U.S. patent application number 09/779618 was filed with the patent office on 2001-09-27 for process for separating and purifying eicosapentaenoic acid or its ester.
Invention is credited to Asami, Yuji, Fujita, Shiro, Ikeda, Toru.
Application Number | 20010025112 09/779618 |
Document ID | / |
Family ID | 18559381 |
Filed Date | 2001-09-27 |
United States Patent
Application |
20010025112 |
Kind Code |
A1 |
Fujita, Shiro ; et
al. |
September 27, 2001 |
Process for separating and purifying eicosapentaenoic acid or its
ester
Abstract
Eicosapentaenoic acid or its ester is separated and purified
from a mixture containing a highly unsaturated fatty acid or its
ester effectively at low cost by a process, which comprises
developing the mixture by a mixed solvent of an ether solvent and a
hydrocarbon solvent, using a column filled with a silica gel having
a particle diameter of 1-100 micrometers.
Inventors: |
Fujita, Shiro; (Saitama-ken,
JP) ; Asami, Yuji; (Ueda-shi, JP) ; Ikeda,
Toru; (Ueda-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, McCLELLAND, MAIER & NEUSTADT, P.C.
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Family ID: |
18559381 |
Appl. No.: |
09/779618 |
Filed: |
February 9, 2001 |
Current U.S.
Class: |
554/191 ;
554/198; 554/199 |
Current CPC
Class: |
C07C 67/56 20130101;
C07C 59/64 20130101; C07C 67/56 20130101; C07C 69/587 20130101;
C07C 59/72 20130101 |
Class at
Publication: |
554/191 ;
554/198; 554/199 |
International
Class: |
C11B 003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 14, 2000 |
JP |
2000-035140 |
Claims
What is claimed is:
1. A process for separating and purifying eicosapentaenoic acid or
its ester from a mixture containing a highly unsaturated fatty acid
or its ester, which comprises developing the mixture containing the
highly unsaturated fatty acid or its ester by a mixed solvent of an
ether solvent and a hydrocarbon solvent, using a column filled with
a silica gel having a particle diameter of 1 to 100
micrometers.
2. The process of claim 1 wherein the ether solvent is a dialkyl
ether and the hydrocarbon solvent is an alkane hydrocarbon of 5-8
carbons.
3. The process of claim 1 wherein the mixed solvent is developed at
a linear velocity of 1-10 cm/min in the column.
4. The process of claim 1 wherein a precision distillation is
carried out after the development by the mixed solvent.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a process for purifying a highly
unsaturated fatty acid or its ester. More particularly, the
invention relates to a process for purifying eicosapentaenoic acid
or its ester from a mixture containing the highly unsaturated fatty
acid or its ester, by a silica gel column chromatography.
BACKGROUND OF THE INVENTION
[0002] Highly unsaturated fatty acids or their esters such as
eicosapentaenoic acid (hereafter called "EPA") and docosahexaenoic
acid (hereafter called "DHA") contained in fish oil such as sardine
oil have been used in the field of drugs and health foods, since
they have a pharmacological effect in vivo and are effective
against diseases such as arteriosclerosis obliterans and
hyperlipemia.
[0003] Known processes for the purification of these unsaturated
fatty acids include a urea-addition process, a precision
distillation process, a column chromatography process and a
supercritical fluid extraction process. These processes have been
used singly or in combination for purifying highly unsaturated
fatty acids.
[0004] However, the urea-addition process is not satisfactory as a
purification process for removing eicosatetraenoic acid (hereafter
called "ETA") or its ester, because of its being poor in
urea-adduct forming ability. The precision distillation process
needs an operation at high temperature, and may easily cause
denaturation by polymerization or isomerization. In the
purification of EPA or its ester, the process has a great
difficulty in removing ETA or its ester, because ETA and EPA have
the same carbon numbers and make a small difference in boiling
point. Further, since the supercritical fluid extraction process
deals with high-pressure fluid, it has a difficulty in installation
of equipment and it is unsuitable for mass separation and
purification on an industrial scale.
[0005] As a process for the purification of highly unsaturated
fatty acids using the column chromatography method, Japanese Patent
Kokai 5-222392 discloses a combination of precision distillation
and reverse phase partition chromatography using a silica gel on
which an octadecyl group is held. Further, Japanese Patent Kokai
9-151390 discloses a purification process using a carrier having a
silver salt carried on silica gel. These purification processes can
separate and purify EPA and its ester from mixtures of highly
unsaturated fatty acids. The former process is excellent in the
separation and purification, but it has the problem that the cost
of filler becomes high because the octadecyl group is held on the
silica gel surface. The latter process has the problem that the
cost is high due to use of the silver salt, that the method for the
preparation of the carrier carrying the silver salt is complicated,
that the extraction operation is complicated with repeated
agitation and filtration, and that the product may be contaminated
with a silver compound by using a particular solvent.
[0006] Usually, ETA and its ester are contained in the raw material
of EPA and its ester. However, ETA and its ester are impurities
very difficult to separate and remove in the purification of EPA
and its ester, as mentioned above.
[0007] Thus, there has been a demand for a process for the
purification of eicosapentaenoic acid or its ester, by which the
problems encountered in the prior art can be overcome and the
contents of ETA and its ester can be remarkably reduced by
selectively removing impurities.
SUMMARY OF THE INVENTION
[0008] Accordingly, an object of the present invention is to
provide a process for selectively separating and purifying
eicosapentaenoic acid or its ester effectively at low cost.
[0009] A further object of the present invention is to provide a
process for selectively separating and removing ETA or its ester by
a normal phase column chromatography using the silica gel under a
specific condition.
[0010] The present invention provides a process for separating and
purifying eicosapentaenoic acid or its ester from a mixture
containing a highly unsaturated fatty acid or its ester, which
comprises developing the mixture containing the highly unsaturated
fatty acid or its ester with a mixed solvent of an ether solvent
and a hydrocarbon solvent, using a column filled with a silica gel
having a particle diameter of 1 to 100 micrometers.
DETAILED DESCRIPTION OF THE INVENTION
[0011] In this process, the silica gel used as a filler of the
column is required to have a particle diameter in the range of
1-100 micrometers. If the silica gel of small particle diameter is
used, pressure loss will become large, but the amount of raw
material loaded and the purifying capability will be increased due
to its increased surface area. On the other hand, if the silica gel
of large particle diameter is used, pressure loss will become
small, but the amount of raw material loaded and the purifying
capability will be reduced. Thus, it is required to choose the
silica gel of suitable particle diameter in view of the amount of
raw material loaded and purifying capability. For the selective
separation of ETA or its ester, it is preferable to use the silica
gel providing small pressure loss and having as large a specific
surface area as possible. In the present invention, the form of
silica gel may be spherical or fragment as far as the silica gel
has the above-defined particle diameter, but spherical form is
preferable. In addition, it is preferable to use porous silica gel
having a pore size of not less than 4.times.10.sup.-3
micrometers.
[0012] The amount of this silica gel used varies depending on the
quantity of raw material to be processed and/or size of a column,
etc., but it is usually within the range of 5-50 grams, preferably
10-40 grams per gram of raw material, i.e., the mixture comprising
the highly unsaturated fatty acid or its ester.
[0013] In the present process, the mixture containing the highly
unsaturated fatty acid or its ester is charged in the column filled
with silica gel, and then developed with an organic solvent serving
as a developing solvent. As the developing solvent, a mixed solvent
with the highest purifying capability can be selected by varying
the polarity of solvent. According to the present invention, the
mixed solvent of a hydrocarbon solvent and an ether solvent is
advantageous for the separation and purification of
eicosapentaenoic acid or its ester.
[0014] The hydrocarbon solvents in the mixed solvent can include,
but are not limited to, hydrocarbons of 5-8 carbons, preferably
alkane hydrocarbons, for example, n-pentane, n-hexane, n-heptane
and n-octane. The ether solvents can include, but are not limited
to, dialkyl ethers, for example, diisopropyl ether, diethyl ether
and methyl tert-butyl ether. In the present process, the developing
solvent can be used in any combination of the hydrocarbon solvent
and the ether solvent, but a mixed solvent of n-hexane (hereafter
called "n-Hex") and diisopropyl ether (hereafter called "IPE") is
especially preferable. A mixing ratio of these solvents can be
suitably decided so as to provide optimum conditions for the
purification of the desired EPA or its ester. For a mixed solvent
of n-Hex and IPE, for example, the mixing ratio is a volume ratio
of n-Hex:IPE=90-99:10-1, preferably 95-99:5-1.
[0015] In the preferred embodiment of the present invention,
eicosapentaenoic acid or its ester can be separated from the
mixture of the highly unsaturated fatty acid or its ester by a
column chromatography, which comprises developing said mixture with
a mixed solvent of n-Hex and IPE as a developing solvent, using a
column filled with silica gel having a particle diameter of 1-100
micrometers, at a linear velocity of 1-10 cm/min.
[0016] From another viewpoint, the present invention relates to a
process for selectively removing ETA or its ester from a mixture
comprising a highly unsaturated fatty acid or its ester containing
eicosatetraenoic acid (ETA) or its ester, by the above-described
column chromatography.
[0017] The column used in the process of the present invention is
prepared by suspending silica gel in an organic solvent to slurry
it, filling the slurried silica gel in a medium pressure column and
flowing an organic solvent about 1-10 times the volume of the
column to stabilize the column. A preferable organic solvent used
when filling silica gel in slurry is acetone. It is preferable that
an organic solvent used in the stabilization of column is the same
as the developing solvent used in the purification.
[0018] The optimum amount of the mixture containing the highly
unsaturated fatty acid or its ester which should be charged in the
column can be varied depending on the composition ratio of each
fatty acid in the raw material, but it is in the range of 2 to 20%
by weight, preferably 3 to 15% by weight, based on the weight of
silica gel in the column. Preferably, the raw material, i.e., the
mixture containing the highly unsaturated fatty acid or its ester
is dissolved in a developing solvent at most 5 times, e.g., twice
the weight of the mixture, and thereafter this solution is flowed
through the column.
[0019] For the column used in the process of the invention,
preferable is a medium pressure column with the column length
diameter ratio of 2 or more and the theoretical plate number of
15000-20000 plates/m. The pressure resistance of column may be in
the range of 1-6 MPa, depending on the length and the linear
velocity of column. The column is preferably operated at
10-30.degree. C., e.g., at room temperature. In the separation of
ETA or its ester, it is preferable that the mixture of raw material
is developed while applying the pressure so as to provide a linear
velocity of 1-10 cm/min in the column.
[0020] The mixture containing the highly unsaturated fatty acid or
its ester used as a raw material in the present process can be
obtained from fish oils extracted from mackerels, sardines,
codfishes, etc., but is not limited especially. This raw material
mixture contains ETA, EPA, DHA and other components such as fatty
acids of less than 20 carbons.
[0021] The invention is further illustrated by the following
non-limiting examples and comparative examples.
EXAMPLE 1
[0022] (1) 39.2 g of spherical silica gel having an average
particle diameter of 20 micrometers (manufactured by Soken Chemical
& Engineering Co., Ltd.) were slurried with acetone and this
slurry was filled under pressure in a stainless steel medium
pressure column (20 mm in inside diameter.times.250 mm in length).
Then, a mixed solvent (a volume ratio of IPE:n-Hex 3.2:96.8) 3.4
times the volume of the column was flowed to stabilize the
column.
[0023] (2) The composition of the highly unsaturated fatty acid
used as a raw material in this example is shown in the following
Table 1. The highly unsaturated fatty acid in the raw material was
subjected to ethyl esterification beforehand.
[0024] 2.0 g of this raw material were dissolved in 4 ml of a mixed
solvent (a volume ratio of IPE:n-Hex=3.2:96.8) and this solution
was flowed into the column as prepared in (1).
[0025] First, the column was developed by 198 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.23 g of oil as the first effluent fraction with the
composition shown in the following Table 2.
[0026] Second, the column was developed by 7.4 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.24 g of oil as the second effluent fraction with the
composition shown in Table 2.
[0027] Third, the column was developed by 278.6 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 1.48 g of oil as the third effluent fraction with the
composition shown in Table 2.
[0028] Finally, the column was developed by 117.8 ml of acetone at
a linear velocity of 7.0 cm/min, and the resultant effluent was
condensed under reduced pressure in an evaporator, thus giving 0.06
g of oil as the fourth effluent fraction, with the composition
shown in Table 2.
[0029] 1.48 g of the oil obtained as the third effluent fraction
was further subjected to high-vacuum precision distillation using a
packed column type precision distillation tower having a
theoretical plate number of 8 under the conditions: a degree of
vacuum of not more than 0.02 mm Hg at the top of the tower, a
degree of vacuum of not more than 2.0 mm Hg at the bottom of the
tower and an average distillation temperature of 165-210.degree. C.
1.18 g of oil having the composition shown in Table 3 was
obtained.
[0030] The gas chromatograph used in the analysis was GC-17A Gas
Chromatograph manufactured by Shimadzu Corporation.
[0031] The column condition is shown below.
[0032] Column: 0.25 mm (inside diameter).times.30 m (length)
[0033] (Trade name: DB-WAX manufactured by J&W)
[0034] Detector: FID
[0035] Column temperature: 210.degree. C.
[0036] Inlet temperature: 250.degree. C.
[0037] Detector temperature: 260.degree. C.
[0038] Carrier gas: helium
[0039] Flow rate: adjusted so as to retain EPA ethyl ester for
about 20 minutes.
[0040] The composition of the fatty acids in the raw material used
in Example 1 is shown in Table 1.
1 TABLE 1 Content of ethyl ester of each fatty acid (%) ETA EPA DHA
Others Raw material 4.20 73.48 11.65 10.67
[0041] As a result of the chromatography in Example 1, the yield
and the composition of each effluent are shown in Table 2.
2 TABLE 2 Content of ethyl ester of each fatty acid (%) Yield (%)
ETA EPA DHA Others First effluent fraction 11.2 32.52 20.27 10.60
36.61 Second effluent fraction 11.8 5.69 68.53 17.10 8.68 Third
effluent fraction 74.1 0.95 80.32 10.28 8.45 Fourth effluent
fraction 3.0 1.30 66.18 9.56 22.96
[0042] The yield and the composition of the oil obtained by
distillation of the third effluent fraction in Example 1 are shown
in Table 3.
3 TABLE 3 Content of ethyl ester of each fatty acid (%) Yield (%)
ETA EPA DHA Others Product purified by 79.7 1.34 98.31 0.0 0.35
distillation
[0043] The result in Example 1 shows that ETA or its ester
contained before purification was largely reduced by the process of
the present invention and also that EPA of high purity of 98% or
more could be obtained in the subsequent purification process by
distillation.
COMPARATIVE EXAMPLE 1
[0044] (1) 39.2 g of spherical silica gel having an average
particle diameter of 110 micrometers (manufactured by Fuji Silysia
Chemical Ltd.) were slurried with acetone and this slurry was
filled under pressure in the same medium pressure column as used in
Example 1. Then, a mixed solvent (a volume ratio of
IPE:n-Hex=3.2:96.8) 3.4 times the volume of the column was flowed
to stabilize the column.
[0045] (2) 2.0 g of the same highly unsaturated fatty acid mixture
as used in Example 1 were dissolved in 4 ml of the mixed solvent (a
volume ratio of IPE:n-Hex=3.2:96.8) and this solution was flowed
into the column as prepared in (1).
[0046] First, the column was developed by 198 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 1.61 g of oil as the first effluent fraction with the
composition shown in the following Table 4.
[0047] Second, the column was developed by 7.4 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.22 g of oil as the second effluent fraction with the
composition shown in Table 4.
[0048] Third, the column was developed by 278.6 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.05 g of oil as the third effluent fraction with the
composition shown in Table 4.
[0049] Finally, the column was developed by 117.8 ml of acetone at
a linear velocity of 7.0 cm/min, and the resultant effluent was
condensed under reduced pressure in an evaporator, thus giving 0.03
g of oil as the fourth effluent fraction with the composition shown
in Table 4.
4 TABLE 4 Content of ethyl ester of each fatty acid (%) Yield (%)
ETA EPA DHA Others First effluent fraction 80.5 4.70 72.05 11.72
11.53 Second effluent fraction 11.0 1.43 79.92 10.79 7.86 Third
effluent fraction 2.5 1.98 79.55 10.78 7.69 Fourth effluent
fraction 1.5 1.70 29.73 5.02 63.55
[0050] The result in Comparative Example 1 shows that ETA or its
ester was insufficiently separated by using the silica gel of large
particle diameter and also that the yields of the resultant second
and third effluent fractions were extremely low. High-vacuum
precision distillation of the oil in these effluent fractions could
not result in EPA of 98% or more purity.
EXAMPLE 2
[0051] (1) 39.2 g of spherical silica gel having an average
particle diameter of 40 micrometers (manufactured by Soken Chemical
& Engineering Co., Ltd.) were slurried with acetone and this
slurry was filled under pressure in the same medium pressure column
as used in Example 1. Then, a mixed solvent (a volume ratio of
IPE:n-Hex=2.0:98.0) 3.4 times the volume of the column was flowed
to stabilize the column.
[0052] (2) The composition of the highly unsaturated fatty acid
used as a raw material in this example is shown in the following
Table 5. The highly unsaturated fatty acid in the raw material was
subjected to ethyl esterification beforehand.
[0053] 1.0 g of this raw material was dissolved in 2 ml of a mixed
solvent (a volume ratio of IPE:n-Hex=2.0:98.0) and this solution
was flowed into the column as prepared in (1).
[0054] First, the column was developed by 118 ml of the mixed
solvent at a linear velocity of 1.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.29 g of oil as the first effluent fraction with the
composition shown in the following Table 6.
[0055] Second, the column was developed by 23.6 ml of the mixed
solvent at a linear velocity of 1.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.14 g of oil as the second effluent fraction with the
composition shown in Table 6.
[0056] Third, the column was developed by 240.2 ml of the mixed
solvent at a linear velocity of 1.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.48 g of oil as the third effluent fraction with the
composition shown in Table 6.
[0057] Finally, the column was developed by 78.5 ml of acetone at a
linear velocity of 1.0 cm/min, and the resultant effluent was
condensed under reduced pressure in an evaporator, thus giving 0.03
g of oil as the fourth effluent fraction with the composition shown
in Table 6.
[0058] 0.48 g of the oil obtained as the third effluent fraction
was further subjected to high-vacuum precision distillation using a
packed column type precision distillation tower having a
theoretical plate number of 8 under the conditions: a degree of
vacuum of not more than 0.02 mm Hg at the top of the tower, a
degree of vacuum of not more than 2.0 mm Hg at the bottom of the
tower and an average distillation temperature of 165-210.degree. C.
0.40 g of oil having the composition shown in Table 7 was
obtained.
[0059] The composition of the fatty acids in the raw material used
in Example 2 is shown in Table 5.
5 TABLE 5 Content of ethyl ester of each fatty acid (%) ETA EPA DHA
Others Raw material 2.76 48.74 8.86 39.64
[0060] As a result of the chromatography in Example 2, the yield
and the composition of each effluent fraction are shown in Table
6.
6 TABLE 6 Content of ethyl ester of each fatty acid (%) Yield (%)
ETA EPA DHA Others First effluent fraction 29.0 0.06 0.0 0.0 99.94
Second effluent fraction 13.5 15.3 31.76 12.35 40.59 Third effluent
fraction 48.0 0.92 77.68 12.28 9.12 Fourth effluent fraction 2.5
0.0 2.73 0.0 97.27
[0061] The yield and the composition of the oil obtained by
distillation of the third effluent fraction in Example 2 are shown
in Table 7.
7 TABLE 7 Content of ethyl ester of each fatty acid (%) Yield (%)
ETA EPA DHA Others Product purified by 83.3 1.28 98.35 0.0 0.37
distillation
[0062] The result in Example 2 shows that EPA, the content of which
was relatively low in the raw material, was separated and purified
effectively by the column chromatography of the present invention
and that ETA or its ester was largely reduced. Further, EPA of high
purity of 98% or more could be obtained in the subsequent
purification process by distillation.
EXAMPLE 3
[0063] (1) 39.2 g of spherical silica gel having an average
particle diameter of 60 micrometers (manufactured by Soken Chemical
& Engineering Co., Ltd.) were slurried with acetone and this
slurry was filled under pressure in a stainless steel medium
pressure column (20 mm in inside diameter.times.250 mm in length).
Then, a mixed solvent (a volume ratio of IPE:n-Hex=3.2:96.8) 4.2
times the volume of the column was flowed to stabilize the
column.
[0064] (2) The composition of the highly unsaturated fatty acid
used as a raw material in this example is shown in the following
Table 8. The highly unsaturated fatty acid in the raw material was
subjected to ethyl esterification beforehand.
[0065] 2.0 g of this raw material was dissolved in 4 ml of the
mixed solvent (a volume ratio of IPE:n-Hex=3.2:96.8) and this
solution was flowed into the column as prepared in (1).
[0066] First, the column was developed by 220 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.07 g of oil as the first effluent fraction with the
composition shown in the following Table 9.
[0067] Second, the column was developed by 7.4 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.19 g of oil as the second effluent fraction with the
composition shown in Table 9.
[0068] Third, the column was developed by 322.6 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 1.73 g of oil as the third effluent fraction with the
composition shown in Table 9.
[0069] Finally, the column was developed by 154 ml of acetone at a
linear velocity of 7.0 cm/min, and the resultant effluent was
condensed under reduced pressure in an evaporator, thus giving 0.02
g of oil as the fourth effluent fraction with the composition shown
in Table 9.
[0070] The composition of the fatty acids in the raw material used
in Example 3 is shown in Table 8.
8 TABLE 8 Content of ethyl ester of each fatty acid (%) ETA EPA DHA
Others Raw material 3.22 96.03 0.0 0.75
[0071] As a result of the chromatography in Example 3, the yield
and the composition of each effluent fraction are shown in Table
9.
9 TABLE 9 Content of ethyl ester of each fatty acid (%) Yield (%)
ETA EPA DHA Others First effluent fraction 3.4 49.85 37.84 0.0
12.31 Second effluent fraction 9.5 19.53 78.47 0.0 2.00 Third
effluent fraction 86.6 0.62 98.85 0.0 0.53 Fourth effluent fraction
1.1 0.0 57.17 0.52 42.31
[0072] The result in Example 3 shows that EPA, the content of which
was high in the raw material, was separated and purified
effectively by the column chromatography of the present invention
and that ETA or its ester was largely reduced. Further, EPA of high
purity of 98% or more could be obtained in 85% or more yield.
EXAMPLE 4
[0073] (1) 39.2 g of spherical silica gel having an average
particle diameter of 20 micrometers (manufactured by Soken Chemical
& Engineering Co., Ltd.) were slurried with acetone and this
slurry was filled under pressure in a stainless steel medium
pressure column (20 mm in inside diameter.times.250 mm in length).
Then, a mixed solvent (a volume ratio of IPE:n-Hex=3.2:96.8) 3.4
times the volume of column was flowed to stabilize the column.
[0074] (2) 2.0 g of the same raw material as used in Example 1 was
dissolved in 4 ml of a mixed solvent (a volume ratio of
IPE:n-Hex=3.2:96.8) and this solution was flowed into the column as
prepared in (1).
[0075] First, the column was developed by 227.6 ml of the mixed
solvent at a linear velocity of 10 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.10 g of oil as the first effluent fraction with the
composition shown in the following Table 10.
[0076] Second, the column was developed by 7.8 ml of the mixed
solvent at a linear velocity of 10 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.18 g of oil as the second effluent fraction with the
composition shown in Table 10.
[0077] Third, the column was developed by 455.4 ml of the mixed
solvent at a linear velocity of 10 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 1.59 g of oil as the third effluent fraction with the
composition shown in Table 10.
[0078] Finally, the column was developed by 157 ml of acetone at a
linear velocity of 10 cm/min, and the resultant effluent was
condensed under reduced pressure in an evaporator, thus giving 0.05
g of oil as the fourth effluent fraction with the composition shown
in Table 10.
[0079] 1.59 g of the oil obtained as the third effluent fraction
was further subjected to high-vacuum precision distillation using a
packed column type precision distillation tower having a
theoretical plate number of 8 under the conditions: a degree of
vacuum of not more than 0.02 mm Hg at the top of the tower, a
degree of vacuum of not more than 2.0 mm Hg at the bottom of the
tower and an average distillation temperature of 165-210.degree. C.
1.28 g of oil having the composition shown in Table 11 was
obtained.
[0080] As a result of the chromatography in Example 4, the yield
and the composition of each effluent fraction are shown in Table
10.
10 TABLE 10 Content of ethyl ester of each fatty acid (%) Yield (%)
ETA EPA DHA Others First effluent fraction 4.9 40.77 7.48 3.94
47.81 Second effluent fraction 9.2 23.84 41.58 15.85 18.73 Third
effluent fraction 79.7 0.81 79.44 11.49 8.26 Fourth effluent
fraction 2.4 0.00 13.60 2.31 84.09
[0081] The yield and the composition of the oil obtained by
distillation of the third effluent fraction in Example 4 are shown
in Table 11.
11 TABLE 11 Content of ethyl ester of each fatty acid (%) Yield %
ETA EPA DHA Others Product purified by 80.5 1.32 98.29 0.0 0.39
distillation
[0082] The result in Example 4 shows that in the case where the
linear velocity in the column was high, EPA was separated and
purified effectively by the column chromatography of the present
invention and that ETA or its ester was largely reduced. Further,
EPA of high purity of 98% or more could be obtained in 80% or more
yield in the subsequent purification process by distillation.
COMPARATIVE EXAMPLE 2
[0083] (1) 39.2 g of spherical silica gel having an average
particle diameter of 20 micrometers (manufactured by Soken Chemical
& Engineering Co., Ltd.) were slurried with acetone and this
slurry was filled under pressure in the same medium pressure column
(20 mm in inside diameter.times.250 mm in length) as used in
Example 1. Then, a mixed solvent (a volume ratio of ethyl
acetate:n-Hex=1.5:98.5) 3.4 times the volume of column was flowed
to stabilize the column.
[0084] (2) 2.0 g of the same highly unsaturated fatty acid mixture
as used in Example 1 was dissolved in 4 ml of a mixed solvent (a
volume ratio of ethyl acetate:n-Hex=1.5:98.5) and this solution was
flowed into the column as prepared in (1).
[0085] First, the column was developed by 78.6 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.13 g of oil as the first effluent fraction with the
composition shown in the following Table 12.
[0086] Second, the column was developed by 196.2 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 1.16 g of oil as the second effluent fraction with the
composition shown in Table 12.
[0087] Third, the column was developed by 94.2 ml of the mixed
solvent at a linear velocity of 7.0 cm/min, and the resultant
effluent was condensed under reduced pressure in an evaporator,
thus giving 0.64 g of oil as the third effluent fraction with the
composition shown in Table 12.
[0088] Finally, the column was developed by 651.6 ml of the mixed
solvent and 157.0 ml of acetone at a linear velocity of 7.0 cm/min,
and the resultant effluent was condensed under reduced pressure in
an evaporator, thus giving 0.06 g of oil as the fourth effluent
fraction with the composition shown in Table 12.
12 TABLE 12 Content of ethyl ester of each fatty acid (%) Yield %
ETA EPA DHA Others First effluent fraction 6.6 14.10 45.27 14.14
26.49 Second effluent fraction 57.9 5.36 73.03 11.22 10.39 Third
effluent fraction 32.1 0.85 79.53 10.74 8.88 Fourth effluent
fraction 2.8 0.00 18.13 3.15 78.72
[0089] The result in Comparative Example 2 shows that ETA or its
ester was insufficiently separated by using ethyl acetate as an
ester solvent in the mixed solvent as a developing solvent, and
also that the yield of EPA in the third effluent fraction was
extremely low. High-vacuum precision distillation of the third
effluent fraction, however, could result in EPA of 98% or more
purity.
[0090] As described above, the process of the present invention is
characterized by using a medium pressure column filled with
spherical silica gel having a particle diameter of 1-100
micrometers and developing the column by a mixed solvent with a
volume ratio of n-Hex:IPE=90-99:10-1, preferably 95-99:5-1, by
which EPA or its ester can be separated and purified effectively
and also ETA or its ester can be removed selectively. According to
the present process, EPA or its ester can be separated and purified
at low cost by using only silica gel as a filler in the column. The
process can largely reduce initial and running costs for the
separation and purification of EPA or its ester on an industrial
scale.
[0091] Obviously, additional modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
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