THE SIMULTANEOUS DETERMINATION
OF MOISTURE AND LIPIDES IN
PLANT MATERIAL

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

Floyd V. Monaghan
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THE SIMULTANEOUS DETERMINATION
OF
MOISTURE AND LIPIDES
IN

PLANT MATERIAL

,LIL/
. LIJ

by i
FLOYD. VI'MONAGHAN

A ThESlS
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'

1941

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ACKNOWLEDGEMENT

Grateful acknowledgement is made to
Professor C. D. Ball for assistance in
planning this research and for his val-

uable advice in the writing of this

thesis. 2‘ ‘11 19'

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II
III

IV

VI

Introduction .

Historical . .

Experimental .

Moisture and Crude Lipides .
Barium Hydroxide Method .
Hieserman Method .

Recovery of Fatty Acids .

Discussion . .
Conclusions .

Bibliography .

TABLE OF CONTENTS

11

12

14

18

20

21

INTRODLCTION

Early workers in the field of plant analysis were
interested in the lipide content of the material because
of its caloric value. A knowledge of this was, of course,
essential to an estimation of the energy available from a
given plant source. Consequently, early work was centered
around the problem of extracting all of the lipide mater-
ial available. This extract was reported as "crude fat".

As later workers discovered the biological importance
of some of the carotenoid pigments and some of the sterols,
interest was aroused in the unsaponifiable fraction of
plant lipides. Methoos of extraction were altered, and
means of fractionation into the saponifiable and the im-
portant unsaponifiable fractions were worked out.

More recent work has shown the importance of certain
of the unsaturated fatty acids in nutrition. As a conse-
quence the fatty acid fraction is assuming considerable
importance.

With increasing work, attempts have been made to
clear up the various ambiguities of terminology. In con-
formity with the clearest differentiation, fat shall herein
mean only glyceryl esters of fatty acids. ihe term lipids
shall be used in accordance with the classification of
Bloor (l) to include all substances soluble in fat solvents
as ether, chloroform, petroleum ether, and having a common

insolubility in water.

-2-

The problem detailed in this thesis is concerned
with a simplification of the determination of both moisture
and lipide materials in order to shorten the time required.
Work was also undertaken to simplify separation of the

saponifiable and unsaponifiable fractions.

-5-
hlSTORlCAL

The problem of lipids analysis may be conveniently
divided into three phases: the determination of the mois-
ture content of the sample, the extraction of the lipids
material, and the fractionation of this lipide material
into such fractions as the saponifiable, and the unsapon-
ifiable.

Accurate determination of moisture is necessary for
accurate comparison of materials since moisture content
may vary considerably over a large number of Samples, and
the presence of moisture also affects the extraction of
lipides. The Official and Tentative Methods of Analysis (2)
gives the following methods for use on materials such as
grains and feeds. They are: drying at 950- 100°C under
a pressure not to exceed 100 mm of mercury for about five
hours; drying in an air oven at 105°C; drying in a vacuum
desiccator over concentrated sulfuric acid; and the toluene
distillation method.

A modified air-oven method was brought out by
Sandstedt (5) in 1938. In this method an aluminum plate
a half (%) inch thick was placed on the lowest shelf of an
air oven which was adjusted to give a plate temperature of
140QC. The sample was dried in an uncovered aluminum dish
for fifteen minutes and the cold cover then placed on the
dish. It was then removed to an identical plate held at

room temperature, allowed to cool, and weighed.

_ e _

The following year (1940) Ofelt (4) ran comparison
tests of this method with the A.O.A.C. air-oven method
and found the two to agree within limits of experimental
error. He found that the aluminum plate results were less
affected by position of samples in the oven than were the
air-oven methods results.

Another method gaining in favor because of its
rapidity and freedom from errors due to oxidation or loss
of volatile constituents is the distillation method. Re-
sults have been reported by Thielepape and Fulde (5) in
1958 for the use of perchloroethylene in place of toluene.
This solvent is said to give results equal to oven-drying
with a distillation period of from thirty (50) to ninety
(90) minutes.

An improved apparatus for moisture distillation
has been developed by Beckel, Sharp, and Milner (6) having
a construction that automatically eliminated adhesion of
water droplets to the condenser walls. This deSign is
also said to prevent the formation of milky suspensions in
the receiver. Moisture determinations on soy bean meal
samples gave results varying 0.21% between maximum and
minimum values with a water volume of approximately 1 m1.
Determination time is from one to two hours.

The use of the Karl Fischer reagent (iodine,
liquid sulfur dioxide, methyl alcohol, and pyridine) was

originally reported by Fischer in 1955 (7). Its use was

-5-
reviewed in 1939 by Smith, Bryant, and Mitchel (8).
They found it to be satisfactory for determining moisture
in the following types of compounds: alcohols; hydrocar-
bons, saturated, unsaturated, and aromatic; esters and
carboxylic acids except formic. Aldehydes and ketones
could not be titrated satisfactorily due to the formation
of acetals and ketals with the large excess of methanol
present in the reagent with resulting liberation of water.
By reduction of the metnanol content and increasing the
pyridine content, ketones may be titrated in the absence
of the lower alcohols. The iodine of the reagent was re-
ported to show no tendency to react with ethylenic double
bonds.

In 1940 hays, Leibnsr, and Connor (9) described an
apparatus, and reported a aslvent for the continuous deter-
mination and extraction of moisture and neutral fats from
fecal material. moisture was distilled out with isopropyl
ether in about two hours after which the same solvent was
used as an sxtractant for ten to fifteen hours to remove
the lipids material

In 1958 work by hulman in Russia described a methodflo)
in which the sample was immersed in a weighed quantity of
011 in an iron crucible and heated at 1e5°- 185°C on a
sand-bath. The amount of moisture was determined by the
loss in weight. Results from such widely varying samples
as flour, dough, and bread were found to be within 0.2 to

0.3% of those with the air-oven method at 105°C.

-6-

The second phase, lipids extraction, has been
given more attention. One of the more obvious methods
of extraction was that using ethyl ether. Moisture and
ethyl alcohol dissolved in the ordinary grade of this
reagent permitted the extraction of a small amount of
non-lipids material. Of a consequence the anhydrous
solvent has largely displaced the U.S.P. grade for use
in lipids extractions. That non-lipids material was re-
moved was shown by Chibnall and Channon (11) in 1927.
These workers demonstrated that inorganic salts, carbo-
hydrates, and some amino compounds might be extracted
along with lipids material. heiserman (12) was able to
show that U.S.P. ethyl ether extracted on an average of

.37% more material from soy bean meal than anhydrous

ethyl ether. The official A.0.A.C. method (2) provided
for the use of the anhydrous solvent on dried samples,
thus avoiding this objection. Even with this precaution,
all lipids material was not completely removed as has been
adequately shown by numerous workers(15, 14, 15, lo, 17).

The first positive step toward more efficient ex-
traction was made by Waldemar Koch (18) in 1904 in connec-
tion with studies on animal tissue fats. Koch recommended
an ethyl alcohol extraction both before and after the ether
extraction then in use. A later worker, F. C. Koch (19)
modified the original method in such a way that it could

be used on plant tissue. This method marked one of the first

- 7 -
attempts to use fresh materials for extraction rather than
dried samples. By this method it was found to be possible
to preserve material for from one to three months without
significant changes occurring in composition.

Solvents other than ethyl ether and ethyl alcohol
that have been suggested are: (l) ethyl alcohol followed
by chloroform, Shlssinger (20) and Rosenfeld (21); (2)
carbon disulfids; (5) carbon tetrachloride by Bryant (22);
also benzene, trichloroethylsne, and acetone. Alcoholic
soda was suggested by Rather in 1915 (16). Isopropyl ether
has been used by Kaye, Leibnsr, and Connor'(‘9).

In recent years, increased interest in the nutri-
tional value of the lipids portion of plant material has
lent added impetus to the third phase of the problem,
studies on its fractionation. The simplest fractionation
has customarily provided for separation of the saponifiabls
and unsaponifiabls material.

One of the earliest uses of saponification as an
analytical procedure in lipidevvork was that of Liebermann
and stkely (25) in 1898. These workers saponifisd the
entire sample. After acidification the acids and other
lipids material were extracted with ethyl ether and dried
to constant weight. A more complete method of analysis,

based upon the experiments of these earlier workers, was

that of Kumasawa and Suto (24) published in 1908. This

- 8 -
method provided for saponification of the entire sample

by means of aqueous-alcoholic potassium hydroxide. Fatty
acids and unsaponifiable were extracted after acidification,
dried to constant weight and recorded as crude fatty acids.
Re-solution of this material in boiling alcohol and treat-
ment with alcoholic potassium hydroxide converted the acids
to soaps. Addition of petroleum ether, with shaking, ex-
tracted the unsaponifiable material from the soap mixture.
The alcoholic soap solution was then acidified and extracted
to remove fatty acids. The weight of fatty acids multiplied
by a factor for conversion of fatty acids to glycerides gave
the weight of neutral glycerides equivalent to the acids.

A new method for determination of unsaponifiable,
developed by the Fat Analysis Committee of the Division of
Industrial Chemists and Engineers of the American Chemical
Society and Commonly known as the F. A. C. method (23), was
adopted as official for unsaponifiable by the Association
of Official Agricultural Chemists in 1920. In this pro-
csdure the aqueous alcoholic saponification mixture was
repeatedly extracted with petroleum ether. After evapor—
ation of the solvent on the steam bath the residue was test-
ed for solubility in petroleum ether. Any insoluble mater-
ial was filtered off, the solution evaporated and dried to
constant weight.

In 1956, Horwit, Cowgill, and Mendel (25) developed

a method for determination of both the unsaponifiable and

- 9 -
fatty acids in plant tissue. The procedure was essentially
that of humagawa and Suto outlined earlier. These workers
were able to show that in the case of some plant materials,
as high as 50% of the lipids fraction extracted by ether
was nutritionally useless.

Hieserman, in 1959 (12) studied theevarious methods
of analysis and proposed a combination of several to give
a more accurate and complete method including the best
points of the others and avoiding as many of the undesirable
features as possible. The method provided for the use of
the modified Koch initial extractions (alcohol-ether-
alcohol), saponification with alcoholic potassium hydroxide,
and separation of the unsaponifiable by the F. A. C. tech-
nique before acidification of the soap mixture. The petro-
1eum ether extract was washed three tires with l % ethyl
alcohol and these washings added to the extracted alcoholic
saponification mixture. Fatty acids were extracted from the
combined , acidified, alcoholic extracts with ethyl ether,
the ether evaporated and the residue redissolved in petro-
leum ether. The resulting solution was filtered through a
cotton plug, the petroleum ether evaporated and the residue
evaporated to constant weight.

Data published in 1940 by Holwech (25) showed that
increased concentration of soaps in the aqueous phase
increased the solubility of the sterols and fatty alcohols

in the aqueous layer over that in the petroleum ether layer.

- 10 _

As high a dilution of the soaps as could be achieved with-
out causing emulsification was found to reduce the number
of extractions required to remove all unsaponifiable
materials.

Grossfeld (27) found that it was possible to further
separate the unsaponifiable into a sterol and fatty alcohol
fraction, and an hydrocarbon fraction by controlled dilution
of the alcoholic soap solution. This was possible because
the hydrocarbons were readily extracted from concentrated
soap solutions by petroleum ether while fatty alcohols and

sterols were extracted only on further dilution.

_ 11 _
EXPERIMENTAL

Due to the somewhat cumbersome procedure involved in
the method for fractionation of total lipides into fatty
acids and unsaponifiable as outlined by Hieserman (12),
it seemed desirable to shorten and simplify the method.
With this aim in mind, the work divided itself into the
following parts: (1) the use of the apparatus designed by
Kaye, Leibnsr, and Connor for extraction of moisture and
lipides, (2) the use of barium and hydroxide octahydrate as
a saponifying agent, (5) the use of isopropyl ether as extras-
tant and the subsequent examination of the extracted lipides
by means of the hisserman procedure.

The materials upon which the determinations were run
included the following: carrot tops, carrot roots, spinach,
and cabbage. All samples were oven dried, ground to forty
mesh in a Wiley mill, allowed to come to equilibrium with
the air in the laboratory, and then bottled. Moisture con—
tent had been previously determined by the official vacuum-
oven method of the Association of Official Agricultural
Chemists (2). Total "crude fat" was determined by the
official ether extraction method (2). The amount of unsap-
onifiable and the amount of saponifiabls were determined as
in Hieserman's method (12) using the modified Koch alcohol-

ether-alcohol extractions. These results are shown in Table

I.

The procedure for the extraction and determination of

_ 12 -
moisture and lipides using the apparatus of hays et a1.
is outlined in the following paragraph.

Triplicate two gram samples were weighed into Soxhlet
thimbles (Green's 55 X 94 mm) and placed in separate set-ups
of the apparatus of Kaye et a1. as shown in Figure 1.

Water enough to fill the moisture trap (a) to a definite
level was admitted, sufficient isopropyl ether was added
through the condenser (b) to fill the extraction unit (c) to
such a level that siphoning was almost permitted through tube
(d) into the receiving flask (e). Beakers of boiling water
(f) (g) were placed under the extraction unit (c) and the
receiving flask (e). [The hot plate (h) was turned to the
high heat position and the water in the beakers kept boiling
vigorously for two hours.' Moisture contained in the sample
in the extraction unit (0) distilled along with the isopropyl
ether, passed up the side arm (1) of the moisture trap, con-
densed in the condenser (b) and fell back into the moisture
trap (a) where its volume was read in the graduated tube (3).
At the end of two hours the beaker (f) under the extraction
unit was removed, more isopropyl ether was admitted through
the top of the condenser while any water—droplets adhering

to the walls were broken loose with a copper spiral. The
water volume was read to the nearest tenth and estimated to
hundredths of a ml. When sufficient isopropyl ether had

been added, the contents of the extraction unit siphoned

Taq. I

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- 15 -
through tude (d) into the receiving flask (e). The unit
then continued to function in the same manner as a standard
Soxhlet extractor. At the end of a fifteen to twenty hour
extraction period, the solvent was distilled off by opening
the stopcock on the bottom of the moisture trap and allowing
the solvent to drain off as it collected.

The automatic siphon (k) (made from a "Cutter Safti-
flask") served to maintain a constant water level in the
beaker (g) under the receiving flask.

In order to check the operation of the apparatus,
synthetic mixtures were prepared according to the formula
given in Table II. Extraction of moisture was checked both
by the water added to the synthetic mixtures and by adding
a known volume to a completely dried and extracted sample of
plant material. In both cases the moisture was recovered
completely in two hours but not before.

The sugar of the synthetic mixtures, along with the
potassium oxalate remained as a glassy amlid residue in the
bottom of the extraction thimbles. heating the extracted
lipides with boiling water was used as a means of removing
any soluble material (i.e. glucose or potassium oxalate)
which might have been extracted by the isopropyl ether.
Qualitative tests showed no glucose to be present and no
oxalate ion could be detected as calcium oxalate.

Cholesterol, the fatty acid and the glyceride were

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- 14 _
completely extracted. From the color of the glucose--
potassium oxalate residue, some of the chlorophyll was not
removed. These tests were qualitative in nature and not
quantitative.

When the solvent ability of the isopropyl ether had
been demonstrated, the next step undertaken concerned the
use of barium hydroxide octahydrate in 85% acetone as a
saponifying agent. The use of the barium hydroxide is not
an inovation, its use having been previously employed by
Fremy (28) and by Petering et al. (29) but for different
purposes.

Preliminary to work on extracted lipides, it was
thought advisable to determine the solubility of the barium
salts of some of the more commonly occurring higher fatty
acids in 85% acetone. Accordingly the following acids were
obtained: myristic, stearic, oleic, linoleic (as methyl
linoleate), and palmitic. Of these, the stearic, oleic,
and palmitic were technical, commercial products. The
myristic acid was prepared by hydrolysis of trimyristin
obtained from nutmegs (50). The methyl linoleate (51) was
prepared from cottonseed oil. The oil was saponified to
free the acid which was separated as its insoluble tetra-
bromide. This compouh was debrominated and esterified by
the action of methylalcoholic hydrochloric acid on zinc.

The ester was subsequently purified by vacuum distillation.

- 15 _

The salts of the acids were prepared by refluxing
the acid for two hours with a calculated excess of barium
hydroxide octahydrate in 85% acetone. In practice this was
achieved by dissolving 0.1 gm. of the acid in eighty-five
ml. of C. P. acetone and then adding fifteen ml. of cold
saturated barium hydroxide solution rapidly while swirling
the flask and contents. 250 to 500 ml. flask was found
to be very convenient for this reaction. At the end of two
hours, the solution was cooled, filtered through an oven-
dried Gooch crucible, treated with carbon dioxide and refil-
tered through the same crucible. The treatment with carbon
dioxide served to remove any dissolved barium hydroxide as
the very insoluble carbonate. A ten ml. aliquot of the fil-
trate was then evaporated to dryness and the weight of the
residue multiplied by ten taken as the weight of barium
soaps soluble in the aqueous-acetone. Results are shown in
Table III.

While these results indicated a strong possibility
that this method of saponification was not desirable, it
was thought advisable to check the process on actual samples.
To do this a series of samples of spinach, prepared as des-
cribed above, was extracted in the apparatus of Kaye et al.
for a period of ten hours. Moisture was determined as des-
cribed above, and the extracted lipide material was purified

by solution in petroleum ether.

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~16-
The solution was filtered through a plug of extracted and
dried absorbent cotton into a tared beaker, the solvent
evaporated and the residue dried to constant weight in a
vacuum oven at 50°C. This residue was designated as crude
lipides. These crude lipides were transfered quantitatively
to a 250 ml. erlenmeyer flask with exactly eighty five ml.
of boiling acetone. Fifteen ml. of cold, saturated barium
hydroxide were added to the flask from a pipette while the
mixture was swirled vigorously. This action provided a sus-
pension of very small crystals of barium hydroxide octahy-
drate in 85% acetone. The mixture was then boiled for four
hours under a reflux condenser. At the end of this time the
mixture was cooled, the barium soaps filtered off on an oven-
dried Gooch, and the filtrate treated with carbon dioxide.
The resulting precipitate was filtered on the Gooch contain-
ing the barium soaps. The filtrate was extracted with six
successive fifty ml. portions of petroleum ether, the sol-
vent evaporated on the steam bath in a current of rapidly
moving air. The air current aided evaporation and prevented
creeping of the solution on the side of the beaker. The res-
idue was dissolved with three twenty ml. portions of petroleum
ether and filtered through a cotton plug into a tared beaker.
The solvent was evaporated and the residue dried to constant
weight in a vacuum oven at fifty degrees, and weighed as un-

saponifiable.

- 17 _

The barium soaps were dissolved by allowing thirty
ml. of one to one hydrochloric acid heated nearly to boil-
ing to filter thro ugh the Gooch containing the soaps. The
resulting solution was transferred quantitatively with hot
water to a 500 ml. separatory funnel and extracted with two
sixty and two thirty ml. portions of U. S. P. ethyl ether.
The contents of the Gooch crucible and the filter flask were
also washed with U.S.P. ethyl ether and the washings added
to the extracts. The solution was evaporated on the steam
bath as above and extracted with three twenty ml. portions
of petroleum ether. These extracts were filtered while hot
through a plug of dried, extrac=ted, absorbent cotten into a
tared beaker, the solvent evaporated and the residue dried
to constant weight in a vacuum-oven at fifty degrees centi-
grade. Results are shown in Table IV.

The results as shown in Table IV indicated a failure
of the alkali to affect saponification. Therefore it re-
mained necessary to compare the efficiency of the extrac-
tion using isoprOpyl ether with the modified Koch extrac-
tions. For this purpose, the Hieserman procedure (12) was
used with the exception that the iSOprOpyl ether extraction
for fifteen to twenty hours in the apparatus of Kaye and
associates was substituted in place of the Koch alcohol-

ether-alcohol extractions.

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- lg -

Lipide material was extracted as indicated above,
in the Kaye apparatus. Crude lipides were determined by
extraction of the dried total lipides with skellysolve B
rather than petroleum ether. This solvent was adopted in
place of petroleum ether since its more narrow boiling
range (2%» was thought to indicate a more nearly pure pro-
duct. The crude lipides were dissolved in lOO ml. of 95%
ethyl alcohol and the flask rinsed with two twenty m1.
portions of U.S.P. ethyl ether. These extracts were com-
bined in a 400 ml. beaker and twenty ml. of ION. potassium
hydroxide added. The alcohol was evaporated in a current
of air on the steam bath and saponification continued until
total time on the steam bath was one and one-half hours.
The sample was then cooled and transferred quantitatively
to a separatory funnel using 95% ethyl alcohol to a volume
of forty ml. and hot water followed by cold water to a
total volume of eighty ml. The beaker was then rinsed with
fifty ml. of skellysolve B and this added to the contents
of the funnel which were shaken as vigorously as possible
for one minute. Five more extractions were made in a sim-
ilar manner. All extracts were combined in a 500 ml. sep-
aratory funnel and washed with three successive twenty-five
ml. portions of 10% by volume ethyl alcohol. The washings
were added to the alcoholic saponification mixture. The

skellysolve extract was run into a 400 ml. beaker and the

. - 19 -

solvent evapo rated in a current of air on the steam bath.
The residue was dried in a vacuum-oven at 50°C. for four
hours. At the end of this time the unsaponifiable material
was dissolved in fifty ml. of boiling skellysolve B and
filtered through a dry, fat-free cotton plug into a tared
beaker. The solvent was evaporated and the sample dried to
constant weight in a vacuum-oven at 50°C. An initial heat-
ing of one hour with successive heatings of thirty minutes
followed by fifteen minute cooling periods and subsequent
weighing was found to be the most satisfactory procedure.

The combined alcoholic extracts were acidified with
hydrochloric acid (20 ml. of 12 M. dil. to 50 ml.) and ex-
tracted when cool with two sixty ml. and two thirty ml.
portions of U.S.P. ethyl ether. These extracts were com-
bined, and the solvent evaporated as above, the last traces
being removed by heating four hours in a vacuum—oven at
50°C. At the end of this time, the sample was extracted
with fifty ml. of skellysolve B and the extract filtered
through dry fat—free cotton into a tared beaker. The sol-
vent was evaporated as previously described and the fatty
acids dried to constant weight in the same manner as the
unsaponifiable. This was recorded as fatty acids, or as
saponifiable. Results of this work and further determin-

ations of moisture are reported in Tables V and VI.

g-..) Vw--_-o....a ‘ ..-..J I

ml 7-! 1 who or” 11 Ivy-”~71 n-w r1173 Ufm "-H-‘o'ra’ '1"
*a‘LBaA J aik"IBJ- '9 r‘ \v|” PL. :4. 541- u_-&-LLJ —‘i CqD

cm 1' Haves?
-ILIHLILUI.‘

'r m': for: who"? ”earn
7- .L-‘ .Ll -b‘v.’ -J‘cfiJ

 

 

grams ct. volume percent my wt. Deviations of per-
haterials of of of cantcae moisture

 

- ,.. - '1
sample Vfitpr water from storage thine
- . c: n.
Carrot tons 2.0300 0.15 7.50 1 1.-8

 

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to

 

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mp '1 o ,
u. Jib-17 (3.1“ L/on) +0.3.)

y.- a I '7
7.01.3 0.17 v.45 + 0.93
3.9??? 0.07 4.9: ”(3.33

 

Averafie 6.14 * 0.53

 

flfi
Carrot too? 5.0o07 c.“n 5.79 * I.-7

I .1

5.0010 0.99 5.59 f 0.07

 

 

.500an Riot); 5019 ~ 0.3::
r‘
Averare 5.53 v.00 —

 

Total AFOPRTB 3.09 + 0.48

 

{'74
1

013 V (cont'd

\

)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

irons Vt. voluwe percent bf Vt. Deviatio- of oer-

Xaterials of of of cent-he moisture
so ole voter Hater frod avers e value

Carrot roots 5.000? _.”5 5.05 +'1.03

5.0000 0.70 5.99 + 0.97

5.0000 0.00 5.00 1 0.00

5.0001 0.00 7.0- ' 1..?

5.0000 0.00 7.9? ' 1.0

5.00~3 0.00 3.09 ' 1.0c

5 0008 0.00 5.00 "1.03

5.0000 0.00 5.99 "1.05

5.0014 0.00 a 00 ' 1.03

5.0005 0.13.“: 5.79 + 0.77

4.9007 0.00 5 00 *'0.78

4.0007 0.00 5.30 +'0.7P
Avere;@ 5.0? 0.00

 

TA 33 v (cont'd)

 

{jrnsrzs wt. VOl‘t‘ZG percent by ’.-.'t.

 

" ' .2 '1 .4" .1" .f‘
1hr.:\_.rd.an3 0.4. 0-. OJ.
'1 L» ,- 4- .
S" '1". 1 1?? VP. o r' l“ '1 -.". L. {‘1‘
3 3 P. f- .-’ '3 n / '7
7333'”??? .3.!W " 3 o n 0.9-

 

r: , (\f‘f- r‘rv A 5-
1.2.0 {V‘O O.~:\3’ Ooxpg

 

5.0005

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'31
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'3

31
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01
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5.0018 O.C7 5.73
5.0010 0.7? 5.39
5.fl001 0.373 0.79
5.0001 0.3? 5.73
5.0007 0.32 5.39
5.9038 0.33 Q.YC
5.0701 0.37 9.39
=.fl’1(‘l To]? 00:0
s coco .7“ 6.70

 

f: ,l f.“
‘k‘: (3.130,. e L) . ".. '.)

TABLE v {cont'd)

 

volume porcont by wt. Devia
of centa
“Pter f.

4,
"P.er

043‘

D
Ll

2e mdlstr‘e

'1.
1.

ans of per-

rnm avornje va

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Slinach c.3397 C.?5 4.99 ' 0.77
5.971: “.55 4.99 - 0.77
a 000; 0.93 4..T ' 0.77
S.Q¢Ol 0 ?5 4.9? ‘ 0.3
5.9“”5 0.7“ 4.;9 '37.97
5 OCUG 0.35 1.9? ' ..27
5.0011 0.25 3.19 -¢.O7
5.0flLé T.?S v.19 - 0.07
5.C907 0.77 5.39 * 0.13

Q.m gm.

nyristic said 5 Q?00 0.39 5.90 * 0.54
5.°018 0.79 5.90 + 0.64

 

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_Jb‘./ CI {Tye

 

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oquoa uwum m.9)oq 0.3unu v.33 0.03m 0.03m 0.0mon 3.00
m.QDHO 0.chm n.0u b.umm 0.mun Q.rmoq #.o

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m.oopm ..mrwomo .n.3m c.0uum o.mqm a..mmo H.3m

.13.... ..uH >.mon H.3m
own-.. no... m.uuoo n.9uqu o.mqn 0.033q : a].
w.ooam o.onmm onflam 0.93.3 o.mnu
m.oo.. o.omo$ 9.003 0.03.9 o.m3m
1.0».3 1| 0.33.9 a..m. 3.9).. 0.3..
m.oq3. ..pwwmuz o.mqo a awuq 3.. n

- m a... o ngx .1. o.mmw 0.3.00 0.33m

 

 

 

 

m.oo.o 0.0mu3 3.33 a .omow o.mou 9.3nuo 0.3mm
m )3.0 3.03.3 3.3..

m.333~ Juan m o.qmm o .03.3 _.muu o.o~ou 3.433
m.onoq o.ou.. 9.33m 3.030m o.no3

 

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A Goad 3.0JHQ D.Awm 0.0ku O.uum

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u .II | Lil {

abuum 4H Anoma.mv

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ow muJon owsmm ijwmam owgmm Hmemmm ow finnuJ. QUnmJ. ow nnJ. mag.

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r .er....,.nw PJOQ.QHJH DON/fou- HOOQ O. A. ”a...” .LOCQC Ooopmruul OOHJJQ
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(.FOQ/QVN 300.. .1..-“ Hod‘r 000.3 \yJHJ. .x. l..(.r.l. \IJOQPL‘NO Diomn‘

 

 

 

 

 

 

m.u on o.onQ3 3.q3 o.omom a.mua 0.03m3 3.0mm
w.ouo3 onnquo wrmo
m.;qu 3.9moo 3.0
I. m.o))w 3.0uma 3.90
m.oao3 3.300q 3.00 n.0u30 0.00m 0. man 3.03m

 

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baowwtw H.q¢ 3.30c 3 so

v;~.

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- 20 -

As a further check on the method, a 0.2 gm. sample
of myristic acid was added to each of three samples of spin-
ach. Extraction, saponification, and separation of the sa-
ponified extract into its saponifiable and unsgaonifiable
components were according to the procedure outlined above.
The results are shown at the conclusion of Table VI.

In order to determine the purity of the myristic
acid used to test recovery of added lipide material, trip-
licate 0.2 gm. samples were weighed into tared 100 ml. beak-
ers. Each sample was analysed for unsaponifiable and sapon-
fiable by the hieserman procedure detailed above. The results

are reported in Table VII.

TABLE VII AS. was s or 1:215:10 ACID syrm

 

 

 

 

 

 

 

 

 

 

 

 

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‘1 ‘1 if. : - 4‘ ', ~I‘.‘ 1 Q ‘ p

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'..‘. . . J .“n' \J o _"'_'..’J 1 O “4.; 0 «”73 9C) 0 PM.)
A nqml f‘. (\an 1 "r: n 16"”1 an [:1

..:¢_ ‘s'ol._.Jo Lot/L. ‘J.-I~Vl—- -K)._
A H H _. A A
“verge 1.;5 (Tat?)

 

 

- 21 -
DISCUSSION

As previously mentioned, the materials chosen for
this work were carrot tops, carrot roots, cabbage, and
Spinach. They were chosen because all were believed to
contain a relatively large amount of non-glyceride material
in the "ether extract". These materials were also readily
available throughout the winter months. Carrot roots were
selected because they were high in carbohydrate, low in
nitrogenous substances, and moderately high in unsaponifi-
able material. The tops were used chiefly because they were
at hand and provided another green material. The cabbage
provided a material of moderate carbohydrate content, a
fairly large amount of nitrogenous compounds and in addition
to unsaponifiable and saponifiable materials had been shown
by Chibnall (32) to contain the calcium salt of phosphatidic
acid. It was felt that this substance might provide some
complications in extraction and separation. Whether the
rather erratic results obtained on cabbage are due to this
or other factors is not known. The spinach served to pro-
vide a green material high in chlorophylls, and fairly high
in unsaponifiable material.

The solvent used, isopropyl ether, was chosen because
it was reported to have several desirable properties. Whit-
more ($3) claims that the higher boiling point and lower

mutual solubility with water should make it a better extract-

- 22 -
ing agent than ethyl ether. Kaye and associates (9) have
shown that it provides an immiscible solvent for the dis-
tillation of the moisture contained in the sample, and a
better solvent for lipides than alcohol-ether mixture or
ethyl ether alone. While this may have been the case in
the extraction of the fecal material with which this group
worked, the present results obtained on plant materials did
not confirm this greater solvent action. By examination of
the data contained in Table VI and Table I it may be seen
that the amounts of crude lipide material extracted in twen-
ty hours are consistently lower with the iso-propyl ether
than with anhydrous ethyl ether. In addition, the solvent
must be purified before each set of extractions to remove
any peroxides which may have been formed. If this is not
done, the peroxides form eXplosive mixtures. However, when
the precaution of removing dissolved peroxides is observed,
no trouble is likely to occur. It has been found that the
removal of peroxides is conveniently accomplished by distil-
lation with metallic sodium. A drop of water added to the
distillation flask, by reaction with the metallic sodium
sets free nascent hydrogen. This serves to reduce the per-
oxides. The small amount of added moisture as well as any
dissolved in the solvent itself is removed by this treatment.

The data shown in Table I, and used as a reference

against which other results were checked, was kindly sup-

plied by John Dill. The moisture content of all samples
was approximately the same with the exception of the cab-
bage which was unusually high. Consistent duplication of
the value showed that any error must be in the method and
not in technique. Examination of the dried residue showed
the material to be brown and quite gummy. This may have
been due to decomposition of carbohydrates or nitrogenous
compounds with resulting loss in weight. There may have
been.some loss also due to the volttilization of substances
such as essential oils. Any or all of these taken together
may have accounted for the high apparent moisture content.
I In considering the synthetic mixture formula of Table
II, it must beI‘emembered that it is exceedingly difficult
if not actually impossible to prepare a synthetic mixture
which Will closely approximate the composition of a plant
material. Of the substances shown in the formula, only
cholesterol has not been shown to occur in plants. The
amounts of individual substances must be kept fairly large
to reduce errors in weighing to a minimum. The results re-
ported in the B part of Table II tend to show that even with
such a simple mixture containing nonrotein, some form of
combination may occur between constituents giving results
which cannot be readily explained.

Table III includes the solubilities in 85% acetone

of the barium soaps of some of the more commonly occurring

- 24 -

higher fatty acids. These were determined in an attempt

to show whether or not the barium hydroxide octahydrate
would function as a saponifying agent. The values aiown
should not be considered as absolute, since the acids from
which they were prepared were mostly of technical purity
only. The method used for the preparation of the soaps

was the same as that used in saponifying extracted lipide
referred to in part II. The use of the octahydrate in ace-
tone was suggested by the work of Petering (29) in which it
was used to saponify the chlorophylls in acetone extracts
from alfalfa-leaf meal.

The high apparent solubility of the barium palmitate
may have been due to the large amount of impurities present
in the only sample of the acid available. The very high
apparent solubility of the barium linoleate prepared from
quite pure methyl linoleate may be due to the unsaturated
structure of the acid, or it may have resulted from forma-
tion of lower acids by oxidation at one or both of the double
bonds.

Although these results strongly indicated that this
method of saponification was not satisfactory for this pur-
pose, it was thought advisable to try it using extracted
lipide material. Table IV contains the results of this ser-
ies of experiments on the extracted lipides of carrot tops.

From these results it may be seen that the values for the

-25-

unsaponifiable fraction were excessively high in comparison
with the results reported in Table I. In fact, at times the
weight of the unsaponifiable fraction was nearly equal to
that of the extracted crude lipides.

Variations in the efficiency of saponification may
be due to variations in particle size. Since saponification
can take place only on the surface of the particle, increase
in particle size is equivalent to a decrease in the amount
of alkali available for saponification. Particle size is,
in this case, a function of the rate of addition of the cold,
saturated barium hydroxide solution to the acetone, the temp-
erature of both solutions, the degree of stirring of the ace-
tone as the barium hydroxide is added, and the amount of free
fatty acids present in the solution. On the basis of the re-
sults shown, and the obvious impossibility of standardizing
all of the above conditions, further work on the method was
discontinued.

The moisture content of these and following samples
were determined using the method of Kaye and associates (9).
The results of these determinations are reported in Table V.
It was found, using this method, that all of the moisture
present in a given sample could be distilled out in two hours.
This is shown in Table II, parts B and C. It was also ob-

served that over a longer period of time there was a slight

\

-20-

but continuous increase in the volume of moisture collected
in the graduated trap. This may have been due to the grad-
ual seepage of moisture into the system through the corks
used at the various connections, or it may have been due to
the partial decomposition of some of the substances present
in the plant material under long continued heating. In other
instances, high results may be attributed directly to a high
humidity at the time samples were weighed. It diould be
pointed out that there is a possible error in reading the
collecting burette of from .01 to .02 ml. When a small vol-
ume of moisture is to be determined, this amount represents
an appreciable error. Therefore, when working with air-dried
material, samples should be chosen as large as can be conve-
niently extracted. It will be noted in Table V that the per-
centage of moisture is calculated directly from the volumes
read on the colleCLing burette. This was allowable since
corrections based on the density per unit volume at 21°C
(d.= .99802 gms./ml.) gave variations within the limit of
error in reading the burette.

The results of the lipide extraction corresponding
to these moisture determinations are detailed in Table VI.
Where crude lipides were determined they are reported as
such. The fractionation of these crude lipides into sap-
onifiable and unsaponifiable is also reported. Examination

of the data in comparison with the average shown in Table I

- 27 -
indicates that iso-propyl ether extracts less of both
saponifiable and unsaponifiable than the modified Koch
extractions using ethyl alcohol followed by ethyl ether, and
then again by ethyl alcohol. The isopropyl ether also ex-
tracts less total crude lipides or "crude fat" than anhydrous
ether. The alcohol of the Koch extraction probably breaks
down protein-lipide and carbohydrate-lipide complexes in
the tissue leaving the lipide material exiosed to the solvent
action of the following ether extraction. The isopropyl
ether might not be expected to have such an effect; however,
Kaye and associates (9) working with feces, found it to be a
better solvent than a mixture of alcohol and ether. In spite
of the results of these workers and the predictions of thit—
more previously mentioned, the isopropyl ether failed to ex—
tract as large an amount of material as did the anhydrous
ethyl ether. This failure may be due to the increased mole-
cular weight and molecular volume of the isoprOpyl ether as
compared with the ethyl ether which decreases the penetra-
ting and dissolving power of the higher ether sufficiently
to account for the difference. Other than this, no reason
is immediately apparent.

That recovery of added fatty acids is complete is
shown by the last three items of Table VI. The added ma-
terial consisted of 0.2 gm. of myristic acid prepared as

previously indicated.

- 28 _

Analysis of the data on recovery of the acid as
contained in the last three samples shows an average re-
covery of 100.3%.

The increase in the weight of the unsaponifiable
fraction in the samples containing added myristic acid is
difficult to explain. Analysis of the acid to determine
the amount of unsaponifiable present showed that this could
account for only a very small part of the increase, roughly
one-third. This analysis showed the acid to be 98.65% pure.
It is possible that the large excess of free fatty acid may
have exerted a solubility effect taking some additional un-

saponifiable out of the sample when removed by the solvent.

CGKCLUSIONS

From the foregoing results the following conclusions

may be drawn:

(1) Provided adequate precautions are observed, the

(3)

method detailed herein, and suggested by Kaye and
associates, is an accurate and rapid means of de-
termining moisture content of samples of plant
materials.

The use of isopropyl ether as a solvent for plant
lipides cannot be recommended due to its lower
efficiency and higher cost than anhydrous ethyl
ether.

The use of barium hydroxide octahydrate in 85%
acetone cannot be recommended as a saponifying

agent in the analysis of plant lipides.

(l)

(2)

(4)

(5)

(6)

(7)

- 30 _
BIBLIOGhAFhY

Bloor, W. H., Biochemistry of Fats. Chem. Rev.
2:245-900 (1925)

Official and Tentative Methods of Analysis of the
Association of Official Agricultural Chemists.
Washington, D. C. Fifth Edition, pp. 353-4 (1940)

Sandstedt, R. h., The rapid determination of moisture.
Cereal Chem. 153813—15 (1938)

Ofelt, C. W., A comparison of the A. O. A. C. air-
oven method and the aluminum plate method .

Cereal Chem. 17:050-2 (1940)

Thielepape, E. and Fulde, A., Determination of mois-
ture in feedingstuffs by distillation with per-
chloroethylene. Z. Wirtschaftsgruppe Zuckerind.
as Tech. Tl. 904-8 (1938) Cited in Chem. Abs.
33:8848 (1939)

Beckel, A. 0., Sharp, A. G., Milner, R. I., An
apparatus for moisture distillation. Ind. Eng.
Chem. (Anal. Ed.) 11:425-e (1939)

Fischer, Karl, Neues Verfahren zur massanalytischen
Bestimmung des Wassergehaltes von Flussigkeiten
und Festen Korpern. Angew. Chem. 48:394 (1935)

Smith, D. M., Bryant, W. M. D., Mitchell, J. Jr.,
Analytical procedures employing the Karl Fischer

reagent. J. Am. Chem. Soc. 61:2407-12 (1939)

(10)

(12)

(15)

(15)

Kaye, I. A., Leibnsr, I. W., Connor, E. 8.,

Apparatus for the continuous drying and extraction
of biological material. Application to the extrac-
tion of the neutral fat fraction of feces.

J. Biol. Chem. 52:195-207 (1940)

Kulman, A. G. M. et. al., Vesoyus Nauch. Issledovatel
Inst. “dlebOpeharnoi Prom. S. S. S. R., (1938)

36 pp. Cited in Chem. Abs. 3431085 (1940)

Chibnall, A. C., and Channon, H. J., The ether-
soluble substances of cabbage leaf cytOplasm.
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