Full text of DOI:10.1007/BF02663968

Liquid
of the Hydroxy-,
and Oxo-Stearic Acid Methyl Esters'
A. P. TULLOCH, National Research Council of Canada,
Prairie Regional Laboratory, Saskatoon, Saskatchewan
Abstract
hydroxystearates using a polyester column. Kitagawa.
et al. (17) also examined a few methyl hydroxy-,
acetoxy- and oxostearates using a silicone colmnn.
Itowevcr, it appeared that no systematic investigation
of all the isomeric hydroxy- and oxostearates had been
made.
Carbon numbers have been determined for all
the 17 isomeric methyl hydroxy- and aeetoxy-
st~arates and for 15 of the 16 isomeric methyl
oxostearates using silicone SE-30, silicone QFd,
and ethylene glycol sueeinate (EGS) as liquid
phases. The carbon mnnbers of the ismners in-
erease with increasing distance of the point of
substitution from the earboxyl end of the fatty
acid chain (the increases for the 4- and 5-hydroxy
esters are unexpectedly large). The change in car-
bon number from one isomer to the next is great-
est when the substituents are attached near
either end of the chain. The best separation of
isomers with the substituent near the earboxyl
end is obtained for oxo esters using the QF-1 col-
umn, and that of isomers substituted near the
methyl end is obtained for acetoxy esters using
the EGS column. Ismners with substituents at
carbons 9-13 are not distinguishable on any of
the colmnns used, but the other isomers are partIy
or completely separated. The quantitative aspects
of the separations have not been investigated.
In early work, using silicone eohmms, it was found
that the 17- and 18-hydroxy esters were readily sepa-
rated (32). Later it was observed, during the eharac~
terization of a 3-acetoxystearate (33), that while the
carbon mnnbers of 3- and 8-acetoxystearates were
fairly close to each other those of 8- and 17-aeetoxy-
stearates were much furl!her apart. The investigation
was then extended ~o the other isomers and it was
found that the separation of the acetoxystearates fin-
proved as more polar colmnns were used. A n ethyl-
ene glycol phthalate on C-22 firebrick column (6) was
first used, but the work of Craig (7) indicated that
ethylene glycol suecinate on Chromosorb W was a
more polar combination and this colmnn was used
in the present investigation. The fluorinated silicone
QF-1 was used particularly to achieve a better sepa-
ration of the oxo esters, since compounds with an oxo
function have greater retention times on this column
than those with hydroxy or ester functions (34). The
silicone XE-60 and |he polyamide resin Versamid 900
Introduction
.
, . , , , , : ,.
sT
there has been an in-
(
22) were also tried as liquid phases but were not
(
teasing number of reports of the isolation of
found to be as useful
hydroxy fatty- acids from natural sources, particularly
from baeteria mul funN. Unsaturated hydroxy adds
with 18 carbon atoms occur in plant seed oils, the
hydroxyl group of these usually being at positions
When this work had been completed, O'Brien and
Rouser (23) reported the retention times of all the
isomeric methyl hydroxypalmitates using Apiezon
and polyester columns and of the methyl acetoxy.
pahnitates using m'dy the polyester column. They
found that the retention times differed appreciably
from each other if the substituent was near the ends
of the chain, but were very similar to each other if :it
was at positions 6-12.
8
,9,12 or 18. Mixtures of hydroxy acids are also
found among the products of autoxidation.
Most of the m a j o r physical methods have been used
in attempts to identify the isomeric monohydroxy
fatty acids of the same chain length. X-ray crystal-
lography has been applied to the hydroxy- and oxo-
stearic acids (2) and the isomers in which the hy-
droxyl group is near the ends of the chain can be
distinguished but those in which it is at positions
Materials and Methods
Preparation of Corn.pounds Used. BergstrSm et al.
2) described the synthesis of all the methyl oxo- and
(
5
-14 cannot; however, all the oxo acids can be
hydroxystearates, but since many of the intermedi-
ates used in their syntheses were not readily available,
new syntheses were devised for some of the isomers
using more common starting materials. 2-H,ydroxy-
stearie acid was obtained by the method used for 2-
hydroxypalmitic acid (30) and converted to the
methyl ester with diazomethane. Methyl 3-D-hydroxy-
stearate (33), (-) methyl 8-hydroxystearte, methyl
identified by this method. It also has been reported
that the oxo esters can be distinguished by Ig sI)ec~
troscopy (10), but this method has not been success-
fu[ with the hydroxy esters. Mass spectrometry can
apparently be used to determine the structure of any
one of the methyl esters of the hydroxy, or oxostearic
acids (25).
UsuMly physical methods require a faiNy pure
1
2-hydroxystearate, methyl 17-L&ydroxystearate, and
sample to identify an isomer and cannot be used to
measure the composition of mixtures or be applied
directly to natural oils which contain only small amt
of oxygenated acids. Thus it appeared that GLC might
have some advantages over the other methods. GLC
had been used previously With a few hydroxy esters
and it was shown that they could be distinguished
from non-oxygenated esters by their quite different
emergence times on non-polar and polar columns (31,
methyl 18&ydroxystearate had all been obtained in
previous work (32). Methyl 3-oxostearate was pre-
pared as described by StNlberg-Stenhagen (28), and
the methyl esters of 2-,8-,12- and 17mxostearates were
prepared by chrmnie add oxidation of the correspond-
ing hydroxy esters (2,11.).
Diethyl n-dodecylmMonate and 3-earbomethoxy-
butyryl chloride were used to prepare 5-oxostearate,
and pentadeeanoyl chloride and diethyl 2-ethoxycar.
bony!suecinate were used to prepare 4-oxostearate;
both syntheses were by the generalmethod of Bowman
2
0,11). Morris et al. (21) appear to have been the
fi~xst to show that isomeric hydroxystearates could
be separated by (}LC when they separated 2- and 12-
(
3).
Hydrogenation over W-6 Raney nickel at 25C
1
Presented at the AOCS M'eeting in bfinneapolis, 1961/. Issued as
and atmospheric pre~ure (2) yielded methyl 5-by-
NRC Ne, 8095~
8
33

834
THE JOURNAL O F T H E A M E R I C A N O I L C t t E M I S T S ' S O C I E T Y
T A B L E
VOL. 41
I
C a r b o n N u m b e r s of M e t h y l t t y d r o x y - , Aeetoxy- a n d O x o s t e a r a t e s U s i n g
D i f f e r e n t L i q u i d s P h a s e s
M e t h y l h y d r o x y s t e a r a t e
M e t h y l acet0xystearate
M e t h y l o x o s t e a r a t e
S u b s t i t u e n t position
S E - 3 0
Q F - 1
E G S
S E - 3 0
.
Q F - 1
E G S
S E - 3 0
1 8 . 9 5
Q F - 1
E G S
2
.......................................................
.......................................................
.......................................................
.......................................................
.......................................................
.......................................................
.......................................................
........................................................
....................................................
.......................................................
.......................................................
......................................................
......................................................
........................................................
.......................................................
........................................................
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 9 . 2 5
2 0 . 1 0
2 3 . 5 5
2 0 . 3 0
2 0 . 3 5
2 0 . 4 0
2 0 . 4 5
2 0 . 5 0
2 0 . 5 0
2 0 . 5 0
2 0 . 5 0
2 0 . 5 0
2 0 . 5 5
2 0 . 5 5
2 0 . 6 0
2 0 . 6 5
2 0 . 7 5
2 0 . 9 5
2 1 . 1 5
2 1 . 9 0
2 1 . 9 5
2 2 . 3 5
2 2 . 4 5
2 3 . 5 0
2 3 . 9 5
2 4 . 4 0
2 4 . 4 5
2 4 . 5 0
2 4 . 6 0
2 4 . 6 0
2 4 . 6 5
2 4 . 6 5
2 4 . 7 0
2 4 . 7 0
2 4 . 8 0
2 4 . 9 0
2 5 . 1 0
2 5 . 5 0
2 5 . 9 5
2 7 . 4 0
2 1 . 0 0
2 2 . 5 5
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
1 9 . 5 0
1 9 . 8 5
2 0 . 8 0
2 4 . 5 5 b
2 4 . 4 5
2 7 . 1 5 e
a
1 9 . 4 0
1 9 . 5 0
1 9 . 6 5
1 9 . 6 5
1 9 . 6 5
1 9 . 7 0
1 9 . 7 0
1 9 . 7 0
1 9 . 7 5
1 9 . 8 0
1 9 . 8 5
1 9 . 9 0
1 9 . 9 5
2 0 . 0 0
2 1 . 9 5
2 2 . 4 0
2 2 . 8 0
2 2 . 9 0
2 3 . 0 0
2 3 . 0 5
2 3 . 0 5
2 3 . 1 0
2 3 . 1 5
2 3 . 1 5
2 3 . 1 5
2 3 . 1 5
2 3 . 3 6
2 3 . 7 5
2 4 . 7 0
2 4 . 8 5
2 5 . 2 0
2 5 . 2 5
2 5 . 3 0
2 5 . 3 0
2 5 . 3 5
2 5 . 3 5
2 5 . 4 0
2 5 . 4 0
2 5 . 5 0
2 5 . 6 0
2 5 . 9 0
2 6 . 4 0
2 0 . 1 0
1 9 . 8 5
1 9 . 9 0
1 9 . 9 5
2 0 . 0 0
2 0 . 0 0
2 0 . 0 0
2 0 . 0 0
2 0 . 0 5
2 0 . 0 5
2 0 . 0 5
2 0 . 1 0
2 0 . ] 0
2 1 . 0 0
2 5 . 1 0
2 1 . 7 0
2 1 . 7 5
2 1 . 7 5
2 1 . 8 0
2 1 . 8 0
2 1 . 8 0
2 1 . 8 0
2 1 . 8 0
2 1 . 8 0
2 1 . 8 0
2 1 . 9 0
2 2 . 1 0
2 3 . 0 5
2 8 . 0 0
2 6 . 1 5
2 6 . 2 0
2 6 . 2 0
2 6 . 2 0
2 6 . 2 5
2 6 . 2 5
2 6 . 2 5
2 6 . 3 0
2 6 . 3 0
2 6 . 3 5
2 6 . 5 0
2 6 . 9 5
........
2 2 . 6 0
2 2 . 8 5
2 2 . 9 5
2 3 . 0 0
2 3 . 0 0
2 3 . 0 5
2 3 . 0 5
1
1
1
1
1
1
1
1
1
2 3 . 1 0
2 3 . 1 5
2 3 . 2 0
2 3 . 3 0
2 3 . 6 0
2 3 . 9 0
2 4 . 9 0
T h e c a r b o n n u m b e r of 5~-stearolactone w a s a ) 1 9 . 8 0 ; b ) 2 4 . 5 5 ; e) 2 7 . 1 5 .
droxystearate;
the 4-oxo ester gave v-stearolactone with mp 49C [lit
15) gives 52-53C]. The NMR spectrum (in carbon
tetrachloride), which had no peak at 6.4 ppm rela-
however, under the same conditions
methyl hydrogen adipate; 13-oxostearic acid from 6-
oxoundecanoie acid [mp 53-54C, lit (9) gives 53-54C]
and methyl hydrogen azelate; 14-oxostearic acid from
7-oxoundeeanoic acid [mp 48.5-49:5C, lit (8) gives
51.5-52C] and methyl hydrogen azelate ; 15-oxostearic
acid from 7-oxodecanoic acid [mp 40.5-41.5C, lit (19)
gives 42-43C] and methyl hydrogen sebacate; and 16-
oxostearie acid from 6-oxocaprylic acid (14) and ethyl
hydrogen dodecanedioate. The methyl oxo esters were
prepared by treatment with diazomethane. The methyl
esters of 6-,7-,9-,10-,11-,13-,14-,15- and ]6-hydroxy-
stearic acids were prepared from the oxo esters by hy-
drogenation over Raney nickel using the conditions
described by Skogh (26). The oxo- and hydroxystearic
acids and esters had melting points which were al-
most the same as those reported by BergstrSm et al.
(2).
(
tive to tetramethylsilane= 10.00 (]3), and the IR
spectrum (0.7% in carbon tetrachloride), which had
carbonyl absorption at 1783 em-1 characteristic of a
5
-membered lactone ring, confirmed that the product
was the lactone and not the methyl ester. Hydrolysis
of the lactone with aqueous sodium hydroxide followed
by aeidfication with 2 N hydrochloric acid gave 4-
hydroxystearic
acid which was converted to methyl
4
-hydroxystearate (rap 56.5-57.5C) by treatment with
diazomethane.
6-Oxostearic acid was prepared as de-
scribed by Hfinig and Lendle (14) and 7-oxostearie
acid as described by Hiinig et al. (15).
9-,10-,11-,13-,14-,15-
and 16-Oxostearie acids were
prepared by the following general method:
Methyl 4- and 5-acetoxystearates were prepared by
treating the hydroxy esters with excess acetic anhy-
dride and pyridine (1:1) at zero C for 18 hr and
then removing the reagents at 0C and 0.1 mm (27).
The acetates of all the other hydroxy esters were
prepared by refluxing with acetic anhydride for one
hr and then evaporating the excess at 70C and 10 mm.
Gas Liquid Chromatography. The GLC units were
of conventional design using thermal conductivity
cells for detection. It was found to be important to
use solid supports which did not have a basic reaction
for preparation of the columns. Chromosorb W, which
had not been acid washed, was sufficiently alkaline to
cause complete decomposition of all the hydroxy-
stearates and also of 3-aeetoxystearate which is very
sensitive to alkali (33). The columns used were a 30
in. x 1~ in. copper column packed with silicone SE-30
on 60-80 mesh acid washed celite (1:6 w/w) operated
at 220C and a flow rate of 40 ml helium/rain; a
6 ft x 3/16 in. copper column packed with silicone
QF-1 on 60-70 mesh Anachrom AB (1:6 w/w) op-
erated at 202C and a flow rate of 40 ml helium/min;
and a 12 f t x 3/16 in. copper column packed with ethyl-
ene glycol succinate on 60-80 mesh acid washed Chro-
mosorb W (1:4 w/w) operated at 224C and a flow
rate of 60 ml helium/min. The injectors were main-
tained at a temp 40C above that of the column. The
recorder chart speeds were 1 cm/30 see for the SE-30
column, 1-in./5 min for the QF-1 column and 1 in./6
min for the polyester column. Since sample size has
a considerable effect on the emergence of peak maxi-
mum (1,24) the retention times were measured from
the start of the air or solvent peak to the point of
intersection of the base line with a tangent drawn
to the forward slope of the peak. Carbon numbers
were determined as described by Woodford and van
O H 3 - - ( C H 2 ) y - - C 0 - - (CH, z)~,, r ~-- C 0'~H ~- H 0 . ~ C - - ( C H e ) ,--C0.~CH:~
CI-I3 ( C I ~) y-- C O - - (CI-I2) x + ~o r 5-- CO.~CH.~
The appropriate 6- or 7-oxo monoearboxylie acids
were prepared by the methods of Hiinig and co-
workers (14,15) and anodieally coupled with the
appropriate diearboxylie acid half methyl esters using
the conditions described by Greaves et al. (12). By
using both 6- and 7-oxo acids of the same chain length
some half esters could be used for two preparations.
When the coupling reaction was complete, the reac-
tion mixture was acidified with acetic acid, methanol
added to give an approx 10% solution of product
and the insoluble dioxo hydrocarbon by-product was
filtered off. In the preparation of the 9-,10-,11- and
1
6-oxo acids the solvent was removed, the product
distilled and the fraction with bp 150~180C/0.15 mm
collected. The ester was then hydrolyzed and the oxo
acid crystallized from ethanol. In the preparation
of the 13-,14- and 15-oxo acids, the crude product was
saponified and acidic material isolated, the desired oxo
acid was taken up in hexane, filtered from insoluble
long chain dicarboxylic acid and crystallized from
ethanol. The yields of oxo acid obtained in the
coupling reaction were 25-40%.
The oxostearic acids were prepared from the inter-
mediates given below. Where the intermediate 6- or 7-
oxo acid has been previously synthesized by a route
other than that of Hiinig et al. the melting point ob-
tained in the present work is compared with the litera-
ture value. 9-Oxostearic acid was prepared from 6-
oxopentadecanoie acid [mp 75-76C, lit (16) gives 75.3C]
and methyl hydrogen glutarate; 10-oxostearic acid from
6
-oxomyristic acid (4) and methyl hydrogen adipate;
ll-oxostearie acid from 7-oxomyristic acid (29) and

r
l'cLi,oc~
t
: GLC o~~METHYL ESTERS
835
DECEMBER, 1964
Gent (35) using a standard mixture of the methyl
esters of normal Ct(~, Cls, C~o and C~2 acids.
®
Results and Discussion
The carbon numbers obtained are shown in Table I:
It is considered that the figures accurately represent
the carbon number of each isomer relative to its
immediate neighbors. However, :if the isomers arc
run randomly at intervals of a week or more, when
small changes in operating conditions ha~'e occurred,
the carbon nmnbers are only reproducible within ~0.1
of a carbon number for the SE-30 and Q F d eolmnns.
For the polyester colmnn, which has a higher "bleed-
ing" rate, it was found that as the column aged ~]~e
retention times of the oxygenated esters decreased
to a greater extent than did those of the normal esters
used for the determination of carbon numbers. Magid*
man et al. (18) also f(mnd that as polyester columns
aged the retention times of unsaturated esters changed
at a different rate than those of saturated esters. Craig
40
60
BO
I00
~0
140
IB
4
1 5 ~
Z
0
0 0 .......................
40
60
~
lO0
120
140 ~I
T{ME, minu|es
(
7) observed a similar effect when he measured the
Fro. 1. A) GLC sepm~ation of methyl 2,3-,8~ ~l,{t /7d~ydroxy~
relative emergence times of saturated and unsaturated
esters on a number of polyester columns which had
a wide variation in the ratio of liquid phase to solid
support. The figures which were obtained using the
polyester eolmnn are not absolute values but refer
only to a eoImnn of the particular age used in these
slearates usillg tilt ]!,~(]S eolunln, B ) (~I~(, separation of meihy/
2
.,3-,4.,12-,14-,15-,16-,17-
a n d 18.aeetoxystearates usillg the EGS
column. C ) GL(? separation o f methyl 2~,4-,5-,6-,8~,16~ a n d 17-
oxostear~.tes usil~g a 12 ft x :}/i(; in. silicone QF4 column.
was obtained using the polyester column; Figure 1B
shows the separation obtained with some of 1he iso-
II~lers.
experiments
(2-3 weeks at operating temp). A col-
umn which had been in use for six weeks yielded fig-
nres which were all 0.3-0.4 of a carbon number lower
than those in the table, but relative to each other they
had not changed appreciably.
Methyl Hydroxystearates. As might be expected
from the ease of formation of intramotecular hydrogen
bonds (21,17) the 2- and 3-isomers had inueh lower
carbon numbers than the rest on all three columns,
The 4- and 5dsomers had carbon numbers which, in
the case of the SE-30 column, were slightly greater
than those expected and which in the case of the other
two columns were markedly greater than: t h o s e of any
of the other isomers. ,/-Stearolactone gave peaks with
the same carbon numbers as those given by methyl
Methyl Oxostearates. Methyl 3~oxost(arate decom-
posed on all three columns, giving a product with a
carbon nmnber of about 16, using the SE-30 eolmnn.
The IR spectrum of the product, which was collected
when batches of methyl 3-oxostearate were put
through the SE-30 eolumn, did not show the original
ester carbonyl absorption at 1746 cm ~ (10), but only
ketoni( carbonyl absorption at 1710 cm i, which in-
dicated that the product was probably 2-heptadec -
none. The carbon mnnbers increased wih change in
snbstituent position using the SE-30 colunm but the
overall change was quite small. There was a eon~
siderably greater overM1 change using the QF-1 col-
umn, especially for the region of the 2- to 8-isomers,
4
-hydroxystearate.
It would not be surprising if the
which is illustrated in Figure 1C. A considerable
4
- and 5-hydroxy esters were converted to lactones
change in carbon nmnber from the 2- to 17dsomers
also was found using the polyester column but the
change in the region of the 2~ to 8-isomers was smaller
than that found for the QF-1 eolumm Methyl 2-oxo-
stearate appeared to deeompose to some extent using
the polyester column since the peak to which the car-
bon number of 22.55 was assigned was followed by a
smaller broader peak. The major peak a/so may have
been a decomposition peak as its carbon number was
m~expectedly low.
Applications to the Identification and Analysis of
Oxygenated Stearates. Any oxygenated ester can be
distinguished from a non-oxygenated ester which has
the same carbon number on the SE-30 column by re-
analysis on the QF-1 or the polyester eolmnn. In
attempting to identify an unknown isomer it is prob-
ably best to compare it with a known compound such
as 12-ox , hydroxy- or aeetoxystearate, which can
easily be derived from hydrogenated castor oil esters,
rather than to rely on measurements of carbon num-
ber alone. If the unknown ester is a hydroxy ester
only the 2-,3-,17- and 18-hydroxy esters can be posi-
tively distinguished and separated from 12~hydroxy-
stearate on any of the eolmnns. The 4- and 5~hydroxy
esters also are separated from 12-ester on the more
polar columns but their behavior is exceptionl and
may not be the same under all operating conditions.
at the temp of the injector but it is still difficult to
explMn why a ]actone with an appreciably lower tool
wt would have such high carbon numbers. The carbon
numbers of the rest of the isomers increased gradually
as the substiment approached the end of the chain.
However, only the 2-,'1- and 1% or 18-isomers dif-
fered from the others by more than 0A of a carbon
atom. The curve obtNned for some hydroxy esters is
shown in Figure 1A. The 18dsomer did not Nve a
peak on the pNy-ester column perhaps because the
primary hydroxy group reacts with the liquid phase
particularly readily.
Methyl Acetoxystearates. Using the SE-30 column
acetylatiou was found to have a considerable effect on
the carbon numbers of the 2- and 3-isomers since
hydrogen bonding was no longer possible. All the
isomers now formed a series of confinnousIy increasing
carbon nmnber; there was only 0.25 of a carbon num-
ber difference between the 2- and 12~aeetates but the
13- to 18dsome~:s showed an increasingly greater dif-
ference between each other. The total change in car-
bon m~mber from the 2- to the 18-isomer was 1.6 units.
Similar results were obtained using the QF-1 column
except that there was a greater Change between iso-
mers, the overM1 change from the 2- to the 18.isx)mer
was ca. 2.9 units. A still larger change, of 3.9 units,

836
T H E JOURNAL OF THE AMERICAN OIL CHEN[ISTS ~ SOCIETY
VoL. 41
3
4
. Bowman, R. E, J. Chem~ See. 325 (1950).
~ Christie, W. W , t~. D, Ganstone and H. G. Prentice, Ibm. 5768
(I963).
Clutterbuek, P, W. Ibid. 2330 (1924)~
For further identification the unknown hydroxy
ester should be converted to the acetoxy ester. Using
the polyester column and 12-aeetoxystearate as stand-
ard it can be seen from Figure 1B that 14-aeetoxy
ester can be partly resolved from 12-ester if the rela-
tive amt in the mixture are suitable and that 2-,3-,4-,
5
.
6. Craig, B. M~, Chain, Ind. (London), 1442 (1960).
7. Craig, B. M, in "Gas Chromatography," 3rd Symposium, ca. by*
N. Brenner, J. E. Callen and M. B. Weiss, Academic Press Inc., New
York, 1962, pp. 37-56.
8
. Diaper, D. G. M., Can, J. Chem. 83, I720 (1955).
9
. Fiesev, L. F., and a. Szmas~kovic~, J~ Am. Chem. See. 70, 8352
1948).
0. Fischmeister, I., Arkiv KemL 18, S01 (1961).
(
15-,16-,17- and 18-acctoxy esters are partly or com-
1
pletely separated. If the unknown hydroxy ester is
converted to the oxo ester (2,11) further information
can be obtained by analysis on the QF-1 cohmm, par-
ticularly about isomers with the substituent a t posi-
tions 4-8. For this purpose it was found best to use
a 12 ft x ~4'6 in. eolunm, as was used to obtain Figure
11. Gorin, P. A, g. J. F. ~. Spencer and A. P. Tulloeh, Can. a.
ChenL gg, 846 (1961).
I2. (}reaves, W. S., R. P. Linstead, B. R. Shephard, S. L. S. Thomas
and B. C. L. Weedon, J. Chem. See. 3326 (1950).
is. Hopkins, C. Y., JAOCS 88, 664 (1961),
14 Hiinig, S, and W. Landis, Bar. 98 913 (1960).
17. Kitaggwa, I . M Sugai and F. A, Kummerow, JAOCS 89, 217
1962).
(
1
C, instead of the 6 ft x :}{6 colmnn used to o b ~ i n
the carbon numbers. Figure 1C shows that the 2-,4-,
-,I6- and 17-oxo esters M1 are separated from each
18. Magidman, P., S, F. Herb, 1¢ A. Barford and R. W, Riemen~
schneider, Ibid. 39, 137 (1962).
9, Manyik, R. M., F. C. Frostick, J. J. Sanderson and C~ R, Hauser,
J. Am. Chem. See. 75, 5030 (1953).
0. Miwa, T. K., K. L. Mikolajeaak, F, R~ Earle and I. A. Wolff,
Anal, Chem. 82, 1739 (1960).
1. Morris, L, J., F;. T. HoIraan and K~ Fontell, J. Lipid Res. 1, 412
1969).
22. Morrissette, I~. A., and W, E. Link, JAOCS 41~ 415 (1964).
8, O'Brien, J. S., and G. Rouser, Anal. Biochem. 7, 288 (1964).
4. yon Rudloff, E,, Can. J. Chem. 88, 631 (1960).
25. Ryhage, I¢, and E. Stenhagen, Arkiv KemL 15, 545 (1960)~
6~ Skogh, M., Acts Chem. Seand. 6, 809 (1952).
27. Spencer, E. Y. and G. F. Wright, J. Am. Chem. See gg, 1281
1941).
8. Stgllberg~S~nhagen, S., Arkiv Kemi. Mineral. Geol. 2OA, No, 19,
1 (1945),
9. Stork, G., A~ Brizzolara, It. Landesman, J. Szmus~kovies and R,
Terrell, J. Amo Chem~ See. 85, 297 (1963)~
0. Sweet, I¢~ S., and P~ L. Estes, J. Org. Chem. 21, 1426 (1956)~
1. Tulloeh, A, P,, B. M. Craig and G. A. Ledingham, Cam J.
MicrebioI, g, 485 (1959).
1
5
2
other and that 6mxo is partly separated from 8-oxo.
Using this column the 6- and 12-oxo esters are con>
pletety separated, 7- and 12-oxo esters are partly sep-
arated and 8-oxo ester frames a pronounced shoulder
on the 12-oxo ester peak. Thus by using a combina.
tion of the three types of oxygenated ester and the
three columns 2-,3-,4-,5-,6~,7-,8-,14~,15-,16~,17- and 18-
isomers can be identified using the 12-isomer as a
known standard but the 9~,10~,11- and 13dsomers can-
not be characterized in this way.
2
(
(
2
2
2
2
2
8
3
ACKNOWLEDGMENTS
3
2, Tulloeh, A. P. J. F. T, Spencer and P. A, J, Gorin, 0an, J.
Many useful discussions with B~ M. Craig and E~ yon gudloff; L~ L.
Hoffman for ~ble experimental assistance. NMR and IB, spectra by W.
Haid.
Chem. 40, 1326 (1962),
38. Tulloch, A, P. ,and J, F. T. Spencer, Ibid. 42, 880 (1964),
34. VandenHeuvel, W. J, A., E. ~. A. Haahti and E, C. Horning,
J. Am. Chem. See. 83, 1513 (1961).
3
1960).
REFERENCES
5. Woodford, F. P., and C. M. van Gent, a. Lipid Res. 1, 188
(
1
. Bans, E~ M. and W. R. Mcbride, Anal. Chem. 51, 1879 (1959).
. Bergstrgm, S., G. Aulin-Erdtman. B, Rolander, E, Stenhagen and
2
S, (tstling, Aeta Chem. Seand. 6, 1157 (1952).
[Received June 22, I964--Accepted August 10, 1964]
of
Mixtures DEAE
With
Cellulose Column
GEORGE ROUSER, CLAUDIO GALLI and ELLEN LIEBER, City of Hope Medical Center,
Department of Biochemistry, Duarte, California; and
M. L. BLANK and O. S. P R I V E t , The Hormel Institute, University of Minnesota, Austin, Minnesota
Abstract
servations of Borgstrgm (1), Fillerup and Mead (2),
Barren and Hanahan (3) and Lea e t a l . (4). Di-
ethylaminoethyl ( D E A E ) cellulose column cbroma-
togr~)hy was introduced by Rouser et al. (5) to elimi-
slate the problem of the elution of acidic lipids with
other lipid classes encountered with silicic acid col
mnn chromatography, D E A E column chromatography
combined with si]idc acid eohlmn chromatography and
with silieic aeid~silicate column chromatography was
used to obtain separation of nmst lipid classes of brain
(5,6) and other mixtures (6). Recently, Privett and
Blank (7) and Blank et aL (8) have greatly extended
the possibilities of TLC for quantitative applications.
These investigators demonstrated that a spray reagent
composed of HAS04 and potassium diehromate can be
used to char spots obtained by TLC to a reproducible
optical density. Under proper conditions the extent
of charring is independent of the degree of unsatura-
lion of lipid classes such as lecithin and sphingomye-
lin, can be made to give a linear response over a fairly
wide concentration range, and is readily determined
by transmission densitometry.
A
quantitative chromatographic procedure for
the fractionation of complex lipid mixtures is de-
scribed. The method utilizes diethylaminoethyl
(
D E A E ) cellulose column chromatography fol-
h)wed by thin layer chromatography (TLC).
Spots produced in TI~C are charred with Slflfuric
acid~pctassiunl dichromate and heat and are then
measured by quantitative densitometry. Results
obtained with beef brain and beef heart n/ito-
ehondrial lipids are presented, and the close cor-
respondence between column isolation procedures
and the new procedure is demonstrated. Methods
utilizing only column chromatography, column
chromatography and TLC, and one- and two-
dimensional TLC without column chromatogra-
phy are compared.
Introduction
It~MATOGRAPHIC PROCEDUR~ :S are ~21idely used for
C
the determination of lipid class composition. St-
lisle add column chromatography has been the most
commonly utilized approach based on the initial ob~
The present report describes a new approaeh to