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J Am Oil Chem Soc (2008) 85:77–82
3.1.1.3), methyl oleate, oleic acid and alcohols of various
chain lengths, methyl pentadecanoate and methyl palmitate
were purchased from Sigma-Aldrich (St Louis, MO, USA).
Aminopropyl extract-clean cartridges and silica gel G
Uniplates (250 lm) with inorganic binder for thin layer
chromatography (TLC) were purchased from Alltech (State
college, PA, USA).
1.5 h at room temperature. For alcohols with a chain length
longer than 10, the reactants were heated enough to keep
them in a liquid state. The alkali catalyst was generated by
reacting sodium metal with the various alcohols before the
methyl oleate was added. The alcohols were freed of car-
bon dioxide before adding the sodium by sparging with
nitrogen or with reduced pressure. Catalyst was used at
*0.19 mequiv/g of FAME [4]. After the reaction, ade-
quate acetic acid was added to neutralize the catalyst. The
esters were dissolved in hexane and washed with 2%
aqueous sodium bicarbonate solution until the acetic acid
was removed, usually three times. Residual alcohols that
had a carbon chain length less than 5 could be removed by
evaporation under dry nitrogen gas or with a rotary evap-
orator. Longer chain alcohols were mostly removed by
distillation in a water bath at *100 °C and at *0.1 Torr.
Residual alcohol, usually 1–2 ml, was removed by passage
through a silica gel (1 g silica/0.1 g reaction mixture)
column with hexane–diethyl ester (90:10 v/v).
Bench-top Scale-up Procedure for Isolation and
Purification of BCFAs
Methyl esters of lanolin free fatty acids (FFA) were made
by Downing’s procedure [3]. Lanolin FFA was mixed with
benzene, methanol and concentrated sulfuric acid in a ratio
of 5:50:50:1 (g:ml:ml:ml), and refluxed for 4–5 h. The
benzene solution was extracted once with 2% sodium
bicarbonate solution and twice with water. The interme-
diate emulsion phase was separated by centrifugation, and
the benzene solution was filtered through a Buchner funnel
containing sodium sulfate. The benzene was evaporated,
and residual benzene was removed with a stream of
nitrogen gas. The crude FAME was then distilled at
0.5 Torr pressure in a simple insulated still, and the dis-
tillate was collected up to a vapor temperature of 160 °C
using an air condenser to cool the distillate. The distillate
was further fractionated by chain length in a spinning band
column as described previously [1].
Enzymatic Synthesis and Purification of Oleate Esters
When doing transesterifications with a large excess of the
alcohol, it was not economic to remove the residual alcohol
with a silica gel column. To avoid this problem and
improve the purity of products, enzymatic syntheses were
performed with oleic acid and alcohol at a 1:2 molar ratio in
a solvent free system. Candida antarctica lipase was added
at 2% by weight. Molecular sieve in an amount which was
calculated to absorb the water generated in the reaction,
was added at a ratio of 1 g molecular sieve to 1.8 g water at
3 and 24 h after the reaction started. The reaction took
place at room temperature (*25 °C) with constant stirring
and was stopped after 4 days with the addition of water.
The product was centrifuged to remove the enzyme and
molecular sieve, and then extracted with hexane and 2%
sodium bicarbonate solution. Since alcohols were added in
smaller excess than in the chemical syntheses, silica gel
chromatography was applied to the product after extraction
to remove residual alcohol and FFA. If subsequent gas
chromatography (GC) or TLC detected FFA, these samples
were further purified by passing through an amino cartridge
with hexane, and the neutral esters were eluted with chlo-
roform-isopropanol (2:1 v/v).
Urea counter-current distribution of methyl esters was
used to separate branched, normal and hydroxy compounds
with similar boiling points according to their ease of
forming urea complexes [1]. Since *3 g urea is required to
complex 1-g long-chain FAME, we added 6 g FAME
obtained from a spinning band distillation fraction, to a
500-ml filter flask (designated flask #1) and then added 12–
15 g urea and 200 ml of methanol 50%-saturated with
urea. Then the mixture in flask #1 was heated to dissolve
the FAME and urea and slowly cooled it to room tem-
perature for crystallization. A series of 500-ml filter flasks,
each containing 7 g urea, were prepared. A filter stick was
used to transfer the liquid from flask #1 to flask #2. Another
portion of 200 ml 50%-saturated urea–methanol solution
was added to flask #1. The mixtures in flask #1 and #2 were
both heated to dissolve and then cooled to crystallize. This
process was repeated until 15–18 flasks were filled with the
complex. The FAME was released by heating the mixture
at 60 °C, and then extracted with water and hexane.
GC, TLC and NMR
Chemical Synthesis and Purification of Oleate Esters
The purities of oleate esters and BCFA esters were
examined by GC. Esters in hexane solution were injected
into a HP 5890 Series II instrument (Hewlett-Packer, PA)
with a SP-2330 fused silica column (15 m 9 0.25 mm and
Methyl oleate (99% pure) was transesterified with an
excess of the desired alcohol (1:200 molar ratio) for about
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