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THS.

THE PREPARATION AND PROPERTIES

OF PHYTIC ACID AND ITS SALTS

Thesis Ior Ifee Degree of M. S.
Ler James Swift
I936

 

 

 

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THE PREPARATION ADD PROEERTIES
of PHYTIC ACID AND ITS

SALTS

A Thesis Submitted to
the Faculty of
MICHIGAN STATE COLLEGE

OF
AGRICULTURE AND APPLIED SCIENCE
in parfiiel fulfillment
of the requirements
for the degree of
MASTER CF SCIENCE
By
Lyle James Swift

July, 1936

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ACIQIOWLEDGBENT

The writer wishes to express his sincere
appreciation to Professor C. D. Ball, whose
able guidance and helpful suggestions have

made this thesis possible.

105405

TABLE OF CONTENTS

INTRODUCTION
HISTORICAL
Discovery
Structure, Salts, Preparation, and Properties
Analytical
EXPERIMENTAL
Methods of Analysis
Analytical Studies
Phytic Acid Content of Various Materials
Recovery of Phytic Acid Added to Wheat Bran
Influence of Period of Extraction

Effect of Period of Extraction and Heating the
Extraction Mixture

Preparations
Modifications of Posternak's Methods
Preparations of Crude Calcium Phytate
Preparations of Saturated Sodium Salt

Purification of Crude Calcium Phytate by Modi-
fiethnderson Method (36)

Preparation of Barium, Strontium; and Calcium
Phytates from Wheat Bran.

Preparation and Some Properties of Phytic Acid
DISCUSSION

Methods of Analysis

Analytical Studies

Preparations
CONCLUSIONS

BIBLIOGRAPHY

IO
10
ll
11
12

12

13
15
15
15
22

21+

26
31
33
33

3h
M1
he

INTRODUCTION

The term phytin is now generally understood to mean the calcium-
magnesium salt of phytic acid as it occurs in nature. Phytic acid it-
self is the hens-orthophosphoric acid ester of inositol.

Phytin occurs universally in seeds,or more generally, in the
propagating'and.growing parts of the plant. Its function is not known
‘with certainty, but it is supposed‘by same plant physiologists to be
connected in some way with carbohydrate and protein formation. It has
been shown to be particularly abundant in the germ of the seed. When
the seed sprouts the amount of phytin in the germ increases by transfer
from other parts of the grain. Other evidence supports the view that
phytin functions as a specific controlling factor in growth instead of

merely as a reserve material.

 

- 2 .-
HISTORICAL

Discovery

In 1854 Hartig (l) discovered microscopic granular particles in
various seeds which he called "klebermehl" and later ”aleurone grains.”
(2) They were soluble in dilute acids but not in alcohol or ether.
Van none (3) considered them protein carriers or "Proteinkorner" while
Sachs and Gris (3) looked upon them as fat concentrates. Pfeffer (4)
in 1872 differentiated the grains into three groups; calcium oxalate,
a protein substance, and a third group containing calcium, magnesium,
I and phosphorus but no nitrogen. Il‘his last group Pfeffer called "globoid"
because of the spherical shape in which they occurred. In 1893 Palladin
(5) obtained a substance by extracting fat-free ground seeds with sodium
chloride solution. Upon heating a voluminous precipitate formed, but
when the solution cooled the substance redissolved. The substance, filter-
ed from the hot solution, contained phosphorus, calcium, and magnesium, but
no nitrogen and was non-reducing even after hydrolysis. It was soluble in
water and acids and was precipitated by the alkaline earths and heavy metals.
Its phosphorus was not precipitated by amonium molybdate according to
Schulse and Winterstein (6) who believed it to be identical with Pfeffers
”globoid." Winterstein (7) published a paper a year later (1897) in which
he established the identity. linterstein suggested the name "inosito-
phosphoric acid" since, on hydrolysis, inositol (inosite) and ortho-phos-
phoric acid were produced. Pasternak did much work on the substance from
1900 to 1905 and proposed the name "phytine" (8), discarding Iinteretein's

more descriptive "inosite-phosphoric acid."

-3-

tructure, Salts, Preparation, and Properties

At the time these earlier contributions were made Posternak (9)
contended that inositol formed no part of phytic acid, but was formed
from fragments produced upon decomposition by hydrolysis. On the
strength of his analysis he proposed two formulae, the more important
of which is known as the "anhydro-oxymethylene-diphosphoric acid" (9)
(10). In 1907 Suzuki (11) and his associates decomposed phytin by the
action of an enzyme and obtained inositol and ortho-phosphoric acid.
From this they concluded that phytic acid was the hexa—ortho-phosphoric
acid esthr of inositol, the view that prevails at the present time.
Nalberg (12) came to a similar conclusion the following year when he
obtained inositol and furfurol on mixing phytin and phosphoric acid and
distilling under reduced pressure. He proposed a formula in which each
carbon atom of inositol was linked to a phosphorus atom through an oxygen
atom and each pair of the three pairs of phosphorus atoms so linked was
joined together by an oxygen atom. In addition, each phosphorus atom bore
three hydroxyl groups. Starkenstein (13) noted that the behavior of phytic
acid was similar to that of pyrophosphoric acid when titrated with uranium
acetate. Furthermore, its silver salt resembled that of pyrophosphoric acid.
From these considerations, tarkenstein submitted a formula in which three
molecules of pyrophosphoric acid are "added on" to inositol to form a complex
rather than combined as an ester (1M); Contardi (15) demonstrated that phytin
was a salt of inositol-hexaphosphoric acid when he obtained identical analysis
for preparations from rice bran and synthetic preparations from heating anhya
drous inositol and ortho-phosphoric acid together. Carre (16) attempted to

repeat the work of Contardi, but could only obtain a mixture of the two chem-

icalst Anderson (17) produced tetra-ortho and di-pyro phosphoric acid esters,
but could obtain no hexa phosphoric acid ester. In later papers Anderson (18)

described several new salts prepared from naturally occurring phytin, among

-4-

them the tri-bsrium phytste, pants-barium phytete, pants-barium momium
phytste, pants-magnesium phytste, penta-magnesium amenium phytate, tetra-
cupric di-celcium phytste, calcium-magnesium-pstsssium phytste, penta-
cslcium.phytste, tetrs-calcium phytate, pants-magnesium.phytete, octe-

silver phytste, hepts—silver phytate, and hexa-cupric phytate. The start-

ing point for most of these preparations was phytic acid itself or commercisl
phytin. duderson also prepared phytic acid from the tribsrium.phytste by the
method of Patten snd.Hsrt (19) which consisted of decomposing the barium sslt

by sulphuric acid, precipitation of the phytic scid by cupric scetste, suspension
of this sslt in water end decomposition with.hydrogen sulphide, snd.the con-
centration of the filtrate in vacuo. Anderson (20)'wus unsble to isolate

phytic acid or its salts from wheat bran by extraction with 0.2% hydrochloric
scid. However, upon extraction with 1.0% acid he obtained s yield dud concluded
thst the stronger soid inhibited the action of phytsse while the weaker acid
permitted the enzyme to decompose the phytic acid to lower esters and to inositol
end ortho-phosphoric acid (21). In 1919 Pasternak (22) published s paper in
which he claimed to have synthesized phytic acid. His methodfemployed phosphoric
snhydride and host to dehydrste the process of esterificstioininositol by orthoé
phosphoric acid. The reaction.mixture contained, besides phytie scid, some

lower esters snd.mnch.mets-phosphorio acid. The letter was transformed.into
sodium pyro-phosphste by warming with sodium hydroxide and filtered from the

cold solution. Concentration and cooling yielded s new crop of pyro-phosphate
crystsls. The excess sodium.hydroxide was removed by alcohol and decanting.

The syrup was then dissolved in‘water, acidified with scetic scid, and precip-
itated with.cslcium acetate as the celcium.sodium salt of phytic sold, 06 BB 024
P5 Cs2.ls8. This, end the sstureted sodium.sslt prepared from it, were iden-
ticsl with the sslts prepared £rom.nsturslly occurring phytin. Anderson (23)

fsiled to achieve the same result, but Posternsk (24) defended his method in

.. 5 ..
a paper in which he stated that Anderson did not follow his proceedure
strictly. At the same time he announced that improvements of the method
made possible an increase in yield of from 5% to 8%. He also presented
elaborate crystallographical data to support his claim. No further

question of Poaternak'n work appears in later literature. These formulae

suggested are given below:

H /OH

0-0-330

0/11 \OH
\H on
0-00 .0

n \on

Poaternak'a "mare-ownetlwlene diphoaphorio acid" formula

 

 

 

HO 3 n on
o>r-o-c Ct-o-rfo
m \on
no on
OE‘r-o-cn Hc-o-r40
no on
o}r-o-c c-o-rfo

n n on

Formla of Suzuki, Yoshinmra, and Takaiahi

 

 

 

 

”I 3“
HO 3 H n
::>P - o - P c - 0 - 1,0
m / I Na
0 o
/a
no“ -0-3 mac-<0
m | |on
. no on
ac on
I |
0
Ion
:fi\0/O|;OH

Neuberg'l Formula

II 3

ED -.P - OHthC

 

 

 

“O” /
HO-fi-OEECH Ho 03.33-5-03
no 0 n
on HO

0733\0/p\. o

Starkenatein'a Formula

-7-

According to Rose's (3) summary of the properties of phytic
acid it has never been prepared in a crystalline tom. At room
temperature it is a straw-colored syrup which becomes very viscid on
cooling to 20° C. It darkens on heating but this darkening is not the
result of oxidation since it takes place in an inert atmosphere as 1611.
Boating to 125° 0. produces an insoluble dark char. Phytic acid is de-
composed by heating vith strong acids in sealed tubes, but it does not
break down spontaneously into inositol and phosphoric acid. Beating with
strung alkali (20% sodium hydroxide) to 230° 0. causes cleavage, though

heating to 100°

0. in alkaline solution causes no decomposition. The acid
is miscible in all proportions with water and is soluble in alcohol, but

not in other at solvents. In fact, phytic acid is precipitated from alcoh-
olic solution by the addition of other. Solutions of the acid are optically
inactive and pass a semi-pomeable membrane slo'ly.

Phytic acid forms neutral salts, acid salts, double salts, and acid
double salts. Many of the salts tend to crystallise in spherical masses of
very short needles. 'rhe solubility of the salts decrease in the following
orders alkali, alkaline earth, and heavy metal. The solubility of the phy-
tates is greater in cold than in hot water, heating frequently precipitating
them. All salts of phytic acid are soluble in dilute mineral acids. Acetic
acid acid dissolves double salts as well as alkali and magnesium salts, but
not those of the heavy metals. Alkali salt solutions of phytic acid are
solvents for salts of the alkaline earths, double salts forming in the solutions
on standing. All salts but those of the alkalis and the acid magnesium salts

are precipitated from aqueous solution by alcohol.

-8-

Analytical

The quantitative estimation of phytin phosphorus was, for many
years, based on the determination of the difference between the total
soluble phosphorus and the inorganic phosphorus. Soluble phosphorus,
of course, meaning that soluble in dilute acids. The amount was ob—
tained by evaporatflng the extract and destroying the organic matter
with.sulphuric and.nitric acids according to the method of Neumann (25)
after which the usual inorganic phosphorus determinations were made.
Hart and Andrews (25) in.1908 developed an approximately accurate method.
They extracted with 0.2% hydrochloric acid solution. Since ammonium
molybdate did not precipitate the phytin phosphorus Hart and Andrews
used this means of separating'the two kinds of phosphorus compounds.
Using the minimal: amount of nitric acid to give a crystalline precipitate,
they'made the precipitation of inorganic phosphorus with neutral ammonium
molybdate solution. Vorbradt's (2?) method consisted of a triple precip—
itation of inorganic phosphorus, first precipitating~with magnesia mixture
and dissolving the precipitate with nitric acid, heating to 100° c., and
treating with amonium molybdate solution. The yellow precipitate was then
dissolved in ammonia water, and precipitated.with.barium.chloride solution.
The resulting precipitate, after washing, is dried and weighed. Stutser
(28) and.Forbes (29) used acid and alcohol. Forbes precipitated his acid
extract with magnesia mixture, washed the precipitate with amonia and.
alcohol, and separated the inorganic phosphorus from.the phytin by digesting
the precipitate'with 95% alcohol containing 0.2%?nitric acid. The alcoholic
solution.was then filtered, the filtrate evaporated, and the phosphorus de-
termined in the usual way. Starkenstein (13) found that titration of a sol-
ution containing ortho-phosphate, pyro-phosphate, and inosital-phosphate with

uranyl acetate standardised against ortho-phosphate using cochineal as an.in-

.. 9 ..
dicator, gave in each case true values for total phosphorus. With
ferrocyanide as an indicator, the total phosphorus was equivalent to
all the phosphorus as ortho-phosphate, one-half of that as pyro-phosphate
and inositol-phosphate, glycero—phosphate not entering into the reaction
at all. Heubner and Stadler (30) devised a rapid method in which the
acid extract (0.6 hydrochloric) is titrated with standard ferric chloride
solution. Amonium thiocyanate is used as an indicator. An empirical
factor, 1.19, is used to convert amount of ferric chloride into phytic
acid. Lindenfield (31), Tropp and Bolts (32), Averill and King (33),
Harris and Mosher (34), and Andrews and Baily (35) have introduced mod-

ifications of the method of Heubner and Stadler.

‘10..
“ERIEEHTAL
The experimental work of this thesis consists of (1) a brief
examination of the methods of estimating phytic acid in naturally
occurring substances as well as in preparations, (2) a study of estab-
lished methods of isolation of phytic acid or its salts with modifi-

cations.

Methods of Analysis

Because of the rapidity of their determination the search for
suitable methods of anlaysis was confined to modifications of that
of Heubner and Stadler (30). Of these the methods of Averill and King
(33) and of Harris and Mosher (3H) appeared best adapted to the work.
Both methods make use of a ferric chloride solution containing .00195 g.
iron per ml. This was prepared by dissolving 1.95 g. of electrolytic
iron in hydrochloric acid, oxidizing with a small amount of nitric acid,
and making the volume up to one liter after filtering. The amount of
acid used was such as to give an approximately 0.6% hydrochloric acid
solution.

The Method of Averill and King (33)

8.0 g. of finely ground sample are extracted with hydrochloric
acid. Using ammonium thiocyenate as an indicator the filtered extract
is titrated with standard ferric chloride solution. The empirical factor
used in calculation is 1.19 for phytic acid phosphorus. The percentage
of phytic acid phosphorus multiplied by 3.55 gives percent phytic acid.

This method proved unsatisfactory because of the difficulty in
judging the endpoint, due to the obscuring effect.3f the white precipitate.
Different strengths of hydrochloric acid were tried in the extractions,
but little confidence could be placed in the results obtained. Moreover,

in extractions where acid of great strength was used extreme dilution was

-11..
necessary in adjusting the solution to 0.6% hydrochloric acid strength
for titrating. This further increased the difficulty of judging the
endpoint.
Method of Harris and Mosher (3h)

This method is very similar to that of Averill and King (33) except
that the endpoint is over-titrated and the precipitate filtered off.
The red color of ferric thiocyanate in the filtrate is then matched by
adding ferric chloride to a blank and the difference of the titrations
is used as a basis for calculation. The computation is identical with
that of Averill and King. This method worked well giving results capable

of close check.

Analytical Studies
Phytic Acid Content of Various Materials
The substances chosen for analysis were prepared by grinding until
they passed a 20-mesh screen. They were then thoroughly mixed and the

samples weighed into beakers.

Table 1

 

 

i:

Ml.FeC13solution M1.FeCl solution Ml.FeCl3solution Tier Cent

 

 

Substance. Added for Blank Net Phytic Acid
Alfalfa Seed 5.50 1.07 u.u3 1.82
Wheat Bran 13.50 0.65 12.85 5.23
Soy Bean Meal 5.50 1.03 u.u7 1.8M
Yellow Corn Meal 3.00 0.60 I 2.h0 0.99
White Corn Bran 2.20 0.53 1.67 0.69

0at Hulls 1.30 0.60 0.70 0.29

 

- 12 -

Recovery of Phytic Acid Salt Added to Wheat Bran
Some barium phytate samples (student preparations) were analysed
for their phytic acid content
#1 was a preparation from corn meal.
#2 was a preparation from wheat bran according to Anderson's method.

0.1 g. samples were dissolved in 50 m1. of 2% hydrochloric acid.

 

 

Table 11
M1.FeCl Ml.FoCI Ml.FeCl
Sample solution added for El solutio Net. % Phytic.Acid
#1 7.70 0.55 7.15 58.9
#2 7.h0 0.u5 6.95-
7-25 .50 6.75 57-3

 

To one 8 g. sample of wheat bran (one-fourth of which had given a
net Fe013 titration of 12.85 ml. on a previous trial) was added 0.1 g. of
#1 barium phytate preparation which had given a titration of 7.15 ml. To
another 8 g. bran sample was added 0.1 g. of #2 barium phytate which had
given a titration of 6.85 ml. (average) FeClB solution. Since the added
phytates were quartered in aliquotting, the titrations should equal 12.85

1

. 6.95
4 or 1h.6u ml. and 12.85 4 or lu.59 ml. respectively. The analysis

was carried out as usual.

 

 

Table 111
ce.FeCl ce.Fe01 solution ce.FeCl solution ce.FeCl solution
Sample solutio added for lank Net (Deéermined) Net (Predicted)
#1-1st.Aliquot 16.50 0.55 15.95 - )1h.6u
"-2nd. I 16.50 0.50 16.00 lu.6u
#3-lst a 16.00 0.50 15.50 1h.59
”-2nd. - 16.20 0.50 15.70 1h.59

 

Influence of Period of Extraction

In this experiment the 8 g. samples of bran were extracted with 200 ml.

-13...

acid for different periods of time. In other respects the determinations

were carried out as usual.

 

 

Table IV
Period of m1.FeCl solution m1.FeCl solution ml.FeCl solution
Sample Extraction Add d for Blagk Net3
#1 2 hrs. 1h.00 0.60 13.h0
#2 3 hrs. 13.50 0.35 13.15
#3 6 hrs. 13.50 0.h3 13.07

 

Effect of Period of Extraction and 0f Heating Extraction
Mixture.

In consideration of the results of Table IV a further investigation

of the effect of period of extraction and heat treatment of the extraction
mixture seemed to be warranted. In addition to the bran samples a series

of determinations were made on the barium phytate preparation (#2). Heat:
treatment for bran consisted in immersing the 200 m1. of mixture in boiling
water for ten minutes immediately after the acid was added. For the barium
phytate the 50 ml. of solution was immersed in boiling water for five minutes.
Some data, marked with asterisks, were included from proceeding tables for

the sake of comparison.

-111-

 

 

Table V"
ml. FeCl ml.FeCl sol. m1.FeCl sol.

Sample Treatment of Sample solution added for Blgnk Net. 3

s g.Wheat bran Extracted cold 2 hrs. 1h.00I 0.60I 13.h0I
. n n I 3 I 13.50* 0.35* 13.15I
u I I I 6 I 13.50'I 0.u3* 13.07*
a u u I an I 13.20 o.uu 12.76
,I I I I ha I 13.20 o.u5 12.75
I I Heat treated.& extracted cold 6 hrs 12.00 0.M3 11.57?
u u u u a I I eh I 13.00 0.38 12.62
a a n I a I I Is I 12.00 0.30 11.70
0.1 g. #2 In cold solution 2 hrs. 6.50 0.23 6.27
I I I I 3 I 7.20I 0.M5I 6.75*

I I I I 6 I 7.10* 0.55* 6.55*

a t a n 2h I 6.50 0.23 6.27

I u u N ha I 6.50 0.25 6.25“

I Heat treated a in cold solution 6 hrs 6.50 0.23 6.27

i I I a I I I 2h I 6.50 0.27 6.23

I I I a I I I ha I 6.50 0.25‘ 6.2.

-15..
Preparations~
The purpose of the following preparations was to try the effect
of varying different conditions in the methods of Posternak and Ander-
son in an effort to improve the yields and quality of phytic acid and
its salts from naturally occurring sources. Wheat bran appeared to be
the most feasible material and was used throughout.

{0difications of Posternak's Methods
Preparations of Crude Calcium Phytate

Prep. No. l - One kg. of wheat bran was extracted for three hours
with two liters of 2% hydrochloric acid, filtered on a Buchner funnel,
and washed with about 1 L. of water. 2 L. of extract (with washings)
were obtained. The pH of the solution was brought to about M.6 with
strong sodium hydroxide (using bromophenol blue as an external indic-
ator) and saturated calcium acetate solution was added until precipit-
ation was complete. After the precipitate had settled it was filtered
off on a Buchner funnel. The precipitate was dissolved in about twice
its volume of concentrated hydrochloric acid, giving an opalescent sol-
ution or suspension. From this mixture the calcium phytate was precip-
itated by the addition of two volumes of 95% alcohol. After standing
for an hour the precipitate was filtered off on a Buchner funnel, washed
with alcohol and ether, and dried in a vacuum dessicator over sulphuric

acid. The weight of product was 3.97 g.

Prep. N0. 1.- Six liters of 2% hydrochloric acid were used in the
extraction, the proceedure being the same in other respects to the first
preparation until the precipitate from the cabcium acetate treatment was
obtained. To this precipitate 20% hydrochloric acid was added until the
volume reached about 500 ml., giving an Opalescent solution. This solution
was filtered through asbestos on a Buchner funnel and precipitated as before

wit h two volumes of 95% alcohol. The scant precipitate appeared to have a

- 16 -
gelatinous consistency and was discarded. The filtrate was again
treated with an equal volume of alcohol and a voluminous precipitate
was obtained that filtered easily and weighed, after washing and drying

as before, 21.1 g.

(Prep. N0. 3.- The proceedure was identical with that of thesec—
0nd preparation until the precipitate obtained from the calcium acetate
treatment was obtained. This was dissolved in a much smaller volume of
20% hydrochloric acid than before, 110 ml. The alcoholic precipitation
was made with four volumes of 95% alcohol. After filtration on a Buch-
ner funnel and washing and drying as before, the precipitate weighed

29.8 g.

Prep. N0. M— The proceedure was identical with that of the second
and third preparations until the acid extract from the bran was obtained,
having a volume of I800 ml. This was divided into four parts of 1200 ml.
each and each part was completely precipitated by the addition of 80 ml.
of saturated calcium acetate solution. The precipitates were filtered
off separately on Buchner funnels, placed in beakers, and 1abeled.A, B,
C, and D.

Part.A

The precipitate was treated with 30 ml. of 20% hydrochloric acid
and filtered, 10 ml. of water being used to wash the filter. The fil-
trate was then divided into two portions labeled 1 and 2.

Portion 1

This was precipitated with two volumes (75 ml.) of 95% alcohol.

The precipitate, after filtering, washing, and drying, weighed less than
1 g.
Portion 2

This was precipitated with four volumes (150 m1.) of 95% alcohol.

-17-

The precipitate, after filtering, washing, and drying, weighed I g.,
but appeared to be of a gelatinous nature being extremely difficult

to dilter and dry.

Part B
The precipitate was treated with 30 m1. of 20% hydrochloric acid
and 30 ml. of water and filtered, 10 m1. more water being added through
the filter. The filtrate was then divided into portions 1 and 2.
Portion 1
This was precipitated with two volumes (116 m1.) of 95% alcohol.
The precipitate was negligible and gelatinous.
Portion 2
This was precipitated with four volumes (232 ml.) of 95% alcohol.
The precipitate was crystalline and easily filtered. The weight, when

washed and dried, was 3.3 g.

Part C"

This was treated with M5 ml. of 20% hydrochloric acid, filtered,
and 35 ml. water were added through filter. The filtrate was divided
into portions 1 and 2.

Portion 1

This was precipitated with two volumes (122 m1.) of 95% alcohol.

The precipitate was negligible in quantity and gelatinous in nature.
Portion 2

This was precipitated with four volumes Q2MM ml.) of 95% alcohol.

The precipitate was crystalline, easily filtered, and weighed, when

washed and dried, 2.072 g.

Part D

This was treated with 60 ml. of 20% hydrochloric acid, filtered,

and 20 ml. water was added through filter. The filtrate was divided in
two portions, 1 and 2.
Portion 1
This was precipitated with two volumes (126 ml.) of 95% alcohol.
The precipitate was negligible in quantity and gelatinous in nature.
Portion 2
This was precipitated with four volumes (252 ml.) of 95% alcohol.
The precipitate was crystalline, easily filtered, and weighed, when

washed and dried, 1.097 g.

In order to more accurately evaluate the results analysis were
made of the first three preparations and of the second portions of the
B, C, and D parts of the fourth preparation. The phytic acid analysis

gave the following results, using 0.1 g. samples of the salts in all

 

 

cases.
Table V1
ce.FeC1 ce.Fe01 solution ce.FeCl solution

Sample solutio; added for B ank Net 3 % Phytic Acid
Prep. #1 6.25 0.25~ 6.00 I9.38

" #2 9.50 0.38 9.12 75.05*

” #3 9.00 0.25 8.75 72.01

” #H 32 9.00 0.25 8.75 72.01

I I 02 8.50 0.25» 8.25 68.00

I I D 8.00 0.25 7.75 63.78

 

The calcium content was then determined on all of the aboge pre-
parations in duplicate, using 0.05 g. samples for some and 0.1 g. samples
for others. The samples were dissolved in water, 10 ml. of saturated

ammonium chloride was added, and the calcium was precipitated from the

- 19 _

boiling solution by adding 10 ml. of saturated ammonium oxalate sol-
ution. lfter standing two hours the precipitate was filtered, washed,

dissolved in 1-1 sulphuric acid, and titrated against .07896 N potassium

 

 

permanganate.
Table V11
Titration

Sample ml. .07896 KMnOu % Calcium
#1 0.05 g. 2.78 8.78.
' m 2.51 7.93
#2 0.05 g. 3.u8 11.00
I u 3.52 11.12
#3 0.05 g. 3.81 12.0u
” n 3.81 12.0w
32 0.1 g.* 7.23 11.u2
I n * 7.20 11.38~
02 0.1 g.* 6.82 10.78
0 n * 6.63 10.u7
02 0.1 g.* 6.01 9.50
a u * 6.0M 9.5u

 

*Note - The 0.1 g. samples of 32, 02, and D2 were not completely
soluble in 150 m1. of water even when heated. They were taken into sol-
ution by adding dilute hydrochloric acid, neutralized to turbidity with
ammonium hydroxide, and then just clarified by the careful addition of

acetic acid. The precipitation and other operatiohs were as given above.

In order to verify the data on calcium content given above and to

determine whether the phytic acid inhibited its precipitation another

series of analysis was made in which the organic matter was destroyed

-20-

by ignition. .Ash determinations were also made. 0.1 g. samples were
weighed into tared crucibles, ignited, and the ash weighed. The cru-
cibles were then placed in hydrochloric acid solutions and warmed, after
which the crucibles were removed, rinsing them back into the beaker with
distilled water. Ten ml. of saturated ammonium oxalate were then added
and the solution brought to the neutral point of methyl red while being

heated on the steam bath. The calcium was determined as before.

 

 

Table V111

Sample Ash wt. Ash % m1. .07896 KMnOu % Calcium
#1 0.1 g. 0.0950 h5.0 5.02 7.93
I I 0.0u53 u5.3 5.23 8.26
#2 0:1 g. 0.0595 59.5 7.20 11.37
I I 0.0605 60.5 7.50 11.8w
#3 0.1 g. 0.0590 59.0 7.35 11.61
I I 0.0578 57.8 7.2 11.h2
32 0.1 g. 0.0590 59.0 7.55< 11.92
I I 0.0589 58.9 7.60 12.00
02 0.1 g. 0.0572 57.2 7.35 11.61
I I 0.0560 56.0 7.31 11.5h
02 0.1 g. 0.0590 5u.0 6.86 10.83

I I 0.0539 53.9 6.53 10.31

 

«921-

ther analysis were kindly made by Mr. L. White for phosphorus

and nitrogen in B C , and D2 are given below.

 

 

 

 

 

2' 2
Table 1x
Aliquot
Sample Wt. Titrated Net ml. 0.1981 N NaOH % Phosphorus
32 — 0.1002 g. .0h01 g. 28.95 19.1
32 — 0.1006 .0M02 30.75 20.3
02 - 0.103h .0u1u 30.05 19.2
32 - 0.101h .0h06 26.80 17.5
92 - 0.1000 .0u00 2u.85 16.5‘
Table X
Sample Wt. Net. ml. .202 N 301 % Nitrogen
132 .. 0.2997 1.30 1.2
02 - 0.1997 1.05 1.5
02 - 0.2012 1.60 1.6
D2 "' 000755 .90 3.11

 

 

Inasmuch as the above work showed that the method of preparation of

B was superior to the others both in yield and in percentage of phytic

2
acid, this method was ad0pted and four preparations were made following
the proceedure given below.

1 kg. bran was extracted with 6 liters of 2% hydrochloric acid for

3 hours and filtered through folded papers, allowing them to filter over-

night. The filtrate was adjusted to a p H of ”.6 with strong sodium hy-

- 22 -

droxide, using brom0ph¢nol blue as an external indicator, after which
saturated calcium acetate solution (300 - MOO ml.) was added.until
precipitation was complete. After filtering off the precipitate on a
Buchner funnel it was dissolved in about 30 ml. of 20% hydrochloric
acid and a volume of water equal to that of the mixture was added. The
precipitation was made with four times the total volume of 95% alcohol
and, after settling, the precipitate was filtered on a Buchner funnel,
washed with alcohol and ether, and dried in a vacuum dessicator over
sulphuric acid. The yields obtained varied from 30 g. to 35 g.
Preparations of ghe Saturated Sodium Salt

10 g. of the crude calcium phytate preparation above was dissolved
in dilute hydrochloric acid and the phytic acid.precipitated as ferric
phytate by the addition of 10% ferric chloride solution which was added
until the solution was yellow with the excess. The ferric phytate was
centrifuged and the liquid decanted. Water was added and the centrif-
'uging and decanting repeated. Then the salt was suspended in water and
90% sodium hydroxide was added until the precipitated ferric hydroxide
was sufficiently well flocculated that it did not spread when a drop of
the mixture was placed on a filter paper. The ferric hydroxide was then
separated from the sodium phytate solution by centrifuging and filtering.
A volume of 95% alcohol was then added, but only by chilling overnight
could the syrupy sodium phytate be made to precipitate and then only in
small quantity. The alcohol-water mixture was then decanted and about 2
volumes of water added, after which the rest of the alcohol was driven off'
on the steam bath. Crystals separated on long standing in the refrigerator,
but they were apparently contaminated by ferric hydroxide.

A second preparation of the sodium salt was made using smaller vol-

umes, but otherwise like the first. A greater quantity of the syrupy

-23..

material was obtained. This was chilled until frozen without giving

a precipitate. However, upon thawing the solution separated into two
layers which were separated. The lower layer yielded a small crop of
crystals upon standing in the refrigerator, but they were contaminated
with ferric hydroxide. The clearer, upper layer eventually yielded a
small crop of colorless crystals.

In a third preparation the acid solution of the crude calcium
phytate (500 ml. volume) was treated with a tannic acid solution to
remove the proteins. The precipitate was removed by filtration and
the filtrate treated as in the second preparation. The syrupy material,
this time water-white, deposited a fair crop of crystals on standing in
the ice box. Filtration was not feasible because of the great viscosity
of the mother-liquor so the mixture was dried in a vacuum dessicator
over sulphuric acid to constant weight. The yield was about u g.

A fourth preparation was made using potassium hydroxide instead of
sodium hydroxide. The preparation was similar to the others, but the
syrup obtained did not precipitate crystals within the week, though kept
at the temperature of the refrigerator.

An analysis of the dried crystals of the third preparation for phytic

acid gave the following results.

 

 

Table X1
ml.F8013 solution ml.FeCl solution ml.Fe013sol.
Sample Added for Blgnk Net. % Phytic Acid
0.1h53 g. 1u.50 0.90 13.60 77.0w
0.179u 18.00 1.h0 16.60 76.2

 

Posternak (2h) claimed that the aqueous solution of the saturated
sodium salt of phytic acid was alkaline and could be titrated accurately

with acid, using the "rose" tint of methyl orange to indicate the endpoint.

- 2h _

The endpoint occurred when six equivalents of acid had been added,

according to Posternak. Since the sodium salt was saturated, thati
is, contains twelve atoms of sodium per molecule, the titration ob-
tained would represent one—half of the sodium present. The results

of such a titration are given below.

 

 

Table X11
Sample . ml. 0.1015 N HCl . 3% Na
0.1153 g. 10.30 33.1
0.179h g. 11.60 30.2

 

Purification of Crude Calcium Phytate by Modified Anderson's Method
(36)

Ten g. of crude calcium phytate was dissolved in 100 ml. of 2%
hydrochloric acid and the volume made to 500 ml. The solution was treated
with tannic acid and the precipitated proteins filtered off. Then the
calculated quantity of sodium acetate in small excess of the hydrochloric
acid was added and the phytic acid was precipitated from the hot solution
as barium phytate by the addition of hot barium chloride solution. After
settling overnight the precipitate was separated by centrifuging and fil-
tering and resuspended in water. Sufficient dilute sulphuric acid was
added to decompose the barium phytate and the precipitate of barium sul-
phate was allowed to settle and filtered off. Hot, saturated cupric acetate
solution Was then added in excess to the phytic acid solution. After stand-
ing the COpper salt was separated and washed by centrifuging and filtering and
then resuspended in water. The cupric phytate was dissolved by bubbling‘
hydrogen sulphide gas through the suspension. The cupric sulphide was then

filtered off. The phytic acid solution gave a positive though faint test

-25..
for inorganic phosphorus. To further purify the product it was pre-
cipitated as the barium salt by the addition of barium hydroxide un-
til alkaline. The barium phytate was then separated by filtration and
dissolved in the minimum amount of 5% hydrochloric acid. The solution
was filtered and the phytic acid reprecipitated by the addition of
alcohol. The precipitate was separated by filtration and again dissolved
in the minimum amount of 5% hydrochloric acid. The solution was then
neutralized by the addition of barium hydroxide and the precipitate of
barium phytate separated by filtration, after which it was resuspended
in water. The details of the decomposition by sulphuric acid, separation
of the barium sulphate, preparation of the copper salt, and its subseq-
uent decomposition with hydrogen sulphide were as given above. The test
for inorganic phosphorus was negative. The phytic acid solution was then
treated with saturated calcium acetate solution in excess. After settling
the calcium phytate was filtered off, washed with hot water, 50% alcohol,
95% alcohol, and ether. It was dried in a vacuum dessicator over sulphuric

acid. Its analysis follows:

 

 

 

 

 

Table Xlll
ml.Fe013501ution ml.FeCl solution ml.FeCl3sol.

Sample Added for El Net % Phytic Acid
0.1 g. 7.00 0.08 6.92 56.9

0.1 g. 7.00 0.08“ 6.92 56.9
Sample Ash Wt. % Ash ml. 0.07861 N Krnouso1. 8 Ca

0.1 g. 0.0600 60.0 11.97 18.82

0.1 g. 0.0599 59.9 11.95 18.80

 

Drying the salt and correcting the percentages of phytic acid, calcium,

and ash gave:

-20..
Phytic acid 65.5%

Calcium 21.7
Ash 69.1

Preparation of Barium, Strontium, and Calcium Phytates
from Bran

The crude calcium phytate was prepared from 2 kg. of bran accord!
ing to the method.used on page 21. The crude salt was filtered off and
washed and suspended in water. To the suspension glacial acetic acid was
added until an Opalescent solution was obtained. At the same time a pre-
cipitate of markedly different dharacter than the suspended salt was ob-
served to form. This was easily filtered off and reserved for later exp
cmination. From the acetic acid solution the free phytic acid was pre—
pared by Anderson's Method as given on page 2p; Its volume was 1860 m1.
and it gave a negative test for inorganic phosphates. Its analysis is

given below.

 

 

Table XIV
s 1 ml.FeCljsolution ml.Fe013solution g. Phytic Acid g.Phytic Acid
ample 4gdded Blank fist in 50 ml. in 1800 ml.
50 ml. 62.0 0.50 61.50 0.506 18.82
50 ml. 62.0 0.73 61.2 0.50M 18.77’

 

The volume of phytic acid solution minus that taken for the above
tests was divided into three equal parts of 587 ml.-each. To each of
these was added a hot, concentrated, filtered solution of barium acetate,
Strontium acetate, or calcium acetate in excess. After settling overnight,
the precipitated salts were separated from their mother liquors by centrif-
uging and filtering. After being washed successively with 50% alcohol, 95%
alcohol, and ether the salts were dried to constant weight over sulphuric

acid in a vacuum dessicator at 90 - 10000, The yields were as follows:
Barium Phytate 10.u1 g.
Strontium " 8.6M
Calcith n 6.H3

-27..

The salts gave negative biuret and Millon tests for proteins, but
did give positive tests for inorganic phosphorus. The salts were
analyzed for inorganic phosphorus by taking O.h g. samples, suspend-
ing them in 50 ml. of water, and adding 2 ml. of concentrated nitric
acid to effect their solution. The solutions were then treated as
specified in Methods of Analysis of A. O. A. C., third edition, page

16. (11 10a). The results are given below.

 

 

Table XV
Sample ml. .lOO3N NaOH ml. .lOl5N HCl Net.ml. .1000N NaOH %Inorganic P
Ba phytate 50.00 20.10 29.75
a I 50.00 20.15 29.70
Average 1.00
Sr n 30,00 10.80 19-13
I I 30.00 10.70 19.23
Average 0.65
Ca I 10.00 3.80 6.17
I I 10.00 3.20 6.78
_ Average 6.M7 0.22

 

For the total phosphorus determinations on the salts 0.1 g. samples"
were weighed into crucibles and treated with an aqueous solution of mag-
nesium nitrate as prescribed in the Methods of Analysis of A. O. A. C.,
third edition (p.15, 11.5e). The moisture was carefully evaporated and the
mixtures ignited in an eleCtric furnace. The crucibles were then placed in
beakers and their contents dissolved by adding 10 ml. of concentrated nitric
acid and 100 ml. of water and warming. After removing the crucibles the
contents of the beakers were analysed for phosphorus by the method recom-
mended on-page III of Methods of Analysis of A. O. A. C., third edition

(p 11, 12.7).

- 28 -

The results are given below.

 

 

Table XVI

Sample MgePEOY‘WS. % Total P
Ba phytate .0533*g
" " -0533

Average .0533 1u.85:
Sr ” .0628 g.
u n .0631

Average .0629 17.53:
Ca ” .07h6 g.
u a .0752

‘ Average .07h9 20.37

___._._. A inn—‘4... A4-

The barium, strontium, and calcium content of the salts was
determined by weighing 0.1 g. samples into 250 ml. beakers and dis-
solving them in 200 ml. of water and 5 ml. of concentrated hydrochloric
acid. The solutions were heated on a steam bath. The calcium and
strontium were precipitated by adding to the hot solutions 10 and 20
ml. of saturated ammonium oxalate solution respectively. The hot sol-
utions were then brought to the neutral point of methyl red by the
addition of ammonium hydroxide. After standing on the steam bath for
several hours the oxalates of calcium or xtrontium were filtered off,
washed with cold water, and dissolved in dilute sulphuric acid. They
were then heated to about 700 C. and titrated with standard potassium
permanganate, the papers being added at the end of the titration. The

results follow.

-29..

 

 

 

 

 

Table XVll
Sample ml. 0.0782 N KMnOu solution % Strontium
Sr phytate 10.ho
. ” 1o.u2
- Average10.ul 35.70
Table XV111
Sample m1. 0.0782 N K2910LL solution‘ % Calcium
Ca phytate 1M.10
u I 1M.08
‘ Average 1h.09 22.05

The hot barium phytate solution was treated by adding 10 ml. of
20% sulphuric acid and allowing it to stand for a time on the steam
bath. The barium sulphate was then filtered off, washed, ignited in

an electric furnace, and weighed. The results are given below.

 

 

Table XlX
Sample Ba soLL Wt. % Barium
Ba phytate .076h g.
u n .0762:

Average .0763 hu.90

 

-30-

The phytic acid content was determined as usual, 0.1 g. samples

being taken. The results follow.

 

 

Table XX
ml. FeCl solution

Sample Added lank Net % Phytic acid P % Phytic acid
Ba phytate 6.007 0.26 5.7”

u u 6.00 . 0.26 5.7M 13.31 u7.30
Sr ” 7.00 0.22 6.78

n a 7.00 0.22 6.78 15.72 55.90
Ca " 8.50 0.26 8.2M

" n 8.50 0.26 8.2M 19.10 67.90

 

The air—dried precipitate that was observed to form when the glacial
acetic acid was added to the water suspension of the crude calcium phytate

was examined. Its response to the following qualitative tests is given be-

 

 

low.
Table XXl
Test Applied Result' ' Remarks
Biuret Positive Pink oo1or developed
Millon's Positive White precipitate becoming pink
YanthOproteic Positive Typical
Molisch Positive Faint but positive on standing
Reduced Sulphur Positive Typical

Phosphorus-Organic & Inorg. Negative

 

The quantitative determination of nitrogen was effected by a modified
Kjeldahl-Gunning method in which the evolved ammonia was absorbed in M%

boric acid which was subsequently titrated with standard sulphuric acid

-31...
to give its original color with methyl red, a reference solution be—
ing used for the comparison. 0.5 g. samples were used and the follow—
ing results were obtained, the percentage of protein being calculated

by multiplying the percentage of nitrogen by 6.25.

 

 

Table XXll
5:jgemple ml. of 0.1002 N EaseLL 3% Nitrogen yd. protein
Precipitate 15.10

 

‘

The Preparation and Some PrOperties of Phytic Acid

The preparation was identical with that employed in the preparation
of the salts. 2 kg. of bran were used.and the crude calcium phytate
treated by Anderson's method. Some crude calcium phytate from an earlier
preparation was added. The phytic acid solution was concentrated by dis-
tilling in a vacuum below MOO C. as long as this was possible. The color-
less, slightly opalescent syrup was then transferred to a weighing bottle
and further dried by placing it in a vacuum dessicator over sulphuric
acid and finally over phosphorus pentoxide. The color darkened through
yellow to brown during these Operations. The consistancy became extremely
viscous as the drying proceeded until finally it approached that of an
amorphous solid. After the weight decrease became very small (.0295 grams
for a 28.h285 gram.sample after 16 hours in a vacuum desicator over sul»
phuric acid at room temperature) a 3.2030 g. sample was weighed out, dis-
solved in water, and made to a volume of 100 ml. The weighing was made as
rapidly as possible because of the extremely deliquescent nature of the
material. Aliquots of the 100 ml. were taken for analysis. The phytic
acid was determined on 5 ml. aliquots and the results are given in the

following table.

_ 32 _

Table XXllI

 

—_—-—

ml. FeCl Solution
Sample Added E ank Net % Phytic Acid P % Phytic Acid

‘—

 

Phytic acid 0.1601
g. 19.25 0.37 18.88

u n a 19.25 0.37 18.88 26.u0 97.20

 

The total phosphorus was determined on 10 ml. aliquots. These were
pipetted into crucibles and 5 ml. of the magnesium nitrate solution re—
commended in the Methods of Analysis of A. 0. A. C. (p 15, 11 5e) was'
added. The solutions were then evaporated to dryness in an oven-and ig-
nited in an electric furnace. Their subsequent treatment was identical
with that used in the determination of total phosphorus on the salts. The

results are given below.

 

 

Table XXV
Sample MgZPZOY' % P
Phytic acid 0.3203 g. 0.2677 g.
u I I 0.2671
Average 0.267M 23.26

 

The phytic acid gave negative results with biuret and Millon tests.

It was thought that a potentiometric titration would prove instructive
so 13 ml. of the solution was titrated with 0.1003 N sodium hydroxide using
the Coleman glass electrode instrument to measure the pH values. Readings
were taken after the addition of each 0.1 ml. of the base until 33 ml. had
been added. Then 0.25 ml. were added between readings for the next ml. and
0.5 ml. additions were used from 3M to 36 ml. inclusive. The last four

additions consisted of 1.0 ml. each, bringing the total to #0 ml. The curve

is given in Figure l.

 

 

 

 

.,..moaz 282. As.

1 4

Fig.1.

L ..

 

DH":

.. 33 ..
piscussmr

Methods of Analysis

All methods of phytic acid determination that are modifications
of that of Heubner and Stadler (30) have the inherent weakness of
employing an empirical factor in their calculation. Harris and Mosher
(3%) expressed their doubt concerning the accuracy of the factor 1.19
and further evidence will be presented in this paper to support their
contention. However, the fact that the factor is empirical does not
help greatly in explaining the discrepancy encountered in the experiment
dealing with the recovery of phytic acid. Probably the most feasible
explanation would be based on the disturbance of the acidity by the
proteins of the brans so that additional phytic acid was not precipit-
ated within the empirically determined Optimum pH value. It is possible
that at the higher pH an iron phytate containing more iron, and therefore
giving a higher titration, is formed. The investigation of the relationship
between the pH at which the ferric chloride is added and the composition of
the ferric phytate precipitated might be instructive.

Analytical Studies

The data obtained in Table 1? indicates that with wheat bran phytic
acid content falls with the time of exposure to the cold 2% hydrochloric
acid. This might be due to the hydrolytic action of the acid or of the
enzyme phytase. However, in Table V, it is apparent that on heating the
mixture at the beginning of the exposure the destruction of phytic acid is
inhibited. This would indicate the action of an enzyme capable of breaking
down phytic acid (or phytin) even in 236 hydrochloric acid. At the same
time it would eliminate the hydrolytic action of the acid as an explanation,
although the heating apparently causes some breakdown, possible greater be-

cause of the presence of acid. The latter part of the table bears out the

-3Ll-

conclusion that 2% hydrochloric acid does not hydrolyse phytates. The
fact that in this case the phytates are not hydrolysed without the heat
treatment simply means that active phytase is not present; that is, did
not survive the operations of Anderson's purification.
Preparations

Posternak (22) did not describe his work in great detail so it was
necessary to make several trials using different details of proceedure
in order to obtain maximum yields. Perhaps the use of barium acetate
as a precipitant would be better since, as Patten and Hart (19) point
out, the barium phytate is less soluble than the calcium salt. Be that-
as it may, the subsequent solution of the calcium salt in hydrochloric
acid never gives a clear solution, considerable opalescence persisting
even when great quantities of acid are added. The data obtained show
clearly that excessive quantities of acid are to be avoided. Further-
more, two volumes of 95% alcohol were found to be insufficient for the
subsequent precipitation, four volumes greatly increasing the quantity
of the precipitate as well as rendering it much easier to filter.
After the precipitate was obtained it was found advantageous to grind
it with the alcohol and ether in a mortar, returning it each time to
the filter for the separation of these wash liquids. In this way the
water and alcohol are more thoroughly displaced and drying is much easier.

Whether or not the organic matter of the calcium phytates was de-
stroyed or not previous to the precipitation of the calcium oxalate
seemed to make little difference in the result. Apparently then the
phytates are true salts and the organic matter does not interfere with
their determination. As would be expected, the percentage of ash appeared
to be proportional to the percentage of calcium. From the phytic acid

determinations it appears that excessive use of acid in dissolving the

-35..

crude calcium phytate precipitated by calcium acetate in the prepar-
ation lowers the phytic acid content of the precipitate obtained when
alcohol is added later. It also lowers the calcium content. Conseq—
uently, this use of excessive acid increases the percentage of the
crude material unaccounted for. From the data on nitrogen content

it seems probable that part of this increase is due to protein matter.
The remainder of the increase is perhaps due to water associated with
the protein.

The saturated sodium salt preparation seems hardly feasible as a
means of obtaining phytic acid as a stable salt because of the scant-
iness of the yield and the difficulty of their separation. Posternak
(2M) describes two sodium salts, C6 H6 02h P6 Na12 . 3 H2074 MM HBO
which is effloresent and C6 H6 021; P6 Na1L2 . 3 320 + 35‘Hé0 which is
not efflorescent. The writer did not spend sufficient time on the pre-
paration to verify these results and must decline to comment further.
The phytic acid percentages are far too high for either of these salts
and the titration with acid using the "rose" tint of methyl orange as
an endpoint failed to add much information. -

In the adaptation of Anderson's modified method (36) to the pur-
ification of crude calcium phytate much trouble was experienced with
the tannic acid treatment of the acid solution. The tannic acid is
added until the precipitation of protein is complete, but the difficulty
lies in judging the point at which a sufficient quantity has been used
and no appreciable excess is present to contaminate the solution. The
same objection applies with even more significance to the tannic acid
treatment of a bran extract. Furthermore, the precipitate produced by
tannic acid and protein does not always flocculate and in such cases it

is nearly impossible to separate it by filtration and centrifuging. Since

-36..

Anderson's method specifies that the barium precipitation be made

in acetic acid (obtained by adding the calculated quantity of sodium
acetate to the hydrochloric acid solution) the crude calcium phytate
from former preparations was suspended in water and glacial acetic
acid was added.unti1 the solution became opalescent. At this point

a precipitate, differing from the suspended matter, was observed to
form. It was filtered and reserved for examination while the barium
precipitation was carried out on the filtrate. No further departure
from the prescribed method was attempted in the purification itself,
but the examination of the substance precipitated by the acetic acid
proved instructive. The protein tests indicated its nature and the
nitrogen determination gave a protein value of 26.55% (Table XXll)

for the protein content of the air-dried substance so this figure

would probably be considerably increased if calculated to a dry basis.
When it is remembered that there was no phosphorus present it becomes
apparent that there is no loss of phytic acid or phytates. These facts
taken in conjunction with the negative tests for proteins in the purif-
ied salts and acid seem to indicate this as an easy and rapid method
for the complete elimination of proteins. The writer feels that this method
is superior to the tannic acid treatment because of its simplicity and
the fact that it cannot possibl y introduce troublesome contaminating
substances.

The precipitations of the salts were made under identical conditions
and no difficulty was experienced in separating them from their mother
liquors. It was found that repeated centrifugings, decantations, and re-
suspensions in the various wash liquids was advantageous due to the rapid-
ity and thoroughness of the washing. Eventually, of course, the salts were

brought upon a suction filter.

-37..

The presence of inorganic phosphates in the dried salts indicated
that possibly 1000 C. in a vacuum was somewhat too vigorous drying
treatment for them to withstand. Why the greatest stability occurred
in the calcium salt and the least in the barium salt is difficult to
understand although, of course, it is apparent that there is an inverse
correlation between the stability of the salt and the atomic weight of
the metal if this has any significance.

From the analysis of the salts it should be possible to compute the
atoms of metallic element per molecule provided the products are pure
compounds and not mixtures and provided further that the molecular weight
of phytic acid be known. The most commonly accepted formula for phytic
acid, that of Suzuki, Yoshimura, and Takaishi (ll), gives a molecular
weight for phytic acid of 660 while the formulae of Starkenstein (l3)
and Neuberg (12) give the molecular weight as 71h. Taking first the
molecular weight as 660 and computing the percentages for the tetra-

metallic salts gives the following results.

 

 

Table XXVI
Calculated Found
Tetraecalcium Phytate 19.75% 22.05%
TetraPStrontium Phytate 35.00 35.70
TetrarBarium Phytate 35.75 uh.90

 

The agreement, while fair in the strontium and barium salts, is
poor for the calcium salt. Taking the molecular weight of phytic acid
as 71M and computing for the penta-metallic salts gives the following

results.

- 38 _

 

 

Table XXVll
Calculated Found
Pentapcalcium Phytate 22.1uw 22.05%
Penta-strontium Phytate 38.35 35.70
Pentapbarium Phytate M9.37 HH.9O

 

It is evident from Table XXVll that the agreement for the calcium
salt is excellent while that of the strontium and barium salts is phor.

It is difficult to reconcile these figures. However it may be re-
membered that the molecular weights 660 and 71h differ by 5M which is
three times the molecular weight of water. Now, by assuming that the
barium and strontium salts are tetra-compounds and are anhydrous while
the calcium salt, on the other hand, is a penta—compound hydrated with
three molecules of water per molecule the data can be made to conform
to theory as far as the metallic (pntents of the salts are concerned.
However, when the phytic acid content of the salts is considered, there
is in every case a discrepancy in the total percentage, the sum of the
percentages of the metallic and phytic acid constituents falling far
short of 100. Perhaps it is due to water held somewhat as that of crya
stallization. In any case, such theoretical speculations are beyond the
scope of this paper, especially since there existed in the preparations
a certain amount of inorganic phosphates.

When the inorganic phosphorus percentages are subtracted from the
total phosphorus percentages and the differences multiplied by 3.55 to
convert to phytic acid, the percentages of phytic acid (mol. wt. 660)
are greater than those obtained by the titration with ferric chloride
solution, although not enough to make up the discrepancies noted above.

However, assuming the phytic acid figures obtained from the difference

-39..

between total and inorganic phosphorus to be correct, a means is

afforded for checking the accuracy of the ferric chloride titration.

 

 

Table XXVlll
Difference Phytic acid Phytic acid-H_ 0:
Sample Total P Inorganic P Phytic acid P by Difference (FeC13) Difference
Ba Phytate 1’4.85 1.00 13.857 19.2 117.3 1.9
Sr N 17.53 0.65 16.88‘ 59.9 55.9 1+.0
Ca H 20.87 0.22 20.65 73A 67.9 5.5

 

From the table given above it would appear that the values obtained
by the ferric chloride titration should be multiplied by a factor of about
1.06, or better, that the empirical factor 1.19 be changed to 1.26. How-
ever, this factor would change the percentage of the phytic acid preparation
from 97.2 to 103 so probably the change is not warranted.

The absence of inorganic phosphorus in the phytic acid preparation
affords more evidence that heat is responsible for the breakdown of phytic
acid and its compounds.

The potentiometric titration curve showed slight breaks when 19.5 and
26.5 ml. of .1003 N sodium hydroxide had been added. Since the 13 ml. of
phytic acid solution used represented O.N16 g. or .0006} moles (mol. wt.660)
and the quantities of base corresponding to the breaks in the curve equaled
.00195 and .00265 equivalents respectively, it was evident that, per mole
of phytic acid, the breaks occurred when 3.09 and n.22 equivalents of base
had been added. The fact that a break in the curve occurs explains Poster-
nak's (2h) titration of the saturated sodium salt with acid.using the
”rose" color of methyl orange as an endpoint. However, the appearance of
the curve indicates that phytic acid is very weakly dissociated and suggests
that perhaps its naturally occurring salt, phytin, may exert a buffer action

in the plant organism. It should be remembered that the titration for this

_b,o..

curve was not complete; that is, twelve equivalents of sodium hye
droxide per mole of phytic acid were not added. The difficulty of

accurate measurement of the high pH proved to be the limiting factor.

U1
0

7.

— Ml -
CONCLUSIONS

The estimation of phytic acid by the method of Harris and Mosher
is not conshtant in its results.

The activity of phytase is apparently not entirely inhibited by 2%
hydrochloric acid.

Hydrolysis of phytic acid is not effected by 2% hydrochloric acid
in the cold, but is apparently effected by acid of this strength
when heat is applied.

In Posternak's method of calcium phytate preparation great excess
of acid is to be avoided and four volumes of alcohol rather than
two should be used.

Calcium phytate is a true salt and the calcium may be precipitated
and determined without the preliminary destruction of the organic
matter

The method of precipitating protein matter by the addition of glacial
acetic acid to the water suspension of the calcium salt seems pre-
ferable to the use of tannic acid added to the acid extract. The
advantages of the method lie chiefly in its simplicity, completeness
of precipitation, and freedom from the possiblity of introducing con-
taminating substances.

Phytic acid is weakly dissociated.

(1)

(2)

(3)

(h)

(5)

(6)

(7)

(8)

(9)

(10)

(ll)

-42 -
BIBLIOGRAPHY

Hartig, Th.
Ueber das Klebermehl
Botan. Zeit. 13, 881 (1855)

Hartig, Th.
Weitere Mittheilungen, das Klebermehl (Aleurone)
betreffend.
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Rose, A. R.
A.Resume of the Literature on Inosite-phosphoric Acid,
with Special Reference to the Relation of that Substance
to Plants

Biochem. Bull. 2, 21 (1912)

Pfeffer, W.
Uhtersuchungen uber die Proteinkorner und die Bedeutung
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Jahrb. wiss. Botan. 8, h29 (1872)

Palladin, V. I.

Beitrage zur Kenntniss pflanzlicher Eiweisstoffe
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Schulze, E. and Winterstein, E.
Ein Nachtrag zur Abhandlung uber einen phosphorhaltigen
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z. Physiol. Chem. #0, 120 (1903)

Winterstein, E.
Ueber einen phosphorhaltigen Pflangen bestandtheil,
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Posternak, S.
Sur la composition chimique et la signification des
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Compt. rend. luo, 322 (1905)

Posternak, S.
Sur la constitution de l'acide phospho-organique de
reserve des plantes vertes et sur la premier produit
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Compt. rend. 137, #39 (1903)

Posternak, 3.

Contribution a l'etude chimique de l'assimilation
chlorophyllienne. Sur le premier produit d'organ-
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a chlorophylle. Awec quelques remarques sur le role
physiologique de l'inosite.

Rev. Gen. Botan. 12, 5 (1900)

Ibid. 65

Suzuki, U., Yoshimura, K., and Takaishi, M.
Uber ein Enzym, Phytase, das "Anhydrooxymethylen-

(12)

(13)

(in)
(15)

(16)

(17)

(18)

(19)

(20)

(21)

-43-

diphosphorsaure" spaltet.
Bull. Coll. Agri., Tokyo 7, M95 (1907)
Ibid. 503

Neuberg, C.
Zur Frage der Konstitution des "Phytins"
Biochem. z; 9, 557 (1908)

Starkenstein, E.
Das Verhalten der Uransalze zweibasischer Phosphorsaure
gegen Indicatoren
Biochem. z. 32, 235 (1911)

Starkenstein, E.
Die biologische Bedeutung der Inositphosphorsaure
Biochem. z. 30, 56 (1911)

0011th1 , A. O
Atti. accad. Lincei. 18 (1O sem.) 5 ser., 6M 1909
19 (1 sem.) 5 ser., 823 1910

Carre , P.
Sur 1es ethers polyphosphoriques de la mannite, de
la quercite, du glucose et de l'inosite de Contardi.
Bull. soc. Chem. (u) 9, 195 (1911)
Uber die Contardischen Polyphosphorsaurcester des
Mannites, Quercits, der Glucose, und des Inosits.
Chem. Zentr. 1, 1196 (1911)
Anderson, R. J.

Phytin and Phosphbric acid Esters of Inosite.
N.Y. (Geneva) Agr. Exp. Sta. Tech. Bull. 19 (1912)

Anderson, R. J.
Phytin and Phosphoric Acid Esters of Inosite.
N.Y. (Geneva) Agr. Exp. Sta. Tech. Bull. 19 (1912)
Anderson, R. J. - _
Phytin and Phosphoric Acid Esters of Inosite.
N.Y. (Geneva) Agr. Exp. Sta. Tech. Bull. 21 (1912)

Patten, A. J. and Hart, E. B.’
The Nature of the Principle Phosphorus Compound in
Wheat Bran.
N.Y. Agr. Exp. Sta. Bull. 250 (l90u)

Anderson, R.J.
The Organic-Phosphoric Acid Compound of Wheat Bran:,
Preliminary Report.
N.Y. (Geneva) Agr. Exp. Sta. Tech. Bull. 22 (1912)
Anderson, R. J.
Organic Phosphoric Acids of Wheat Bran.
N.Y. (Geneva) Agr . Exp. Sta. Tech. Bull. 36 (l9lh)

Anderson, R. J.
Concerning the Organic Phosphorus Compound of Wheat
Bran and the Hydrolysis of Phytin.
’ N.Y. (Geneva) Agr. Exp. Sta. Tech. Bull. no (1915)

(22)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

Posternak, S.
Sur deux sels cristallises du principe phospho-organique
do reserve des plantes vertes.
Compt. rend. 168, 1216 (1919)

, Anderson, R. J.

Synthesis of Phytic Acid
J. Biol. Chem. M3, 117 (1920)

Posternak, S.
Sur la Synthese de l'acide inosito—hexaphosphorique.
Hel. Chim. Acta. M, 150 (1921)

Neumann, A.
Einfache Veraschungsmethode (Sauregemischveraschung)
und vereinfachte Bestimmung von Eisen, Phosphorsaure,
Salzsaure, und anderen Aschenbestandteilen unter
Benutzung dieser Sauregemischveraschung.
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Hart, E. B. and Andrews, W. H.
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N.Y. Agr. Exp. Sta. Bull. 238 (1903)

Vorbrodt,J‘.
Bull. de l'Acad. des Sci. de Cracovie. ser..£, hlh (1910)

Stutzer, A.
Untersuchungen uber den Gehalt vegetabilischer Stoffe an
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Ohio Agr. Exp. Sta. Bull. 215 (1910)

Heuhner, W. and Stadler, H.
Uber eine Titrationsmethode zur Bestimmung des Phytins.
Biochem. 2. 6M, M22 (191M)

Lindenfield, K.
Uber die Bestimmung von anorganischer und Phytinphosphor-
saure in Phytinpraparaten
Biochem. z. 257, 8 (1933)

Tropp, M. Y. and Soltz, L. M.
Farm. Zhur. 11-12, 332 (1932)

Determination of phytin and calcium glycerophosphate.
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Averill, H. P. and King, C. G.
The Phytin Content of Foodstuffs
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-45..

(3h) Harris, R. S. and Mosher, Q. M.
Estimation of Phytin Phosphorus
J. Ind. Eng. Chem. (Anal. Ed.) 6, 320 (193M)

(35) Andrews, J. S. and Bailey, 0. H.
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Ind. Eng. Chem. 2”, SO (1932)

(36) Morrow, C. A. and Sandstrom, W. M.
Biochemical Laboratory Methods. 2nd Edition.
John Wiley and Sons, New York, 1935

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