Remove excess of copper from the filtrate by HgS â€”
warm to expel excess of H3S and concentrate the liquid if
Test portions of this solution for peptones and albu-
moses by : â€”
(1) Biuret test [an equal volume of strong soda
(NaOH) solution and adding one or two drops only of
dilute copper sulphate solution].
Peptones and albumoses give a characteristic colour of
a redder tint than biuret.
(2) Sodium phosphotungstate and dilute sulphuric
Peptones and albumoses give a white precipitate.
(3) Saturated alcoholic solution of mercuric chloride.
Peptones and albumoses give a white precipitate in-
soluble in water when once thrown down.
If peptones and albumoses are present, remove by
adding alcoholic mercuric chloride as long as it causes a
precipitate â€” filter â€” evaporate off alcohol from filtrate and
remove excess of mercury from solution by H^S. After
warming to remove H2S, exactly neutralize solution with
dilute soda and test portions for amides by : â€”
(1) Addition of freshly precipitated and well washed
Amides form a deep blue liquid with solution of
the hydroxide â€” if this liquid is carefully evaporated and
CH. X] NITRATES. 253
allowed to stand (preferably in vacuo over sulphuric acid),
characteristic crystals of copper oxide compound of amide
may be obtained.
(2) A well-cooled mixture of potassium nitrite and
dilute sulphuric acid.
Amides evolve nitrogen.
(3) Boiling for some time with dilute acid.
Amides give ammonia in solution which can be tested
for in the usual ways, best by heating with excess of mag-
nesia and testing for evolution of the gas.
Ammonia^ Nitrates^ Nitrites, may be tested for
by ordinary methods in separate portions of the aqueous
Ammonia, see Dragendorff, Â§ 97, p. 81.
â– vr-i. -x ( ' ^^Â® Fresenius, Qualitative Analysis, 10th ed.
pp. 228, 230.
The most useful reagents for these substances are
metaphenylene-diamine and diphenylamine. Brucine in
strong sulphuric acid, and starch solution with zinc iodide
and acetic acid are also useful.
If it is required to examine further the nature of the
proteids etc., this may be done by decomposing the
precipitates caused by addition of metallic salts. Precipi-
tates obtained from addition of copper, lead, and mercury
salts may be decomposed by HoS, those from sodium
phosphotungstate by excess of baryta (Ba(OH)o) and sub-
sequent removal of excess of Ba(0H)2 in filtrate by current
254 ESTIMATION [CH. X
Amides can be recrystallised from dilute (50 p. c.)
Estimation of Proteids.
The methods based on weighing the precipitated pro-
teids are seldom satisfactory, as the precipitates are gener-
ally impure. The quantity of proteid in the precipitate
can most conveniently be estimated by determining the
nitrogen present and multiplying by the appropriate factor
for the method employed.
The estimation of nitrogen may be made by any of the
methods used in organic analysis, but Kjeldahl's method
and Wanklyn's albuminoid ammonia process possess the
great advantage that the precipitate or residue does not
require to be powdered or intimately mixed with a solid, a
process always difificult in such cases and frequently almost
The soda-lime method^ of Will and Varentrap much
used at one time for the determination of nitrogen in
organic residues, has been largely replaced by Kjeldahl's
For the estimation of peptones and albumoses, the
nitrogen in the dried sodium phosphotungstate precipitate
may be determined by Kjeldahl's process ; or the precipi-
tate may be decomposed by alkali and the peptones etc.
in solution estimated by Wanklyn's method ; or by com-
parison of the colour obtained in biuret reaction with
the colour given by standard peptone solutions under
1 A full account of the soda-lime process is given in Fresenius,
Quantitative Analysis, 7th ed. vol. ii. pt. i.
CH. X] OF PROTEIDS. 255
similar conditions. Since it is not common to find any
considerable quantities of these substances in vegetable
tissues under normal circumstances the colorimetric
method will generally suffice.
Estimation of Proteids in alkaline solution.
[As the other nitrogenous substances have been ex-
tracted by the previous treatment with water, all the
nitrogen in this solution may be taken to be in the form
Evaporate to dryness a portion of the alkaline solution,
weigh the residue and determine the nitrogen, in the whole
or a weighed portion of it, by Kjeldahl's method.
For a full account of all necessary details of manipula-
tion and precautions desirable see Sutton, Volumetric
Analysis, 5th ed. pp. 68 â€” 70.
Nitrogen found x 6*3 = Proteids.
or Ammonia x 5*2 = Proteids.
Add so much of the solution as will contain not more
than 5 â€” 10 milligrams of proteids to 500 c.c. of pure
distilled water, free from ammonia, in a large retort, and
distil after addition of 50 c.c. of alkaline permanganate
Determine the ammonia in the distillate byNesslerising.
For a full account of the details of the process see
Wanklyn, Water-analysis (Trlibner and Co.), or Sutton,
Volumetric Analysis, pp. 389 and 397.
[The second 50 c.c. of distillate should be Nesslerised
first and if it is found to contain much annnonia, the first
256 PROTEIDS. [CH. X
50 c.c. of distillate should be diluted to 500 c.c. before
being Nesslerised. The first 50 c.c, if Nesslerised with-
out dilution, will often yield a precipitate or colour too
deep to be accurately compared with the standard.]
It is absolutely necessary that all the apparatus and
water employed should be quite free from ammonia.
These operations are best performed in a room kept for
the purpose where the apparatus and pure distilled water
can be stored.
If 600 c.c. of water nearly free from ammonia are
placed in the retort before beginning the determinations
and 100 c.c. are distilled off and thrown away, the 500 c.c.
of water will be quite free from ammonia and the whole
apparatus perfectly clean.
The rapidity with which a number of estimations
can be made by this process renders it very suitable
for comparative experiments involving determination of
Ammonia x 10 = Proteids.
Estimation of soluble Proteids.
The nitrogen in the precipitate caused by copper
acetate may be determined by using Kjeldahl's method on
the dry precipitate, or, the precipitate may be suspended
in water, decomposed by HaS and the proteids estimated
in solution (after expelling ELgS) by the albuminoid
Estimation of Peptones and Albumoses is seldom
required, but methods by which it can be made are men-
tioned above, and the details can easily be worked out
CH. X] AMIDES. 257
from a knowledge of the processes described for proteids.
For the colorimetric method it is necessary to make
standard solutions of pure peptone : any good commercial
peptone may be used for this purpose, but it should be
carefully dried before weighing out the quantities needed
for the standard solutions.
Estimation of Amides.
Amides are estimated in a solution from which proteids
and peptones and albumoses have been removed by the
processes described for qualitative testing. The separa-
tion of amides, if several are present, is a matter of much
difficulty; it is generally sufficient to estimate the total
' amides ' together and calculate the result as asparagin ;
results obtained in this way should be stated as ' amides
calculated as asparagin.'
This method (decomposing amides with potassium
nitrite and sulphuric acid, measuring the volume of
nitrogen evolved) gives very good results but is rather
difficult to manipulate and, unless great care is taken at
all stages of the operation, gives results seriously too high.
For the details of Sachsse's method see Dragendorff,
Â§ 241, p. 245.
With asparagin the reaction is
CHsN.O^ + 2HNO2 = C.HeO, + 4N 4- 2H,0.
Therefore in this process 56 gi's. nitrogen = 1 32 grs.
D. A. 17
258 NITRATES. [CH. X
Method based on the action of dilute acids.
The solution is boiled for about one hour with 5 p.c.
sulphuric acid in a flask with reflux condenser, and the
ammonia produced is estimated either gasometrically by
sodium hypobromite in a nitrometer, or by distillation with
magnesia into a measured volume of standard acid.
With asparagin the reaction is
C4H8N,03 + H,0 = C,H,N04 + NH3.
Therefore in this process 14 grs. nitrogen = 132 grs.
anhydrous asparagin (or 17 gi's. ammonia = 132 grs.
For details of the estimation of 'combined ammonia/
see Sutton, Volumetric Analysis, pp. 59 and 482.
[The gasometric process in the nitrometer is carried
out as in the estimation of urea by sodium hypobromite.]
Estimation of Nitrates and Nitrites.
(a) Evaporate 100 c.c. to 5 c.c. (about) on the
Decompose in the nitrometer with strong sulphuric
acid over mercury and measure the volume of nitric
oxide. This gives the NO from nitrites and nitrates
[reduce volume to 0Â° and 760 mm.].
[See Sutton, pp. 226 and 362.]
(y8) In another portion of original solution (diluted
with pure distilled water if much nitrite is present),
estimate the nitrite by Griess' colorimetric method.
Calculate the vol. of NO from nitrite (at 0Â° and
760 mm.) in 100 c.c. of original, and subtract this from
CH. X] EXPERIMENTS. 259
the total NO observed in (a) : the difference is NO from
[See Sutton, p. 366.]
(1 CO. of NO (at 0" and 760 mm.)
= -00170 grs. NA or '00242 gl^s. NA).
Ammonia and other nitrogenous compounds will not
seriously interfere with these reactions.
For estimation of ammonia present in aqueous solution
see Dragendorfif, Â§ 97, p. 81.
Experiments on nitrogenous Metabolism.
Make an aqueous extract of young plants of Onohrychis
sativa^ which have been grown in the dark and extract the
residue (after exhaustion with water) with 2 p.c. NaOH
(see p. 245).
Examine the NaOH extract for proteids insoluble in
Examine the aqueous extract for soluble proteids,
peptones and albumoses, amides.
Examine another portion of the aqueous extract for
ammonia, nitrates, and nitrites.
Compare the amounts of insoluble proteids, soluble
proteids, peptones and albumoses (if qualitative examina-
1 The seeds should be put to germinate 8 â€” 10 weeks before the
material for these experiments is required.
260 EXPERIMENTS. [CH. X
tion has shewn these to be present) and amides in
extracts (made as for qualitative testing) of
(1) seeds of Onohrychis sativa,
(2) young shoots of Onohrychis sativa grown under
(3) young shoots of Onohrychis sativa which have
been gro\vn in the dark or kept in the dark for several
days before extracting.
Compare the amounts of ammonia, nitrates and
(1) normal young shoots of Onohrychis sativa
which have grown in sand,
(2) in normal young shoots of Onohrychis sativa
which have been kept for several days soon after germina-
tion in the dark in absence of free oxygen,
(3) in normal young shoots of Onohrychis sativa
which have been grown in sand watered with a solution
containing ammonium nitrate ('5 p.c).
OILS AND FATS â€” GLYCERIN.
Particulars of the chemistry of vegetable oils and
fats may be obtained from
Spon's Encyclopaedia, ' Oils and Fatty Substances.'
Beilstein. Handhuch der organischeR CJiemie, 3rd
ed. vol. I. ; Pflanzenfette.
KoNiG. Ghemie der menschl. Nahrungs- und Genuss-
miUel, Berlin, 1889.
Read : â€”
Green. Proc. Roy. Soc. xlviii. (1890).
SiGMUND. Sitzh. k Akad. Wien (1890).
Muller. Ber. d. d. hot. Ges. viii. (1890).
SuROZ. (Abst.) Bot Cent, Beiheft i. (1891).
Oils and Fats.
These substances will all be in the benzene or
petroleum ether extract â€” the extract may also contain
some volatile oils, terpenes, resins, etc., but except in the
case of barks the amount of these is generally incon-
262 OILS AND [CH. XI
siderable. The oils and fats will always be mixtures of
glycerides of fatty acids or of free fatty acids or of both.
It is not necessary to attempt the separation of the
acids, which is a difficult and complicated operation ; it will
suffice to determine the amount of substances which can
be saponified by potash, and the glycerin produced in
Oils and Fats.
Qualitative examination (of benzene extract).
Distil off the greater portion of the solvent, transfer
the residue to a dish and completely evaporate the
remainder of the solvent on a water bath or in a steam
Note the character of the residue, whether liquid, solid,
or semi-solid, etc.
Warm the residue on a water-bath with strong potash
solution for about one hour, dilute with water and filter if
necessary (the portion of the residue which remains
undissolved is probably resins and terpenes). To the hot
solution add hydrochloric acid until litmus shows acidity,
and allow to cool.
The free fatty acids will generally solidify in a cake on
cooling but may remain liquid, in either case they can
easily be separated by filtering through a wet filter-paper.
The acid filtrate is examined for glycerin (it will
contain considerable quantities of potassium chloride) by
evaporating to the smallest possible volume on a water-
bath and applying the following tests for glycerin to
portions of the residue.
CH. Xl] FATS. 268
(1) heat with fragments of acid potassium sulphate
for several minutes.
Glycerin gives a very pungent acrid characteristic
smell (smell of acrolein).
(2) Add a few drops of copper sulphate solution
and then excess of potash solution.
If glycerin is present a deep blue liquid is produced
instead of precipitate of cupric hydroxide.
If much glycerin is present in the residue it may be
recognised by its physical characters.
[In the case of palmitin, one of the constituents of
palm oil, the changes would be represented by the follow-
ing equations, which may be considered typical for
vegetable oils and fats.
Palmitin (glyceryl tripalmitate) = C3H5(Ci6H3i0.2);j
Pot. palmitate (soluble in water).
C3H,(C,eH3,0,)3 4- :3K0H = 3C,eH3,0,K + C3H, (0H)3
CeHoiO^K + HCl = C,6H3iO,H + KCl.
Palmitic acid (insoluble in water).
If saponification does not take place easily with
aqueous potash, alcoholic potash may be substituted,
and the heating must be done under a reflux condenser :
in this case the alcohol must be distilled off before
acidifying with hydrochloric acid.]
Determination of total oils and fats.
Proceed as in qualitative examination, but weigh the
264 OILS AND [CH. XI
residue obtained on evaporating off the solvent ; as soon
as the weight is constant, this may roughly be taken
as the weight of oils and fats.
If any considerable amount of unsaponifiable residue
remains after treatment with alkali, this must be washed,
dried and weighed, and its weight subtracted from the
total residue before calculating as total oils and fats.
Determination of free fatty acids.
The weight of these may be obtained by weighing the
dried cake, or residue insoluble in water, after acidifying
the products of saponification.
Determination of glycerin.
A convenient and fairly accurate process, applicable in
these cases, is based on the power of glycerin to dissolve
cupric hydroxide in alkaline solution.
The acid filtrate, from which free fatty acids have been
removed, is neutralised with soda, and then rendered
strongly alkaline by the addition of 10 c.c. of a strong
soda solution ; a dilute solution of copper sulphate is then
run in with constant agitation until a permanent precipi-
tate of cupric hydroxide is obtained.
Similar experiments are then made with the same
quantities of water and soda to determine (by means of a
standard solution of pure glycerin) how much glycerin
corresponds to the solution of the amount of cupric
hydroxide noticed in the original experiment, which will
be the amount of glycerin in the products of saponi-
[Various modifications of this process are used, but the
method described is one of the simplest, and is quite
CH. Xl] FATS. 265
accurate enough for comparative experiments where the
differences in the amount of glycerin are likely to be con-
Experiments on Oils and Fats in germinating
(1) Determine the oils and fats in dry seeds of
(2) Determine the oils and fats in seedlings (dried
at 100Â° C.) which have germinated and grown for about
(3) Determine the oils and fats in seeds (dried at
100Â° C.) which have just commenced to germinate.
TANNINS AND GLUCOSIDES.
Full particulars of the chemistry of tannins may be
obtained from the following works :
Watts. Dictionary of Chemistry IV.
Trimble. The Tannins. Vol. i. Philadelphia, 1892.
Vol. il 1894
Procter. A Teoct-hook of Tanning, Spon, 1885.
On the Physiology of Tannins, read : â€”
Kraus. Grundlinien zii einer Physiologie des Gerh-
stoffes (Leipzig, 1887).
Reinitzer. Bemerkungen zur Physiologie des Gerb-
stofifes, Ber. d. deut. hot. Ges. vii. (1889).
Gardiner. Function of Tannin in Vegetable Cells.
Proc. Camh. Phil. Sac, 1884.
Waage. (Distribution, etc. of Phloroglucin.) Ber.
d. deut. hot. Ges. viii. (1890).
On the solubility, etc. of glucosides consult :
Beilstein. Handhuch der organischen Ghemie, 2nd
ed., vol. III. ; Glykoside.
CH. XII] TANNINS. 267
Tannins and Glucosides.
These substances will be completely extracted by
alcohol of '850 Sp. G., and will therefore be present in the
alcohol extract. (Extract No. II.)
After evaporating off the alcohol, taking up with
water and filtering, the solution has to be examined for
tannins, glucosides, and sugars, but the whole of the
sugars will rarely be present in this solution.
Some proteids may pass into this solution but they
are generally rendered insoluble by the treatment, and if
they are found to be present after removing the tannins
they can be precipitated by alcoholic mercuric chloride
(as in removing peptones) before examining for sugars :
small quantities of amides if present may be neglected.
It sometimes happens that the solution is rather
strongly acid, in this case it should be exactly neutralised
with dilute soda before commencing examination.
Under the heading of tannins and glucosides are
included a large number of different compounds which are
in many respects widely different in properties, although
possessing certain characters in common, and it is conse-
quently rather difficult to give general instructions.
It is assumed, in treating of these compounds, that it
is not desired to make experiments concerning their rela-
tions to metabolism ; but from any point of view it is of
the greatest importance that the processes for their com-
plete removal from solution should be thoroughly studied.
Tannins and most glucosides readily give the reduction
of Fehling's, Sachsse's, and the other solutions commonly
268 TANNINS AND [CH. Xll
used as tests for sugars ; and since they are liable to split
off glucoses under the action of acids, etc., it is not too
much to say that it is hardly ever possible to ascertain
certainly whether free glucoses were, or were not, origi-
nally present without first removing tannins. Micro-
chemical tests for reducing sugars are useless unless it
has first been shown conclusively that tannins are absent ;
all tannins, not only those which are glucosides, readily
reduce Fehling's, etc., solutions.
It may in some cases be of interest to ascertain
whether a given tannin is a glucoside or not, since there
can be little doubt that the glucose which can be split off
from such tannins is plastic material. This is not quite
so easy as might seem apparent, but a satisfactory and
fairly simple process is given on p. 273, by which this can
If it is required to compare the amounts of tannins in
two extracts, one of the modifications of the permanganate
process can be used â€” full details are given in Trimble,
The Tannins, pp. 48 â€” 51, or Sutton, Volumetric Analysis.
It is generally sufficient to ascertain whether tannins
are present, and then to determine which of the methods
given is best adapted for their removal.
The addition of basic lead acetate will almost certainly
carry down the whole of the tannins, many glucosides,
and any proteids, etc. present, but the precipitate is very
liable to contain the glucoses as well, and it is not at all
easy to wash them out with alcohol or water. The use of
large quantities of water for washing the precipitate is
particularly to be avoided, as the precipitates of tannins
CH. XIl] GLUCOSIDES. 269
with metallic salts appear to be more or less decomposed
by pure water. It is better therefore not to resort to this
method unless absolutely necessary.
Most of the natural tannins resemble in their proper-
ties the substauce described as mimo- tannic acid (tannin
from catechu and various species of Acacia), while they
differ from commercial gallotannic acid, which gives
several rather peculiar reactions and is not a good type of
the general characters of vegetable tannins.
Phloroglucin is present in many cases in woody tissues
(an account of its distribution is given by Waage, loc. cit.
p. 265), but it is not probable that it is a plastic sub-
It would occur in the alcohol extract if present, and, as
it reduces Fehling's, etc. solutions, should be tested for
where woody tissues have been extracted.
It is easily removed if present by shaking with ether
before examining for tannins and glucosides, which are
not soluble in ether.
After the removal of tannins and proteids (if present)
portions of the solution may be tested for glucosides if it
is supposed they are likely to be present.
The reactions and solubility of the various glucosides
likely to occur must be studied, and if they can be
removed by shaking with an immiscible solvent it is best
to proceed in this way (the process should be carried out
in the same way as in removing tannins with acetic ether).
If a glucoside should be present which is not removed
270 TANNINS AND [CH. XII
from aqueous solution by any immiscible solvent it must
be precipitated by some appropriate reagent and the
excess of reagent removed.
Before examination, the original extract must be
evaporated till all alcohol is completely driven off and the
residue taken up with water, and filtered if necessary.
Any residue insoluble in water may be assumed to be
resins, etc., and neglected.
If the solution is acid it should be exactly neutralised
with dilute soda before testing.
Qualitative tests for Tannins.
Test portions of the neutral aqueous solution with : â€”
(1) A few drops of 'neutral' ferric chloride (large
excess must be carefully avoided).
Blue-black or dull green coloration shows tannins.
(2) A few drops of solution of potassium ferri-
cyanide and ammonia.
Reddish-brown coloration changing to brown shows
(3) Gelatin solution.
Dirty white precipitate shows tannins.
(4) Lime-water (Ca(0H)2).
Blue, brown, or red colour or precipitate shows
(5) Uranium acetate.
Brown precipitate, or reddish-brown or brown colour
CH. XIl] GLUCOSIDES. 271
Qualitative tests for Phloroglucin. .
[Where a woody tissue has been extracted, phloroglucin
should be tested for in the aqueous solution obtained from
the alcohol extract as described above.
Shake a portion of aqueous solution with ether, sepa-
rate the ether layer and evaporate off the ether, take up
the residue with water.
Test portions of the neutral aqueous solution so ob-
(1) Ferric chloride.
Deep violet colour shows phloroglucin.
(2) Freshly cut pine wood and hydrochloric acid.
Reddish-violet colour shows phloroglucin.
(For further tests for Phloroglucin see Beilstein, 2nd
ed. vol. II. Phloroglucin.)
If phloroglucin is present the whole of the aqueous
solution may be shaken with ether : the lower layer drawn
off and warmed (to expel ether) can then be used for
testing for tannins, etc.]
Removal of Tannins before examining for
As explained above all vegetable tannins do not
behave in the same way to reagents, excepting perhaps to
gelatin and lead acetate ; it is therefore advisable to try
the reactions of a small portion of the solution before
deciding which method to adopt for the removal of the
The methods may be tried in the following order