ment of this sort :
Time
above
Time
above
a.m.
horizon
p.m.
horizon
'. 17, 8.57
27°
Apr.
17, 12.44
9^
9.43
14
1.30
14
10.18
9
3.30
19
11.45
18
4.40
5.20
15
23
The leaf made 3 complete oscillations in 8 hrs. 23 m.
(261) Autonomous movements : Averrhoa.
If Averrhoa hilimbi is kept at a sufficiently high
temperature (e.g. 27° C), the movements of the leaflets
are easily seen. Each leaflet moves independently of the
rest, it falls suddenly from, e.g. 20' below the horizon to
40° below, and slowly rises in about 20 minutes to its
former position. If the temperature is increased to
31° — 32° C. the oscillations become more rapid and smaller
in amplitude, at the same time the mean position
of the leaflet falls to something like 50° below the
horizon instead of 30*^ as at flrst. These clianges are
232
DESMODIUM.
[CH. VIII
graphically i-epresented in the Potuer of Movement in
Plants, p. 334, fig. 135. The ordinates represent the
angle made with the vertical by the leaflet under obser-
vation ; a fall in the curve thus represents a drop of the
leaflet, 0° representing a vertically dependent position.
The dotted line represents temperature, and is to be read
in connection with the numbers 76° F....90° F. on the right
hand of the diagram.
(262) Autonomous movements: Desmodium gyrxms.
The leaf of Desmodium gy7^ans represented in fig. 44
consists of one large and a pair of very
minute leaflets. It is these which
execute the movements for which the
plant is celebrated. The point of each
leaflet describes a rough sort of circle
or ellipse, but the movement being
exceedingly jerky and irregular the
circular course is not obvious. The
movement does not occur unless the
temperature^ is about 22^ C, and may
be remarkably stimulated b}^ a higher
temperature, thus at 40^ C, the circles
are made at the rate of about two in
three minutes. The experiment may
be performed by immersing a leaf in
cool water which is gradually heated.
Fig. 44. Exp. 262.
From the Power of
Movement.
^ This is only true of full-grown plants. Seedlings move at lower
temperature : see Power of Movement in Plants, p. 362.
CH. VIIl] PERIODICITY. 233
(263) Periodicity : Bellis {light and darkness).
Three or four among a patch of daisies on a lawn are
to be darkened by covering them with an inverted flower-
pot : the hole in the pot must be plugged and a layer of
earth placed over the plug to make sure that no light
enters. For the same reason a ring of earth should be
placed round the junction of the rim of the pot with the
ground. If the daisy is kept darkened for two days it will
cease or almost cease to open or shut, the flower-heads
taking on a permanently half-shut condition. This shows
that the alternation of light and darkness is necessary for
continuance of " sleep " movement.
If, however, the plants are covered in the evening the
flowers will open the following morning, showing that a
certain inherent periodicity exists.
If the plants are covered in the morning the
periodic movement will probably begin to be irregular
by the next day. For instance the waking movement
will only occur late and the closure at night will also
be irregular.
(264) Periodicity : Bellis {temperatvre).
The flower-heads of the daisy are to some extent
influenced by changes of temperature, but they behave
very differently from the crocus or tulip. When they are
naturally closed in the evening a rise of 15' C. in tempera-
ture does not open them ; nor does a corresponding foil
close them in the morning. But, according to Pfeffer, if
they are warmed in the morning or cooled in the evening
234 CONTRAST. [CH. VIII
by about 15° an opening or closing (as the case may be)
is produced \
The experiment in which the temperature is raised is
the only one which we have confirmed. It is simply
necessary to gather 3 or 4 closed daisies in the morning,
place their stalks in water and put the bottle near the fire
in a warm room : similar flowers being placed for compari-
son in a cool place out of doors.
(265) Contrast: Bellis.
If the flower-heads of the daisy or dandelion are
kept shut throughout the day by exposing them to a
temperature of 2° to 3° C. they may, according to Pfeffer,
be made to open in the evening by bringing them into a
temperature of 17'— 20° C.-
A similar experiment may be made in another way.
Daisies kept in the dark for two days are brought into a
warm and lighted room in the evening together with
control specimens growing under natural conditions.
Both sets may be gathered and placed with the stalks in
water. In our experiments the temperature out-of-doors
was 11° C, in-doors 21° C, rising to 24° C. The daisies
from the dark opened wide in 20 minutes. The control
daisies showed no opening in 20 minutes, and had hardly
opened after 2 hours. The degree of opening was how-
ever simply noted by means of rough sketches.
1 Physiologische U liter suchung en, 1873, p. 195.
2 Ibid. p. 197.
PAET II.
CHEMISTRY OF METABOLISM.
CHAPTER IX.
INTRODUCTION. SOLVENTS. METHODS OF EXTRACTION.
GENERAL NOTES ON APPARATUS AND MANIPULATION.
Introduction.
The practical study of the transformations which
plastic substances undergo in metabolism is an application
of organic chemistry : the immediate problem is generally
to determine whether certain substances are present or
absent, and, if present, in what amounts, in particular
tissues.
For the qualitative testing of insoluble substances, such
as proteids, starch, etc. and of soluble substances which
give well-marked colour reactions, such as phloroglucin,
inulin, etc. microchemical methods are invaluable ; but for
quantitative work, and often for the satisfactory identifica-
tion of certain compounds, it is necessary to make extracts
with appropriate solvents and to submit these extracts to
a systematic examination.
It is the object of the physiologist to employ the
simplest methods which will give accurate results as
regards the compounds to which attention is being given,
238 INTRODUCTION. [CH. IX
but it is not often that extracts can be prepared which
will contain only those compounds to which the attention
is directed, consideration may therefore necessarily be
given to substances occurring in the extracts, although in
themselves comparatively unimportant.
The necessity for quantitative results in experiments
on metabolism is obvious, and even for qualitative work it
is sometimes necessary to employ rather complicated
chemical methods.
The arrangement followed in these sections is based
on practical convenience, those substances which are com-
monly extracted together being placed in the same section.
Each chapter contains a short general explanation of the
methods to be used, followed by instructions for performing
the qualitative and quantitative experiments selected.
Attention is chiefly directed to substances which are
either themselves 'plastic' or are believed to have im-
portant significance in metabolic processes — the study of
other compounds is only introduced in so far as these are
liable to interfere with the examination of the above.
It is seldom necessary to attempt a complete analysis
of all the constituents of a tissue, but it is essential to have
due regard to those which may interfere with the recogni-
tion or estimation of a particular compound (e.g. tannins
in the detection and estimation of sugars).
Before beginning the chemical examination of a vege-
table tissue, it is of the greatest assistance to consider
carefully what substances are likely to be present: a
knowledge of the general distribution of the commoner
plastic substances should suggest the method of pro-
CH. IX] INTRODUCTION. 239
cedure, but knowledge of this kind must not be relied
on to the extent of substituting it for (qualitative
examination.
For descriptions of the practical details of many of the
quantitative estimations, reference to standard works on
chemistry has been freely used. A full description is
given here only in cases where processes are required
which are not in general use, or where there is much
difference of opinion as to the best method of operating.
The principles on which the methods of estimation
are based are generally explained in each case ; where
references only are given, care should be taken to under-
stand exactly the reasons for all the steps described in
text-books ; otherwise those who have not received an
adequate chemical training are liable to use instructions
of this kind in a merely mechanical way, and thus to
lose all the value of the work as an introduction to the
practical study of this branch of physiology, — where the
success of an operator largely depends on his ability to
modify methods to meet particular cases.
Frequent references will be found in the text to the
following works.
Sutton. Volumetric Analysis. 5th edit. (Churchill,
1886.)
Fresenius. Qualitative Analysis. 10th English
edition. (Churchill, 1887.)
Fresenius. Quantitative Analysis. 7th (or (ith)
English edition. (Churchill, 1876, 7th.)
Beilstein. Handbuch der organiscJien Cheniie. 2nd
or 3rd edition.
240 PREPARATION OF [CH. IX
Among general works which may be consulted for
descriptions of the apparatus and manipulation used in
the experiments are :
Sachsse. Die Chemie und Physiologie der Farbstoffe,
Kohlenhydrate und Proteinsuhstanzen. (Leipzig, 1877.)
Frankland. Agrictdtural Chemical Analysis. (Mac-
millan and Co., 1889.)
Dragendorff. Plant Analysis. (Trans, by Greenish.)
(Bailliere, Tindall and Co., 1884.)
In references to original papers the ordinary abbrevia-
tions are used.
Preparation of the material to be examined.
For extraction with benzene, ether, etc., the material
must be dried as completely as possible, and as the residue
will generally be treated with boiling alcohol before
extraction with cold water, the original substance may be
dried at 100° C. in the steam-oven, till it ceases to lose
weight. Fixed oils and fats, glucosides, tannins, carbo-
hydrates including starch, will not be seriously altered by
drying at 100°.
On the other hand proteids and ferments would be
completely changed by drying at 100°, and in these cases
either the undried material is used or material which has
been dried at a temperature not exceeding 30° — this is
commonly spoken of as air-dried material.
Where comparative experiments only are being made it
is not a matter of much consequence whether the percent-
ages are calculated for material which is fresh, or has been
air-dried, or dried at 100°, but if determinations of several
CH. IX] MATERIAL. 241
constituents are made with differently treated portions
of the original substance, it is better to calculate all
results in p.c. of material dried at 100'', i.e. in p.c. of
dry weight.
The moisture (i.e. loss on drying at 100'') having been
determined, the calculation from one state to the other
involves very little trouble.
Before treatment with any solvent the substance
should always be as completely disintegrated as possible
— this is a point which requires very careful attention
and too much importance cannot be given to it.
Ordinary dry tissues, such as leaves, portions of her-
baceous stems, etc. can generally be reduced to a fine
powder without much trouble, but hard tissues are often
difficult to extract satisfactorily. Unless very tough —
which will seldom be the case — grinding in a small mill,
such as is used for grinding coffee berries, will generally
bring the substance into a suitable condition.
Fresh tissues are more troublesome to work with :
they can be treated in a large mortar with a small quan-
tity of the solvent, and thus rubbed up into a perfectly
homogeneous paste. The addition of some sand, or very
finely powdered glass, facilitates the process and is seldom
objectionable : indeed its use, in preventing the solid
material 'caking' during extraction, often more than
counterbalances the inconvenient increase in bulk which
it causes. If it is desirable to weigh the residue at the
end of the successive extractions an exactly weighed
quantity of sand or glass can be used and its weight
deducted from that of the total residue.
D. A. 16
242 EXTRACTS. [CH. IX
Preparation of extracts.
Non-nitrogenous plastic substances.
For non-nitrogenous plastic substances a given portion
of the original material should be treated with successive
solvents in the following order : (1) ether or benzene,
(2) alcohol of '85 specific gravity, (3) cold water,
(4) dilute acid.
In each case the extraction must be continued till a
fresh portion of the solvent fails to extract anything more.
10 grs. of material and about 250 c.c. of solvent will
generally be found convenient quantities.
I. Extract the dry substance with boiling ether,
benzene, or petroleum ether (with boiling point not
above 75° C).
Extract (No. I.) contains oils and fats, ethereal salts of
organic acids (esters), resins, terpenes, chlorophyll and
colouring matters, etc.
This extraction is best performed with Soxhlet's appa-
ratus for fat-extraction.
The material in the inner tube is weighed before and
after the experiment, dried at the same temperature in
each case.
When the extraction is complete, the inner tube may
be placed in a steam-oven to dry the residue before
weighing.
II. Extract the dry residue from I. with boiling
alcohol '85 specific gravity (about 55 p.c).
Extract (No. II.) contains tannins, glucosides, and
part of the sugars, etc.
CH. IX] EXTRACTS. 243
This extraction may also be performed with Soxhlet's
apparatus so that after the final weighing in I. it is only
necessary to place the inner tube in another apparatus.
When nothing more can be extracted by the solvent, the
inner tube is again dried in the steam oven and weighed.
III. Extract the dry residue from II. with cold
water.
Extract (No. III.) contains dextrins and soluble carbo-
hydrates not extracted by '85 alcohol.
The residue from II. is put into a stoppered bottle
with a portion of solvent and shaken for some time in a
continuous-agitation machine. The liquid is completely
decanted off and a fresh portion of solvent having been
added the process is repeated.
The residue is finally filtered off and thoroughly washed,
the washings being added to the extract. The residue need
not be dried but is ready at once for No. IV.
[A bottle fixed on to a vertical wheel which is rotated
by a band from a laboratory turbine answers very well for
this purpose. A rather slow rotation of about 12 — 15
revolutions per minute is the most effective. An appa-
ratus for this purpose made to work with Rabe's turbine
is sold by Gallenkamp and Co., London. A simple
machine for continuous agitation is also made by the
Scientific Instrument Company, Cambridge.]
IV. Extract the residue from III. with 1 p.c. sulphuric
acid at 100°.
Extract (No. IV.) contains the products of the action of
dilute acid on starch (dextrins and reducing sugars).
IG— 2
244 EXTRACTS. [CH. IX
The residue from III. is placed in a flask fitted with
a reflux condenser, acid added, and heated on a water-bath
for two hours, or until a drop of the solution ceases to give
any iodine reaction.
Instead of using dilute acid it is often better to use
solution of diastase (active malt-extract) to extract the
starch from the residue No. III. (see Chap. xiv.).
The temperature for extracting with diastase must not
exceed 60° C. and the extraction takes considerably longer —
it is best to allow the action to continue (with addition of
more malt-extract from time to time if necessary) till the
solution ceases to give any well-marked iodine reaction.
It will generally be found sufficient to use 100 c.c. of
an extract, made by completely exhausting 300 grs. of
malt with water and making up the product to 500 c.c.
for each 10 grs. of substance originally taken.
It is not generally required to examine further the
residue from IV., but if it is desired to estimate the cellu-
lose in it this may be treated in the manner described on
p. 293.
The original drjdng at 100° C. and treatment with boil-
ing ether or benzene and boiling '85 alcohol tend to alter
the proteids and render them insoluble in cold water and
dilute acids, so that it frequently happens that the
extracts III. and IV. are almost completely free from
proteids.
Preparation of extracts.
Nitrogenous plastic substances.
For nitrogenous plastic substances (proteids, amides,
CH. IX] FILTRATION. 245
etc.) another portion of the original material which has
been dried at a temperature not above 30° C. is extracted
(1) with cold water, (2) the residue from (1) with dilute
alkali, 1—2 p.c. soda (NaOH).
If much oil or fatty matter is present this should be
first removed by agitation with benzene in the cold ; the
residue washed with ether and dried below 30° can then
be treated with (1) water, (2) dilute alkali.
Extract 1. (Cold water) contains soluble proteids,
peptones and albumoses, amides, nitrates and nitrites,
ammonium compounds, etc. etc.
Extract 2. (Dilute alkali) contains proteids insoluble
in water but soluble in dilute alkali.
These extractions (and the agitation with cold benzene)
may be made with the apparatus used for the cold water
extracts of non-nitrogenous substances (see p. 243).
Filtration.
It is often very difficult to filter clear the extracts of
vegetable tissues ; the use of some of the special kinds of
paper made by Schleicher and Schull will frequently give
a clear filtrate where ordinary filter-paper Mis, but filtering
through asbestos is very effective in troublesome cases.
For filtering with asbestos it is necessary to use a
filter-pump, and the most convenient method of working
is to use a perforated porcelain filter plate with a hardened
paper (such as no. 575 in Schleicher and Schlill's cata-
logue), on to which the asbestos is poured whilst suction
is applied below, the asbestos having previously been
stirred into a cream with warm water ; after the water
246
EVAPORATION.
[CH. IX
has drained away the felt-like layer of asbestos can be
pressed with a pestle to any degree of tightness required.
The asbestos and paper can be washed and used again.
Evaporation of solutions.
Much time is always necessary for this tedious opera-
tion, but work can generally be so arranged that it can go
on whilst other experiments are in progress.
Evaporations should always be conducted on the water-
bath, but even so a considerable amount of 'charring'
usually occurs, and this discoloration of the solutions,
not being easily removable, renders the subsequent exami-
nation much more difficult.
By concentrating solutions under reduced pressure the
difficulty is largely avoided. By the use of a good water
filter-pump aqueous solutions can be concentrated fairly
rapidly at 50° — 60° C. and alcohol can be distilled off below
50° C.
Fig. 45.
A convenient form of apparatus for distilling off liquids
CH. IX] DECOMPOSITION. 247
under reduced pressure can be readily constructed with
distilling flasks and a Liebig's condenser. The diagram
Fig. 45 shows such an apparatus with the parts connected
ready for distilling. Care must be taken that all the con-
nections are air-tight.
Changes occurring in solutions on keeping.
It is necessary that the examination of the solutions
obtained by dissolving in water the residue from the
alcohol extract (II.), by extracting with cold water (III.),
and dilute acid (IV.), etc., should be examined as soon as
they are prepared.
Such solutions undergo change very rapidly from the
action of micro-organisms, and in a few days will often be
full of fungoid growths. If the solutions have to be put
aside before they can be examined, some strongly anti-
septic substance must be added to prevent such growth.
Chloroform is very convenient, as it can be readily
expelled by warming before the examination is made.
The addition of 1 c.c. of chloroform per liter of extract
will generally be found sufficient.
Small quantities of thymol may also be used for this
purpose — the small amount of thymol necessary will not
interfere with the subsequent examination, but its use is
not applicable where any part of the solution is to be
used for fermentation (see sugars).
If the alcoholic or cold-water extract is found to be
acid in reaction when first prepared it should at once be
exactly neutralised, because heating with acids may cause
changes in some of the constituents (e.g. cane-sugar may
248 ACIDITY. [CH. IX
be inverted by citric acid). Dilute soda is most convenient
for this purpose.
When lead acetate, normal or basic, mercuric chloride,
etc., are used in excess for precipitating, and the metal
is removed from the filtrate by HgS, free acid (acetic,
hydrochloric, etc.) is produced in the solution. This should
be neutralised as soon as the HoS has been expelled by
warming [acetic and hydrochloric acids are not removed
by heating dilute solutions].
CHAPTER X.
PROTEIDS, AMIDES, AMMONIA, NITRATES, ETC.
Full particulars of the chemistry of vegetable proteids,
amides, etc., may be obtained from the following works.
EiTTHAUSEN. Die Eiweisskorper der Getreidearten.
Bonn, 1872.
ScHWARZ. Die morphologische und chemische Zusam-
mensetziuig des Protoplasmas. Breslau, 1887. (Cohn's
Beitrdge, Vol. v.)
Read papers by
Palladin. Ber. d. d. hot Ges. Vols. vi. and vii.
(1888—1889.)
E. ScHULZE. Landw. Vers.-Stat Vol. xxxvi. (1889)
and Landiu. Jahrb. xxi. (1892.)
Vines. Journal of Physiology. Vol. in. (1881.)
Proc. Roy. Soc. No. 191. (1878.)
Green. Phil Trans. Vol. 178. (1887.)
Serno. (Distribution of Nitrates.) Landw. Jalrrb.
XVIIL (1889.)
Osborne. Amer. Chem. J. xiv. (1892.)
250 CLASSIFICATION [CH. X
For convenience of practical study we may consider
nitrogenous plastic substances as divisible into
(1) Proteids insoluble in water hut soluble in dilute
1 — 2 p.c. alkali, [e.g. the gluten of cereals.]
(2) Proteids soluble in water.
Substances which give all the characteristic proteid
reactions and are precipitated by potassium ferrocyanide
and acetic acid, trichloracetic acid, cupric acetate, and
normal lead acetate, etc., etc.
They may or may not be precipitated by boiling after
addition of acetic acid, or by adding excess of 90 p. c.
alcohol, [e.g. the ' soluble proteids ' of cereals and legu-
minous seeds.]
(3) Peptones and albumoses.
Soluble in water but not precipitated by any of above
reagents : give a characteristic reddish tint with the biuret
reaction : are completely precipitated from neutral solu-
tions by alcoholic mercuric chloride, and from slightly
acid solutions by sodium phosphotungstate ; are slightly
diffusible through membranes, [e.g. vegetable peptones
and albumoses of leguminous seeds.]
(4) Amides.
Amido -derivatives of organic acids, [e.g. asparagin,
glutamin, betain, etc.]
(5) Ammonia, nitrates and nitrites.
The examination of these constituents is made on
special extracts of original material obtained as described
CH. X] OF PROTEIDS. 251
on p. 244 (which are referred to below as the alkali solution
and the water solution) and not on the extracts used for
non-nitrogenous substances.
QUALITATIVE EXAMINATION.
Proteids insoluble in water^ soluble in dilute
alkali.
A portion of the alkali solution is carefully neutralized
with dilute acid, a precipitate (soluble in excess of acid) is
formed if proteids are present. This precipitate should be
filtered, washed, and portions of it tested by the ordinary
reactions for proteids (xantho-proteic, Millon's, biuret, etc.)
Proteids soluble in water.
Portions of the solution should be tested by boiling
after addition of a drop of acetic acid and by the addition
of 90 p.c. alcohol — if precipitates are produced they
should be filtered off, washed and tested for proteid
reactions.
Whether precipitates are caused or not by the above
add to fresh portions of the solution : —
(1) Potassium ferrocyanide and a drop of acetic acid,
(2) Aqueous solution of trichloracetic acid.
Both these reagents give precipitates with proteids
and they will frequently cause precipitates when the
solution does not change on boiling or on addition of
alcohol.
If proteids are present add cupric acetate (aqueous
solution) as long as it causes a precipitate, and filter.
252 AMIDES. [CH. X
Peptones and Albumoses.