SOME FACTORS INFLUENCING THE
LE'I'ERIVIINATION OF HYDROCYANIC
ACID IN PLANT MATERIALS

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SOME FACTORS INFLUENCING THE DETERMINATION OF

HYDROCYANIC ACID IN PLANT MATERIALS

by

Charles Leonard San Clemente

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
1910

TLOIZOIB

C \CI ’I

Acknowledgment
The author sincerely appreciates the stimulating
guidance given by Professor C. D. Ball during the Course
of this work, and will keenly remember his doctrine of

honest and critical approach in science.

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CONTENTS

Page

Introduction..................................................... 1
Historical....................................................... 2
General stages of procedure................................. 2
Digestion and maceration............................... 2
Enzymes................................................ h
Anesthetics............................................ h

Acids and Alkalis...................................... 5
Distillation........................................... 8
Absorbents and titration............................... 9
Turbidimetric and colorimetric methods......................10
Cyanogenesis by bacteria....................................13
Preservation of cyanogenetic p1ants.........................13
Experimental"...................................................1b,
Studies in the i‘ieldlLL
Method of determination................................1b,

Seasonal variation.....................................16

Diurnal variation......................................17
Leaf-stem variation....................................17

Second growth..........................e...............17

Some factors influencing the determination..................25
Effect of chloroform...................................25
Destruction of chloroform during distillation.....25

Immediate distillation............................25

Delayed distillation..............................26

Effect Of buffer systems......u..un...u.......ouo.26

Picramate method.....................................26
Absorption studies..............................29

Calibration curve...............................§O

Color stability.................................30

Preservation of sample..........................50
Discussion.....................................................56
Conclusions....................................................hh

Literature citedOO...OOOOOOOOOOCOOOOOOOOO00....OOOOOOOOOOOOOOOOué

INTRODUCTION

Experimentally, this thesis is divided into two portions. The first
part, studies in the field on the variation in hydrocyanic acid content
of sudan grass, is essentially a continuation of work initiated at this
laboratory by Lawrence C. Whlker (1) in 1935. Obviously, experimentation
dependent upon seasonal factors requires repeated study over a period of
several years. In carrying out this phase of the work, walker's acid-
titration method for the determination of hydrocyanic acid had to be
employed in order to obtain comparable results. However, it soon became
evident that this particular procedure was not without certain disadvan-
tages, such as time for hydrolysis of the plant material, maceration,
detection of the end point, and the possibility of loss of hydrocyanic
acid during several stages of the process. For these reasons, emphasis
‘was shifted to some of the factors that affect the detemmination of prussic
acid in plant material. The second part of this thesis dwells on these
factors, especially on those concerned with the development of a rapid
colorimetric determination of hydrocyanic acid.

The review of literature is restricted to a discussion on procedures
and methods, as the various agricultural ramifications have'been previously

discussed by walker (1).

HISTORICAL

written accounts show that hydrocyanic acid was known to the ancients.
Doubtless, the alchemists of medieval times possessed qualitative methods
to detect this fatal poison. In.more recent times, especially with the
develOpment of the agricultural sciences, methods of quantitative deter-
mination were introduced; so that today the literature of this field is
replete with figures concerning the prussic acid content of many plant
products. The results of these determinations are extremely variable
and inconsistent;1noreover, the degree of incongruity cannot be explained
merely by the accepted error inherent in all biological quantitative work.

The literature contains a number of methods for determining hydro-
cyanic acid, and equally as many variations in the several salient stages
of the procedure. Instead of presenting the review of literature chrono-
logically, it has been deemed more effective to arrange this review in the
order in which the principal stages of the analytical procedure occur, and
then follow this arrangement by several other important considerations.
First, under general stages of procedure there will be discussed: (1)
digestion and maceration; (2) enzymes; (3) anesthetics; (h) distillation;
and (5) absorbents and titration. This part is followed by: secondly,
turbidimetric and colorimetric methods; thirdly, cyanogenesis by bacteria;

and lastly, the preservation of cyanogenetic plants.

Digestion and Maceration

 

In general most analysts (1,2,5,h,5,6) agree that digestion of the
sample in water was necessary; yet the time of maceration, temperature, or
manner of previous treatment are factors that still require clarification.
Askew (7) found that the highest titration figures were obtained by diges-

tion in water for 2h hours, or heating at h5° for four hours. Bagachi and

-3-

Ganguli (9a)state that four hours in neutral media gave the highest values.
Bartholomew and Raby (8) found that a difference of temperature from 20° -
3h° in the macerating of flax seed meal had no apparent effect in the amount
of hydrocyanic acid.extracted. hydrocyanic acid was lost by volatilization
during maceration when an open distilling flask was used as macerating
vessel.

In this connection Alsberg (2) made the important observation that the
time required for complete liberation of hydrocyanic acid from.plant tissues

is dependent upon the species. One hour was sufficient for Andropogon

 

sorghum.while Prunus virginiana required four hours. Vigorous boiling did

 

not increase the rate of hydrocyanic acid set free. The amount of hydro-
cyanic acid was diminished by macerating the leaves of Prunus before dis-
tillation, the gas apparently being destroyed or converted into a form not
separable by hydrolysis. Alsberg further noted that hydrocyanic acid added

to Andropogon could be recovered only partially after maceration, the tissue

 

apparently exerting the same action on added cyanide as upon the hydrocyanic
acid set free from.its glucoside. The disappearance was chemical or physical
and not dependent upon the presence of living protoplasm.or enzymes as cyanide
disappears rapidly even in the presence of previously heated macerated sorghum.
But this phenomenon was not universal - no disappearance of potassium.cyanide

occurred in Sambucus canadensis. Neither can it be attributed to nitrile

 

formation because glucose was without influence. He suggested, therefore,
that the determination be made by'maceration, by direct and separate dis-
tillation of both the fresh and macerated tissue, and by distillation after
extraction; in this way the method yielding highest amounts of hydrocyanic

acid could be determined for each species.

AskeW'(7) believed that distillation in large amounts of water (a liter

for a 50 g. sample) avoided formation of undesirable compounds.

Enzymes

The addition of enzymes has been frequently advised. Viehoever (h)
found that emulsin did not increase the yield; neither did pepsin plus
hydrochloric acid according to Leeman (9), and Roe (10) also found no
apparent difference in the use of enzyme. Finnemore and Cooper (5)
however, analyzed 32 samples of Heterodendron oleaefolium.and Obtained
values ranging from 0.005 to 0.068% without enzyme, and from 0.009 to

0.521% When enzyme from almonds was added.

Anesthetics

 

The effect of anesthetics has been studied by several workers with
rather interesting results. Sullivan (11) reported that immediate dis-
tillation with chloroform gave as high results as possible, in fact higher
than if autolyzed for 2heh8 hours. he omitted grinding and the addition
of'water to the sample. Boyd and coworkers (12) added 500 ml. of water
and 5 ml. of chloroform to an o g. grass sample which had previously been
chopped into one-half inch lengths. The solutions were distilled immedia-
tely. They did.not compare their results with other methods. Leeman (9)
stated that heating the sample to 59° (wilting temperature) or 70’, re-
leased as much hydrocyanic acid as by the addition of chloroform. He found
that hydrocyanic acid was lost when extracted with.h2.5% alcohol, but not
with 95% alcohol. Apsit and Gain (13) have shown that seeds may remain so
long in the resting stage as to lose their germinative faculty but might
still retain enzymatic activity. Seeds subjected to anesthesia induced by
ether and thereby deprived of power of germination still possessed the power
of amylase and peroxidase activity. Briese and Couch (1h) found that 50-95%
alcohol inhibited eyanogenesis.

hfillaman (l5) eXposing Sorghum.leaves to vapors of chloroform, ether,

-5-

and alcohol found that the yield of hydrocyanic acid was increased. The
anesthetic stimulates both the hydrolytic and synthetic action of the
glucosidic enzymes, which he declared was a case of demonstrated enzyme
synthesis in 1132. Enzyme powder prepared from chloroformed leaves was
about 25 times as active towards amygdalin as powder of untreated leaves.
Frosting also caused an increase probably because of cell rupturing and

disturbed enzyme equilibrium.

Acids and Alkalis

The use of acids and alkali has been studied in many laboratories.
As might be expected the evidence is contradictory and confusing. Among
the acids employed, there may be listed sulfuric, hydrochloric, hydro-
bromic, phosphoric, selenic, lactic acid, tartaric acid, and acetic acid.
Krieble and Peiker (16) found that the rate of hydrolysis of hydrocyanic
acid was independent of the concentration of hydrocyanic acid. Velocity
constants for the hydrolysis of hydrocyanic acid with various concentra-
tions of hydrochloric and hydrobromic acid as catalysts were determined
at u5° and 65°. The velocity increased rapidly with increase in acid con-
centration. The principal catalyst was the undissociated acid molecule;
at 65° sulfuric acid was a poor catalyst compared with the other two acids,
especially at the higher concentrations. The relative difference in velo-
city between.the hydrochloric and hydrobromic acid concentrations was in!
dependent of temperature. The addition of sodium.chloride and potassium
chloride to hydrochloric acid and of sodium.bromide and potassium bromide
to hydrobromic acid increased the velocity markedly - in some cases, five
fold.

Working with McNally, Krieble (17) quantitatively studied the rates
of hydrolysis of amygdalin and hydrocyanic acid when catalyzed by hydro-

chloric acid and sulfuric acid. For 5N concentration, hydrochloric acid

hydrolyzed amygdalin ten times taster than sulfuric acid. For hydrocyanic
acid alone the difference was even greater. When the concentration of
hydrochloric acid was varied from 1.95 N to 7.8h.N, the velocity in hydro-
lysis increased about 1,000 times. Aih N hydrochloric acid solution was
more than six times as effective in hydrolyzing hydrocyanic acid as a
corresponding hydrobromic acid solution.

Cobb and walton (18) studied the rates of formation and properties of

cyanide complexes with sulfuric, phosphoric, and selenic acid; giving the
formulae for the first two; HCN . H2804 and HCN . H3P0., respectively.
Their rate of thermal decomposition to yield carbon.monoxide was maximum
wdth.concentrations of 78-79% sulfuric acid or 100% phosphoric acid. The
rate-determining step in the decomposition appeared to be the thermal de-
composition oi’the formamide salt formed as an intermediate.

Bartholomew (6), Leeman (9), Viehoever (h), and Bagachi (9a), Roe
(10, 19) and respective coworkers did not find the use of’acids advantageous.
Bartholomew Obtained more hydrocyanic acid by steam.alone than by acid plus
steam. On the other hand, Morris and Lilly (20) report that in determining
hydrocyanic acid from sulfuric acid solutions no retardation was caused by
the presence of chloride ion. Loss of hydrocyanic acid occurred unless the
rubber stoppers were protected by tin foil. Ferrocyanide was a common
impurity in "GP" cyanides and when present high results were obtained. ‘With
ferrocyanide-free cyanides, the method of Pagel and Carlson (21) to be dis-
cussed presently was accurate. The rate of hydrocyanic acid hydrolysis was
a function of the acid concentration.

Roe (l9) declared that removing hydrocyanic acid by boiling was ob-
jectionable because some ammonium.formate was fommed in the distilling flask
by hydrolysis. He prefered aeration, and liberated dissociable cyanides by

adding an excess of tartaric acid. ‘Moreover, steam distillation might cause

-7-

a too rapid evolution of hydrocyanic acid for complete absorption in
alkali. Pagel and Carlson (21) refuted Roe's objections by presenting
data showing that with proper precautions and with suitable apparatus,
accurate results could be obtained by treating:mixtures of cyanide and
chloride, bromide, or sulfate with.moderate1y strong sulfuric acid and
distilling. Bartholomew, Sinclair and Jones (22) found that 0.15 ml. of
concentrated sulfuric acid for two liters of solution, or 2% tartaric acid,
was satisfactory for fumigated citrus tissues. Rigg, Askew, and Kidson
(23) suggested adding a few ml. of 2 N sulfuric acid just prior to distilla-
tion of various grass samples which had been digested in water for 20-2h hours.
More specifically Bosin and Gintsburg (2h) treated 100-200 g. of food product
with EOO-hOO ml. hot water, 10 g. of freshly precipitated lead carbonate and
25 ml. of 10% tartaric acid. The addition of 1-19 mg. of cyanide to:fruit
juices, flour, beer, and other products resulted in satisfactory recovery on
analysis with a maximum.error of h.5%. But with 0.2-0.8 mg. of cyanide in
water, the analyes were 10-66% too high.

Saredo (5) ground 25 g. of flaxseed in a steel motar, macerated h5
minutes at h5° with 500 ml. of water, and followed by two distillations:
one with 5 ml. and the other with 50 ml. of hydrochloric acid. Riet and
Echenique, (25) reported that 2% solutions of acetic acid, sulfuric acid,
hydrochloric acid, or tartaric acid followed by water gave strong reactions
for hydrocyanic acid when used to moisten Sudan grass. Bagachi (9a) obtained
maximum hydrocyanic acid from neutral, or slightly acid solutions.

As a general rule all experimental results point to the fact that
alkalis, weak or strong, always yield consistently low results (h,6,9,25)

Viehoever, Johns, and Alsberg (4) found that macerating Tridens flavus in

 

'water containing a known amount of potassium cyanide caused much loss unless

tartaric acid was present, in which case all the cyanide could be recovered.

~8-

In the presence of sodium.bydroxide, however, the cyanide disappeared

completely.

Distillation

 

After liberation of the prussic acid, the next problem.was concerned
with the distillation of the gas into a suitable absorbent. Common
practice has been to use steam distillation. Bartholomew and Raby (6)
Boyd, Aamodt, Bohstedt, and Truog (12), Viehoever, Johns and Alsberg (b)
all used steam. Among the adherents of aeration, Roe (19) has been pre-
viously quoted. In determining the hydrocyanic acid.content of amygdalin,
Roe (10Jemployed 0.05 emdlsin for 0.01 g. of amygdalin and after incubating
at h5° for 15 minutes, he aerated for three hours. In the case of mercuric
cyanides, Roe (19) previous to distillation, treated the solution with hydro-
chloric acid and stannous chloride to liberate the cyanide. But Bartholomew,
Sinclair, and Jones (22) working on fumigated citrus tissue recovered only
15% of the total hydrocyanic acid content after an aeration of 15 hours; and
none after hh hours. They believed that citrus fruits and leaves fixed or
altered a portion of the hydrocyanic acid during fumigation. Amounts recovered
varied with the amounts placed in the fumatorium. When leaf and fruit distill-
ates which contained hydrocyanic acid were allowed to stand, some unknown sub-
stance in.the distillate continued to combine slowly with the hydrocyanic
acid so that it would not react when treated with standard silver nitrate.
In a later paper Bartholomew and Baby (26) reported that aldehydes, sugars,
or citral were not entirely responsible for the disappearance of large amounts
of hydrocyanic acid. By the end of a LO minute fumigation period, a given
volume of green fruit contained 10-lb.times as much hydrocyanic acid as there
was in an equal volume of air in the fumatorium. Hydrogen sulfide from.fumi-

gated fruits was prevented from interfering in the silver nitrate titration

\

-9...

by placing 50 g. of cadmium.sulfate in the distilling flask, or by use of

a second distillation.

Absorbents and Titration

With regard to an absorbent there are comparatively few choices:
acidulated silver nitrate, weak alkali, usually 2-5% potassium hydroxide,
and alkaline ferrous sulfate (for the prussian blue method). For qualita-
tive purposes ammonia alum with lead acetate, and mercurous nitrate might
be used; likewise alkaline picrate which has been developed for quantitative
purposes. Several methods are available if the end point is to be determined
by titration. In the case of the silver nitrate acid solution, the silver
cyanide was filtered off, and the filtrate was titrated with standard potassium
thiocyanate in the presence of ferric ions as indicator. Clark (27) has sug-
gested a means of detecting an insoluble silver halide in the presence of
the cyanide. To the solution there is added nitric acid, silver nitrate,
and an excess of a 5% solution of mercurous nitrate. The black mercurous
cyanide will dissolve leaving a permanent precipitate of silver halide if
it is present.

In the alkali titration (5, 25) standard silver nitrate was added to
the cyanide solution. At first the silver was dissolved completely because
of a complex ion formation, but when enough silver had been added to unite
with all the cyanide in this way, any more reacted with this complex salt,
precipitating silver cyanide.

NaAg (cm),a + AgNOa - Ag (Ag (mm) + NaIIOg

The formation of this precipitate indicated that the endpoint had been
reached. Not only silver cyanide but all other silver salts except the
sulfide were readily soluble in excess of a solution of an alkali cyanide;

therefore, chloride, bromide, and iodide ions did not interfere. The only

-10..-

difficulty in obtaining an endpoint was that the curdy precipitate does
not readily dissolve. Bosin and Gintsberg (2h) overcame this difficulty
by adding a little ammonia in which the precipitate was soluble. To show
the endpoint, it was necessary to add a little potassium.iodide, which
formed a slight opalescent precipitate of silver iodide, soluble in
potassium.cyanide but insoluble in ammonia. In a somewhat related manner
Lubatti (28) has developed a micro-determination of hydrocyanic acid.by
distilling the cyanide into 0.2 N sodium.hydroxide and titrating with
iodine. Bosin and Gintsberg (2h) and Bartholomew and Baby (6), advan-
tageously used freshly precipitated lead carbonate to remove organic
material which frequently obscured the endpoint.

Iofinova and Gurvitz (29) based their titration method in alkaline
solution onthe following reaction which they found satisfactory down to
0.005 mg.

Nagsgoa + NaCN + 2 NaOH = NaCNS + Nazsaoa + Nazso, + H30

Turbidimetric and Colorimetric Methods

 

Recently, photoelectric methods have increased rapidly because of
the appearance of suitable and reasonably priced apparatus. Mfiller (50)
reviewed this field quite completely. Bartholomew and Baby (8) devised
a photoelectric turbidimeter to determine the endpoint in the Liebig
method of cyanide analysis in dilute solutions. The use of two photronic
photoelectric cells directly opposed circumvents the difficulties en-
countered with a single cell. The determinations made were accurate to
wdthin 0.027 mg. of hydrocyanic acid.

Colorimetric methods have mostly been based on the old Guignard
reaction (51) of hydrocyanic acid upon alkaline picrate solution.

Schieblich (52) used such impregnated filter paper for qualitative de-

-11-

tection. Adriano and Ynalveg (55) devised a semi-quantitative rapid
field test by hydrolyzing the sample in a test tube by heating for five
minutes in the presence of a uniform.filter strip which was compared to
standard strips. Paraffin was used to preserve them.

waller (5h) reported using a color scale for estimating hydrocyanic
acid in vegetable and animal tissues. This scale was obtained by incuba-
ting at 2h? known amounts of hydrocyanic acid and equal volumes of sodium.
picrate solution. Tints corresponding to amounts of hydrocyanic acid
smaller than 1 p.p.m. can be read positively. The color, he said, was not
appreciably affected by direct sunlight or by boiling.

Boyd, Aamodt, Bohstedt, and Truog (12) have recently made uaaof this
color reaction by placing 5 ml. of the hydrocyanic acid alkaline dis-
tillate and 5 ml. of alkaline picrate in a boiling'water bath for five
minutes. The red color developed.was compared.with.standards similarly
treated.

Louden and Aantrobus (35) placed a linseed cake meal in a special
closed container in which it was hydrolyzed. The hydrocyanic acid evolved
was absorbed by an alkaline picrate solution. They found that the rate of
hydrocyanic acid development was maximum.in four hours. A 0.5 g. sample
yielded 0.1 - 0.5 mg. hydrocyanic acid.

Limitations of the Guignard reaction do not appear to be realized by
many workers. Chapman (56), however, in 1910 made a very intensive study
of the reduction products of picric acid. The possibilities were (1) mono-
amino (the picramate); (2) the diamino; (5) the triamino (the colorless
derivative); and (b) the isopurpurate, a compound first reported by
hlasievetz (56a) in 1859. This last compound, hi6 dinitro - 2 - amino
5:5 dicyano - phenol was obtained by heating concentrated potassium.cyanide

at 60° with strong picric acid. Instead of being orange -red like the

-12-
picramate, it is red—purple. The formation of the purpurate required
excess potassium.cyanide, and was destroyed by excess alkali. The
coloration of alkaline picrate by hydrocyanic acid was not specific.
Any volatile reducing substance like aldehydes, acetone, hydrogen sulfide,
and furfural would do the same. At least one plant species, Mhndina
domestica yielded hydrochloric acid and acetone, as well as hydrocyanic
acid upon distillation. Chapman further studied the most suitable temp
perature for color formation. At ordinary temperatures the reaction was
slow, many hours being necessary for maximum development. At 70° the
results appeared to be irregular; and finally it was found that a temr
perature lying between h0-50° gave the best results, the full coloration
developing in about ten to fifteen mdnutes. Strict color proportionality,
he declared further, occurred only in solutions containing 2-8ng. hydro-
cyanic acid when caused to react with 20 ml. of saturated picric acid
containing 10% sodium hydroxide. Under the same experimental conditions
0.02 mg. of hydrocyanic acid could be detected with certainity, and
possibly less night be detected by varying somewhat the strength or
volumes of the solutions used.

In detecting gaseous hydrocyanic acid, hennig (57) used test papers
moistened with fresh mixtures of equal parts of one solution containing
2.86 g. of cupric acetate in a liter of water, and of another solution
containing L75 ml. of a saturated (at room temperature) aqueous solution
of benzidine acetate plus 525 ml. of water. He found that if no color
deve10ped in seven seconds, there was no danger of hydrocyanic acid
poisoning. Using the same reagent Sieverts and Rehn (38) determined that
small errors in weighing, mixing, and the presence of acetic acid were un-
important. Any white filter paper was satisfactory. Other tests conditions,
such as temperature, had little influence. It was important that the color

shade be ascertained immediately after the test.

-13-

Cyanogenesis by Bacteria

 

At this point it seems pertinent to consider the report of Clawson
and Young (59) concerning the production of hydrocyanic acid by bacteria.
Chemical and bacteriological studies showed that B. Byocyaneus, B.f1uor—

escence, and B. violaceous produced hydrocyanic acid from protein material.

 

An extracellular enzyme did not set free hydrocyanic acid nor was it done
in the absence of oxygen. Certain liquefying bacteria seemed to be able
to produce hydrocyanic acid. WOrk done on the occurrence of hydrocyanic
acid in protein containing substances like grains, beans, linseed meal,
and sorghum.would have been vitiated by the activity of some hydrocyanic

acid producing micro-organism.instead of an enzyme in the plant.

Preservation of Cyanogenetic Plants
With regard to the preservation of cyanogenetic plants for analysis
Askew (7) stated that cool storage of samples up to six days did not
decrease the yield. Briese and Couch (1h) have investigated the problem
rather completely. In brief they found that:
1. Fresh plants stored at ordinary temperature lost 15-85%
of hydrocyanic acid in 1-6 days.
2. Maceration of refrigerated plants caused great loss.
5. Acid solutions caused rapid loss although salicylic acid
offered possibilities.
h. Alkaline solutions caused 52-95% loss; organic bases caused
less loss than inorganic ones.
5. Alcohol was not reliable.
6. Mercuric chloride when used in 1%rconcentrations based on the

weight of fresh plant gave excellent results.

-m-

EXPERIMENTAL
As was previously stated in the introduction of this thesis, the
experimental portion is divided into two distinct parts: (1) studies
in the field on the variation of hydrocyanic acid content of Sudan
grass; and (2) studies on some factors influencing the determination of

hydrocyanic acid in plant tissue.

STUDIES IN THE FIELD

l. flethod of Determination

 

In order to obtain comparable results, walker's (1) modification of
the acid titration method (b6) was used throughout. The sample of grass
was cut in approximately half-inch lengths and thoroughly mixed. Tri-
plicate samples of thirty grams each were quickly weighed and ground in
an iron mortar for a few minutes. About 50 to 100 ml. of distilled water
'were added and the grinding continued for about one minute more to complete
the extraction. The remainder of about 500 ml. of water was now used to
transfer the pulp to a liter round bottom.flask by means of a 12 inch
funnel and a 9 mm. glass rod. After swirling the contents around and
taking care to have no blades of grass above the water surface, the flask
was immediately attached to the condenser and set in a tin can mounted on
two movable blocks, as illustrated in the accompanying photograph, (Fig.1).

Usually to avoid loss, the condenser adapter has been previously sub-
merged below the collecting solution in a hoe ml. beaker. The collecting
solution was composed of 10 ml. of 0.02 N silver nitrate and 10 ml. of
6 N nitric acid. These beakers were covered with a cloth during the two
hour maceration period to prevent photochemical action.

The source of steam.for distillation came from.two liter balloon flasks
each heated by Meker burners. Each of these steam.sources will supply
enough steam.for three samples simultaneously. All rubber stoppers and

rubber tube connections were freed of sulfur and other impurities by treat-

-15-

 

MULTIPLE STEAM DISTILLATION APPARATUS

1.

FIG.

-16-

ing with hot dilute alkali and rinsing with dilute acid and water.

About 150-180 ml. of solution were distilled into the acidulated
silver nitrate. The insoluble cyanide was filtered off, and the fil-
trate was collected in a 500 ml. Erlenmeyer flask. Next, there were
added five cc. of indicator composed of 200 ml. of 6 N nitric acid and
100 g. of ferric alum.per liter. The excess silver was titrated with a
0.02 N solution of potassium.thiocyanate to the first permanent pink.
This end point could be reproduced best by using a reference flask.

One ml. of potassium thiocyamte is equivalent to one ml. of silver
nitrate, which in turn is equivalent to 0.5h.mg. of hydrocyanic acid.

This method will henceforth be referred to as the acid titration

method.

2. Seasonal Variation

 

walker (1) studied the effect of stage of growth in the summer of
1955. There is now reported a continuation of this study for the summers
of 1958 and 1959. Material for analysis was taken from.the southwest
portion of the first dairy pasture field immediately east of Farm.Lane and
just south of the Red Cedar river. A ccmplete series of analyses was made
fronithe time the grass was about six inches tall until the time it began
to head. All samples were taken.at 10 a.m. The plants were taken from
a wide area over the field, cut close to the ground and conveyed immediately
to the laboratory. After the plants were cut into half-inch lengths and
thoroughly mixed, samples were weighed out for analysis and for dry weight.
The samples fer dry weight were weighed into covered aluminum.cups of 250 ml.
capacity and dried to constant weight at 100° in an electric oven. The
height of the material was obtained from.the average of 20 representative

plants. The number of milligrams of hydrocyanic acid per 100 plants was

-17-

calculated from.the average weight per plant and the total number of
milligrams in the sample used. It should be mentioned here that prior
to the sewing of the seed in 1959, the field was fertilized with 500 _
pounds of ammonium.sulfate per acre.

The data obtained for 1958 are presented in Table I and Fig. 2,

and for 1959, in Table II and Fig. 5.

5. Diurnal Variations

 

In general, an attempt was made to choose a clear, sunny day to
study the diurnal variations of hydrocyanic acid content in Sudan grass.
The plot was the same as mentioned above. Samples were analyzed at two
hour intervals between 8 a.m. and h p.m. The acid titration method of
analysis, already described under Method of Determination, was used.

Data obtained are presented in Table III and Fig. h.

h. Leaf-Stem Variation

 

An attempt was made to study the relative concentration of hydrocyanic
acid in the leaf as compared to the stem. Unfortunately, this study was
made only during a limited period; the grass averaged about three feet in
height. In the laboratory the stem portion of the plant was carefully
separated from.the leaf, and each'was cut into half-inch lengths. Dry
weight determinations were made on each portion, and the percentage of
hydrocyanic acid based on dry weight was made on each respectively. The

data obtained are presented in Table 1V.

5. Second Growth

 

By the end of July the grass averaged almost four feet in height; it
was quite mottled, partially desiccated, and almost completely headed.
A portion of the field was mowed down close to the ground and the plot

was raked over to remove all lirst growth. In about a week, green second

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-25..

growth, less than a foot in length and mostly leaf in structure, was
carefully cut from.the first growmh stumps and analyzed. The data

obtained are reported in Table V.

SOME FACTORS INFLUE'L‘ICING THE DETERMINATION OF HYL‘ROCYANIC ACID

1. Effect of Chloroform.

 

In this study the acid titration method was modified in several stages.
Since it was believed that loss of hydrocyanic acid could occur during the
grinding and macerating of the sample, this step was omitted. The grass
sample was cut into one-half inch lengths, thoroughly mixed, weighed,. and
immediately washed into the round bottom flask with 500 ml. of water. The
chloroform was added, and the whole mixture was shaken in a rotatory manner
after the flask had been attached to the condenser. Steam was used to dis-
till the gas into acid silver nitrate as usual. All determinations were
made in triplicate.

(a). Destruction of Chloroform Durinngistillation

 

The effect of steam distillation upon chloroform was deter-
mined by setting up six samples. To round bottom flasks there were added
300 mu. of‘water and 20 ml. of chloroformyand attached to condensers. By
means of steam 180 m1. of solution was distilled into the customary acid
silver nitrate absorption beaker. Back titration with the 0.02 N potassium.
thiocyanate showed that an average of 0.35 ml. of 0.02 N silver nitrate
(equivalent to 0.189 mg. of hydrocyanic acid) was lost from solution. This
error, or a proportional amount depending upon the volume of chloroform used,
was accordingly corrected for in all calculations.

(b). Effect of Chloroform in Immediate Distillation

 

In this study variable weights of sample and.variable volumes

of chloroform.were employed. In all cases the chloroform‘was added last.

-26-

As soon as the flasks were attached to their respective condensers, the
mixtures were shaken vigorously, and steam was led in immediately. Care
had to be exercised to control the steam pressure in order to prevent
sudden volatilization of the chloroform and attendant back suction in
the condensers. Most of the chloroform.came over in the first few
minutes. Table VI shows data obtained from this series of experiments.

(0). Effect of Chloroform.when Instillation was Delayed

 

All sampleS'were prepared in the customary manner,
attached to the apparatus as illustrated in the photograph, and allowed
to stand. It should be noted that if any hydrocyanic acid was liberated
during this period, it was absorbed by the silver nitrate solution.

Results of a few trials are tabulated in Table VII.

2. Effect of Buffer System

 

Stock buffer solutions were prepared according to Clark (hl) and Clark
and Lube (h2) using potassium.dihydrogen phthalate-sodium hydroxide and
potassium.di-hydrogen phosphate -sodium.hydroxide mixtures, covering a pH
range from.h.to 8. The fresh grass sample was cut into half-inch lengths,
weighed (30 or to grams), and washed into the distilling flask with 250 ml.
of water plus 50 ml. of the above buffer solution. The apparatus was set
up completely and a two hour period for maceration was observed. Some
results are recorded in Table VIII. All determinations were made in tri-

plicate.

5. Picramate, or Colorimetric Method

 

Materials and reagents.
(1). Cenco-Sheard-Sanford photelometer.
(2). Alkaline picrate solution: 5 g. of picric acid and
50 g. of sodium carbonate were dissolved in 1000 ml.

of distilled water, heated nearly to boiling to bring

 

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-28-

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-29-

about solution, and filtered.

(3). 2% potassium hydroxide: 2 g. of potassium hydroxide
were dissolved in 100 ml. of water.

(A). Copper sulfate solution: Benedict's quantitative sugar

reagent was used.

The "center" cell was filled with alkaline picrate solution, and
the "zero" point (100 on the micro-ammeter scale reading) was set against
it. A cell filled with Benedict's reagent was used as the light filter.
It is generally agreed that accurate photelometry requires that:
(l). The colored solution possess at least one absorption
band or zone.
(2). A spectral filter must be available which transmits
light only in the region of one absorption zone of
the material under test.
(3). A method must be available to obtain a suitable standard.
Part of the following studies were necessary to fulfil these conditions.
Determinations with the photelometer were made as follows: 10 ml. of
alkaline cyanide solution.were added to lOInI. of the alkaline picrate solu-
tion in a test tube. The tube was placed in a water bath at h0-60° for
10-15 minutes. After cooling to room temperature, the picramate solution
was poured into a clean cell, and its light transmission determined. The
scale reading was compared to the standard curve, and the total hydrocyanic
acid calculated therefrom.

(a). Absorption Studies

 

In order to determine the best type of light filter,

absorption curves by means of a spectrophotometer were made of the alkaline

picrate solution, the picramate solution, and of Benedict's reagent. Data

-50-

calculated from these observations are listed in Table IX, and illustrated
by curves combined in Fig. 5.

(b). Calibration curve

 

The standard reference curve shown in Fig. 6 was obtained
by averaging the results of three sets of readings. The strength of the
standard hydrocyanic acid solution was determined by titration against
0.02 N silver nitrate according to the regular method.

(o). Stability of the Colored Picramate Solution

In order to learn whether it was necessary to make a reading
of the picramate solution immediately, or whether it could be delayed, and
to what extent of time, a study of the stability of the colored solution
was made both at room.temperature, and in the ice box, ca. 5°. In the
case of the latter, the test solutionS'were always allowed to attain room
temperature before being compared in the photelometer. Table X indicates

the results of this experiment.

A. Preservation of Sample

In any analysis of fresh.material one soon becomes confronted with the
problem.of the stability of the sample from the time it is taken from the
field to the moment when the analyst is ready to work upon it. Some comment
on this phase was made in the review of literature.

It seemed that Briese and Couch (1h) had found a suitable preservative
in mercuric chloride after making rather complete studies of various organic
and inorganic acids and bases, alcohols, aldehydes, and saccharides. They
found that a one per cent concentration of mercuric chloride based on fresh
tissue gave Optimum.results. All samples were stored in glass jars at room
temperature. The mercuric cyanide complex was decomposed by stannous chloride
just prior to distillation. To test the efficacy of this method in our labora-

tory, a number of trials were attempted. The results are reported in Table XI.

-31-

TABLE

II

Absorption Curves of Alkaline Picrate,

Picramate, and Benedict's Quantitative Reagent

 

 

 

 

 

 

 

 

 

‘Wave Alkgléggogéprate 0.538733%;6 enedietii.g;antitative
Length 4 Log IQ/I Log 14.11 fif‘ g Ig/I T
700 - — 0.15 15,000 - -
680 - - 0.15 15,000 - —
670 0.17 5&0 - - - -
660 - - 0.20 20,000 - -
éuo 0.0h 80 0.20 20,000 - -
620 0.05 100 0.16 16,000 - -
600 0.02 be 0.05 5,000 1.00 0.555
580 0.00 o 0.00 0 0.78 0.h55
560 0.05 60 0.05 5,000 0.50 0.278
5&0 0.02 be 0.00 o 0.18 0.100
520 0.07 1L0 0.00 0 0.16 0.089
500 0.28 560 0.0h u,000 0.09 0.050
h90 0.80 1600 0.10 10,000 - -
ABS - - - -
heo - - 0.16 16,000 0.11 0.061
L70 - - 0.55 55,000 0.10 0.050
L65 - - - e 0.15 0.072
héo - - 0.95 95,000 0.01 0.005
h55 - - 1.50 150,000 - -
hSO - - 1.80 180,000 0.08 O-Ohh.
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-55-
DISCUSSION

Method of Determination

 

walker (1) studied three methods of analysis: the alkali titration,
the Prussian blue, and the acid titration methods. For various reasons
he eliminated the first two and decided to use the third one. The present
author tried the colorimetric method of Francis and Connell (hS) but was
compelled to discard it because the conversion of cyanide to thiocyanate
was always attendant by interfering colloidal sulfur.

The acid titration method was modified by walker (described in the
first portion of the Experimental section), and subsequently used as the
procedure of reference with other methods, turned out to be rather un-
fortunate. Initial suspicions that losses of hydrocyanic acidvvere occurr-
ing during the process of grinding and maceration were confirmed. In
practically all cases where this stage was omitted and the use of chloro-
form, or buffer systems was introduced, the percentage of hydrocyanic
acid was greater than that detennined by the regular method. However,
the greatest disadvantage of the use of the acid titration was the imp
possibility of correctly interpreting the wide variation that occurred
in the percentage recovery of hydrocyanic acid by the modified methods
when compared with the usual acid titration method. Are the discrepancies
to be attributed entirely to one method, or to both? In either case the

consistency of any of the modified procedures cannot be verified.

Seasonal Variation

 

The maximal content of hydrocyanic acid for each of the two summers
of 1938 and 1939 diffeHSgreatly. This fact may be due to fertilization

in 1939 in the form of 500 pounds per acre of ammonium.sulfate. Meteoro-

logical conditions were variable, too. For June normal sunshine in this

-37-

region is 70% and the rainfall is 3.51 inches. In 1938 the sunshine was
69% and the rainfall was 2.89 inches; in 1939 the sunshine was 66% and
the rainfall was 3.77 inches. The seed was sown on June 2nd, 1938 and on
June 9th, 1939. When the prussic acid content is calculated on the basis
of dry tissue, Table I and II with accompanying graphs show that the con-
tent increases somewhat at first but slowly and continuously falls udth
maturity. This observation is consistent with Walker's (1) findings in
1935. Yet, when the data are calculated on the basis of content per 100
plants the picture is slightly different. walker found that after about
hO-b5 days of growth that the content fell off abruptly. Present data show
that the content still remains high even after 57 days of growth. Possibly,
the size of the plant at maturity is increaseing considerably not in propor-
tion to the decrease in hydrocyanic acid content.

According to Boyd et a1. (12) Sudan grass is dangerous to pasture if
the content of hydrocyanic acid exceeds 75 mg. per cent. If this value
is true, our field was dangerous in 1939 only for a feW'days, at a time

when the height of grass was still too short for pasturization.

Diurnal Variation

 

The data from Table III are rather inconsistent, but in general it
seems that the cyanide content rises slightly in the early forenoon,
especially in young growing plants. After falling off during the day, the
content usually rises again the later part of the afternoon. The degree of
variation is ordinarlly not sufficiently marked to warrant any definite

conclusions.

Leaf-Stem.Variation

 

Dry weight determinations for leaf and stem.were made on leaf and

stem.samples respectively, and not on the total plant. For this reason

-53-
it is impossible to declare correctly which of the plant portions contains
the greater amount of cyanide unless the basis of determination is given.
Paradoxically enough, data from Table IV indicate that the cyanide content
of the leaf exceeds that of the stem on a fresh weight basis, but the re-

verse is true on a dry weight basis.

Second Growth

 

Table V shows that Sudan grass growing from.first—growth stumps regains
considerable cyanide. walker (1) found that if the cyanide content were
compared to the first growth with respect to size rather than age that a
similarity existed. Present data appear to confirm this finding. It
should be mentioned that with second-growth grass, uniform.samples are
difficult to prepare because of the presence of some first-growth dry

matter, and the variability of the length of the blades.

Effect of Chloroform.in.Immediate Distillation

 

It is commonly known that anesthetics increase the permeability of
cellular membranes. “Rather they exert a synthetic, or analytic action is
not always clear, but Willaman (15) believes that both activities are
possible. Table VI presents data concerning an attempt to substitute
chloroform for the grinding and macerating of the sample. In this way the
danger of the loss of cyanide could be averted. That the chloroform does
liberate cyanide is evident, yet, in spite of many trials, the percentage
recovered never quite equalled that obtained by the regular method. Sullivan
(11) in a publication that appeared after our experimental work was completed,
found that the addition of water was not necessary, and that its omission
hastened the steam.distillation. This observation may partly explain our
low results. A slight error due to the conversion of, or to the chloroform

itself was corrected for in all calculations as explained in the Experimental

-59..

section. The data show, moreover, that the weight of sample or amount of
chloroform are not exceedingly important. Care must be taken, on the
other hand, that the chloroform.does come in contact with the entire sample.
For instance, the first two items in Table VI show a very wide variation

in percentage recovery; yet, the recovery of the second item.compares well
with a number of subsequent analyses where three to four times the amount
of chloroform was used.

It must also be remembered that the wide variations in the percentage
recovery may not be due entirely to the chloroform.modification, but rather
to irregularities in the acid titration method. As mentioned previously,
this fact must be kept in.mind in all cases where the regular method is used

as a reference standard.

Effect of Chloroform.in Delayed Distillation

 

Chloroform used in this manner gave consistently higher values of
cyanide content than the regular method. In the two exceptional cases,
the cyanide content is so low that the experimental error may account for
the inconsistency. In spite of the huge amount of water present, the time
delayed may have permitted the chloroform to reach the entire sample, and

thus permit maximal liberation of prussic acid.

Effect of Buffer Systems

 

The effect of a buffer system.in the vicinity of neutrality appears to
be very favorable. Results in Table VIII were consistently higher than those
of the regular method. When the pH of the buffered solution.was lowered, the
results were affected directly. It is also interesting to note that regard-
less of the strength of the buffer system, the Sudan samples consistently
exerted a strong buffering action of their own. No correction was made for

possible salt effects upon the hydrogen ion concentration.

-ho-
The use of buffer systems appears to be highly encouraging and should

be investigated.iurther.

Picramate Method

 

Difficulty in detecting the thiocynate endpoint in.the regular method
directed efforts towards finding an improvement. The familiar Guignard
reaction.was adapted by Boyd et al. (12) to a colorimetric method using
standard solutions in.test tube for reference. This method of comparison
is subject to obvious error. To remedy this defect, the photelometer was
employed.

Absorption Studies

 

In order to obtain a proper filter for the instrument, a study of the
absorption curve of the picramate solution was made by means of a visual
spectrophotometer. The picramate salt solution showed a definite absorption
in the region of too millimicrons which increased decidedly towards the
ultraviolet. After some trials, it was found that Benedicts' quantitative
c0pper sulfate reagent possessed rather good transmission in this region.
The extinction coefficients for these solutions were plotted against wave
length as illustrated in Fig. 5. For the sake of comparison the absorption
of the alkaline picrate was also plotted. These curves are not directly
comparable because the percentage concentration is not the same. The ex-

tinction coefficient, E, was calculated according to the following formula:

1
E=-5E I log Io/I

where Io = intensity of incident light

I = intensity of transmitted light
d - thickness of absorbing solution in cm,
c = percentage concentration of the product

.141-

Cells having a width of 10 cm. were used for the picrate and
picramate solutions, and a cell of one cm. for he Benedict's solution.
The percentage concentration of the product is given in the data of

Table IX. The ratio, log Io/I, is read directly on the spectrophotometer.

Calibration Curve

 

The standard calibration curve shows that the picramate solution
observes Beer's laW'only within certain limits. Cyanide solutions should
not be used which contain more than 2.0 milligrams of hydrocyanic acid
per 100 ml. unless they are previously diluted. Likezise, cyanide solu-
tions containing less than 0.03 milligrams of hydrocyanic acid per 100 ml.
cannot be accurately determined colorimetrically. It is also apparent
from.the graph that there is a greater sensitivity with smaller concentra-
tions of cyanide. Chapman's (36) excellent study of the picramate reaction

has been briefly presented in the Historical section.

Color Stability

 

lemperature is a factor in determining the stability of the picramate
solution. According to data in Table X this solution may be kept at room
temperature without appreciable change for at least two days. If the
solutions are kept in the refrigerator, the period of keeping may be
extended to possibly a week. Therefore, for reasons already given, an
aliquot of cyanide solution should be heated as soon as possible with

the alkaline picrate, but the reading of the picramate in the photelo-

meter may be delayed for a time depending upon the method of storing.

Preservation of’Sample

 

The results of using one per cent mercuric chloride based on fresh
tissue for preservation are given in Table XI. The number of variates

is far from sufficient to warrant any generalization; yet from.1imited

-hg-

experience it may be suggested that the low recovery in the first trials
may be due to adding insufficient stannous chloride to<iestroy the mer-
curic cyanide complex completely. In agreement with Briese and Couch
(1h) we found that the mercuric salt did not stop cyanogensis. The
effect of the mercury upon the rate of cyanogensis was not studied.

The last two items in the Table indicate that the mercuric cyanide
complex is relatively stable.

The value of this particular study could be improved by using mercuric
chloride and the usual modifications in the determination of the original
cyanide content. In this way variations due to method can be eliminated,
and comparable results obtained.

In contrast to the results obtained with buffered solutions, the pH
of samples treated with mercury was about h.2 prior to distillation and

about 2.1 after distillation.

Genera1_Considerations

 

It may be assumed that no method is worthwhile unless it takes into
consideration first, the peculiar nature of hydrocyanic acid as an organic
compound; and secondly, the possible forms in which this acid may occur in
plant tissue. Concerning the first consideration, prussic acid is a tau-
tomeric substance which occurs in.two forms (Leemann (9)).

HCN and HNC

When the hydrogen is replaced in the first isomer, the nitriles are
obtained; and when the hydrogen is replaced in the second isomer, the iso-
nitriles. Aqueous solutions of prussic acid are unstable and either isomer
could be converted to ammonium.formate. This similarity of action.would be
indicated by an electron study of hydrocyanic acid. The pure acid is rapidly
decomposed by concentrated hydrochloric acid with the production of formic

acid and ammonium chloride. With concentrated sulfuric acid, carbon monoxide

4,3,-

is formed. (Partington (A3). Likewise, all salts of prussic acid
undergo decomposition when boiled in aqueous solution. At 22°, Sinozaki
and coworkers (Ah) found that hydrogen cyanide has a vapor pressure of
65h4h.mm. This high vapor pressure explains the great difficulty involved
in maintaining a solution of cyanide. Most analytical workers in this
field have not considered means to stabilize hydrocyanic acid during the
process of analysis. Failure to appreciate the lability of this compound
undoubtedly accounts for much of the irregular experimental data.
As to the manner in which hydrocyanic acid occurs in the plant, Acharya
(AC) thinks that there are at least three forms:
(1). "Tree prussic acid" formed by enzymic hydrolysis
of "labile" prussic acid, destroyed by 10 per
cent sulfuric acid and steaming.
(2). "Labile prussic acid" liberated by simple steaming,
and destroyed by 10 per cent sulfuric acid.
(3). "Bound prussic acid", liberated by enzyme action,
destroyed by heating and 10 per'cent sulfuric acid.
Willaman (15) says that prussic acid exists in the glycoside and non-
glycosidic form. The glycosides isolated from grasses are amygdalin and
dhurrin. There is a close resemblance between the two. However, in
dhurrin the glucose is coupled with the nitrile of p-hydroxy benzaldehyde,
while in amygdalin a disaccharide, gentiobiose, is coupled with the nitrile
of benzaldehyde. Emulsin and acids will hydrolyze both glycosides with the
liberation of prussic acid. hfith alkalis, dhurrin is converted to dhurrinic
acid and ammonia. All these factors must be considered in any quantitative

work.

l.

10.

ll.

-hh,

CONCLUSIONS
The acid titration.nethod still leaves much to be desired in the
way of accuracy.
The cyanide content of'conmercial Sudan grass rises slightly at
first and then gradually decreases with.maturity.
In 1938 the Sudan grass grown at Michigan State College did not
contain enough cyanide to be dangerous at any time. In 1939 the
cyanide content was definitely dangerous for a very short time.
The total amount of cyanide per plant appears to increase with
vacillations until sometime after heading has begun.
The hydrocyanic acid content varies during the day, being usually
highest in the forenoon.and late afternoon, and lowest in the
middle of the day.
The cyanide content of Sudan grass for second growth compares with
that of the original growth on the basis of size.
The leaf content of prussic acid exceeds that of the stem.on the
basis of fresh weight respectively. The converse is true on the
basis of dry weight respectively.
Chloroform is partly destroyed or converted during steam distillation
so that it interferes in the silver nitrate absorption solution.
Immediate distillation with chloroform.does not necessarily result in
the complete liberation of prussic acid, especially in the presence of
much water.
Delayed distillation with chloroform usually yields maximum values,
even in the presence of much water. A 10-15 minute delay is sufficient
for a 30 g. sample in the presence of 300 ml. of water.
Tie use of buffers in the vicinity of neutrality appears to give maxi-

mal yields of hydrocyanic acid.

l2.

13.

15.

16.
17c

18.

19.

-h5-

Buffer systems of low pH cause much destruction of cyanide.
Picramate solution shows maximum.absorption in the region of h60
millimicrons and increases decidedly towards the ultraviolet.
Benedict's quantitative copper sulfate reagent shows maximum.
transmission at about h58 millimicrons.

Aliquots containing more than 2.0 mg. of hydrocyanic acid per

100 ml. should not be determined by the Guignard alkaline picrate
method; neither should aliquots containing less than 0.05 mg. per
100 ml. be used.

Greater sensitivity is found with lower concentrations of cyanide.
Picramate is developed more regularly at h0-60 degrees for 15
minutes than at the temperature of boiling water for 5 minutes.
The color of a picramate solution is stable for at least two days
at room temperature, and for almost a week at 5’.

The use of mercuric chloride, one per cent based on fresh tissue

was confirmed as a preservative of cyanide in plant material.

4,6-
LITERATURE CITED
1. walker, L. C. The hydrocyanic acid content of Sudan grass.
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added hydrocyanic acid from.certain plant tissues, and its
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3. Finnemore, H., Joyce M. Cooper. Hydrocyanic content of Lotus Australis

 

and Heterodendron oleaefolium. Australian Vet. J., _1_h_, 153—7 (1958).

 

h. Viehoever, A., C. 0. Johns, and C. L. Alsberg. Cyanogenesis in plants.
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6. Bartholomew, E. T., E. C. Raby. Photoelectric turbidimeter and the
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l2.

l3.

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.../4'7 .-

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Boyd, F. T., 0. S. Aamodt, A. Bohstedt, E. Truog. Sudan grass
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Apsit, J., E. Gain. Seeds killed by anesthesia preserve their
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Briese, R. R., J. F. Couch. Preservation of cyanogentic plants
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Willaman, J. J. The effect of anesthetics and of frosting on the
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Krieble, V. K., and J. G. McNally. Hydrolysis of hydrocyanic acid
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Roe, J. H. The estimation of simple soluble cyanogen compounds,
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1875-9 (1923)-

Morris, 8., V. G. Lilly. Distillation of hydrocyanic acid from
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21.

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25.

28.

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31.

'MB-

Pagel, H. A., W} Carlson. The accurate determination of cyanide by
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Bartholomew, E. T., W} B. Sinclair, and B. E. Janes. Factors affecting
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Rigg, T., H. D. Askew, E. B. Kidson. Occurrence of cyanogentic
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Clark, R. E. Rapid detection of Ag X in the presence of AgCN. J. Chem.
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111 .

-h9-

Schieblich. J. Investigations of the breeding of Sudan grass
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113 .

115 -

-50-

Partington, J. R. A text book of inorganic chemistry. Macliillan
and Company, London, Fourth Edition (1933).

Sinozaki, Heima, R. Hara, S. liitsukuri. The vapor pressures of
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of Official Agricultural Chemists. Washington, D. C. (1935).

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CHENHSTRY DEPT.

114612.015 3194

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T612 .015

Some factors influencing the
determination of hydrocyanic
lant materials.

 

 

_ 31293 02446 7106 .

 

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