3 mL″ ― hexane to eluate,shake,and cendfuge.Transfer up‐ a用 “ 抑 rs rolsto second screw‐ capcen宜 血ge tube.Re‐ perlayercontaining sに ο t t α 0 “ S a r r a . ― celite 545,cr equivatent. a)DJ″ cを ttube with two 4 mL PortiOns pett extraction ofDMS0 1ayerin aぃ ‐ -2 μ s脅万 どsra″ ″ 。 ″α ″sθ !″ rfθ z.一 g p_sitostero1/μ L (b)βSfrθ ″‐ Lxane‐bewnc(lⅢ l),Caninly mnsfering upperlayerto second CHC13・ P r e p a r e f r o m A l l Al pt pe lc i卜e d S d e n c ae b■o r a t o r i e s , I n c . , ttlbe each dme.Vigorous,shake pooledupperlayers ttid1 3 mL H20 standardo5%p_sitosterol,5%campesに rol)(■ 010ngeravailablo. andmHttge untilc臨 ほ Removeupperiayer andevaporate underN2 徐■助 脇第御 Sは I Pure grade over KOH.(a″ ″す例 f挽 2Aμ 02‐ or虚lLIed air in 30 1五 L beaker on stean btt T― fer ttdue to sondistillation,potassiumhydroxide,naHlm体 pendixB,safety noに O.5 dFanSCrewrcapvi』 wtttw00.8mLportionsCH03・ ARerevapo‐ ble solvents,and hexane.) ming sOlventwitlN2 0raltered airoversteam防 向 redissolve sterols in O.l mL CHC13fOr GC mlytts. a APParatws にmperamに s (a)Cω 説 "θttrag叫 放 ― Operating conddon革 C>COluHln225-245andtteCdOnPoFtandiamelonizationdecc‐ (°
見 D e r _ f n a r f O n ect 2-8卜 Lexmctedsmpleandcdculatep_sitosterol by con―
珂 tor265-285.AdiustN2Cを 口tergasnowca50r60正 Wmin)to6btain vedng peak area to weight,using dお ly standard curve. following retendon dmes(min〉 Ch01eSterol 16r18,cmpesterol ‐ 22-24,and β SitOstero1 28-30.Use l.8m(6 ftj x4 mm id colunn ‐ mg β SitOSteroyl∞ g butter oil= c o n t a n i n g 3 % J X R s i l i c o n e L 1o 2n 0 l m側e s h C aC sh ‐ rom Q and ニ珂ected)X(1007g sample) (燿frOm curve/1000)x(100/ロ c o l l d i d o n c o l u m 2 4 h a tC2 5w0i° t h 1 5 - 2 0 p s i4(-110338。 kPajN2・ を M O n i t o r p e r f o m a n c e o f g a s c h r o m a t o g r aIpdhe n d f y p e a k s f r o m b u t t e F O i l S m p l e s b y c o m p a r i n g h e i r r ( b ) 乃r r a r m t t ,c 一 tion tiHles by noは ng separationofcmpesterol and sitosterolexpressedas peakto retention times ofknown 30mpOunds.Relative reten‐ r e s o l u t i o n = 2 D / K C + 3 ) , W h e r e D = d i s t a n c don e bdmes e t arei w e e cholesterol n t h e 2l.0,campestero1 peak 1.4,p_sitosterol l.7.
Hlaximar C=campesterolpeakbasewidth,andB=p― sitosterolpeakR e f e r e n工D ce革 aFり Sci叡 ら1 7 弧1 9 6 7 ) . スOAC 52,600(190)i53,535(197《 め.
base widh.Peak resolution should be≧ 1.6.
え″肋 叱 ″. 二W m 1 0 μ L H 航 l t o n m i c r o snガ ge, 01可 CCtお d r a w l a●i r i n t O b a r e l , i n s e r t n e e d l e i n tCAS‐ o s83‐ o l4 6‐ u 5(p_sittsterol) don,and draw SiFed alnount into barel.Remove needle fronl solution and draw l口しair intO barel,Notevolumecnscale and adiusttO desiredvol― 41.1,45 une,if necessary.
de‐
AOAC O荷 ictai Method 970.51
α″rゎ″ゲ Jttα〃 C″ ″4-Prepare standard soludon (O Pttβ Fats(Animaけ in v9getable Fats ‐ of2 μ sitOStero1/11L CHC13・ [Detemine composidon ofstandard g β a n d O i t s ( D e t e r m i no at te is ot ne r co fゅ C ぃ asin 970.50Bc)G2241.1,43).]Obtよ n standard curves ly d嵐 cover‐ G a g c h r o m a t o g rM ae pt hh に od i n g r a n g e l -g1 0p _μ sitosterol,using3 olls 13° C(cottOnseet com,Orsoybeanoils)presence ofpeanutollis in‐ at one time.Standards containing known amounts ofにa seed oilin dicated.ConflHn by 871.01(S2241.1.49). 1937). αりsr 62,9“ Referencett A″ JAて ,AC 28,293(1945),32,363(1949).
oliveollthatgivelitdeornoplnkwihhistestshouldberunsimulta― n e o u s l y w i t h s a w l e . P r e l i m i n t t y F 0 0 m t e m p e m t u er se it ne ds it ‐g 市 c t t o n o f s c a n d a r d s t t b e u s e Hd 2 0h mi ec te h‐o d .
CAS‐ 8002‐03‐7(peanut Oil)
RefeFenCeS:JAθAC 19,493(193o;2は 418(1937).
41.1.51
41.1.53 AOAC O付 lclat Method 893.01
AOAC Otticia:Method 929.08
0H(SeSamO in OiiS and Fats
Sa:ad 01:s(Refined,Winttrized)
Mod3fted Vi:3avecchia Test First Actlon 1893 Finat Actton
Coid Test procedure Ftrst Actlon 1929
Add2 mL furRralto 100 mLalcchol.Thoroughly EtX O,l mLof this solution with 10 mL HCl and 10 mL sample by shaking h test tube 15 s.Let mixture stand 10 min,observe color,add 10 mL H20, inett Cracked ice and add H20 undlit dses to top ofbOtde.Keep shake,and agAn 6bserve color.Ifcrimson color dsappears,sesalne bucket ilにd solldly withice tt removhg any excess H20 and add‐ cil is absent.(As furfural gives violettint witlHCl,itis necesstt to leandexarnineoll.If lngice whennecessary.After5.5 hremovebo■ it is properly wintered,smple wili be brilliant,olear,and limpid. use the very dilute solution sPecifled`) nl1 4 oz oil test sample bottle widl oil at C,coFk 25° dghtly,and ly submergebottlehbucketcontaining
sea wittparaffln.Compleに
Reference: 」は似 C12,203(1929).
References:ユ Sθa C確 醜.I閉 成 12,67(1893);13,69(1894). JAθAC島 441(1923). CAS‐ 8008‐74-0(seSanle oll)
41.1.52 AOAC Omciai Method 036.12
0 ‖t T e a s t t d ) l n O i l V e o ‖ Qualltatlve Color Test t Actacn 1936 Fir● Finat Actton
41.1.54 AOAC Otttcial Method 920。 163 Fats(Fore:gn)Contatning Tristearin in Lard Melt3ng Pcint Method Ftrst Acticn 1920 Final Action 1989
For prelimintty qualitative test use following F00m temperatuFe method:Measure exactly O.8 mL ttedc tthydride,1.5 mL CHCi3, A P r r r r P r e ,MiX, and O,2 mL H2S04intO testtube(18x150-is convenienり Presence ofbeef的 ,ta1lows,and similar fats,as well as hydroge‐ andcooltoroom temperature.Add7dropsofol tobetesteddirectly cにdby deに o measure test dl,use glass tdb‐ nated andinterestenfledPOrk ttinpork fats and lard,is to reagents,mix,and cool agaln.ぼ detemining difFerence between mp ofcrystauized tFiglycerides and rso‐ ing,4 HInod,andca2 11mid;7 dropsshouldweighcaO.22gぅ mpoffatty acidsderivedfromthesetriglycendes.This valueisiarge lutionofollinreagentsiscloudyanermixingandcooling,addacedc for pure Pork fats and smali for beeffats. 飼 呼d r i d e d r O p w i s e , s h a k i n g a f t e r e a c h a d d i d o n u n d i s o l u t i o n s u d ‐ denly dears.Appreciable deviations from these amounts,Particu‐ a mr_rnafrm l a r l y i n H 2 S 0 4 , C a u S e d i s d n c t v a r i a t i o n s i n c o l o r i n t e Weigh n s m e s e5 sg i melted nce and flltered lard into stoppered glass‐ graduate mixedreagentdeterioratesslowly,donotmlxinadvanceoftesting, and add 20 nL warm acetone.Mix well,taking care dlat solution is
dearandhastempemmre>30° C.Letstand 16r18hatconstantに m‐ ARer5 mln,add 10 mL absolute edler fron graduate and mix im‐ mediately by inverting once.Tea seed oll foms brown solution C.Fine mass of crystals occuPyhg≦ 3 mL should perature of 30° c h a n g i n g t o i n t e n s e r e d w i td sl ri en dm ri en ao cr hs eo s. mコa x i m u dml e n b e f o u n d a t b o t t o n o f g r a d u a t e . S h o u l d v o l u m e o f c r y s initial green and then fades slowly within few min,01ive cil fo― terially exceed 3 mL,take smaller amounl of lard(3-4g)for new solutktton additionofetler.This colorfades slowly tobrown‐ test.If crystals obtained from 5 g lard are insufflcient,increase gray, occasionally passing through faintpink stage.Botholive cil and tea weight iard and volume acetone pFOpOrtionately. seed oil evenmally fade to pemanentlight brown,Mixtures of tea Decant supemate acetone solution ton crystallized glycerides. seed oll and olive oil show characteristic tease9d oll colors propor‐ Add血 ●e5mL PortiOns wam(30-35° C)acetOne trom small wash はo n a l i n i n t e n s i t y t o a m o u n t o f t e a s e e bottle,taking d o i l p r ecare s e nnotto t . break up depositin washing,and decant frst
2 pO由 Ons.Achvely agitate third Portion in graduate an lnovemnt transfer crystals to smali fllter paper.Using wash wash crystals with 5 successive sm1l potlons of tle warln acetone, andremove excess acetone by sucttn.Spreadout pap breaking up any large lumps,and Ar dry atroon temperame. addcoldetheroaking carethatnoH20falls intotesttube),and mix. ne mp of crystals in is im‐ H20 bath and let colors develop while i↓ Thoroughly commnute mass and dete航 Retum tube tt ice― .163. closed l Hlm tube,using apparatus ttHHlaF tO that ofFigure捌 mersed in ice― H20・ COlors develop slowly and reach maximum HeatH20inbeakerrapidlytoca55° C andmintainthistemperamre within ca 5 min,
For approximate quantitative estimadons,drop oll into reagents as descibed above and let remain at F00n tempeFature S min.In After meantime,cool 10 nL portion absolutt ether inH20・ ice‐ H20in1 ice‐ min, 5減 n,place test mbe cOntaidng oil and reagents
0 2000 AOAC tNTERNAT10NAL
0!LS AND FAT
AOAC OFFIC,AL METHODS OFANALYSiS(2000)
chapter 41,p.52
acids ‐ ofβ おn,275.14.r smpleis impure,Elean MW paldttdsに should approach山 筑 offatty ttds mm ttsに 航 n,284.49. R e f e r e n c e s i t / S D AぇAJ″ ″′ れが 五 閉 広 Circ.132.
JAθAC 3,2ほ 2 (1920j:4195(1920j;19,417(193o: 59,323(1976). れ arysr 65,623(1940j. CAS‐555‐ 43‐1(triSiearin)
41.1.55 AOAC O付 lolal Method 974.20 Detectton of Fish and Marine Aninla1 01:s prccedure 1974 (APplicable in Presence ofvegetable oils and in absence ofmetallic salts.) ″f ざ22 Appendix B,safety notes on bromine. 磁 ″jθ Dissolve 30 drops oilin 8 mL CHC13in 50 mL beaker and add 10mLWiis sOlution,9郷 .159A(彪 241.1.lo.Add from buret,widl constant stiring,Br2 CHaC00H(1+3-4)to endpoint where solu‐ hon appears tobleachout,andthen addstightexcess.Mix,inmedi― ately pour into flatも otton test tube,and let stand l-3 h until clear supemate is obtained.Measure height of騨町 lpltate with mm mle and compare with standards pFepmd With known amounts of ish oil,including O,Anount of insoluble bromides is PropOrtional to amountofash01l up toca 15%and as little as l%ish oil can be de‐ 悔cted by slight precipitate on standing ovemight. ●Rin0 Figure 920。16← AppaFatuS tbr deterHlining 8■ Point.
Cithen heat again until therlnometer∽ =ying mp tube registers 50° C.Renove and dse temperature ofouterbath ratler quickly to 67° bumer.MPisreachedwhen fusedsdbstancebecomespeFfeCdyclear and transparent.
Reference革ユAれ 0"Cれ 卸 .Sて だ。 47,23く 1970> 泌 OAC S7,1005(197o.
41,1.56 AOAC Ofrictat Method 945.102 0‖ (Minerat)in Fats FtFSt Actlon 1945
68.6° C,Pres‐ Whenmp ofglycerides obtttnedby this ElethOdisく A.Orarrratrye rest ―P r o c 」 63.2° C ence ofbeeffatorotherfat shouldbe suspected,and mpof≦ ure i s e v i d e n c e t h a t s m p l e i s n o t p u r e l a F d . C O n d u c t d e t e m i n a t Pi lo na wc iet hl m L o i l o r m e i t e d f a t h E r l e m e y e r ; a d d i n L K O H s control sample ofpure lard. tion(3+2)and 25 nL alcohol.Boll under refluxお r condens句 Conflm presence of foreign fat by taking mp of fatty acids pre‐ shaking occasionally,undl saponiflcation is ccmplete ca 5 H遠 ゅ. Add25 mL H20 and mix.In presence of)幻 .5%mineral oil,disunct pared from glycerides.After detemittng mP,transfer crystallized turbidty appears. 克監ぁ add25 mL caO.5M alcoholic KOH,and glycendes to 50 mL敬 solutioninto ユ h e ■o n s t t a m b a t l u n t i l s a p o n l i c a t i o n i s c,oPmopulre に Owantratrye Mah何 ― FrnarA●ffon seParator conttning 200 nL H20'aCid,,add 75 mL e血 拓 shake, and iet stand.Drain aqueous acd layer and wash etler solution
(Ca″rわ″f品%Appendix B,safety■ otesondisullatiOn,flammable
≧3 dmes withH20・ Transferethersolutontocle‐ dry 50mLbeaker, solvents,and petroに um ehert) ° evaporaに ether on steambath,and inally dry acids at X洵 C.Let ac‐ Treatunsaponiflableresidue,933n8G2241,1.39),withH2S04 aS ids r卸 減 n atroom temperame ca2h andtttemine mp,Ifmp ofgly― below.When very small anlounts ofmineral c e n d e s , p l u s t w i c e d i f f e r e n c e b e t w e e n m p o f g l y c e F iunsaponiflable d e S a n d m p residue of for test may be obtained as fo1lows: 鮎 t y a c i d s , i3 s° C守 , t l e l a r d i s r e g a r d e d a s a d u l t e m t e d . Saponify 100 g fat by refluxing under air condenser 2 h wih
55 mL KOH solution(3Ⅲ 2)and 240 mL alcchol,wtth occasional shaking.Cool,add300mLpetroleumetheroP35-60° C),andtrans‐ b e to f o separator.Rinse r e u s e , a n d nask wit1240mL alcohol andaddrinsingsto fer separator.Add 480 mL H20 and Shake vigorously.Letlayers sepa― weightfatty acids x 1000/tmLxlnol述 ty KOItluse● .IFsampleispure rate,血 in lowerlayer,and transferupper layer to anodler separator. lard,meanMWoffattyacidsshouldcorespondcloselyttthatoffatty Repeat extraction of saponifled fat with 300 mL petroleun ether, ConcluslonsmaybeconflrIIledfmherbyprecisedek】 HunttnsOf mean MW of separated fatty acids.Dissolve ands in cOlodess, r e d i s t l e do 施 hol,caremlly neutralizediElmettately dtrate with O.5‐ ●.2M KOH,using phenolphthalein.Mean MW=
◎ 2000 AOAC iNTERNAT10NAL
dl
a
AOAC OFFiCiAL MttMODS OF ANALYSiS(2000)
OILS AND FAT Chapter 41,p.53
a n d c o m b i n e e x t r a c t s . W a s h e x t r a c t t w i c e iwointSh H6200 ,n L Table P o ■966.17 Repoatability and reproduoib‖ Ry waluo3 KOH,fol‐ u s i n g g e n t l e a g i t a t i o n . R e p e a t w a s h imnLgO 。 w5iMh ω O/c Hydrocarbon content lowed by vigoFOuS agitation wm successive m mL po鵡 ons H20 atlevel of tractto smali volume and p o reaxに u n t i l w a s h i n g s a r ef r ae le k. aE lv ia ― Deterrninations dry with anhydrous NttS04・ Two sin91e deterrninations in l
0.09
0.14
0.33
Filter petroleuEl ether solution trough small cotton plug int0 laboratory should dttr≦ Babcock milk‐test botde,989.04A(a)Gを233.2.27),add few small Singie deterrninations in difrerent 0。 10 0.14 0.40 n sに p l e c e s b r o k e n nP ,o ar nc de l r拭e m o v e s o l v e n t t t h ae la It Ii n g oiaboratories should differ≦ bath while passing curent of air mough bOttle.Cool,add 5 mL H2S04,miX,andkeepbOtdeinboilingH20bath30min,shaking oc‐ caslonally.Remove bottle fFOmbath,cool,and nll with H2S04until Gende air strealn will aid solvent removal.Transfer to weighed m surfaceFiSeS Wellinttgraduatedneck.CentFifuge 5 min at 1200中 250 mL Soxhlet aask,insing witt three 20 mL pOrtions petroleum andreadvolume ofurreactedresidue.Ifenoughmineral ollis avAl‐ ether.ReFnOVe relnaining solvent by evaporation on wam surface 48(sでを40.1.Oo,using smali SPrengel able,obtain density asin 95住 w itlgende airstream,keeping solutiOnbelowbp.Cooltoroomtem― tube.Weight mineral oll can be closely approximated by mul位 ply‐ or and welgh. perature in desicc■ ing volume by O.88.Refracdve index ofcolorless residue should be く1.500 at 20° C. Reference:泌 θAC 28,285(1945).
Repeat with additbnal he述 昭 periods of 20 min9 co01ing,and Wei」 ng undi change in weightis 3.5 mg. Conduct blank deteminaは on without test Portion.
CAS-8012-95-l Kmineral oll)
Hydrocarbons,%=(S―
D x100/C
41.1.57 AOAC Officia:Method 966.17 Hydrocarbons(Saturated)in Glycerides A:umina Column Chromatographic Method Ftret Action 1966 Fina3 Action 1970 AOCSrAO四
0角に納oJ
A.ApParartJs anど Reagents . 一W i t h T e d o n s t o P c o c k , 2 5 - 3 5 m m c ねb 夕 ( a ) C んr O 初αr 9 g r a piた diameter and 400 mln long. ― Activated alui拭na,AlccaP grade F‐20, ●)A筋 ″れ柳 拭 能、 8い2m mesh(Aluminun Co.of America,1501 Alcoa Bldg,Pitts‐ burgh,PA 15219,USA,or equivalenり .Dry alumina≧4 h at200°C before using,Store in sealed botdes in desiccator.Alumina must foい o rettn glycendes. h a v e a l o l s t unrte ocも% ntに
whereS=gsampleresidue,3=gblankresidue,and Ci gsample. a Prcrgrm Following 95%confldence limits my be exPected(磁 ble 966.17):
g Ta_
Conim presence ofhydrocarbons or mineral oil tt ofIR specmm Ofresidue with that ofPure hydroclbons or min oil,USP.
Referencei JttAC49,71,232(196o,
41.1.58 AOAC Ottlctal Method 961.11☆ Chick Edema Factor(Dloxins):n CttS and Fats Bi0383ay Method
ユ DerrmrnarrOn
Firet Action l弱 1 Fina:畑 ,on 1962 Surplu3 1974
Weigh 10± 0.01 g melted,well‐ H工xed test podon into 125 mL Erlenmeyer,and dissolve in 50-100 nL petroleun ether,waming
gently,if necessary. Scを28.113r28.117,12th Ed` Preparecolumnbytampingplugofglasswoolintobottomoftube so dlatsone ofgiass wooliSin constncted Portion above stoPcock.
Hll tdbe ca%full with petroleun ether(nsher scientittc Co.
41.1.59 No.B‐ 139,or equivalent)and add 200 g alumina ttmugh powder AOAC Otticial Method 968.23 funnel.Tap side oftube to aidin packing,Cover witt ca l cm anhy‐ Chick Edema Factor drous Na2S04・Elute atrateof80-90 drops(3.0-3.5 nLj/min,using (Dioxins)in(Dils and Fats stopcock for control. Gas Chttmatographic Method When solventheaddropsto ca l cHl,add sanple solution.Let sol‐ Ftrst Action 1968 vent layer drop to ca l cm above aluIIllna, collecung elutte in Finat Action 1989 5(沌mLErlenmeyer.Rhsesampleflaskwith 50mLpetroleumeher A . P r r n c r p r e and add to column.Repeat 3 times,adding each rinse to column wttn solvent headdrops,ocalcm,and wash dbwn sides ofcoluBm Fat,oll,fatty acid,or lipid is treated with H2S04 and eXtracted on transfering.Continue adding Pctroleun etherto colum untilto‐ With pe位oleum ether.Extractis puFifled on A1203 C01umn,further t a l o f 4 0 0 m L h a s p a s(sIendv etrhtreodu ぶ H2S04,and examned by electron capture GC.Peaks v o l u m e t r i c ntreated a s k c with an s e r v e a s c o n v e n t t n t r e s e r v o i r f o r f l n a l a d d i t i o n o f s o witt l v e n tretention .) dmes relative to alttin(Rか be:ween 8 and45indcate Evaporate eluate on steaFn bah to 50二 75 mL.Sti拭 ng rod placed in flaskwill help prevent superheating andcOnsequentbciling over.
presence of chick edema factors(heXa‐ octachlorodibenzo,p―dioxins).
, hepta‐ , and
◎2000 AOAC iNTERNAT10NAL
AOAC OFFDAL METHODS OFANttYSIS(2000)
01LS AND FAT Chapter 41,p.54
島 Reagents anJAPParatws
itteCt S口L(equiVttent to 50 mg oiginal toxic fat)intO gas
use.Do not chromatogram should exhibit sttes of はh S e J l g l a s s w a r e w i t h a p p r o w a t e s o l v e n t s b e f o r echrom筑ograph,Resulting store solvents in polyethylene contalners,Solvents availablepeaks fromwtt Raca 8-45.Peak heights should be between 10 and 25% Burdick and Jackson Laboratories,or equivalent。 ) 閉 2 協夕∴― D i s t n e d i n g l a s s , b p C3 .0 - m ° ( う P 夕! 乃! 2 ″ (b)酌
り ↓夕rttrル
ra肋
瓶 岡 統 胸 脇 所θg均 ヵ ンー Ehrc%alCOrnlj
r
側 1岡de deflection.Peaks■ 8-13 are dueめ heXachlorodibenzoBP‐ di‐ oxln isomers:2p併 逮s at 17-22,to ttE 2 hq脚独iSOHErs,and peak at 35-45,to octa‐ is倒 田eL
a b s o l u t e. 0e1h%earl(c幻o h o l ) . ・ (O HC滋 ″夕 れ2.―Disは1led in glass. θ C″ (0/Sθ
(b)P″ わだ″ り S″rrZr,c″ ″ c`em2P3 Dissolve 2.5 g test PatOn h 10 mL hexane in 500 mL glass‐ stcPpered Erlenmeyer. Add 10mLH2S04,StOpperP and shake 30 s.Add 125 nLpetroleun e巡 一■.05 μg/mL isooctane. 、stopperP and shake vigorously ca l min.Let separate and de‐ ど θ 払 ″ ざ θ ガ (e)Attri4 sraz`ね cantupperlayerinto 500 mL Erlenmeyer,avoiding transferoflower 1 . 5 % . θ 肥 夕M 呼 ル. 放 冴2 肥 ″わw β s ' 昭 ガ 挽 材 0勧 racゎ extraction widl additional 125 nL portiOn petroleun P r e p a r e f r o m c o n c e n t r a t e d r e f e r e n lc ae b lt eo x mi mc t t v iK _a layer`Repeat Vホ eher.Evaporate combined petroleun ether extracts lo S nL. sionofPesticides andlndusdalChemicals,UoS.FoodandDrugAd‐
or
midstration,200 C S`,SW,Washington,DC 202敬 与USA)in USP f Do not contact toxic CottOnseed or other vegetable ガわれ dl.(6α fat.) →FisherSdendttc Co.No.A‐ 540i do not 筋И.― (g)Acriya,caα比協円 substtute.Acuvate loog poldonsby heating4hat260° C.Transfer vity of withoutcooling to dry container and close dghdy.Check acは
o Ar蜘 商昭 統 の み _BefOre use,dry ali solvents町 shaking with anhydrous Na2S04・ Transfer evapomted petroleum
etherextractto aluminacolumn,(め ,using tota1 0f lomLpetroleun ether.晩tdraintojuttaboveLvelofNa2S04・ KecPing liquid level above Na2S04筑 all dmes,elutt wid1 100mLpetroleumether(tac_ はon l),50 mL 5%ehyl ether in petroleun edler KfraCは Onり ,and 100 mL25%ethyletherinpetroleumether tfmCtiOn 3),tFlowntes AL03by analysis oflow PositVe reference smple,o,eXalnlnlng of 8-9 mmin are satisfactory.)Discard fFaCtOns l and 2 and col‐ AL03 8aCtions 2 and 34 Wih Sufrlciently activated A1203,ChiCk edema factor elutes predominandy or entirely h fraction 3 as indi‐lectfraction 3 in 125 nLErlenmeyer.Add several bolling chips a a n s f e r r e距 s週 wihsHlallpor‐ e V a p O F a t e t O c a 2 n L o n s t e血 a lTnr悦 cated by gas chromatograms.KChromatoBFanS Should show senes o n s p e t r o l e l m e h e 1 r 0 的 m L g i a s s t s o ‐ p p e r e d P duate andmer 伍 ofpeaks widl ttbetWeen ca 8 and45.) ― e v a p o r a t e t o 3 m L u n d e r N 2 ・ .― れ ,c cθ J″ 加 ″ TO dry tube, α ごんrottarθ (h)Ar″加'″ g,α″
／ ′︲ ヽ 、
苫わM2S″ ル riC ac″】2叫 み― Add 2 mL H2S04tO pe‐ 17(oO x250mm,ntted ttbottom with ccarse porosity mttedglass OA″ trolttnethersdttn,stopper,andshake宙 dskandTenonstOpcOck(mbewitloutdiskbutwithglasswoolplug gorously 30品 臨 sepa‐ dried lled petroleum ehe■ rate and decant upper layerin的10 mL bea監 ぁ avoidng transfer of at bottom may aso be usedj,add redisは Add2 nLpetroleumetlertowduate,swirl vigorously, before use wihお 町drOus Nが04,until mbe is%mll.Transfer 15 g any H2S04・ AL03的 mbe h smll ttons,tapplngめsene.After last portion haslet separate,and decant upper layer into beaker.Add O.5 g solid
t a d dy ‐ 5 g‐ rcaO.5 min.Lttstand5minanddecantpe‐ NaHC03tObeakerandsは s e t d e d a n d t t b u b b l e Ss 面施 s t o Pf s no sl iv ne gn め Drain excess pemlem etler until i isjust above suretroleum etleriayerinto 2 or4 dram v胡 .Wash NaHC03 Wld12 nL drous Na2S04・ face ofNa2S04・ petroleum ether and decant washings into vial.Evaporate solvent “閉″,一Glass, 1.8-2.7m θttα!οgrαPんjc cο′ (1)CaSご 力″ (6r9ftj X4 mm id,packed wid1 2.5%SE 52 or odler Polysiloxane
Justto dryness at room temperame in vial under N2・
ttarθ Dissolve residue in 250 μL (e)CaS Cれ ,θ graPれメ.――
atOmceOus eコ 組twashed and silanizedい onatt phase on抵 staは 氏 isooctane,stopper vial,and rotate to wet stts with■solvenは ject 120 wihnarowrangeo5 μゆgrainsizebetween 125-250,m(6い 5 卜L solution (equivalent to 50 ng test portion)intO gas meSゆ.COat support with substrate as followtt DissolveC h2.5=slll‐ 45 having same rettntion F O m a t O g r a p h , c ) . P e a k s owfi t8h-■
heat,Add C O n e g L t t r u b b e r i n 3 0 0 m L C H 2 C 1 2 t t O l ule)nWei(hl Ⅲ 5 mm reference toxic fat(O indiCate pr d m e a s p e a k s 8o-f4■ 97.5gGas,ChromQandletstand10minwi血 occasionalgentlestiFe ence ofchck edem factor. dng.Dry in rotttr evaporator held Cinbatt 50°Apply vacuum to PerfOm Feagent blank他 的問血ぶOn Wh each set ofsmples. chromatographic tube and add small amounts of ocated support . Smoodl baseline should be obtttned in8尊region■ whiに tapping mbe at packing level after each addition.F11lto widlh 48,構3(1965〉 entrmce side and f11l remaining Rettrences:泌 O AC毎 ,384(1963】 2.5 cin on exit side ttd 7.5oncI■ 口由ngpres‐ 50,874,1338(1967);51,940K1968)i S3,628(1970); space withsilanizedglasswool.ConditioncoluIIlnatol湾 C. SuFe 2-5 days at 250° は ,528(1981). 二 With 63Ni electron capture or 3H con― 地 位″ 22tb,702(1968). G)働 S協 ″初 rOg切元 centric― type electron capture detector.Operate instmmentin accor‐ R2ッなaオ Marchメ 9% dance with instructions of manuFacturer. Ob能 意n stable baseline beforeusetChooseoperatingvoltagethatwincausebetweenO.6 and fuliscale denecdon for O.l ng aldrin oル standardalttin soludonj at sensidvity setting of l x 10 9 amp full scale.Keepc01umn temper‐
41.1.60 AOAC O打
:●la:Method 942.19
ature at 200± 1° C and attust N2 f10W So that aldrin elutes h Synthetic Co:ors tn 01ls and Fats L l-1.5 min d)25-0.33血 .E6r8mm]/min chart speedj.Iniect 2 μ Spectrophotometnc MethOd First Action 1942 alttin standard soluton before each reference or test sample. Final純 tlon
a DerermfHatrOn
α rys活 α r研た,拡―Dissolve 2.5 g referenceA (a)A″ ザ 呼ヵ″″ l . 5 t / o ctfoa対t i n 1 0 m L C C 1 4 i n 5 0 0 m L gs it ao sp sp ‐ eredErlenmeyer
and proceed asin(Dく d).Dissolve residue Linisooctane 250 μ and ◎ 2000 AOAC iNTERNAT10NAL
P e a g e n t s
″ス ニ Mix l L CH3COOH With200 mL HCland o Ac″ sοttr:θ 100 mL H20・
AOAC OFFICi札
01LS AND FAT Chapter 41,p.ss
METHODS OF ANALYSIS(2000)
″ヱーCautiously add 400 mL H2S04t0 100 mL θttrfθ (b)Ac材 ざ H20・When cool,add 900 mL CH3COOH and mix. 一 (C)Sο冴:閉 研 乃Ifル Sθル筋 払 Approximaに ly 25%.Dissolve 250 g NaOH in H20 and dihteto l L.
in acid extracts and as green residue on papers after flltration of troleunl ether solutions. References:立 A θAC 25,726(1942)!M,235(1951).
a sePararrOn a何 どrdentrFcatrOn
41.1.61 力:SceAppendix B,safety notes ondistillation,aammable (6α″rjο AOAC Off`oiat Method 965.35 solvents,and petroleuHl ether.) G:ycerides Place125mLoiland250mLpetroleumetherineachof6separa― in Monoglyceride Concentrates 50 wi血 mL Soludon X and,as soon as tors.Shake contents of flrst Cotumn Chromatographic Method iayers separate,transferlowerlayertoflaskccntaining250ELH20・ Firet Actlon 1965 Mix,and i― ediately extFaCt this diluted acid solution by passing Final Action 1969 successively throughtwo500nLseparators,eachcontaining 75 mL A . A P P a r3a , 日 Petr01eum ether.Shake vigorously,let layers separate,and discard l o w e r a q u e o u s l a y e r . R e p e a t t h i s p r o c e d u r e w i t h e a c h o f o t h e r 勧初確rOgrapれ た例肱―ReseFVO士 ,250 mL widl Tenon ω 5 sep‐ S frOm the di‐ a r a t o r s , u s i n g s a l n e p e r o l e u me xettrhaecr,tcoo lrocF‐ stopcockattacheduroughstandardtaper ipdpinnerjointto 19/22位 luted acid solutions.Combine dle 2 petroleum ether extracts,wash colum■ ,19 GO X290 mm,with outer standard taper 19/22joht with three 25 mL portions H20,and fliter. top and witl ccarse mtted glass disk and inner standard t pOr‐ Extract combined petroleun ether solution with two 25L 1■ ng of 19722jointatbottom.Bcttomjointconnectstoadaptercon dons Solution X.Treat each acid extract separately by nixing wih k・ outer standard taper 19/22 30int comected to Teflo extracttg quicktt by paSSing trough two 150 mL H20 and配 ‐ 12.) (Available from LuFeX SCientinc,from PFint No.580241‐ 250 mL separators,cach containing 50 mL Pe位oleum ether.CoHl‐ f ttewith 15mL騨 ― ons,washacid‐ unehesepetroleumedlersoluは eyTwin shell Blendett orequiV ( b ) M t t n ―P a t t e r sK oe nn ‐ dish はOnS H20'and evaporate to dryness on stealnbath.Do not he■ Ke■ey Co.,Div.of vAlable frottPatterson‐ formixng adsorbent.は & C Y e l l o w Harsco, 100 Burson St, PO Box 458, East Str6udsburg, a f t e r r e m o v a l o f s o l v e n l . R e s i d u e m aEyx tcroanctta lD■ No.9 or No.10(fOrlnerly FDaC Yellow No.3 or No.4,resPcc‐PA 18301‐0458,USA.) Hvely)and pOSSibly trace ofExtract D&C Orange No.4 Kforme的 E Prwaratron Jsrfrca cer
FD&C Orange No.2)if latter dye was oFittndly present in iarge amounts.Identify color spectrophotomencally as in chapter on color addittves. ShakeFlrstseparatorcontainingttlutedollwith25mLSoludonL let separate 20 min,and transfer lower layer to flaskning contお nd remove 2 0 0 m L 2 5 % N a O H . M i x , a n d a d d 2 0 0 mCL0 0H12,0a・
.No.S‐679,grade Place ca 10 g silica gel KFlsher Scientinc C。 923,100-200 mesめ in tared weighing bottle and cap immdiady. Weigh to nearest mg and subtracttare wdght.Remove cap and t町 2 h at 200° C.Remove from oven,cap immedately,and let cool 30 min atroom temperature.Raise caP momenttly to equ temal pressure wih atmosphereo Weigh,reheat 5 min at C, 200° color from tts alkaine s01ution by passing successtte 100 mL Poト cool,and reweigh.Repeat 5 min dryhg cyde unti1 2 consecudve ough two 250 mL separators,each containing 75 mL petro‐ tions tど weights agree within 10 mg.Calculate H20 COntent a leum ether.Discard ex位 【ted alk:岨ne soluはon.Continue his Solutton/treamentofollin other5 separators,andttnally combhe ●e50 mL portions H20 the2petroleumetherexmcts.Washwith血 ng tt layers and extract with two 20 mLPortiOns Solution leは sepa‐ rate 5航 n.Drain lowOr layers into flask containing 300 mL 25% C001,andremove color from tlis NaOH,mix,mdadd300 mL H20・ alkaline solutton by passing successive 100 mL w胡ons trough
s解尋 品
耐二
Adiust H20 COntent ofonginal sllica gei to 5%as fo 】 5 H 2 0 t O b e a d d e d ig i gn d o 占 d la地 g e l x ( 5 )- わ
2 separators,each containing 75 mL petroleum ether.Combine pe― Weigh silica gel to be attusted in blender and add calculated t r o l e u m e t h e r s o l u d o n s , w a s h f r e e f r o m a2l0k,atlria nwsiftehr耳t o a m o u n t H 2 0 t O g l v e i n a l H 200. 1C%O.nBtleenntd olf h5t± o en e v a p o r a t i n g d i s h , a n d r e m o v e s o l v e n t o n s t e a l n b a t h . R e ssure i d u e complete may H20 diSdbution and store in sealed container.Deter‐ c o n t a i n E ax cばt D & C O m g e N o , 4 a n d D & C G r e e n N o . 6 . R e n o v e mine H20 COntent of attusted silica gel as above,and rea fomer by dissolving in 3-5 mL PoHionS 60%alcohol and Fllttng necesttly. each portion trough small paper.Identify color spectrophotometri‐ a PreparatrOn Or Test sampre cally as in chapter on color additives.
(TO aVOid rearrangement of Partial glycendes,use extreme cau― Dissolve residue on paperin 2-5 mL portiolls petroleuneher,coト t i o n i n a p p l y i n g h e a t t o t e s t s a mC P. l) e s . D o n o t h lecdngiltrateinorittnal evapoFatingdish.Remove solvenlon stealn ° ヶ war″ れg αr batl.Blue residue よぅ ates i駅 D&C Creen No.6,Dissolve residue in(a)nttr sa“ メ2s″″″れど う2わッ 5θ C一 現宮加 ゼ 15 mL alccholand 10 mL H20,and add O.5 nL CH3C00H・ color Hnk
spectrophotometFiCaly colorin
the
various
acid
as
Iよ加ti母 in
extractt
く50°Cル rsれθtt Pヶ わ`体rヨ θ閉あ 加a厳協″切″).
Cttdnd ca 10 g ill mortar c h a p t t r (b)挽 o n cSro sa々 l o pル r さaれ2"宅 d d i dα vあeθッ sタ50° . samples in solld C02・ u s u a l l y and i n dPestle.If i c a t e s necesstty,chili synhenc
dye.However,com oilsomedmes produces faint pinkcolorin these
Weigh O,9-1.l g Prepared test PortiOn to l mg in 100 nL beaker. Add 15 mLCHC13andWarmifnecesstt forCOmplete soludon.Use heat and do not heat y40° C. p h o t o m e t r i c a l l y . C h l o r o p h y l l m a y a P p e a r a s g r e e n s c u n a t honly t e r fminimum ace extracts.This is readily differentiatedfronsynthett colors spectro‐
◎ 2000 AOAC tNTERNATtONAL
AOAC OFFtCIAL METHODS OF ANALYSlS(2000)
0 にS A N D F A T Chapter 41,p.56
, P r e P a r a f f o n
o r C O r t r m a
Monoglyceride,%=g monogiycende x 100/g test portion
Assemble chromatographic mbe but witlout nservdr.Do no4
ReFerences:ユ A秘.0″ 9Lθれ Scc.35,325(1958). grettejdnts.Weigh30 gptpared stta gelinto 150 mLbeaker and ンにXC 48,々 “(1965), add 50rm mLpetroleumether.Str slowly wihglass rodundi all air り. bubbles are expelled.Place powderfunnelintllbe andmnsfer sl囲 O p e n s t o P c o C k a n d l e t l i q u i d l e v d d r o p t o c a 2 c m a b o v e s i l t t a g e41.1.62 l.To mnsfer any silica gd slury remlmg in beakerDinvert beaker over AOAC Offtctal Method 966.18 powder fumel at 45°C and wash into tube witl wash bode,uttng 1‐ Monogけcendes minimurn amount petroleum edler.闘 nse funnel and sits of mbe: :n Monogtycer:de Concentrates when solvent Lvel dFOpst0 2 cmabove silica gel,close bottom stop‐ Oxidation Method cock.Remove Powder mnnel and carettly add smple.Open stoP‐ Firat Actlon 1966 cockandadiuStaowt。 2 mWmin.Rinsebeakerwid15 mLCHC13and Final ActBon 1982 ove silica gel. add ttnse to colum wttn level drops to 2 cmお (Keep temperature ofwork areabelow bp ofmostvolatile solvent C).Higherに mperature will cause separation used,ethyl etler(34,6° ofcoluHln Packing and pemit solventto channel.
AOCSrAOAC MernoJ A . P r f n c r P r e l‐ Monoglycendes aredeteminedfromH104COnSunedindlecx‐
Neverletcolumnbecomedry ontoP,andmttntaln 2 mL/min flow idationofadiacenthydroxyi groups.2,Monoglycerides are not oxi‐ Fate mOughOutelution.Ifnecesstt tOintemptelution,dosowhen MChOdis applicable tomonog,ceFide COncentrate dzedby H104・ leglyceFide iS passing httughbottomstopccck.Avoidsuch very li“ 15%1‐ monoglyo胡 dei not appl的品le to samples con‐ contAning≧ interuptions,since solvent above bottom stoPCOCk may cause pres‐ taining CHC13‐S01uble substances withと 2 adiacent hydttxyl surebulldup and resultinleakage through stopcock or cracks in sil‐ ica gel pacttng.)
groups.
i swararron or crycerfreg
a Feagentg
(Ca″rわれiSC2Appendix B,safetynctesondistill江拘n,fl― ble solvents,toxic solvents,benzene,and diedlyl ether.) Attach reservoir to column,add 200 mL benzene,and collect oh〉When ali ben‐ eluate in tared 250 mL nask(triglycende fracは
山 所:θ 払― Reagent grade(available tom GFS (ゆ 乃だ材をacir sθ Chemicalslnc.).Testasfollowtt ToO.5rO.6g glycerolin50mLH20,
zene has been added from sepamor and level in coluIIln drops to 2 cm above sllica gel,add 200 HL 10陽(v/V)ether in benzene and collect eluate in second tared250 mL nask(diglyceridefree Ⅲ fatty acid[FFA]fFaCtiOnj.When ali benzene‐ edler solvent has been 2 cmabove siltta addedmm separatOr andlevelin cohmndroPs的 障d tared 250 mL flask,and col" gel,add 200 mL edler,lhange to d村 lect monoglycedde fraction.
50mL r e p m Ыt t m d d 鴫 a d d t t n L H 1 0 4 S O l u t i o n t ot mP 距 ■ terofsolution containing H20・Let s的岨 301Bin andtttrate asinD。 glyc卸ol伸ter Ofblank=0,StO.76 rreagent is satisfactoFy. Dissolve 5.4 g H104in l∞ mL H20,add!1900 mL CH3C00H, stoppered bottle in dark. and mix dlorougHy.Store in giass‐ _Make h6mogeneous paste of ,wr″ われ (b)Srarchれ ガ'c″θ
(
Addto 100 mLboiling H20,Strrap‐ l.O g soldble starchwidl H20・ 乱 Ⅲm a y b e a d d e d a s p r e ‐ 1a2c5■ g r l ∞ i d l y , a n d c o o l . S a i c y l i c0 。 servative.Soluttm keeps longer if stored in rettgerator.Cclor of on diluted wid1 100 mL H20 COntttning O,05 mL (Attlition ofehyl etherto coluElnoften creates intemal pressure, 2 HL starch soluは 05 nL O。lM Na2S203・ Discard if lM L must be discharged町 0。 O・ ng in increased flow rate and cracks in sllica gel packing be‐ resul位 end point from blue to cdorless is no longer shu・ fore fraction is completely eluted.Avoidby slightly separating FeS‐ 二 onwitl f l o w i n t o c o l u m (C)働 わ沌力 月比 ReagentorUSP.BlanksonH104S01uは e r v o i r f r o m c o l u m n f o r c a 3 0 nsg asnodl vleenttは 狂 same rate tt duateおcdに cted.)
and without CHC13 muStCheck wtthn O.5 mL.
To ensure quandtative separation oftacdons,inse dP ofcolumn a PrepantfOn orSamPres
into receiverwithsame solventusedineluttonjustbefore changing (Do not subiect Samples to excessive temperatures or nasks for next eluate.
monoglyceride content may be reduced.)
Evaporate collecにd ti―,d‐ Plus FFA,and lnolloglyceride frac‐ tions on stealn bath under sttcam ofN2 0r dry air.Let aasks c001 at roon tempeFatuFe≧ 15mよ n and weigh.Reheat smples on steam bath 5 minunder N2 0r dry ar,Ltc001 15航 n,andreweigh.Repe斑 5 Hlin evaporation,cooling, and reweighing unt1 2 consecutive weights agree within 2 ng.
(a)肋 脇 れル 印
rpLttMよ wihout nelti理.
10° C above mp,mix thor‐ ●)肋 riA″ ,れ,α確 力 rPL―Melt at玉 ougtty,and take smple.Do nol俺 stsmples condning so much free glycerol hatit separates on sohdiflcation. 泳 α″ ′ ). (OS初 筋 ′ ″ 泳 . ―P r O C e e d t t h ●
A n y f r e e f a t t y a c i d p r e s e n t i s e l u t e d w i t l d i g l y c t t d e faIた r a c trermrnatrOn ion.Tc d e t e m i n e f r e e f a t t y a c i d c o n t e n t o f w e l g h e d d i g l y c e r i d e , aAccurately d d 2 5 m L weigh O.3-2gに 並 pOrtiOn caculated mmequ筑 10n:g warm neutral alcohol and l drop phenolphthalein indicator,and ti‐ =31J/%monoglyceride h smple,dssolvein CHC13,and ttansfer to trate
with
O,05M
NaOH.
見 carcura的 何。
′
standuntiltters sepanteandCHCLlaye v i g o r o u s lny. 晩 lt航 .Ifemulsions fom,causingpoor dearoronly slighdycloudy(1-3め ′ ｒ ヽ ヽヽ
FFA(as oleiC),%=mL NaOH x Molarity x28.21g test pOrtion
100 mL glass―stoppered volumetric nask.Dllute to volune witl stoppered CHC13 and航 X.Transferentire solutton to 500 mL giass‐ Erlenmeyer(dOnot五 nse andadd 100 mLH20・ StOpper,and shake 、
Diglycende,%=(g diglyCeide x 100/g testportioo-7o FFA
◎ 2000 AOAC,NTERNAT10NAL
、
Triglycedde,79=g ttglycende x loo/g test portion
separttonDrepett deに rmin■ lon,using 100 mL CH3COOH(5+95) insにad of H20・
OILSAND FAT Chapter 41,p.57
AOAC OFttC,AL METHODS OF ANALYSIS(2000)
Pipet 50 mL H104 SOmtiOn htt sttes of tt mL beakers.Add glycerol and HsI06‐ methyl alcohol solution oxidizes monogiyceride plus glycerol. 1」 nused H5106 iS deterHlined 50 mL CHClato 2 and 50 mL H20 tO third as blanks tBc)].Hpet iodometrically by reachng with KI and titrating with O.0125M shn_ 50 mL CHC13 Sample soludon into fo曲 ,avdding any aqueous dard arsenite sclution. ・ 05°F[32°C], (S01utiOns mustbe cooledto“ phase,and shake gendン ifnecessaFy.)cOVer wih watch glass and leCsmd 30 min. Calculation ofmonoglycende is based on amount ofH5106 COn‐ Add20mL 157o KIsolution,IIllx by gentle shaking,andlet stand sumed in oxidation of monoglyceFide plus glycerol cOrected for amount consumed in ox的 鯨m of free glycerol alone. ≧l min but≦ 5 min,away from strong sudight.Add 100 mL H20, Na2S203,Stirring continucusly Metlod is applicable to fats,olls,shoHenings,monoglycttd andはtrate with standardized O.1〃
with lnechanica stirer.After L CO10r disappears from aqueous and blends
containingl smpleisrun on salne day widlsame reagents,averageは trationof2 blanks whichcheck withinO.l mLcanbe usedh allcal‐ A ApParatws culations,If2blanksdonctcheckwidMnO.lmL,thirdblankmustbe
and
挽2 9 6 5 3 5 A ●α4 1 . 1 . 6 1 ) . Tl)isく 狗%,降‐ run.欧cessHsI06muStbe20-100%.If100Tttpl― 島 PrcParatrOn Orsfrta cゴ ,repeat wi血 peat witt smller saHIPlet if excess is much>100枠 m e 的 l d C C h o l . T o c o m p l e t e H 5 1 0品 6 ″96SaSB Gec 41.1.61).CheCk each ttw lot of adscrbent for u s e L s s H 5 1 0 「 largersmpleげ oxidation, Hlinimum of 20% excess is required. If excess H5106 methyl alcchol is much>1007う , high apparent sult. monoglycerlde mtty I●
satisfactory separation of glycendes.Collect last 15 mL of each f residue after evaporation is>2 mg,in‐ eluate h wdghed nask.ェ crease amount ofeluate until last 15 mL collected contains(2 ng. OnCe aE10unts of eluates needed for compleに separation of
呼 ω F″ gt燿 減 ― Free g,CerOl nust tt extracted comple悔 g,Ceridettactionsareestablished,only occasionalcheckingofeach 8om DMF layer into aqueous layer,If glycerol is not in aqueous lot of silica gel is Fequired. iayer when Hs10「 H20 iS added,or during smding dme,results a PrepararOn Ormmpre my below. to avoid overheating,and Melt entire sample in hot H20 atC 50° Pipet50mLaliquotofwelghedtestportionfrome)into 500 mL mix wdl.Weigh 4.9-5,l g prepared shortening to l mg in 100 mL Erlenmeytt Recodに st POrtiOn wttght in dttuol岱W2・Prepare beaker.Add 10 mLbenzene andwagnifnecessary forcomplete so‐ 2 blanksin similar nasks by adding 10 mL 25%DMF and 40 mL lution.Use caly minimum heat and db■ot heat>50°C. C H C 1 3 ・ a Pppara怖 孤 加 励 Addca100mLH20tOtestsolutionandblanks.Swirlca l min,re‐ 41.1.61)and adiust aow tO Prepare coluHln as in 96535D(s資 versing direcion several dmes.Pipet exacdy 25 mL H5106 H20 2 mL7min.Beghcollectingeluateinweighed4001nLbeaker.Rinse intt each nask.Letstand atroom temperature 30min,swirling vlg‐ beaker with 10 mLbenzene and add to column when Lvel dropsto orous,と 30s■ begilttng and every 5 min duttg this脚的 d.Add 2 cm above ttlica gel.Repeatinttng wih 10 mL pOrtions benzene, 40 mL KIsolutton from graduate,swin,and ld stand l航 n.Titate using total of40 mL. w i t h O . 0 1 2 5 M s o d i u m 1 2a r es ne dn i pt Oe i nt to as st a ir nc h c―) , i n ‐ Observe precautions of iast 2 Paragraphs of 96S.35D Gを タ adons.Record test solution dtration as T2 and aVeF‐ c l u d i n g bには l宜 調 4 1 , i 6 1 ) . % i s t t m i s s i b l e f o r a g e o f b l a n k t tA rn ay t ie ox nc e as s > 32 20 ・ E L P a r a r r o n翻 徳 a r 卸 H510「 H20・
(
E Carcwrarran Monoglyceride,7)=
【乳-1)―tW1/L)幌 ―孔河X4Mx〃 W x100 Wlx2 x1000 u n a r s e n i t e s o l u d,oaは 側 w h e r e 〃 i s m o l a F i t y O f sほ n d 〃W i s m o l e c u ‐ lar weight ofmonoglyceride.
Attach reservoiF SepaFatOr to colum,add 300 nL benzene,and collect eluate in weighed 400 mL beakerくtriglyceFide缶抵tioゅ. When ali benzene has beenaddedand levelincolulmdropsto 2 cm above silica gel,add 250 mL 10%(v/V)etherinbenzene and collect eluate in second weighed 400 mL beaker(diglyc筑能 fracttonj. When all benzeneredler solventhas beenadded andlevelincolumn dropstoca2cmabovesilicagel,add200mLetheFandC01lecteluate
de ttactioけ in third weighed 400 mLbeaker(monOg,ceぶ ObserVe 夕 241.1.61). on ofsecond paragraph,%53SE● precauは T o e n s u r e q u a n vt ei t sぷe p a r a t i o n o血職 f f r 笹n s e 的
,よ ,ofcOlum Monoglycende content ctt be calculated as monostearin.Using eighed into receiver before changing beakers for next eluate.げ exactly O.0125M sodium arsenite,358 as MW ofmonosteaFin,and 300 nL Soxhletflasks can be used fbrcollecting fracdons,but2 will sample weight of free glycerol lqual to twice alnount used for be required for triglyceFide duate.) monoglyceFide plus glycerol,fo1lowing fomula can be appliedi
Monoglyce由 拒 (as mOnostearin),%= [(38-ra)一Q5(亀 ―T2)]XQ895 比
◎ 2000 AOAC,NTERNATiONAL
酌 aporate d‐ ,di‐ ,andmonoglycttde tactions on sに alnbadlun‐ derstreamofcleanN2 0Fdry Ar.晩 tbeakeA coolttroomtemperture 15 min and weigh.Replace beakers on stealn bath 5 min,remove, cool≧15 min,and reweigh.Repeat 5 min evaporation,cooling,and reweighing unti1 2 consecutive weights agree within 2 mg.
AOAC OFFICiAL METHODS OF ANALYStS(2000)
0 1 L S
A N D
F A T
Chapter 41,p.59
i carcwrarrOns
ユ A p p a r a r w s
Tiglycedde,%=g triglycttde x 100/g smple
―E q u i p p e d w i t h s p l i tOinn iOerC は ( a ) C C S y s , 2.閉 ocno‐ lulnn in‐ ng,and flame‐ lonizattOn de‐ Jecはon,oven temperature pttgra―
Diglyceride,7o=g diglycel宝L x 100/g smple
tector.Operating conditiOntt split itteCti飢 (split Fati0 1,1041:5o; drectiniecdOn(splitless,hold for l min),itteCtiOn Port,320° C(Or for on‐ column inieCtOn,60° C)i C01um,initial,80° C(or fOr
%Monoglyce他
=g monoglycedde x 100/g smple
on‐coluHln,3)°C);program rate,10° 《 y血 ni td temperature, H i g h t t e f a t t y a c i d c o n t e n t t w i l l g i v ,e s ih ni cg eh r ae ps pu rl o低x l ‐ 360°C,hold 15 mini detec的 ,350° C;cz口trgas flow,5 mL He/min m a t e l y 2 0 % o f f r e e f a t t y t t i d i s e l u t e d w i t h e a ca hn d o f t h(at e t80° r iC‐ );iniecdOn vOlune,1-5 μ L.o西θ確:For on‐cOlumn inieC‐ monoglyceride fracは ons and 60%witl diglycende fraction.With はon,or dttct inieCtiOn,dilute 50卜 L reaction mixture widl i mL mos:shortenings,effect is insigniicant,since level offree fatty ac‐ hexane and iniect l μ L.When aPplying on‐ c01umn inieCtOns,a ids is“ 3.2%. precolumn my be used to lenghen colum life.On‐ colum iniec‐ N o n h y d r o x yd‐e r i v e d g l y c e r i d e s , w h i c h H l a y b e f o r m e d b y don t h egives r ‐ nore consistant resPonse Facttrs.) mal exposure,may interfere.側はs material is found Primarily in rを ″rjθ "g″ ,α,″りrgたor″ぇた れrcgraゎL (b)監 C。だJ″ g pθ diglyceride f岡 じはono Signiflcant alnounts are not present in fresh (C)CC∽ r四“″._o.25-0。35 mm id x15-25 m glass or ttsed sil‐ shortenings.Wm used fats,digly∝ide ccntent ofthat fraction can ica,surace fully deacttvated by silylation agent,dinethyisll通 one be obtained mm hydrOxyi value.
sP‐2100j or phenyinethyldinethylsilicone,10%phenyl(OV‐ 3) coating(orOtherphase withsimilarpolariけ ltO.2卜 mnlmdlick_ ),0。 n e s s ,t障 c : U S e c O l u m n i e n g d l a s r e q Ou rl r e d d i g l y c e F i d e S . I n d i v i d u d u n s a t u r a taendd mdoingol‐ yceFideS my not separate from saturated or less‐ unsaturated mono‐Or diglycttdes,■ 直n layer chromatophy on sllica gelimpregnated widl boric acit i― editly prioF tO derivitization,can be used to resolve glycerol‐ 2‐ monoesters ton giycerol‐ 1‐ monoesters.)
Reference:泌 OAC 49,812(1966).
41.1.65 AOAC Off:olal Method 993.18 MonoH and Di口 :ycerides in Fats and 01:s Gas ChttmatographBc Method First Actlon 1333 Fina,Act3on 1998
口りAガ θ,施ric s卯 夕■― Optiod.orθ ttf For automatic sam‐ pltts with 2 mL crimp‐ top vials,double sample and reageni anounts.)
brnc」
rt/PACヽA00SrAOAC‖
W‐Capッ ねh-2.5mLtor2.OmLcrimpHtop vials forauto
(O SC″
にto detenninぶ on of mono‐ a nd diglyceFideS in concen‐ s m p l e ,めw i h T e n o n _ f a c e d s e p t a e (Applicお trates and fats and oils.Other emulsiflers and components of fats ―Capable 6f maintaining α!:れ ッ:cc rbr ッ :a′ s.― (f)″を g ″● may be converted to and oils lgiyCer01,fatty acids,steF01S,etc.】 70± 0.5° C. t i m e 的1 韻 lyに t h e r d e F i V a t i v e s a n d a n d y z e) d b y t h i s p F O C e d u r e 。 a Feagentg
挽2 Tables 993.18A― C for the results ofthe interlaboratoFy study lyl)trinuorOacet倒 耐 de e)siゅ :ar:町ag御 な 一〈r)Bisttrimedlyl豆 supporting acceptatlce ofthe etlod. l■ (BSTFA).o Trimettlchlorsnane tTMcS), A
Store over KOH. (b)ル 勉 :陥 ―‐ 宮初 a C)閉 ‐
P r f n c r p r e
M o n o ‐a n d d i g l y u i m a r e c o n v e F t e d W i h b i s m 距 船 1 メ) 苗n u O r 0 滋 た" _ T e t r a d e c a n e , 9 9 % m i C ) = 確, 肥r s 胸 acetamideい 1■ A ) a n d t i m h y l c H o r s i h e n C s ) h 財 占d t t t O v o l ‐ . 断 v 筑 a r e s e p a W e d " g a s 池 輸闘W S i l y l e m r 鴎 所距JS腕 沼 SOrrtth_Accurately weigh ca 100 mg (0'確 t t v a t i v 岱 c h r o M o p p h y c o a n d t t e c t e d b y n血 a m.en_T‐ ie od ne ic 奴 am ″‐ tetradecane,to nearestO。 2 mg,into 10mLvoluHletric naskanddト i s u s e d a s a n i n t e m a l s m lute to volume with pyridine.
n
purit
T a b l e 9 9 3 . 1 8 A i n t e r g a b o r a t o r y s t u d y r e s u 3 t s f o r d e t e r m i n a,tyicoenr lodfe 3m o3nnc じ c2 aamnnodd『 dd ii 口 gtycer3de concentrates
(eXpre38ed
ag
percent
５ ７ ０ １
16MyriState
１
７
SR
RSDr.% 0.04 0.1 ■9 2.4 3.3
０ ６ ０ ０
６ ０ ０
Palmitate‐ l‐ 3‐ stearate
５ ０ ４ ０ ０
１ ０ ０ ３
１
４
３ ０
１ １
l,3‐ Distearate
eampBej RSD。 .% 9.2 5.7 10.9 8.9 14.1 10,7 15.8
０２ ８２ ８ ４ ７３ 釣２ ︲２ 勢
４ ６
l,3‐ Dipalmitate‐3‐stearate
０ １
１ ３ ０
２
60.1 6.2
９ ８ ０ ０
16Stearate
0fteSt
８ ０ ０ ３
27.2 23.6
ma38
２ ４ ０ ７
l‐ Palmitate
SP 0,04 0,05 7 0。
of
︲ ︲ ８ ４ ５ ５ ４ 。 ３ 。 ３ ．３ ． ．４ ． ．６ ． ．６ ． ．３ ． ． ３ ４ ３ ３ 碑３ ４
Mean・ 9る
0 2000 AOAC iNTERNATiONAL
AOAC
01LS AND FAT
OFttCIAL
METHODS
OF
ANALYSIS(20001
Chapter 41,p.60
T a b l e 9 9 3 . 1 8 B l n t e r t a b o r a t o r y s t u d y r e s u i t s t o r d e t e r m i n a t i o n o f m o n o n a ne dX p dr it gt tS ye cd e ra igにe dぃen3t iOnf 2 f o R l f l e d ma88 0f eamplo
r
Splke・%
Mean rec.・ t%)
1.00
0.96(96.0)
0,03
0.12
3.3
12.0
1.77
1.72(97.幼
0,03
0,23
4.8
13.4
1.00
0.98(98.0)
0.03
0.14
3.4
13.8
2.85
2.78(97.5)
0,14
0.40
4.9
14.5
0.97(97.り
1‐ Palmitate
l‐ Stearate
sn
RSD"%
RSDR,%
0,04
0.24
4.0
24.4
2.06
1.98(96.1)
0.06
0.53
2.8
26.9
t,3‐ Dipalmilate
1.00
0.93(98.0)
0.02
0.19
2.5
20,2
t・ 2‐Distearate
1.00
0,06
0。 19
6.2
1,00
l,2‐ Dipaimltate
0.68
0,97(97.0)
076(11分
0.06
0.20
8.0
二 腔脇rFcatrOn
ras._Glycerol,Palmidc acid,1‐ PaltttOyl (9魚 衆″″2 Srattα dipalmitoyl glycerol, stearoyl glycerol, 1,2‐ 31ycerol, 1‐ 基)9%purity distearoyi glycerol.畑 1,3‐ dipahitoyl glycerol,1,2‐ u Chek PreP,Inc.,Elysian,MN,USA,is sdttble sou距). 仰 _For each reference stantt accu‐ lgj Rて 力″″確 WrZ`醜 ・
Or
19.8
2
26。
融随 wOn,助 a n印 g例
Analyze reference solution undeF Sane Operating ccnditions as fortestsolution ldentify peaksby compansonofretentiondme widl 18. known substances(or apply coupled CCrMS)。 s″ Rgure"ユ A CattrrarfOn
rately weigh,to ttarest O.2 mg,ca 100 mg refeFenCe Smdard,o,
二usingrefeFenCeSolutionchromtogmm, tetradecane,(d),int0 10 mL voluEletric flask and ぇ‐ and ca 100 mg″‐ (a)ReSP9附eracrθ r welghca 100ng ofmixture con‐ dlute to voluEle withpyndine;。 calculate response Facto阜 阜 ,of reference standard vs intemal 胡 n i n g s e v e r a l ( e . g " 5 ) r e f e r e n c e s t a n d a r d s , e a c standard. h component being presentin abouts卸障 quantiけ,and 100ng tttetmmane mt0 2 nL volunetric nask and dluにto volume with P」 戸dne。 随 を:One Cr 氏 = いヽプ鴫 ) X は/ A θ more reference solutions can alsO be prepared without o ns is then caTriedoutas e,Sllylationofreference duは 閉‐ tetradeca■ forsample soludon,D(a),ateradditonofinteFnalStandardsoludon and silylating reagents.]
where岳 =resPOnse factorofreference standardヌ ;鴫 8=ngintema standard,sx=mg reference stmdard x:A】 =peak aFeaCfreference standard】i andAi3=peak aFea OFintemal standard.
(
cmk resPonse ttmls periodically.RespOnse facFS ShOuld be 対 .5.LowerrespoN factorsindicate somelossordecowosition,Use ce and smple solutions. O.S-10 mymLcomponentsinbotllef―
a DerermrnatrOn ル=ioな― Accurately weigh,to nearest O.2 mg,ca (a)Sa印 ル Sθ 10 mg homogenized emulsifler concenmm,or ca 50 mg oils and
fats cont宜 減ng emulsiflers,intt vial,B● ).Add O,2 mL BSTFA, JCWrarヵ ″ザ sttβた Cattθ″打 ∽確 班 ヨ 3dCulate con‐ 0働 Ca)(r),and O.l mLTMCS,C(a)(り ,and dlen o.l nL intemlstan‐ にnt ofSample componentぁ mxl(in ng%)aS f01low蛍 dard solutton,C(0,tO Sample.Cap vial securely and shake vigor‐ ngdevice,Wihout o u s l y . H e a t r e a c t i o n m i x t u r e 3 0 m iCnhiena7t0i° A佑) m'x=(1爪ゆXぶ 、 /mり x(A生′ delay,inieCt l-5 11L reaction nixture into CC tpreVbusly equili‐ brated to stablebaseline).C卸 =円 spOnse factor げOutreacdon2、 duplicateiniectiOns inに斑smplα where m'x=mg%componentメ 馬 ofcomponentxinsmplα m'3 m'“=mg mtemalstandardin smplα =peak =mgtteiAF諄 孤醜OfCompoLntxm― plei andA'“ area of internal standard in test samples
per FeaCtion. RttkをSοれ'ど て 加.工BtttO。 10mLFeference solution,C(9, ●)呼 into vial and add O.2 nL BSttA andO.l mL TMCS.Heat reaction
ofreft m i x t t r e a n d i t t e c t i n t O C UC s ea s ci on n ce e) n・t r a t i o n r a n g eReference:P″
e r e n c e s t a n d a r d s s i m i l a r t o r a n g e o f c o m p o n e n t s t to i fb le e dq ―i n
″ A,ュ C陸 "・63,1153(1991).
ユA O A C れ, 7 7 , 6 7 7 ( 1 9 9 4 j .
sample solution.Check line菰 け by p10ttingresPonse factoF VS COn‐ centration ofreference solutions.
Rをッ な宏 March r997
8un■ OWer of fo問 Table 993.18C interaaboratOry study resuits for determination of lyceride3 monoD and in di。bt3nd dup!tcates fted o3,(eXpressed as percent of ma38 0f Samplo Splke.%
Mean rec,。
(%)
S,
SR
0.14
1.44(93.2)
0.24
0。 33
17.0
1.02(93.6)
0.09
0.11
8。
0.30
1‐ Stearate
1.54
l,2‐Dipat輌 nitate
1,09
l,3‐Dbaimilate
1.39
1.36(97.5)
0.07
110
0,30
◎ 2000 AOAC iNTERNAT10NAL
2.18
_ __
07
1‐ Patm陥 崎
1,2‐ Distearate
RSDp比
0,74(91.9)
2.38て
0・
0.12
0.94
18.5
10.0
5。
11,3
23,4 4 3
36,3
10:4 9.2
《
AOAC OFFICIAと
METHODS OF ANALYSlS(2000)
OtLS AND FAT Chapter 41,p.61
里言費けｇ 占岳０●０
Reterは"Sme tmi0 Figure 093.18-Typical chromatograms oftttmethyisilytether derivatives of monぃ and dig:ycerides:A,reference standardo;B,8nOnO・ and digtycende emutsitter.The sttytation procedure,cotumn epecl■ cation3,Operating oondト tions,and peak identl■ cation are ae f● :icws:(a)SfryraH研 ._sampte sizo,10 mg,reagents,0.18■ L pyridine containing l.O ntetradecane,0.2 mL BSTFA,0.l mL TMCSireaction time,30 min at 70。C.(b)COrtrmn.-25 m xO。 31 mm 3d fused silica:0,17 μ m f8:m thickness(5%pheny,methy,oこ !icon,U:tra No.2,HewiettaPacttrd)。 (c)Operarrng● 。nd随 "s「―in‐ C;carrter gas,He,5 mWmin,80° C.(d)Peat rrerPrrrra何 。何「→S,matetradecane(interna3 standard),1,olyぃ iector 320° orol,2,digtycero:;3,hexadecanolc acld,4,octadecanolc acid;5,口 lycero1 ltetradecancate;6,01ycero: 2‐hexadecanoate,7,gtycerot l‐ hexadecanoatei 8,gtycero1 2‐ ctadecanoate,9,gtycero:1‐ octndecanoate5 10,01yCerol l口 :oosanoate,11,。 lycerol l‐ docosanoates 12,giycero:lctetradecanoate99ワ みand no oll aerosol should be observed rising above tle glass wool dwing ext
Superc付宙cal F3uld Extractlon tSFE)MethOd First Act3on 1999
な「も-10 mL intemal volume. (め mracrゎれα′
AOCS―AOAC tte命oJ
を 一S u i t a b l e f o r e x t a c m n s y S ― O c r a s s a r r a c r 切 ! ″c r f t t w s sな. mustbe capable ofholding≧ 1.O g 10osely packed glass s o y b e a n s , c o t t o n s e e dtem.Vessels , a t i o n o f o i l nctoonfに ( A p p l i c a b l e t or E dI en に wool. .) canola seed,saFaower sect and Sunacwer胡 ― A c c u r a tにo 主 0,0001g. 江 院 比閉竹・ ( d ) A ″け, た ″: SFE uses compressed gases as an extraction fluid.S22 ca″r:θ 鶴 a白″″幽4P.― Maintaning d.rying conditions as (e)｀物C″四"θ け
Appendx storage must be order
B,Laboratory Safety,for safe handling and outlined in 9拓12ぃ o241.1.02)with pettssble oven temperature of conPressed gas cylinders.SFE equipment 20-25°Cお ove bP of H20 at WOrking pressure≦ 100 Hlm Hg consmcted and mlntalned in proPer working C at 100 mm Hg working pressurei (13.3 kPa:72-77° ± 1° to minimize dle chance ofany exPlosiVe decoma C at 50 Hlm Hg working pressure). 5 8 - 6 3と1 °° pression ofextraction auid.Although C021S a nOn‐ C.Use if vacuun o乃 鳳 み αル θ″″.―Operating at i30± 1° toxic gas,adequate ventilation in he lttratory must oven is not availablet of diSPlaCing too be maintained to avoid dle possibiliけ s o be man‐ much oxygen.Adequate ventilation nust』 a脱 ( 胡 配d when using modiners tO avOid dle toxic effects 。 ._welding研 曲 sdd grade宙 血 dP髄 を競 脇 ofbrea皿 ng organic vapors. Pwr grades of C02 may be used,but are not requlred.
挽2 Tables現".tD2A―C Forthe resultt ofthe intedaboratory smdy 血 supporting dle acceptance of dle碇 A.財 ,仰 re
r″ 確._IIPE grade or equivalenti moisture '`あSθ ●)Er施脇伊 contentく1コ唱A嘔.Denatllring solvent type is unimportant. 一 OD脇 `"Cθ 四 “″L gra″ ″ねえ HydromatrixTM(Smple
馴 胡二 1撒網 淵
Oil is extracted from prepared oilseed witl a supercntical fluid ned in a (1lquid C02)and deposited direcdy onto giass wool contお SI.Jose,L M149085,USA),or n apparams.ARerevaporationofttsture andany residua collectお .―Borosllicate giass. on aPPmtus,the weightofex‐ modifler fromglass wool and Collecは (d)orass wθ ′
equivdent,are
Table 999.02A intertaboratory study resultts(17 1aboratories)ofAOCS SFE Method Am 3a96 u33ng Co2a:One for deteHminaば on of oi3 content of o:iseeds
Sttdy samHe dにα対° Labs accepted
No.of ou付 ねrs 3
x
A/M(Soy 4)
14
19.2
E/1(Soy 7)
14
G/R(Sun l)
15
2
38.9
C7P(Sun 3)
15
2
41.6
K/Q(Can 4)
15
D/J(Can 3)
15
Brs(saf 2)
14
H州 (Saf l)
14
3
F汗 (Cot l)
14
3
3
2 2
0.30
19.2
0、
1.66
357
19。
0.33 90
0,45 2.51
2.86 4.62
0,45 0.67
36.6
SR
「
1,02
39.8 37.7
3
革
1.39
R
% RSDR,%
RSD角 16
1.27 0.91
1.68
4.7
2.55
26
4.70
2.30
4.0
6.45
2.4 4.8 4.3 5。 5
1.26
0,90
2.51
1.1
1.88
1.03
2.89
18
2.7
2.25
1.66
4.65
3.8
46
2.4
2.7
0.86
240
1,08
1
0.46
1.28
303
2.4
0.51
2.3
3.0 144
L70(Cot 2) 14 3 18.4 0.34 0,95 0,38 1.06 1.9 2.1 8 0‖ seeds from 1995-1996 AOCS Smalley LabOratory Prondency Program Soy,sOybeani Sun,sunacweri Cant can。 lai Sari samOwe,cOt.cottonseed
◎ 2002 AOAC tNTERNAT,ONAL
suit
AOAC OFFiCIAL METHODS OFANALYSiS(2002)
C)!LS AND FAT Chapter 41,p.67
Table 999.o2B interlaboratory study results t15 1aboratories)ofAOCS SFE Method Am 3‐ deteHninatlon of oit content of ol130ed3 Study samp崎
o‖se筑 3 Labs accepted No`of outl●
A/M(Soy 4)
12
E′1(Soy 7)
13
G′R(Sun l)
12
C/P(Sun 3)
12
時 、 返
3
sr
r
20.5 2
3
20.4
0.58
39。 3
SR
0.43
7
044
430
0。 63
o.77
1.63
o.87
1.22
1.65
1.78
1.64
2.16
2.1
2.43
2.9
4.63
1.1
4.59
42
14
1
Brs(saf 2)
12
3
38.0
0,77
2.15
1.14
3.13
2.0
H7N(Saf l)
12
3
36.7
0.67
1.86
1.48
4.15
1.8
3
o,37 0.86
197
0.18
L70
1
19
1.04
1.32
2.42
1.43
0.50
0.52
0,35
43
1.5
D/J(Can 3)
40.2
3.68
3.8
0.9
4.00
3.0
2.2
1.46 146
%
3.8
12
12
43.5
%RSD酌
RSD“ 1.19
K/Q(Can 4)
F灯 (Cot l)
3
R
96 using Co2 Wlth EtOH modmerfOr
3.6 3.0 4.0
0.9
2.7
1
2`7 ・ Oilseeds from 1995-1996 AOCS Sma‖ ey LabOratory Prondency Program_Soy,soybeani Sun.sunnowe,can,canola:sat samowe,cOt cOttonseed.
a Deremrna節
。"
(a)Ce‐ θ"り ar確 ごrj例 !θs,"ガ αtt P!α″rp″ cをss clrrac_ 肋 ■,―Prepareに st皿 叩 le as desttbed in section A ofthe AOCS dP″ 胡 αS,5血 醐 .,Ameican の C朋 材統 拡 sa財 絶 cθ閉由 Oil Chemists'Socieけ ,CttHwttgn,L,USA,using he IIlethod sPe― ciflc for oilseed being malyコ 働エ
Take≧l g glass w001,C(d),and Pack into each collection vessel B(C),unditop ofresuldng plug is atleast 。 2m.O5fcvme sf―r o m b o 性 sel. Accurately weigh collection vessel with glass wool tO 却 .0001g.o,θ 姥:CaPs or septaon collection vessel are notrequired unless dle SFE hsmment requitts tler use.) InstalleachcollectiOnvessel onthecollecは onreel ofthe extractor
Accurately weigh 2± 0.0001gに st pOrtion into extraction cell, (autOmaはc systero or inStall each collection vessel with he B(b),Wid10utletendcaPinPlaCe.TaPcellSeveraldmes on work sur― restrictor or transFer mbe leading tOm the restFiCtOr inserted as face to pack material htc an evenly Packed plug in Oudetenddeeply ofthe as PoSSお Lin的 悔 giass wool plug(manualloading syst鉱 ゅ. c e l l . U p p e r s u r f a c e o f P l u g s h o u l d b e e v e n . n l l r e mSolneinsmments t t n i n g v o i d i n may requlFe ttuSment Ofhe restricto c e l l w i t h g r a n u l a r d a t o m a c e o u s, Ce(抽 c ) . W i P e a n y s P l l l e d n i l e raddiはonal glass w001 tO ensure deep posidOrung of the restrictor or 間は画 al from outside of extraction cell. transfer tube wihin the glass w001.
Table 999802C Cornpar3son of methOds:sFE versu3 80臣 Oiiseed° Ψ WS〔 汚 u g 1and 8u 81に method Wluu
x
sr
:
sR
ent exhction RSDP,D/c
RSDR,%
4
SFEoC02 a10ne
19.2
0.30
0083
0.45
1.6
2.4
SFEtC02 Ⅲ 15%ethand
20.5
0.43
1.19
0,77
2.1
3.8
AOCS Ac3‐ 44め
19.0
0,03
0.36
0。 2
1
7
SFE‐C02a10ne
19。 2
0.90
2.51
0.91
4,7
4.8
SFE‐C02・ 15%ethanol
20.4
0.58
1.63
0.87
2.9
4.3
1
0.05
0.3
2.0
Sunlower seed l
SFE‐C02a10ne
38.9
1.02
2.86
1.68
2.6
4.3
SFE‐ C 0 2 Ⅲ1 5 % e t h a n o l
397
0.44
1.22
1.65
■2
4.2
0.10
0.28
1.24
0.3
3.2
AOCS Ai3‐ 75う
38.5
AOCS Am 2‐ 93・
41.8
SFE‐C02a10ne
416
1.65
4.62
230
4.0
55
S F E C‐0 2 Ⅲ1 5 % e t h a n o l
43.0
0.63
1.78
1.64
15
3.8
AOAC A,3t75め
42.7
0.09
0.25
0.50
0.2
12
seed 3
AOCS Am a Olseeds ttm 199st1996 AOCS Sma‖
ey Laboratory Prottdency Program.
' Soivent extractbn,But tube extradbn method,AOCS Ottdtt Methods approwate tO。 lSeed.Srrla‖ ey Laboratory Prondency Program resute rOr 1 995r1 996. C Soivent otaclion,AOCS O師 間al Method Am 2‐ 931 FOSFA intemattonal Hlethoc.
0 2002 AOAC tNTERNATtONAL
0にS AND FAT Chapに,41,p68
AOAC OFFICtAL METHODS OFANALYSiS(2002)
rrα 町を2″α冴 had each extraction cell on the instrument reel(automated sys‐ (1982),Vol,3(ediにd by Applewttte)(1985):6θ tem),Or install each extraction cell onto the extracttr(manua1load‐6θrr9酒β″ Prttcrs,Interscience Publishers,Inc.,New York, t i o n i s i n d l e o u d e t eNY(1948). nd。 i n g s y s t e m ) ,e0nは t t h e c e l l s s os tt hpaotrに rysおα材 伽 把c姥挽″ われ ゲ θjh施 な,α材 nH 欧 r a c t f o r 3 0 価n w i t h C 0 2 u n d e r t h e f o l l o w i n g c o n d i t i oBOEKENOOGEN,A″
ミn(2・ 6r3.4g/min Cino航nalliquid aow rate,3.4ぎ 51,7 kPa;100° is pemissible):and resthctor temperature such chat C02 eVOIVes at between 80-100° C(40-130° C is acceptable).The temperature of
Far P陶 ど″cぉ,Vol.1(1964),Vol.2(1968),Joh Wileyも しSons, Inc.,New York,NY. B R t t S H S T A N I D A R D S I N S T R t夕 t施I OおN ,ザ材A ぇa r y s ザ な O f なα材
Fαな,British Standard 684,London,UK(1950). rttcr″ ″ ヴ ,レ fガ S,JOhn Wiley&Sons,Inc.,New CHAPMAN,m2説 alnount of moisture and7or modifler entralned in he extracted oil York,NY(1965). and the subsequenttime required to remove it before the weight can CHRISTE,A益 邸 c奮 れ 白レガ 材2れ。所θ!θ θ″2,■ に Oily Press, =メー be detemined.Overheating can cause loss ofthe more voladle olls. Ayrp SCodand(1992). Consuit instrtlment manufacturer fbr resttctor/collection tempera‐ 畑 昭 rο ゼ とtiPjな CttsTB Cas勧 ,The Oily Press,Ayr, araPり α′ n imize rloisture(and mOdineL ifuseo re‐ ngs that will価 ture scは Scodand(1989). movallimc.A lowerexpanding C02temperamre may be used widl 芝 勧 死加 αrograpり α材 ,,どな , CHRISTIB,HigれP"筋 α"確 とj?″ insmments thatincludc heating ofthe collection vessels.Somein― Pergalnon Press,New York,NY(1987). smments may notrequire subsequentremoval ofmoisl町 9or mod‐ CHRIST配 ,L"″ A″αい な,2nd Ed.,Pergalnon Press,New York,NY ner from the exract,Vedfy by drying the extracts according to (1982). 926.12G2241.1,02)to enSure that no signiflcant weightloss is ob‐ C O C K S & V A N R E D E , 協 防 ″わヮ 打α芝 防 o た拘 r θ】α泌 月α! A ″ り‐ s e r v e d b e f o r e e l i m i n t t i n g hP e. d r y h g s に sお ,Academic Press,New York,NY(196o. the exPandng C02 in the collecdon vessel will direcuy affect tle
Remove each collection vessel froln the instmment and,ifneces‐ DEUEL ttc町 泳 ,VOl,1,Inters,lence Publishers,Inc.,New York, S 叩 , r e m o v e r e s i d u a l m o i s t u r e b y t t i n g t o c o n sNY(1951). ttt weight using vacuum oven,B(e),(preferred),or fOrced‐ なr oven,Bo,with rec_ DEUTSCttE CESELLSCHAF「 問 R nttTWIssENSCHAFF E.V.,後 閉竹昭 Record flnal colに ction ves‐ ommendedに mperatures and pressuresち S 勧 滋 ″ M a 施 お ヵ ″! ル A ″ r y s 体 ザ n な 滅 o r 2 2 , 1 妙施 , sel weightto± o.o001g. (English transiation),Munsに r,RG(1988). “rara a脇匹だ″ 研 rac‐ ω Cθ 2+ゴ 5%を r脇″ :餌 racri伽め sf閉 rJθ L ― Prepare test smple,weigh test poilon into 研 exmcは cell,
EcKEY,“ Vegetable Fats and Oils,"Reinhold Pubushing c。
.,New
York,NY(1954). F I R E S T O N E , PS り i C aα ど 泌 勧卸 泣 r
C h a r a c r施 2 ホ ザ 0 : な, 施 句 αtt W後 確s,AOAC Press,Champaign,IL(1999). Aと ,返 CtOSSaヮ ,The Oil Press,Ays, GuNSTONE&HAMILTON・ terf11ling cell void withdiatomaceous 配h,depress eセ uppeFSufaCe ca 3 HIIn to tighten packing and installinlet endcap.Exmtfbr60 min Scodand(1992). GuNSTOtt A″ あ!Rガ“crわ″!θれを働 を閉前 ヮ aゼ 'focれcttsリ ザ With C02+15%emaol,c(b),under the following condiは on車 挽 fr crycctts,2nd】 丑 ,ChapElan and H』 1 Faり Actts滅 51.7 kPa;100。C;noEinal auid aOw raに ,2.lg/min(1.6r2.6g/min Ltd.,London,UK(1967). is Pemlssible>and restrictor temperature such dlat C02 eV01Ves at 的 TCN&HERSL6F,と tipttA″rysお=A Pracr,c″:App″ 延 れ,Ox― between 80t100。C K40-130°C is acceptable).挽2 cOmments on ford University Press,New York,NY(1992). resmctor/collecよ onに mperature in Procedure for C02‐ Only extrac‐ IIAMILTON&RossEL Attry品 ヴ θJrs a材免 な,Elsevier Applied don.Remove each collecton vessei from insmment and,if neces‐ and i】 ●pare and install extraction cell and coll∝ はon vessel in the S F E i n s t e―n t a s d e s c d b e d a b o v e Of no lr yt h e x mC c0 d2 O‐n . A f ‐
sary,remove residual ndsture by drying to constant weight as descdbed above for C02‐ Only extraction.Record rlnal c011ection vessel weight to±0.0001g.
刈
セく 助 β閉た】 め Ari!“rゎれヴ 肘″“rar Faな ,
4th Ed.,John Wiley&Sons,Inc.,New York,NY(1964j. HOLMAN,Prog″ s s れ, 権勧 を開なリ ザ F a r s a t t θ ! 姥r ん" f お, V O l . 1
Calculate percent oil as follows: =
O N D ・働 ″醒 O g r a P り 乃 r れ c A ″ r y s おげ 砕 施 , c R C P r e s s , Boca Raton,FL(1993).
HILDFrCH&WTlLAMS,ロ
E carcura育。n3
呻
Science Publishers,New York,NY(198o.
的
(1952)Pergamon Press,New York,NY. Hul,3at!り な r材 “srria′θ】 α角″月α,Pぇ ガ“crs,Vols.1-5,Jchn Wiley&Sons,Inc.,New York,NY(1996). INTERNAT10NALUNIONOFPUREANDAPPLIEDCHEMISTRY,Sran沈 舛 ′ ど
∞
ReferencαAOCS ttCj】 筋夕陥 ふ αtt R“θ"切例姥 どPracric2s, 5th Bと,1988,Ameican Oil CheEttStS'Society,Cham‐ pおgn,L61826,USA,Ofrlcial Mchod Am 3‐
96.
R2フな夕″f Marc力 2ω 2 SPEClAL REFERENCES
〃夕r 肋な ヵ r r t t A ″ rysお α肋aPs,7th revised and ザ θJ h F a t t材 enlaFged editon,Blackwell ScientiFlc Publications,Boston,MA (1987);lst Supplement(1992), αりsを どタアFcせ確 “れど F夕rrP″ど″たた,2 Volume, KAUFMANN,A″ Verlag,Berlin,GFR(1958). Springer‐ 】jど αガA″rjο LuNDBERG.A″ rθ α′ jοtt ″ IJtta″ rs,Vol.1(1961);Vol. 2 ( 1 9 6 2 ) , I n t e r s c i e n c e P u b l i s h e r s ,,INnecw。 Y o r k , N Y . MARKLEY,FarryAご
AMBRICAN OIL CHEMISTS'SOCIETY,QttC筋 ど財夕 抗θて な αtt R夕●οれ‐ れで,どをどPracr,ces,Ch倒 阻PAgn,IL(1999 incmding additions and revislons)
比なっ動 をど rCれ を″:H,ProP夕 rr,2島Pr9ど″cr:ο た, α4tr r/sを s,2nd Ed.,Part l(1960):Part 2(1961);Part 3(1964);
Part4(1967),Part S(1968);Interscience Publishers,Inc.,New !York,NY.
脇 r b E y ' s″ 加 なr r i aθ ′ , r 御どF a r P r t t c r s , 4 h E d . , e d i t e d b y S wMcDoNALD&MOSSoBA.打 em, β″ 1膨cん,セ″容 `Mtrン,P'cα ガa,sれ J o h n W i l e y & S O n s , N e w Y o r k , N Y ( 1 9 6 4 j i V o l . 1 ( 1 9 7 9 ) , V oLilp″ l . 2 A″ rysな,AOAC Press,Champaign,IL(1997).
◎ 2002 AOAC tNTERNATiONAL
(
AOAC OFFICtAL MttMODS OF ANALYSiS(2000)
0 1 L S A N O
F A T
Chapter 41,p.69
MBHLENBACHER,何
竹 A,α 研 な ゲ Fars a″ど 0:な ,The Carard Press,
Champagn,IL.(1960),
Dibettrazsれ
M I N & S M O U s R F r a t t r t t β 閉なリ ザ F a r s 研 ″θ! r s , A m e d c a n 0 1 1 Chemists'Society,Champaign,IL(1985). PARDUN,A″
!距夕どを,N品 脇″gsrgr々,vol.16 ofseries R勧 はRdlagen und Fortschritte der LebensHlitteluntersuchung und Lebensmitttitechn。10git,"Verlag Paul Parey in Berlin/Ham‐
burg,GFR(1976). PATISON,乃
恥 夕jrれ あ srrtt APガ ∽ !加 S,Marcd り Actt aゼ Dekker,Inc.,New York,NY(1968).
PERKNS,A″
rysな ゲ Fah θ
RAPPE,CHAUDHARY,&KEITH(Eds)Cれ
,4α
ガ LilP?″
C h e m i s t s ' S o c i,eCけ hmpaign,L(1991).
確 ;西 ,Amencan Oil
MI(1980・
s t
わrれa姥 ″ Dあ ガ四 切 ″ 乃 ,ヮ 2cr挽 ,Le琥 s Publisheぉ ,Inc.,cheisea
協料酬 総摺 仏 , 正
れ A4aい 容 , 当獣翠盈摺路軽笛踏難F滅亜 S確
WEISS,Fθ ο″ θ:な α材 恥 2:rr/scs,Avi Publishing CO.,Inct,
Westport,9「 (197o. WILLIAMS,α :s,r七 馬 αガFar9 F∽ 』s,4th Ed,Amedcan Elsevier Publおhng Co.,New York,NY(19(拓 ).
0 2000 AOAC tNTERNATiONAL
`
`
