SANIPl Na AND ANA YS S Ol (I)MMERCIAL FAISi\ND ()lLS

AOCS Official Method Ce 2'66

RerIp.ovei]:rorj9

Preparation of Methyl Esters of Fatty Acids

DEFINITION
Thls merhod provides a means tor preparnrg nerhll esters ollong-chaln frrv aclds for lirdrer analvsls bl
GLC,
scoPt
Th. m.thd is xpplicrbl. ro conuon fats. oik, and tirft,v acids wldr drc er.priol of.rilk les Gee Norcs.
l). U*aponiliables are not renrrcd but. llprcsenr in l.rgc amounrs. dre) nr), iiterfere rvith subsequert
analyses. Ihe pocdure will resuk li partial or .omplere dcsfucdon of dre foiloving g.oups: rpory,
hvdroperoxv, c,vclopropdyl,.rcloprop)1, and po$ibl), h1'drorf i, and actvlerlc Fativ a.lds, and is n.r
"uir
able lor dLe preparatior ofrncrirvl csLcrs offitq'acids conriinhg thcse groups.

APPARATUS
1. lLasks 50 ud 125 mL flat bortoni boiling 11*ks, o.s olrer rccks.
2. Waro coolcd condcnrrs Licbig or \Vcsr design, s rri, "!o
xxrr iinr.
3. Scparatory turnds 250 !rL.
4. Boilidg flask 100 mL, lor solvcnt rcnovrl.
i. Boilnig cliips iicc ofl:rr.
REA6TNTS
l. BF. mcrhxDol r.asd1i. 129/0 to 159/0, arailable.o,rncrcially $ 1,1% and 50% solurion Gee Nores, Cr,r,,, Noks, 5l
(l2r e Bf- pu llrrr oa f'crhJn. ) a v n labl. comn ercially, or d ay be prepared usins Bf. eas xnd methanol [see Secrion 3
J,,l,Arr.ritr So.i.L, f.r T.{i,rA rd IlJ rerirls (ASTM)Method D-1983-64T, or Rcfcicnccs, ll.
2. Sodium hrdroiidc INaOH) tt.5 M in methanol.
.. \-J,, . ,1o, l.r\ri' ..:,,.d.o, o|,.....
4. le[oleun edrer redisLi]lcd, bp 30 6[t'C (see Notes, Czaaaz).
5. Hcptanc gas dromaro$aphlcally cleen (see Notes, L-,,r,,1.
6. Soditrm sulture (Nr)sor) anhvdrous, reagenr grad..
7. Merhyl red n icantr 0.]!n id 60% c mol.
8. Nit..gen $Js high purirr.

PROCEDURE
1. ,A.c.urate weighnrg is !(tr reqdred. T.st porrion size need be known only to derermine the size of Ilask and atounts ot
reagens tlur sLouLi be uscd rccording to tlie fbllowing uble:

NiOH BF. merhrr.i

Te$ po(ion, Fla\k,

100 2t0 i t
250 i00 t0 6
i00 ^:t0 t:t
750 1000 12t t0

2. For fatg acids

(a) Inirodu.e rhe fatry acids into the i0 o. ll5 lrl reactlon fltrsk. Add the specltied anountulBF-mcn,a,", rrge,r,
rtrachrcordcnscrandboilfor2minGecNorc\l).Add2 5 hLofhelrane through the condenser and boillor I nnr
loryer Remorc fron heat. rcnovc condenser and edd,bout 15 mL olsaruiared sodium chloride solurio. (Reagents, s).
stopp* drr liask md stukc vigorouslv for 15 sec (ti1e the solutio. is srill repid. Add suflicie"r sanrared sodl"m chlo
llor the heptarc solutior olrhe methvl estrs inrc rhe neck ofrhe llask (References, 2). Tnnsfer abour
ride solurtun n)
1 mL olrhe heprane solLrtion into a tcst rube and add a sdrll amounr ofanlydrous sodnrm ullire. The dry heprue
solurion may rhei l,e injected dirccrll iDro a gas .hroniatograph. (Sce Notes, 2.)
(b) Io recoveL drl esten, tnnsfer rhc salt solurion and heprane phasc ro a 2i0 mL separarory funnel. Exract tlvlcc with
50 nL pordons ofredisrilled ptroleum ethcr (bp 30-60'C). \lhsh die combnred exuacrs {irh 20 mL poftn,N ofs'"ter

PnSc I oi 2
 SAMPI NC AN! ANATYSIS OI IAI S ANI] O LS

Ce 2-66 . ol M-.thvl Esters of Fattv Acids

'OMMLR'IAL
Preoaration

undl ftcc of a.ids (rcs warci $irh mcrhyl r.d indlcaror), d,l-. wilh sodirm sultrre lid euponre d,e nrhtnr urder l
sricem olnirogen or a steam badi (se. Norcs. 3 and,i).
i. for ftts and oils
(a) Irtrroduc dre fat infu rhr 50 or 125 mL rea.tion ilask. Add the sp.cllird &ounr of0.5 NI merhanoli. sodium h1-drx-
ide ard add a boiling chip. Arach a <r,den*r arJ hear rhe mixrure on r re3nr barh rnril rhe far giobules go inro solu-
tn,n. This srep should nke 5-i0 min. Add rhe specilied rmounr of BFr-herhanol reageot rhLough the condenser, :nd
pru.ted as direcred i. rhr faiil rcid sectn,, (fro.edurc. 2).
.t. Ahernare merhod lir tis and oils (acid valrc <2) (Refc.edc.s. 3) ,{n alreniare nerhod for $e prepararion oi hq acid
m.di).l.sres (|AME) folloss: Accuratelyrcigh a resr portion (approximarely 200 mg) lnro x siofp€red-ghs cenrihrge
is as
rial. Add 2.0 mL ol h.xan. G.c Norcs, a',,,'r,,) or heprane, followed bv 0.I mL of 2 11 merhanollc KOH. Close rhe vial
ard shale well for 30 sec, celtiifq.. rcnovc No drops ofrhe upper layer and dilur. wirh 2.0 mL ofhcxaneor hcptane. The
.on.enration ofihe IAVE ii hexare is rppioxnnatcly 0.5vo. Injecr 0.2 fL Ior cepillary.oiurD GLC anilysis using splir
injectioni nr case ofon-colunxr inj.crioD. dllurc ro 0.05% beli,re injecrion.

NOTES

retoleum edrer, hexme a,rd heptare are erueorelv flamnablc. Use effecrn e fum.-rcmovaL devicc. Avoid srxrlc .l..ri.hy.
Tl,e BF. ,rrrlLar r rc,gcrr hJq ll ired .lrelflife, cvcn r4ren refrigeratcd, and Llie use ofold o. roo-.oncentraled so]udols
.arrusuL r prndL,. tio ,l, ftilr.i' rn d to$$ o a apprcciablc amourrs of polFnsaiuratcd fatt\, ,cids (Refcr€rccs, 4). Bf r is poi
reconnerdtd rlur thc emlvs prepare rhe medraroll. soludm. Preceurn x should
sonous. Fo. rhis .eason ir is nrlr be tikcr ibr
elt frcteanrn a.d for potedon tiom the dang*s of coiiosnt chemial burDs.

NUMBERED NOTES
1. The requlred reacrion rime lppexrs o xrcrere as bot]1 rhe carbon number and dcgrcc of uisarumrion hcrease. lor
longer chair and nore ursaturuted lirtry acids. a gcneral observrr . is rhar the reactiotr should be alosed ro pLoceed jus
unril rherc is no visual evidence ofoil gln>u1es nr c rcacrioD mixmre.
2. Ir is reconmended rhar all methrl esrers br vashed wlth warer (ro rh. disapp.mrce olia rn*hyl rcd cnd poiDr) prior ro
injecttun. An ahc.native is ro paititlon the h*ane mcthvL csrer phase againsi warcr.onralDnrg a uall aDounr olnixed-bed
(indicari.g resin).
3. There is danger of losing somc ol rhe nore volrtlle esros if the solvent-remoraL srcp is prolong.d, or if roo vlgorous a srream
of nirrogen is uscd. lor infrarcd specroscopl,, this srcp should be termlnated as sooo as ali solvcnr is removed. For gan
liquid chromarographv rhe merhod mal.be *e.d.d ro farq'acids with elghr cubon arorts. ifthc solrcit is not complerely

4. Thc mcrhlt aten should be analro.d * ulms possible. Thcy mal be kepr nr atnosphere of nirog.! h a scrcw-cap vial
ar l"C for For loryci $orase rhershould br seeled ln a glass mpulc. subjected firt b r vrcuur !trd rhcn bacl$lled
24lt
with niuogcD and sroicd !r 2tl'C (lleezer).
5. Prcprrc rhe BFr solurion only if,r r ab", urely oecessary $ing Bf3 g gas and rncLh,nol {125 g Bf. pcr llro ofmcthanol).
\xrrnlurs: op(rr( undrr rvtnol,red hood lree Section 3(d) of,{mericdr So.lcry fo.Teniig Dd Maidiils (ASTM) Me$od
D 1948 64T. or Rcfrcnc. ll.
REFERENCES
.. ,ta ,1t..,t\ 1( I;,.
2- J. Chtondtogl 217:63 t,1982)-
3.&d nd Mtthant Jil A atl^ ofoih, Fat' dn./ Deri,arr,.r I e rarlonal UIrtur ofPurc and Applied Chentistir,7rh cdn.,
Bldcks,ell Sclentific Publicitions, 1987, IUPAC Merhod 2-301.
1. INFOkn 3: 1031 (1992)

OTHER REFERENCES
I.An. Oit Chen. So..70:3251993).