——‘——"

 

SOME FACTORS AFFECTING THE

DETERMINATION OF FATTY ACIDS

AND UNSAPONIFIABLE MATTER
IN PLANT MATERIALS

Thesis for the Degree of M. S.
MICHIGAN STATE COLLEGE

John I. DiII
I942

 

 

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SOME FACTORS AFFECTIhG TBA DETERMIHATION OF
FATTY ACIDS AND UNSAPONIFIaBLE MATTER

IN ILANT MATERIALS

by
John J. Dill

A THESIS
Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of

MASTER OF SCIENCE

Department of Chemistry

1942

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ACKN OWLE D GELEIIT

It is a pleasant duty to acknowledge the
invaluable assistance of Professor C. D. Ball
in the planning of the research, and for the
constructive criticism and advice in the pre—

paration of this thesis.

May/5M

140105

II
III

IV

VI

TABLE OF CONTENTS

Introduction

Historical .......................................

EXperimental .....................................
A. A.O.A.C. and Hieserman Method ..............
B. Modifications of Hieserman Method ..........
C. Modified Extraction Procedure ..............

Discussion .......................................

Conclusions ......................................

Bibliography OOOOOOOOOOOOOOOOOOOOOO00.00.000.00...

10
10
16
24
27
38

INTRODUCTION

The research connected with this problem was origin-
ally undertaken to evaluate a procedure for lipide analysis
proposed by C. E. Hierserman (1). As the work progressed
the method was recognized as superior in some respects to
the existing procedures for lipids determination. The work
was then adopted as a thesis problem and an attempt was made
to eliminate certain undesirable points that were encountered,
and to determine what effects some variable factors had on the
determination of the unsaponifiable and fatty acid fractions.

As the terminology of ether soluble, fat-like substances
is quite confusing, one of the better classifications must be
chosen and followed for sake of clearness and uniformity.

The term "lipids" as used in this paper shall include all
substances soluble in such solvents as ethyl ether, chloroform,
petroleum ether; and insoluble in water and which are either
ester-like combinations with fatty acids or capable of form-
ing such combinations (2). The term "fat” shall include only
those fatty substances which are glyceryIIesters of fatty

acids.

HISTORICAL

When plant tissues are extracted with ethyl ether or
any of the other similar solvents, the extract contains a
wide variety of substances some of which yield fatty acids
on hydrolysis. The most of these fatty acid-yielding sub-
stances are the glyceryl esters, sterol esters, phosphatides,
and cerebrosides. Also appearing in the fat solvent extract
are compounds of such varied chemical composition as the wax
alcohols, sterols, essential oils, hydrocarbons, resins, lig-
nins.chlorophylls, and carotenoids.

The choice of the solvent used is one of the deciding
factors of the crude lipide extract. Unfortunately the sol—
vents such as petroleum.ether that extract a minimum of non-
lipide material give a poor yield of true lipides while the
solvents that do give a high yield of lipides generally ex-
tract large amounts of non-lipide material. As no "ideal
solvent" for lipide has been found the trend in recent
years has been to employ the solvent or combination of sol-
vents that give a maximum yield of crude lipides and then to
determine the amount of the desired constituents in the crude
extract.

Hence a logical division for an approach in the field
of lipide analysis is indicated; namely the extraction of
the crude lipide material and secondly the purification and
fractionation of the lipide material.

Ethyl ether is probably the most widely used laboratory

fat solvent for extraction of lipides. The common procedure

is to run a sixteen hour continuous extraction and report
the total yield as "crude lipides" or "crude fat". How-
ever, in this crude extract obtained from.many plant
tissues by the use of ether, the non-lipide substances may
amount to more than the total weight of true lipides (5).
The purity of the ether employed definitely affects the
quality of the crude extract. Chibnall and Channon (4)
state that the presence of alcohol and water will allow the
extraction of small amounts of inorganic matter, amino cmm-
pounds, carbohydrates and possibly tannins. Hieserman (1)
also reported a small but definite increase in the yield of
crude lipides when moisture-containing ether was used. This
source of error is eliminated in the official A.O.A.C. (5)
method for crude lipide determination by the use of C. P.
anhydrous ethyl ether on previously dried samples.

Even the use of C. P. anhydrous ethyl ether has three
decided disadvantages. First, that all lipide material is
not extracted has been shown by numerous workers (6) (7) (8)
(9) (10), second, many non-lipide materials such as pigments,
hydrocarbons, essential oils, alkaloids, and resins appear in
the ether extract (3) and third, it is assumed but without
justification that drying has no effect on the lipides. How-
ever, in total crude lipide determination of most plant
tissue these factors tend to off-set one another. Hence the
values obtained by the relatively simple A.0.A.C. method fre-
quently approximate the values for true fat of the more in-

volved and tedious methods.

Although petroleum ether extracts the smallest amounts
of non-lipide material of any of the fat solvents (3), the
yields of true lipides are quite low. Its use for total
lipide extraction is not recommended.

Carbon tetrachloride, being non-inflammable and because
it extracts most lipides with ease, possesses many character-
istics of a most desirable solvent. Bryant (11) claims that
a two hour extraction with carbon tetrachloride gives similar
yields of lipides to those obtained by a sixteen hour ethyl
ether extraction. However, Wiley (12) found that the last
traces of this solvent are removed with the greatest diffi-
culty and in the presence of even minute traces of moisture
it is partly decomposed into hydrochloric acid which has a
detrimental effect upon extracted lipides. The observation
of Schindelmeiser (13) that many alkaloids are readily sol-
uble in even cold carbon tetrachloride further discredits
its use as a solvent for crude lipide determinations.

Kumagawa and Suto (14) state that the efficient remov-
al of lipides in the method advanced by Rosenfeld (15) is
not due to the more energetic action of chloroform but to
the pre-treatment with alcohol.

Although carbon disulfide is a liquid of high solvent
power for fatty materials it has not come into general use
for quantitative lipide estimation. This is probably be-
cause of its poisonous properties, its unpleasent odor and
its liability to decomposition when used for the extraction
of certain lipides (5).

Acetone has a wide range of solvent power for many
classes of organic compounds including lipide material but
its solvent action is too general to permit its use for
quantitative crude lipide determinations (5).

Kaye (et. a1.) (16) reported that isopropyl ether was
a desirable solvent for fatty materials in feces. Isopropyl
ether, being an immiscible liquid, permits the simultaneous
determination of moisture and lipides. Monaghan (17) attempt-
ed to modify the method for lipide extraction in plant material
but his results indicate this solvent to be very ineffective
for plant tissues. Satisfactory values for the moisture con-
tent of plant materials were obtained, however.

Benzene and trichlorethylene have also been suggested
for crude lipide determination but are seldom used. The last
traces of the former are very difficult to remove from.the
lipide residue whereas the latter has too great a range of
solvent power to be of value.

Where the extracted lipide material is subsequently to
be fractionated and purified, more general solvents, combin-
ations of solvents, or other methods that facilitate a more
complete removal of the lipides can be employed as most imr
purities will be removed by later fractionation treatment.

W. Koch (18) reported that a more complete lipide ex—
traction from animal tissues could be obtained if the ethyl
ether extraction was preceded and followed by alcoholic ex-

tractions. Later F. C. Koch (19) introduced modifications

which adapted the method for analysis of plant tissue. This
modified procedure makes it possible to extract the lipide
material from fresh plant tissues without first subjecting

it to a harsh drying treatment. It also permits the storing
of fresh plant material (without drying) for a period of time
before the actual analysis has to be made. Further evidence
as to the value of a combination of solvents for lipide ex-
traction was reported by Shulze and Stieger (10). Their re-
sults indicate that the phOSpholipides can only be completely
removed from seed material if the ether extraction is follow-
ed by one of alcohol.

Acetone has been recommended (3) in place of alcohol in
the modified method of Koch. The results of Hieserman (1)
however, indicate that this substitution cannot always be
favorably made. In the extraction of lipides from.such a
typical plant tissue as alfalfa it was found that acetone
was definitely inferior to alcohol.

A number of investigators, Leathes (20), Lieberman and
Szekely (21), Rosethal and Trowbridge (22), and Rather (8),
have reported procedures which are based on the extraction
of the fatty acids. The entire sample is saponified with
_some suitable reagent such as alcoholic potash, then acidi-
fied, and the resulting fatty acids are extracted, dried and
weighed.

Early investigators considered an ether extract of most
tissues to consist mainly of neutral fats and other simple

glycerides. Later it was definitely established that a con—

siderable portion of the fat soluble extract was non-lipide
in nature and indigestible in the animal body (23). This

led to the separation of crude ether extract into the saponi-
fiable and unsaponifiable fractions with the interest center-
ing on the saponifiable or fatty acid fraction because of its
energy value in animal metabolism. During recent years the
unsaponifiable fraction has become biologically important be-
cause it contains vitamins E and K, and the precursors to
vitamins A and D. These factors have led to an increased de-
mand for better and more rapid means of fractionation of the
crude lipide extract.

The majority of the methods proposed to date call for
saponification of the sample or crude extract as a means of
separation of the fatty acids. Liebermann and Szekely (21)
in 1898 reported the use of saponification as a means of
lipide analysis. Their proposed method called for the sapon-
ification of the entire sample and following acidification
the liberated fatty acids were extracted with ethyl ether and
dried to constant weight. Kumagawa and Suto (14) modified
and improved the above method in 1908. The product obtained
in the method of Liebermann and Szekely was redissolved in
boiling alcohol and saponified. The unsaponifiable material
was extracted from the soap mixture with petroleum.ether.
After acidification of the alcoholic soap solution the fatty
acids were extracted with ethyl ether, evaporated to dryness,

and dried to constant weight. An approximation of the neutral

glycerides originally present was obtained by multiplying
the weight of fatty acids by an empirical conversion factor.

An improved method for the determination of the unsaponi-
fiable fraction was developed by the Fat Analysis Committee of
the Division of Industrial Chemists and Engineers of the Ameri-
can Chemical Society. This procedure, commonly known as the
F. A. C. method (24), was adopted as official for unsaponifiable
by the Association of Official Agriculture Chemists in 1926.
In brief the method calls for extraction of the unsaponifiable
with petroleum ether following the saponification of a five
gram fat sample. After evaporation of the solvent the residue
is tested for solubility in petroleum ether. Any insoluble
material is filtered off, the solution evaporated to dryness
and dried to constant weight. Hieserman (1) in 1939 adapted
the F. A. C. method for determination of the unsaponifiable
in plant tissue.

In 1936 Horwit, Cowgill and.Mendel (23) introduced
minor modifications in the method of Kumagawa and Suto where-
by they were able to run a positive determination of both the
unsaponifiable and fatty acids in plant tissues. These work-
ers demonstrated that in some cases over fifty per cent of a
crude lipide extract of plant tissue was unavailable for
animal nutrition.

F. Werr (25) in 1938 advanced a simplified method for
determining the lipide content of peppy, rape, hemp, linseed

and other oil seeds. One half gram of the seeds was ground

in a mortar for five minutes by rubbing with twice this

amount of sand and some anhydrous sodium sulfate. The ground
mass was transferred to a centrifuge tube and 5 m1. of benzine
(B.P. 80-900) were added, then shaken for 3-5 minutes and
centrifuged at 3,000 r.p.m. for 5-10 minutes. One ml. of the
clear solution was transferred to a weighed filter paper, the
solventevaporated off and the sample weighed. Herr claimed
the method was sufficiently accurate and especially useful in
plant breeding experiments. Z. Huber (26) in 1940 reported
the results of lipids determination on various seeds by the
werr method and described it as preferable to all others. The
method was checked by the more time consuming extraction pro-
cedures of Soxhlet and others on eight different kinds of
seeds, poppy, sunflower, sinapis, hemp, linseed, soybean, etc.

After studying the various methods available for lipide
analysis, Hieserman (l) outlined a procedure in which the
initial Koch extraction (alcohol-ether-alcohol), the F. A. 0.
method for removal of the unsaponifiable, and finally the
Kumagawa and Suto technic for determination of the fatty
acids are employed. As proposed, the method is somewhat in-
volved but gives positive, reproducible values for the un-
saponifiable and fatty acids in plant tissues.

Results published by Holwech (27) in 1940 following an
extensive series of investigations show that increasing the
concentration of soap in aqueous phase increases the solubil-
ity of the unsaponifiable in the aqueous soap phase over that

of the petroleum ether phase, thereby requiring a greater

number of extractions, or a larger volume of petroleum.ether

or both in order to completely extract all the unsaponifiable.
He states that the soap concentration in the aqueous phase
should be kept below BIper 100 ml. But since a concentration
of but five per cent of this value is rarely reached, in fact
it is usually but one per cent in lipide determination on plant
tissues, this factor can practically be disregarded.

Numerous methods have been proposed for the estimation
of the individual fatty acids present in a given sample of
fat. But since this thesis is limited to the separation of
a crude lipide extract into the unsaponifiable and fatty acid
fractions these methods will not be discussed except to men-
tion two procedures that might be adapted to a crude lipide
extract. The first is the crystallization of the fatty acids
from an acetone solution at temperatures from -400 to -50° C.
Earle and Milner (28) reported in 1940 that very rapid and
effective separation of the saturated from the unsaturated
fatty acids could be accomplished in this way. The second
method involves the use of the adsorption column. Kaufmann
has published a series of articles (29) (30) (31) in which
the use of silica gel, alumina and animal charcoal are re-
ported for the adsorption of fatty acids from solution.
Kaufmann stated that in most cases complete separation of
the individual fatty acids was obtained by the selective

action of the above adsorbents.

- 10 -

EXPERIMENTAL

As this problem was initially undertaken to evaluate
the procedure for lipide analysis in plant tissue as pro-
posed by Hieserman (l), the first part of the work, after
the preparation of samples and moisture determinations,
was concerned with fractionation of the lipide extract into
unsaponifiable and fatty acids according to the Hieserman
method. This procedure was checked in turn by the A.0.A.C.
method for crude lipides. The second part was primarily
an effort to eliminate as far as possible the undesirable
features encountered above. And third, an attempt was made
to shorten the extraction process by running a simultaneous
alcohol-ether extraction.

Part I. The plant materials upon which the deter-
minations were run were as follows: Spinach, cabbage,
carrots, leaf lettuce, peas, green beans, carrot t0ps,
corn, oats, alfalfa meal, and the stock ration used for
the rat colony in the local vitamin assay laboratory.

After thoroughly washing the fresh samples, the carrots

and green beans were sliced and the leafy materials, (cab-
bage, lettuce and spinach) were separated to aid in drying.
Drying was accomplished by placing in a 45°C. room in which
circulation of the air was maintained by an electric fan.
After 36 hours the samfies were removed and ground to 40
mesh in a Hobart electric mill. The samples were then al-

lowed to stand for 24 hours to reach an equilibrium with

the air in the laboratory whereupon they were bottled and
tightly stoppered.

The moisture content was determined as follows; tripli-
cate 2 g. samples were placed in covered aluminum dishes and
dried in a vacuum oven at 90-950 C. for 6 hours and then
weighed. The drying was then continued for 2-hour periods
until a constant weight was obtained. The loss in weight
was recorded as the moisture content. The results are
shown in Tables I, II and I. These moisture free samples
were then carefully transferred to 33 x 94 mm. fat free paper
thimbles and used for the crude lipide determination as out-
lined below.

The paper thimbles containing the dried samples were
placed in individual dry Soxhlet extractors, equiped with
calcium chloride tubes, and extracted for 16 hours with
C. P. anydrous ethyl ether. After distilling off the sol-
vent the samples were dried at 1000 C. for 30 minutes and
weighed. The drying was continued as above for 15-minute
intervals with intermittant weighings until a constant
weight was obtained. The values for crude lipides are re-
corded in Tables I, II and.X.

The fatty acids and unsaponifiable content of the pre-
pared samples were determined by the Hieserman method (1).
Triplicate 5 gram samples of the dried and finely ground
plant tissues were transferred to 33 x 94 mm. paper thimbles
and covered with discs of fat-free filter paper. The paper
thimbles were placed in individual Soxhlet extractors and

extracted for 10 hours with 95 per cent ethyl alcohol.

_ 12 -

The alcohol extraction was followed by an 8-hour ethyl
ether extraction and finally another lO-l4—hour alcohol ex-
traction. Two ml. of hydroquinone solution in ether con-
taining 0.2 mg. hydroquinone were added to the combined
extracts which were then transferred to a 250 ml. beaker
and evaporated on a steam bath, without overheating, to a
volume of about 100 m1. Approximately 20 ml. of 10 N.
sodium hydroxide were added. The mixture was saponified on
a steam bath in an open beaker until the total volume had
been reduced to about 40 m1. At this time 30 ml. of water
were added and the saponification continued until the total
saponification period had been approximately one hour.

The saponified material was transferred to a separatory
funnel and washed to a 40 m1. volume with 95% alcohol. The
transfer was completed first with warm, then with cold water,
until the total volume was 80 ml. After rinsing the flask
with 50 m1. of petroleum ether the rinsings were added to
the contents of the funnel previously cooled to room tempera-
ture. It was then shaken as vigorously as possible for one
minute and allowed to settle until both layers were clear.
The petroleum ether layer was drawn off as closely as possible
and placed in a separatory funnel of 500 ml. capacity. The
extraction was repeated six more times, using 50 ml. of
petroleum ether for each extraction. The combined extracts
.in the separatory funnel were washed three times with 25 ml.
portions of 10% alcohol by volume and shaken vigorously each
time. The petroleum ether extract was transferred to a

weighed beaker and evaporated to dryness on a steam bath.

The beaker with residue was heated for 30-minute intervals
at a temperature of 1000 C. until a constant weight was ob-
tained. The final residue was tested for solubility in 50
m1. of petroleum ether at room temperature. The solution
was filtered and washed free from any insoluble residue and
dried in the same manner as before and weighed. This weight
represented the total unsaponifiable matter.

The combined basic alcoholic extracts from.the above ex-
tractions were collected in a separatory funnel and acidified
to a pH of about 6 with hydrochloric acid. The solution was
cooled, and the free fatty acids were extracted by the use
of four portions of ethyl ether, two 60 ml. and two 30 ml.
portions. Each time the top ether layer was collected in
'a 250 m1. beaker while the alcoholic layer was returned to
the separatory funnel to be re-extracted. The ether was eva-
porated off from the combined extracts on a steam bath. The
fatty acid residue was dried in a vacuum oven at 500 C. for
4 hours. The fatty acids were then redissolved in four
15-30 m1. portions of boiling anhydrous petroleum ether and
filtered through a fat-free filter paper into a tared beaker.
The ether was evaporated off on a steam bath. The beaker
containing the fatty acids was dried at 1000 C. for 30
minutes, cooled in a desiccator, and weighed. The drying
was continued for 15eminute intervals until the weight re-
mained constant. This weight represented the true fatty
acids multiplied by the factor 1.046 gave the weight of the
neutral tri-glycerides. The results are shown in Tables I

and. II.

- 14 _

TABLE I

Moisture, Crude Lipide, Unsaponifiable
and Fatty Acid Determination on
Carrot Top, Cabbage, Spinach

and Carrot

(Recorded as percentage composition on dry weight basis)

    

 

  
   

 

_.... -—_.____._ _UW __...—_.__ -_ cu-.. __._. ._ __— v _

Fats (Gale 0)

 

 

 

 

 

   

 

 

 

 

Crude Unsa- (% f tt id
‘ ' Lipides - a y ac 8
material moisture (A10.A.C. ponifiable x 1.046)
#_ method) (Hieserman's method)
Carrot
tops
Sample 1 5.63 2.66 0.75 1.88
2 5.62 2.48 0.76 2.10
3 5.37 2.54 0.77 2.02
Average 5.54 2.56 0.76 2.00
Cabbage
Sample 1 13.20 1.62 0.75 1.49
2 13.14 1.60 0.69 1.63
3 13.07 1.59 0.77 1.49
Average 13.14 1.60 0.74 1.54
Spinach
Sample 1 5.43 3.74 1.43 2.82
2 5.06 3.64 1.45 2.99
3 5.04 3.71 1.38 2.83
Average 5.18 3.70 1.42 2.88
Carrot
Sample 1 6.93 1.70 0.37 1.04
2 6.58 1.62 0.41 1.09
3 6.83 1.71 0.41 .92
Average 6.78 1.68 0.40 1.02
M

 

 

 

 

- 15 -

TABLE I I

Moisture, Crude Lipids, Unsaponifiable and
Fatty Acid Determinations on Corn, Oats,
Alfalfa, and Stock Ration

(Recorded as percentage composition on dry weight basis)

 

 

 

 

 

 

 

 

 

 

 

Crude Unsa- Fat (calc.)
Material Moisture lipide ponifiable (% fatty acids
(A.0.A.C. x 1.046
method) _(Hieserman's method)
C“— F‘
orn
Sample 1 9.99 4.57 0.43 4.66
2 9.73 4.53 0.46 4.91
3 9.61 4.55 0.41 4.61
Average 9.78 4.55 0.43 4.73
Oats
Sample 1 9059 9.51 0.50 9010
2 9.99 9.45 0.46 9.05
3 9.98 9.39 0.53 9.40
Average 9.85 9.45 0.50 9.18
Alfalfa
Sample 1 5021 3.84 1006 2035
2 5.28 3.96 1.00 2.11
3 5.43 3.89 1.04 2.24
Average 5.30 3.89 1.03 2.24
Stock
ration
Sample 1 7.08 13.45 0.76 12.38
2 7.20 13.32 0.80 12.23
3 7.12 13.38 0.83 11.96
Average 7.13 13.38 0.80 12.19

 

 

 

 

 

 

Part II. In order to determine the efficiency of the
above saponification and fractionation procedures in the
recovery of known amounts of pure fats, .075 g. of trimyristin
and .025 g. of myristic acid were added to individual 5 g.
samples of alfalfa. The extraction and fractionation were

carried out as above. The results are shown in Table III.

TABLE III
The Recovery of Myristic Acid and
Trimyristin by the Hieserman
Method
Alfalfa Moisture -- 5.30%

(Recorded as weight in grams)

 

 

 

 

Unsaponifiable Fatty acids
5 g. alfalfa 5 g. alfalfa
. .025 g. myristic -5 . . .025 g. myristic
5 g alfalfa acid .075 g. g alfalfa acid .075 g.
trimyristin trimyristin
0.0500 0.0459 0.1067 0.2146
0.0473 0.0492 0.0957 0.2152
0.0492 0.0472 0.1016 0.2136
Ave. 0.0488 0.0474 0.1013 0.2145

 

 

 

 

In order to determine the effect of the chlorophylls upon
the fractionation of the crude extract and to determine, if
possible, what role they play in the formation of the difficult
emulsions encountered in analysis of chlorophyll containing
tissues, .050 g. of crystalline chlorophylls were added to

each of three 5 g. samples of corn. The extraction and

- 17 _

fractionation were carried out as outlined above. The re-

sults are given in Table IV.

TABLE IV

The Effect of Chlorophyll upon the
Fractionation of the Crude
Lipids Extract by the
Hieserman Method

Corn Moisture -- 9.78%

(Recorded as weight in grams)

 

 

 

 

Unsaponifiable Fatty acids
5 g. corn 5 g. corn
5 8o corn .050 g, chloro- 5 8- corn .050 g. chloro-
phyll phyll
0.0196 0.0433 0.2012 0.2005
0.0211 0.0414 0.2121 0.2094
0.0184 0.0401 0.1991 0.1949
Ave. 0.0197 0.0416 0.2041 0.2009

 

 

 

 

In an attempt to eliminate the emulsions encountered in
the above determinations several modifications of the Hieserman
method were tried. The first consisted of adding 5 m1. of
concentrated hydrochloric acid to the combined crude extracts
of alfalfa after they had been evaporated to a volume of 100
ml. After heating on the steam bath for 10 minutes, saponi-
fication and fractionations were carried out as in the ori-
ginal procedure except that an additional amount of base,

sufficient to neutralize the hydrochloric acid, was added.

_ 18 -

The results are shown in Table V.

TABLE V

The Effect of Ere-treatment with Aoid on
the Fractionation of the Crude
Lipids Extract
Alfalfa MOisture -- 5.30%

(Recorded as weight grams from 5 g. sample)

 

 

 

 

Unsaponifiable Fatty acids

Original Original
Hieserman Modification Hieserman Modification
method method

0.0500 0.0449 0.1067 0.1092

0.0473 0.0474 0.0957 0.1117

0.0492 0.0445 0.1016 0.1065

Ave. 0.0488 0.0456 0.1013 0.1091

 

 

 

 

The second modification consisted of adding various
amounts of ethyl alcohol to the crude extracts after saponi-
fication. To each of three saponified samples from 5 grams
alfalfa were respectively added 0, 5, and 10 ml. of 95% ethyl
alcohol. Due to persistent emulsions encountered in sample
one, 15 m1. of alcohol were added thereby giving three samples
containing 5, 10, and 15 m1. of alcohol respectively. The
fractionation was carried out as in the original Hieserman

procedure. The results are given in Table VI.

- 19 -

TABLE VI

The Effect of the Amount of Alcohol
upon the Fractionation of the
Crude Lipids Extract
Alfalfa Moisture ~- 5.30%

(Recorded as weight in grams from 5 g. sample)

 

 

Sample Unsaponifiable Fatty acids Remarks
5 ml. 8.100110]. 0.0522 g. 0.1276 Slight emul"
sions
10 " “ 0.0549 “ 0.1302 Practically
- - - no emulsions
l5 " " 0.0508 " 0.1278 Slight emul-
. sions

 

 

 

 

The third modification consisted of evaporating the

alcohol—ether extracts of triplicate 5 gram samples of

alfalfa to dryness before saponifications.

The lipide

material in this dry residue was extracted by heating and

subsequent decantation with five 50 m1. portions of petro-

leum ether.

100 m1. of ethyl alcohol.

To the combined, decanted extracts were added

After evaporation of the samples

to 100., the saponification and fractionation were carried

out as above.

The results are given in Table VII.

_ 20 -

TABLE VII

The Effect of Drying upon the Fractionation
of the Crude Lipids Extract

Alfalfa Moisture -- 5.30%

(Recorded as weight grams from 5 g. sample)

 

 

 

 

Unsaponifiable Fatty acids
Hi . H' , . . .
mgzfiggan Modification égifiggan Modification
0.0500 0.0454 0.1067 0.0964
0.0473 0.0462 0.0947 0.0942
0.0492 ---- 001016 000993
Ave. 0.0488 0.0458 0.1013 0.0966

 

The fourth modification was concerned with maintaining as
low a volume of solution as possible during the extraction of
the unsaponifiable and fatty acids. This was accomplished
by evaporating the combined extracts of 5 g. of alfalfa on a
steam bath to a volume of 20 m1. At this point 3 m1. of 10 N.
sodium hydroxide were added. The heating was continued until
the volume had been reduced to 10.m1. whereupon 10 m1. of
water were added and the heating was continued until the total
saponification period had been one hour. The saponified
material was transferred with the aid of first hot then cold
water to a 250 m1. separatory funnel to which were added 10
ml. of 95% ethyl alcohol. The volume at this point was be-
tween 40 and 45 ml. The rest of the fractionation followed
the procedure as outlined by Hieserman. The results are

listed in Table VIII.

- 21 _

TABLE VIII

The Effect of the Fractionation Volume
upon the Extraction of Unsaponifi-
able and Fatty Acids

Alfalfa Mbisture 5.30%

(Recorded as weight in grams from 5 g. sample)

 

 

 

 

Unsaponifiable Fatty acids
Hieserman . . . Hieserman , . .
method Modification method hodlficatlon
0.0500 0.0621 0.1067 0.1178
0.0473 0.0589 0.0947 0.1228
0.0492 0.0590 0.1016 0.1191
Ave. 0.0488 0.0600 0.1013 0.1199

 

 

 

 

The fifth modification was made to determine further

whether or not the above modification facilitated the ex-

traction of the unsaponifiable matter.

The extraction of

the 5 g. alfalfa sample and the subsequent saponification

of the crude extracts were carried out as described under

the fourth modification. However, in the extraction of the

unsaponifiable material only five extractions with 50 ml. of

petroleum ether were made instead of the seven as prescribed

in the Hieserman method.

.Also a fifth extraction of the fatty

acids using 30 m1. of ethyl ether was made to determine the

completeness of the removal of this fraction in the orginal

procedure.

This fifth extraction of the fatty acid from

each of three samples was combined and the total yield was

determined. The results are shown in Table IX.

TABLE IX

The Efficiency of Five Extractions with
Petroleum ether in the Removal of
the Unsaponifiable

(Recorded as weight in grams from 5 g. samples of alfalfa)

 

 

 

 

Unsaponifiable *Fatty acids
Hi e . . .
Hégifigglan Modification 3123:?“ Inc dlflcati on
0.0500 0.0593 0.1067 0.1212
0.0473 0.0587 0.0947 0.1187
0.0492 0.0612 0.1016 0.1224
Ave. 0.0488 0.0597 0.1013 0.1200

 

 

 

 

*A combined fifth extraction of the fatty acids in the samples
yielded 0.0014 gms. or 0.0005 gms./sample.

For the further check on the second fourth, the fifth
modifications, determination of the unsaponifiable and fatty
acid content of triplicate 5 g. samples of peas, lettuce, and
green beans were.made. These modifications summarized as
follows: After adding 2 ml. of a solution of hydroquinone
in ether containing 0.2 mg. of hydroquinone, the combined
extracts were evaporated to 20 ml. on the steam bath. Then
3 ml. of 10 N. sodium hydroxide were added, the heating was
continued until the volume was reduced to 10 ml. whereupon
10 m1. of water were added and the saponification continued

until the total saponification period had been one hour. The

saponified material was then transferred with first hot and
then cold water to a 250 ml. separatory funnel. After in-
creasing the volume to 35 ml. with water, 10 ml. of 95%
ethyl alcohol were added. The unsaponifiable material was
extracted by shaking five times with 50 ml. portions of
petroleum ether. The combined extracts were washed with
three 20 m1. portions of 10% ethyl alcohol. After evapor-
ation of the petroleum ether extract to dryness the amount
was determined as in the original Hieserman method. The
combined basic alcoholic extracts from the above extractions
were acidified with hydrochloric acid. Extraction of the
fatty acids was accomplished by means of two 60 ml. and two
30 m1. portions of ethyl ether. The evaporation of the
extract and the weight of the fatty acids were determined

as in the original procedure. The results are in Table I.

- 34 _

TABLE.X

Moisture, Crude Lipids, Unsaponifiable, and
Fatty'Acid Determinations on Leaf Let-
tuce, String Beans and Peas by
Modified Method

(Recorded as percentage composition on dry weight basis)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m mm:
Crude (L Unsaponi- Fats (calc.)
Material Meisture Lipide fiable (% fatty acids
(A.0.A.C. 1.040)
method) _ . }
hodified method
Lettuce
Sample 1 17.90 4.84 1.25 5.85
2 17.82 5.02 1.19 5.88
5 17.88 4.85 1.15 5.85
Average 17.87 4.91 1.19 5.85
Peas
Sample 1 12.24 5.41 0.52 5.67
2 12.50 5.59 0.51 5.84
5 12.45 5.41 0.54 5.96
Average 12.52 5.40 0.52 5.82
Beans
Sample 1 16.24 2.46 0.59 2.51
2 16.06 2.52 0.57 2.42
5 16.25 2.59 0.65 2.42
Average 16.18 2.52 0.59 2.58
Part III. The Third phase of the eXperimental work was

an attempt to shorten and simplify the initial extractions by
means of a simultaneous alcohol and ether extraction. For

this purpose a solution consisting of 40% ethyl ether and 60%

ethyl alcohol was used. By trial it was found that a solu-
tion of alcohol and ether in the above ratio gave,on the
partial distillation desired, a distillate composed of
approximately equal portions of each solvent.

Triplicate 5 g. samples of alfalfa were placed in in-
dividual Soxhlet extractors. To the distillation flask were
added 90 ml. of alcohol and 60 m1. of ethyl ether. The samples
were extracted continuously for 16 hours. The crude extracts
were treated by the modified procedure as outlined above. The
results are listed in Table.XI.

Triplicate samples of both alfalfa and carrot tops were
handled in the same manner as above except that the extraction
was continued for 24 hours. The results are given in Table

III.

TABLE.XI

Simultaneous Alcohol and Ethyl Ether
Extraction

Sixteen Hour Extraction

(Recorded as weight in grams from 5 g. samples of alfalfa)

 

 

 

 

Unsaponifiable Fatty acids
EmZEEEEan Modification B;::§§§an Modification
0.0500 0.0502 0.1067 0.1079
0.0475 0.0538 0.0947 0.1096
0.0492 0.0495 0.1016 0.1088
Ave. 0.0488 0.0508 0.1015 0.1088

 

 

 

 

T3 313 XII

Simultaneous Alcohol and Ethyl Ether

Extraction

Twenty Four Hour Extraction
Alfalfa Heisture - 5.50%

(Recorded as weight in grams from 5 3. samples of alfalfa and
Carrot tops)

 

 

 

 

 

 

 

 

Unsaponifiable Fatty acids
Eigigggan Mbdification Hieserman Modification
method
0.0500 0.0614 0.1067 0.1152
0.0475 0.0524 0.0947 0.1164
0.0492 0.0555 0.1016 0.1252
Ave. 0.0488 0.0558 0.1015 0.1179
Carrot Tops Mbisture -- 5.54%
0.0554 0.0557 0.0952 0.0581
0.0558 0.0528 0.0951 0.0612
0.0565 0.0521 0.0915 0.0585
Ave. 0.0558 0.0555 0.0905 0.0596

 

 

 

 

_ 27 -

DISCUSSION

The materials that were used in this work were chosen
because of their importance in animal and human nutrition
and because they were available throughout the year. These
materials also represented a variation in content of lipide,
carbohydrate, pigment, and nitrogenous substances. The
carrots provided a material high in carbohydrates and low
in lipides. The lettuce, peas, beans, alfalfa, spinach and
carrot tops provided a variety of chlorOphyll-containing
substances that contained relatively large amounts of non-
lipide substances in the "ether extract". The cats and
corn were used because of their high fat and low unsaponi—
fiable content. The stock ration, was included because of
its varied content. It was composed of 50% whole milk
powder, 50% cornmeal, 50% oatmeal, 5% bone meal, 4% alfalfa,
and 1% inorganic salts.

The air drying of the above samples was aided by main-
taining constant circulation of the air in the drying room
by means of an electric fan. Grinding of the materials was
first attempted in a Wiley mill but was found to be unsuit-
able because many of the samples gummed up the mill. The
Hobart electric mill was found to be very rapid and satis-
factory for the grinding of all the materials used.

For moisture determination the A.0.A.C. method was
adopted. The high moisture values for peas, leaf lettuce,

and string beans (Table X) were probably due to the high

humidity of the air at the time the samples were prepared.
Because of the brownish chared appearance of the various
samples after drying it is believed that a temperature of
90-950 C. under vacuum causes considerable oxidation and
decomposition.

In the use of the Soxhlet extractors in the subsequent
lipide extractions, it was observed that each extraction
was merely diluted by the following one due to the slowness
with which the solvent passed through the samples. Complete
siphoning of the solvent from the extraction chamber was
accomplished by placing 5 cm. lengths of large diameter
glass tubing beneath the paper thimbles.

In the crude lipide determinations a few minor points
should be noted. Determinations were made on the same
samples that were used for moisture determinations. As the
samples were dried at 90-950 C. before extraction some al-
teration through oxidation might have resulted. The crude
lipide extracts were dried and weighed in the extraction
flasks. It was found that reproducible weighings within
0.5 mg. could be obtained on these 250 ml. flasks. The
ethyl ether therefore could be recovered after the extraction
was complete by simply replacing the paper thimble with a
glass tube of the same design and continuing the distilla—
tion. It was also found that by allowing the flasks to cool
in a desiccator for 5 to 4 hours after drying at 1000 C. that
constant weights could usually be obtained after the second

heating.

-29...

The A.0.A.C. method for crude lipides gives highly re-
producible results (Table I, II and X). However, in nine
of the eleven materials analysed the crude lipide values
were lower than the combined fats and unsaponifiable values
of the Hieserman procedure. By simple observation it was
apparent that the crude lipide extracts contained most of
the chlorOphylls or their breakdown products present in the
original sample whereas in the latter method, the final pro-
ducts were practically free of chlorophylls. Hence any
nutritional studies concerned with the utilization of fat
in plant tissues, based.on feeding of crude extracts as
lipides, would be subject to appreciable errors.

The Hieserman method was found to give relatively high
yields of unsaponifiable and fatty acids (Table I, II).

The unsaponifiable fractions were yellow to orange in color,
indicating an absence of chlorophyll, and were waxy in nature.
The fatty acids had the appearance of colorless oil when heat-
ed, and when cooled, assumed a more or less fatty acid-cry-
stalline nature, thus indicating a fairly high degree of
purity.

However, the method was found to be quite tedious and
time consuming, especially when tissues containing chloro-
phylls were analysed. In the fractionation of chlorophyll-
containing samples, persistant emulsions formed which separ-
ated only when allowed to stand for long periods of time.
Since a total of fourteen extractions were required for the

complete fractionation of the crude extracts, the undesira-

- 50 -

bility of allowing ten to forty minutes for each emulsion
to break is readily apparent.

Hieserman's procedure for handling the combined crude
extracts was indefinite and it was found to be physically im-
possible to follow his detailed directions at this point.

"---—evaporation of the alcoholic and ether extracts
to a volume of 100 ml. To this solution 20 m1. of approxi-
mately 10 N. potassium hydroxide solution is added. The
mixture is saponified on a steam bath in an open beaker
until the total volume has been reduced to about 40 m1. At
this time, 50 ml. of water are added and the saponification
continued until the total saponification.period has been
approximately one hour. The saponified material is trans-
ferred to an extraction cylinder and washed to a 40 m1.
volume with 95 per cent alcohol."

The evaporations called for required over two and one
half hours and still left a final volume of over 40 ml.
Because of this inconsistency in volume, in the determina-
tions recorded in Tables I and II no alcohol was added as
it was thought that adding an indefinite amount of alcohol
would introduce a variable factor with adverse effects on
the results. The 20 ml. of base used for saponification was
recognized as being an unnecessarily large excess. This por-
tion of Hieserman's procedure was taken directly from the
F.A.C. method in which a 5 g. sample of fat was to be saponi-

fied.

- 51 -

The data in Table III indicate that recovery of added
fat is very complete by the Hieserman method. Possibly the
fat that was added had a favorable effect upon the extrac-
tion of the fatty acids thereby giving the higher than theo-
retical yield.

The indication that the chlorophylls were largely re-
sponsible for the persistant emulsions encountered was con-
firmed by the results in Table IV. In the initial analysis
of corn very little trouble was encountered with emulsions
but when pure chlorophylls were added, the characteristic
emulsions of all the green materials analysed were present.
The increase in unsaponifiable was probably due to the phy-
tol alcohol that was split off the chlorophylls. The lower
yield of fatty acids probably was a result of emulsions en-
countered in the fractionation of the crude extract.

In an attempt to alter the chlorophyll molecule and
thereby lessen its emulsifying effect, concentrated hydro-
chloric acid was added to the crude extracts before saponi-
fication. The results (Table V) indicated a small loss in
the unsaponifiable with a slightly larger gain in fatty acids.
However, no lessening of emulsion-formation_was noticed.

The results listed in Table VI indicated that the pre-
sence and the amount of alcohol have a very favorable effect
upon the extraction of both the unsaponifiable and fatty
acids. This favorable action of alcohol was partially due
to the elimination of troublesome emulsions. In most cases
separation of the layers was complete in 5-10 seconds. In

Spite of the limited nature of this eXperiment it was assumed

- 32 -

that approximately 10 ml. of alcohol provided the optimmal
concentration for this particular determination. In con-
nection with the high yield of fatty acids it should be
mentioned that the final product appeared to be quite im-
pure. This could have been caused by insufficient heating
of the crude fatty acid extracts allowing thereby a resol-
ution of chlorophylls and other impurities.

The fourth modification wherein the combined extracts
were evaporated to dryness (Table VII) proved impractical be-
cause a thick gummy mass was formed which made re-extraction
of the lipides difficult. The extracting solvent had to be
decanted as the amorphous nature of the material made filter-
ation impossible. The low yields were undoubtedly due to the
occlusion of lipide material by the gummy precipitate. Fur-
thermore much of the chlorophyll was redissolved.

In some of the previous determinations the volume of
the alcoholic potash solution was increased past the 80 m1.
mark in effecting a complete transfer of the solution to the
separatory funnel. Fractionations made on these solutions
indicated that emulsion formation increased as the volume in-
creased. In the fifth modification (Table VIII) 5 ml. of 10
N. base were used for saponification and the fractionation
volume was reduced to approximately 45 ml. The results show
a very high yield of unsaponifiable with a fairly high yield
of fatty acids. Although the yield of fatty acids was lower
than those shown in Table VI, they appeared to be somewhat

freer from chlorophyll. Furthermore the unsaponifiable

- 33 -

(Table VIII) was much higher indicating that the lower
volume permitted a more complete removal of this fraction.
It was noted that the sixth and seventh extraction of the
unsaponifiable was colorless while the fifth one was nearly
so. The values in Table IX indicate that five extractions
are sufficient to remove the unsaponifiable. The results
also indicate that four extractions with ethyl ether remov-
ed practically all the fatty acids.

The results recorded in Table I with materials of high
chlorophyll content show that in each case the combined
values of the purified fatty acids and unsaponifiable were
higher than that for the crude extract even though the latter
contained most of the chlorophyll present in the original
sample. Furthermore, since only slight emulsions were en-
countered, the determinations were completed in a much
shorter period than would have been possible by the origin-
al Hieserman procedure.

In an attempt to shorten the extraction time requir-
ed by the original procedure, and, to eliminate the neces-
sary of alternating the solvent, simultaneous extraction with
alcohol and ethyl ether was tried. The composition of the
solvent (40% ether, 60% alcohol) was such that would yield
a distillate composed of approximately equal parts of each
solvent. Whether of not this ratio is the most efficient
for the removal of lipides in plant tissues was not deter-
mined. The results listed in Table XI show that a sixteen

hour extraction, when fractionated by the modified procedure,

-34....

gave values that compared favorably with those of the Hieserman
extraction and fractionation procedure. However, upon compari-
son with Table IX it is evident that a sixteen hour extraction
period is insufficient to remove all the lipide known to be
present in this sample of alfalfa. Table XII shows that, al—
though a twenty-four hour extraction gives still higher yields
than the sixteen-hour extraction, the values are still slightly
low for alfalfa (Table IX). The values for carrot tOp were
low, especially for the fatty acids, as to indicate that the
simultaneous use of alcohol and ether could not be universally
used for lipide extraction in plant tissue. Hence a longer
extraction period was not tried. Perhaps a different ratio

of the two solvents might be more efficient. Since many phos-
pholipides are soluble in hot but not cold alcohol, the lower
temperature of the mixed solvent in the extraction chamber

over that when alcohol is used alone might also eXplain the
ineffective solvent action.

From a study of the results obtained in connection with
the work on this problem it is believed that, with certain
modifications in the procedure, the Hieserman method permits
a highly accurate determination of the fatty acids and un-
saponifiable on any type of plant tissue regardless of its
nature or lipide content. Furthermore, the method is suit-
able for the analysis of fresh plant tissues as well as dried
or otherwise altered tissue.

The method in detail, with proposed.modifications, is
as follows: (1) Approximately 25 g. samples of fresh plant

tissues are transferred to beakers and enough 95% ethyl al-

_ 35 -

cohol is added to bring the alcoholic content to about 80%.
The tissue is then boiled for 15 minutes on a steam bath to
arrest enzymatic activity. The fresh tissue is then trans-
ferred to 33 x 94 mm. paper thimbles by fitting the extrac-
tion thimbles into glass filter funnels held in turn in

clean suction flasks. The tissue is washed with a jet of

hot 95% alcohol and covered with a piece of filter paper.

When dried tissue is to be used, the material is finely
ground, then 5 g. are placed in paper thimbles and covered
with pieces of filter paper. The thimbles are then placed

in the extraction chamber of continuous Soxhlet extractors
each of which have been previously equipped with 3 cm. lengths
of large-bore glass tubing. Following 10 hours of continuous
extraction with 95% alcohol, an 8—hour ethyl ether extraction
is made, followed by a second lO-lZ-hour ethyl alcohol extrac-
tion. When fresh tissue is to analysed, the material is freed
from ether and finely ground before the final alcohol extrac-
tion. After adding 2 ml. of a hydroquinone solution in ether,
containing 0.2 mg. of hydroquinone, the combined extracts are
evaporated on a steam bath in an Open beaker to a volume of
about 20 ml. This and subsequent evaporations are made in a
swiftly moving current of air produced by means of a small
electric fan. (This was found to prevent overheating, facili-
tate evaporation, and to prevent creeping on and over the sides
of the beaker). To this solution 3 ml. of about 10 N.sodium
hydroxide are added and the mixture saponified on a steam bath

for 30 minutes. At this time 10 ml. of water are added and

the saponification continued for an additional 50 minutes.
The volume should now be approximately 15—30 ml.

(2) The saponified material is transferred to a 250
m1. separatory funnel with the aid of small quantities of
hot water until the total volume is 35-40 ml. The beaker
is rinsed with 50 ml. of petroleum ether which is added to
the contents of the separatory funnel previously cooled to
room temperature. Ten ml. of 95% alcohol are added and the
separatory funnel is shaken as vigorously as possible for
one minute. When complete separation of the two layers has
taken place, the bottom layer is run into a second separa-
tory funnel containing 50 ml. of petroleum ether and the top
ether layer is transferred to a third separatory funnel (500
ml. capacity). The above extraction is repeated at least 4
more times using 50 ml. of petroleum ether for each extrac-
tion. The combined extracts in the separatory funnel are
washed by shaking vigorously three times with 25 ml. portions
of 10% alcohol. However, if the sample being analysed con-
tains chlorOphylls, persistant emulsions can be eliminated by
gently rotating the initial washing thus removing the emulsi-
fying agents. The washings are returned to the basic alcbhol
solution. The petroleum ether extract is transferred to a
weighed beaker and evaporated to dryness on a steam bath.
The beaker is heated for 30-minute intervals until a constant
weight is obtained. The residue is tested for solubility in
50 ml. of petroleum ether at room temperature then filtered
and washed free from any insoluble residue. The purified pro-

duct is dried in the same manner as before and weighed. This

- 57 _

weight represents the total unsaponifiable matter and should
be free from chlorOphyll.

(5) The basic alcohol extracts are acidified to a pH
of about 6 with hydrochloric acid. Brom cresol purple was
found to be a satisfactory indicator for this reaction. The
solution is then cooled and the free fatty acids extracted
by shaking with four portions of ethyl ether, two 60 ml. and
two 50 ml. portions. Each time the bottom layer is collect-
ed in a second separatory funnel containing the next portion
of ethyl ether and re-extracted. The top layers are collect-
ed in a beaker and evaporated to dryness on a steam bath.
The fatty acid residue is dried in a vacuum oven at 50° C.
until the chlorophyll has been rendered insoluble in petro-
leum ether. This usually requires 4-6 hours. The fatty
acids are redissolved in four 15-50 ml. portion of boiling
anhydrous petroleum ether, and filtered from chlorophyll and
other products thru a fat free filter paper into a tared
beaker. The ether is evaporated off on a steam bath. The
beaker containing the fatty acids is dried at 100° C. for
50 minutes, cooled in a desiccator, and weighed. The dry-
ing is continued for 15 minute intervals until the weight
remains constant. This weight represents the true fatty
acids. The weight of true fatty acids multiplied by the
factor 1.046, assuming that the average.molecular weight of
the fatty acids is 274, gives the weight of neutral trigly—

cerides.

4.

- 58 -

C ON C LUSIONS

The Hieserman procedure was found to give quite ac-
curate and reproducible results. However, very per-
sistant and troublesome emulsions were encountered in
the fractionation of lipides obtained from many tissues.
Since persistant emulsions were encountered with chloro-
phyll-containing plant tissues and because the addition
of crystalline chlorophylls to other samples produced
stable emulsions, it was concluded that chlorophylls
were the main causative agent.

Heating the crude extract with acid, evaporation to
dryness with subsequent heating and re-extraction, and
other attempts to eliminate or lessen the emulsifying
power of chlorophylls were founed ineffective or use—
less.

It was found that persistant emulsions could be elimin-
ated by maintaining a low fractionation volume consist-
ing of approximately 20% ethyl alcohol by volume.

An attempt to shorten the extraction time by employing
a mixed solvent of ethyl alcohol and ethyl ether was
unsuccessful.

The combined values for the purified fatty acids (cal-
culated as triglycerides) and unsaponifiable obtained
by the proposed method were higher than those obtained
by A.O.A.C. method for crude lipides. In view of the
fact that the A.0.A.C. crude extract, according to

Horwitt (et.al.) is known to consist of as high as 55%

_ 39 -

non-lipide material, it must be concluded that the
method is unreliable for lipide determinations.

The proposed method, although quite long is thought to
be superior to existing methods for lipide determina—
tions. It can be used on either fresh or dried plant
tissues and gives reproducible yields of purified fatty

acids and unsaponifiable.

 

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(4)

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_40_

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4!;4 “42-3.: .: s H lzh__fi .

 

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- 43 _

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